virus reviews and research - sbv.org.br · hélio gelli pereira award committee fernando rosado...

AddressUniversidade Feevale, Instituto de Ciências da Saúde

Estrada RS-239, 2755 - Prédio Vermelho, sala 205 - Laboratório de Microbiologia MolecularBairro Vila Nova - 93352-000 - Novo Hamburgo, RS - Brasil

Phone: (51) 3586-8800E-mail: F.R.Spilki - [email protected]

http://www.vrrjournal.org.br

Virus Reviews and ResearchJournal of the Brazilian Society for Virology

Volume 19, September/October 2014, Supplement 2

Annals of the XXV Brazilian Congress of Virology & IX Mercosur Meeting of VirologySeptember/October, 28 - 01, 2014, Ribeirão Preto Convention Center, Ribeirão Preto, São Paulo, Brazil

EditorsEdson Elias da SilvaFernando Rosado Spilki

BRAZILIAN SOCIETY FOR VIROLOGY BOARD OF DIRECTORS (2013-2014)

Officers

President: Dr Eurico de Arruda NetoVice-President: Dr Bergmann Morais RibeiroFirst Secretary: Dr Mauricio Lacerda NogueiraSecond Secretary: Dra Luciana Jesus da CostaFirst Treasurer: Dr Luis Lamberti Pinto da SilvaSecond Treasurer: Dra Clarice Weis ArnsExecutive Secretary: Dr Fabrício Souza Campos

Fiscal CouncilorsDra Maria Luisa BarbosaDra Viviane Fongaro BotossoDra Maria Angela Orsi

Area Representatives

Basic Virology (BV)Dra Paula Rahal, UNESP (2013 – 2014)Dr Davis F. Ferreira, UFRJ (2013 – 2014)

Environmental Virology (EV)Dra Célia Regina Barardi, UFSC (2013 – 2014)Dr Fernando Rosado Spilki, Feevale (2013 – 2014)

Human Virology (HV)Dr Luiz Tadeu Figueiredo, USP-RP (2013 – 2014)Dra Nancy Bellei, UNIFESP (2013 – 2014)

Immunobiologicals in Virology (IV)Dra Sílvia Cavalcanti, UFF (2013 – 2013)Dr Edson Elias da Silva, Fiocruz (2013 – 2014)

Plant and Invertebrate Virology (PIV)Dr Paulo Brioso, UFRJ (2013 – 2014)Dr Tatsuya Nagata, UNB (2013 – 2014)

Veterinary Virology (VV)Dr Paulo Brandão, USP (2013 – 2014)Dra Rita Cubel, UFF (2013 – 2014)

XXV Brazilian Congress of Virology & IX Mercosur Meeting of Virology, Ribeirão Preto, São Paulo, BrazilVirus Reviews and Research, Volume 19, Supplement 2, 2014

Organizing CommitteeBergmann Morais Ribeiro, UNBCélia R. M. Barardi, UFSCClarice Weis Arns, UNICAMP Davis Fernandes Ferreira, UFRJEdson Elias da Silva, Fiocruz ‘Chairman of the Committee’ Eurico de Arruda Neto, USPFabrício Souza Campos, UFRGSFernando Rosado Spilki, Universidade FEEVALE Francisco Murilo Zerbini, UFVLuciana Jesus Costa, UFRJLuis Lamberti Pinto da Silva, USP Luiz Tadeu Figueiredo, USPMaria Angela Orsi, LANAGROMaria Luisa Barbosa, Instituto Adolfo LutzMaurício Lacerda Nogueira, FAMERP Nancy Bellei, UNIFESP Paula Rahal, UNESP Paulo Sergio Torres Brioso, UFRRJPaulo Eduardo Brandão, USPRita de Cássia Nasser Cubel Garcia, UFFSilvia Maria Baeta Cavalcanti, UFFTatsuya Nagata, UNBViviane Fongaro Botosso, Instituto Butantan

Hélio Gelli Pereira Award CommitteeFernando Rosado SpilkiLuciana Barros de ArrudaLuis Lamberti Pinto da SilvaMauricio Lacerda Nogueira


Financial Support

CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCNPQ Conselho Nacional de Desenvolvimento Cientifico e TecnológicoFAPESPFundação de Amparo à Pesquisa do Estado de São Paulo

ExhibitorsQIAGENSARSTEDTSÍNTESESOTELABVECOFLOW

SponsorsSilver Sponsorship - LOBOVBronze Sponsorship - ROCHE and SIGMA ALDRICH

OrganizersOffice Marketing Eventos

General Information

Secretary Office HoursSeptember, 28st - 8am - 8pmSeptember, 29nd - 7am - 7:30pmSeptember, 30rd - 7am - 7:30pmOctober, 01th - 7am - 6pm

Name BadgeName badges will be required for access in all activities, including lunch.

Media Desk (for lecturers only)The media desk will be open as scheduled for the secretary office.Data - files with presentations - must be delivered at the media desk at least 2 hours before the scheduled time for the presentation. Please note that the use of personal computers by presenters will not be allowed.

Lounge AreaA lounge area will be available for lecturers, invited persons and SBV staff.

CertificatesCertificates of attendance will be available on line at www.sbv.org.br 15 days after the end of the meeting.

Travel AgencyFLYTOUR is the official tour operator of the XXV Brazilian Congress of Virology.Booking and information: [email protected](16) 3024.3900

Poster PresentationsThe posters must be exposed from 1pm until the end of the session, and then removed.

Poster Session 1: September 29nd, 6:30-8:30 pm• Human Virology• Immunobiologicals in Virology• Plant and Invertebrate Virology

Poster Session 2: September 30nd, 6:30-8:30 pm• Basic Virology• Environmental Virology• Veterinary Virology


XXV Brazilian Congress of Virology Scientific Program

Sun

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28 2: 00 pm - 4:00 pm

Room TurquesaSatellite Workshop 1 - Enteric viruses

1. Replication and virus-cell interaction of Norovirus - Ian Goodfellow, University of Cambridge, UK 2. Kobuvirus (Aichivirus B) infection in Brazilian cattle herds - Amauri Alcindo Alfieri, Londrina State University, Londrina, Paraná, PR, Brazil 3. Norovirus and sapovirus in day-care children – Divina das Dores de Paula Cardoso, Federal University of Goiás, Goiânia, GO, Brazil 4. Enteric viruses in drinking water, Southern Brazil – Fernando Rosado Spilki, Feevale University, Novo Hamburgo, RS, Brazil (Chair)

Room ÁgataSatellite Workshop 2 - Next-generation sequencing of viral genomes

1. Discovery of novel viruses from insects and plants - Fernando Lucas de Melo, University of Brasília, Brasília, DF, Brazil 2. Next generation sequencing as a tool for discovering and monitoring arboviruses – Renata Dezengrini Slhessarenko, Federal University of Mato Grosso, Cuiabá, MT, Brazil 3. Next generation sequencing technologies for genome and transcriptome studies of Porcine Circovirus - João Pessoa Araújo Junior, São Paulo State University, Botucatu, SP, Brazil 4. Next generation sequencing and metagenomic virome analysis from environmental samples – Tatsuya Nagata, University of Brasília, Brasília, DF, Brazil (Chair)

Room ÔnixSatellite Workshop 3 - Respiratory viruses: from bench to trench

1. Update on newcastle disease virus – Muhammad Munir - The Pirbright Institute, UK 2. Coronaviruses in bats: what is new? - Edison Luiz Durigon, University of São Paulo, São Paulo, SP, Brazil (Chair)3. Targeting cell division cycle 25 homolog B to regulate influenza virus replication - Ana Claudia Trocoli Torrecilhas, Federal University of São Paulo, São Paulo, SP, Brazil 4. Respiratory viral infections in immunosuppressed patients - Meri Bordignon Nogueira, Federal University of Paraná, Curitiba, PR, Brazil

Room TopázioSatellite Workshop 4 - Virology nucleus AUGM (Asociación de Universidades Grupo Montevideo)

1. An overview of the mission and priorities of the Molecular Virology Nucleus (AUGM) - Juan Daniel Claus, Coordinator of AUGM, Universidad Nacional del Litoral, Santa Fé, Argentina 2. Molecular biology of baculovirus: basic and applied studies - Víctor Romanowski, Universidad Nacional de La Plata, La Plata, Argentina 3. State-of-art of viral vector-borne zoonoses in Argentina - Marta Contigiani, Universidad Nacional de Córdoba, Córdoba, Argentina 4. Mechanism of carcinogenesis associated with human papillomavirus infection: role of the domains of protein PDZ interaction - Daniela Gardiol, Universidad Nacional de Rosario, Rosario, Argentina5. Detection of human papillomavirus and co-risk factors for the development of cervical cancer in Paraguay - Laura Mendoza Torres, Universidad Nacional de Asunción, Asunción, Paraguay 6. Development of molecular tools for identifying viral - Adriana Giri, Universidad Nacional de Rosario, Rosario, Argentina 7. Emerging viruses in Uruguay: some yes and others no - Juan Arbiza, Universidad de la República, Montevideo, Uruguay 8. Distribution of vaccinia virus in the South America - Erna Geessien Kroon, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 9. Recent developments in recombinant viral vaccines for veterinary use in Brazil - Eduardo Furtado Flores, Federal University of Santa Maria, Santa Maria, RS, Brazil10. Coronavirus in wild birds in Brazil - Clarice Arns, Campinas State University, Campinas, SP, Brazil (Chair)

4:00 pm - 4:30 pm Coffee break

7:00 pm - 9:00 pm

Room EsmeraldaOpening Ceremony Keynote conference:

• Infections of neurons by herpesviruses: How do they do that? Lynn Enquist, Princeton University, Princeton, NJ, USA

9:00 pm - 11:00 pm Cocktail reception and Visit to Exhibits


Mon

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8:00 am - 9:00 am

Room ÁgataMini-course 1: Introduction to viral replication

• Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Room TurquesaMini-course 2: Proteomic applications in virology

• Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil Room TopázioMini-course 3: Molecular diagnosis in virology

• Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil

Room ÔnixMini-course 4: Viral phylogeny and sequence analysis

• Camila Romano, University of São Paulo, São Paulo, SP, Brazil

9:00 am - 10:30 am

Oral presentations: session 1 to 4 • SESSION 1 – Human Virology I - Room Topázio• SESSION 2 – Plant and Invertebrate I - Room Turquesa• SESSION 3 – Veterinary Virology I - Room Ônix• SESSION 4 – Environmental Virology - Room Ágata

10:30 am - 11:00 am Coffee-break and Visit to Exhibits

11:00 am - 12:00 noon

Room EsmeraldaConference 1:

• Host cell restriction factors for HIV-1 replicationJeremy Luban, University of Massachusetts Medical School, Worcester, USA; Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil (Chair)

12:00 pm- 2:00 pm Lunch-break and Visit to Exhibits

2:00 pm - 3:00 pm


• Emerging and re-emerging virus infections in pigsTanja Opriessnig, University of Edinburgh, Scotland, UK and Iowa State University, USA; Paulo Eduardo Brandão, University of São Paulo, São Paulo, SP, Brazil (Chair)

3:00 pm - 4:30 pm

Oral presentations: session 5 to 8 • SESSION 5 – Basic Virology I - Room Ágata• SESSION 6 – Human Virology II - Room Topázio• SESSION 7 – Plant and Invertebrate Virology II - Room Turquesa• SESSION 8 – Veterinary Virology II - Room Ônix

4:30 pm - 5:00 pm Coffee-break and Visit to Exhibits

5:00 pm - 6:30 pm

Oral presentations: session 9 to 12 • SESSION 9 – Human Virology III - Room Topázio• SESSION 10 – Basic Virology II - Room Ágata• SESSION 11 – Veterinary Virology III - Room Ônix• SESSION 12 – Immunobiologicals in Virology - Room Turquesa

6:30 pm - 8:30 pm Poster Session 1 and Visit to Exhibits / Social Mixer

Tues

day,

Sep

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8:00 am - 9:00 am

Room ÁgataMini-course 1: Introduction to viral replication

• Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Room TurquesaMini-course 2: Proteomic applications in virology

• Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil Room TopázioMini-course 3: Molecular diagnosis in virology

• Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil

Room ÔnixMini-course 4: Viral phylogeny and sequence analysis

• Camila Romano, University of São Paulo, São Paulo, SP, Brazil


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Round Tables 1 to 4 9:00 am - 10:30 am

Room TopázioRound Table 1 - Veterinary Virology - Challenges and progress in disease control

1. Challenges in monitoring avian influenza virus - Helena Lage Ferreira, University of São Paulo, Pirassununga, SP, Brazil (Chair)2. Viruses in wild birds with special reference to avian paramyxoviruses – Muhammad Munir, The Pirbright Institute, UK 3. Porcine epidemic diarrhea virus - Tanja Opriessnig, University of Edinburgh, Scotland, UK 4. Update on FMDV - Rossana Allende, Pan American Foot-and-Mouth Disease Center (Panaftosa), RJ, Brazil

Room ÔnixRound Table 2 - Plant and Invertebrate Virology - Viral metagenomics

1. Polydnavirus: diversity, genome organization, ecological implications and biotechnological potential: CfPDV as a case study - Fernando Luis Cônsoli, University of São Paulo, São Paulo, SP, Brazil 2. Genomic analysis of new baculoviruses isolated in Brazil - Daniel Mendes Pereira Ardisson Araujo, University of Brasília, Brasília, DF, Brazil 3. Bean and associated weeds ssDNA virome – Simone Ribeiro, EMBRAPA CENARGEN, Brasília, DF, Brazil 4. Cell modulation factors are carried along structural proteins in the AgMNPV-2D budded and occluded virions - Carla Torres Braconi, University of São Paulo, São Paulo, SP, Brazil

Chair: Bergmann Morais Ribeiro, University of Brasília, Brasília, DF, BrazilRoom ÁgataRound Table 3 - Environmental Virology - Advances in environmental virology

1. Standard Methods to concentrate viruses from freshwater samples - Garland Shay Fout, U. S. Environmental Protection Agency, Cincinnati, OH, USA 2. Avian parvoviruses as markers for environmental contamination – Silvia Bofill-Mass, University of Barcelona, Barcelona, Spain 3. Virus inactivation in sludge – Maria Elisa Magri, Federal University of Lavras, Lavras, MG, Brazil 4. Virus disinfection in drinking, reuse and sea waters - Célia Regina Monte Barardi, Federal University of Santa Catarina, Florianópolis, SC, Brazil

Chair: Fernando Rosado Spilki, University Feevale, Novo Hamburgo, RS, BrazilRoom TurquesaRound Table 4 - Basic Virology - HIV-host cell interaction

1. HIV replication mechanisms - Jeremy Luban, University of Massachusetts Medical School, USA 2. Diverse mechanisms involved in translation initiation of HIV-1 transcripts - Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3. Mechanisms by which the HIV-1 accessory protein Nef modifies protein trafficking in host cells – Luis Lambert Pinto da Silva, University of São Paulo, Ribeirão Preto, SP, Brazil (Chair)

10:30 am - 11:00 am Coffee break and Visit to Exhibits

11:00 am - 12:00 noon


• Polydnaviruses as symbionts and immunomodulatory gene delivery vehiclesMichael R. Strand, University of Georgia, Athens, GA, USA

12:30 pm- 13:30 pm

Room Ônix• Evolution of canine parvovirus - a need for new vaccines?

Dr. Uwe Truyen, Editor-in-chief of Veterinary Microbiology, Institute for Animal Hygiene, University of Leipzig, Leipzig, Germany

12:00 am - 2:00 pm Lunch break and Visit to Exhibits

2:00 pm - 3:00 pm


• Early events in flavivirus infectionJolanda Smit, The University Medical center Groningen, Groningen, The Netherlands

Round Tables 5 to 8 3:00 pm - 4:30 pm

Room TurquesaRound Table 5 - Basic Virology - Interactions of flaviviruses with hosts

1. Effects of yellow fever virus infection on splicing – Mauricio Lacerda Nogueira, São José do Rio Preto Medical School, São José do Rio Preto, SP, Brazil (Chair)2. Structure of dengue virus proteins – Ronaldo Mohana Borges, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 3. The influence of dengue vírus infection on Aedes aegypti feeding behaviour - Rafael Maciel de Freitas, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil


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Room TopázioRound Table 6 - Human Virology - Human infections by emerging viruses

1. Central nervous system infections by arboviruses - Maria Paula Mourão, Heitor Vieira Dourado Foundation for Tropical Medicine, Manaus, AM, Brazil 2. Clinical pathogenesis of hantavírus pulmonar syndrome - Luiz Tadeu Moraes Figueiredo, University of São Paulo School of Medicine, Ribeirão Preto, SP, Brazil (Chair)3. Chloroquine for dengue virus infections – Benedito Antônio Lopes da Fonseca, University of São Paulo School of Medicine, Ribeirão Preto, SP, Brazil

Room ÔnixRound Table 7 - Veterinary Virology - Molecular diversity of animal viruses

1. Reservoirs of vaccinia virus in Brazil – Erna Geessien Kroon, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 2. Molecular epidemiology and evolution of porcine parvoviruses – André Felipe Streck, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil 3. Emergent viruses associated with gastroenteritis in dogs and cats - Tatiana Xavier de Castro, Fluminense Federal University, Niterói, RJ, Brazil 4. Molecular diversity of canine distemper virus – Alice Fernandes Alfieri, Londrina State University, Londrina, PR, Brazil

Chair: Rita de Cássia Nasser Cubel Garcia, Fluminense Federal University, Niterói, RJ, BrazilRoom ÁgataRound Table 8 - Human virology/Immunobiologic products - Computational virology applied to dengue virus studies

1. Genetic mosaicism in two strains of dengue type 3 - Paolo Marinho de Andrade Zanotto, University of São Paulo, São Paulo, SP, Brazil (Chair)2. Molecular Dynamics of dengue NS3 protease conformations – Jaime Martins de Santana, University of Brasília, Brasília, DF, Brazil3. Early budding stages of a Dengue Virus particle unveiled by Molecular Dynamics - Ricardo Oliveira dos Santos Soares, University of São Paulo, Ribeirão Preto, SP, Brazil

4:30 pm - 5:00 pm Coffee-break and Visit to the Exhibits


Room TurquesaRound Table 9 – CanceledRoom ÔnixRound Table 10 - Basic Virology - Virus-host cell interactions and virus assembly

1. Assembly of icosahedral virus capsids - Juliana Cortines, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil 2. Innate immunity against poxviruses – Daniel Mansur, Federal University of Santa Catarina, Florianópolis, SC, Brazil (Chair)3. Molecular mechanisms of human cytomegalovirus assembly – Alberto Fraile Ramos, INMETRO, Rio de Janeiro, RJ, Brazil 4. Cell interactions of human respiratory syncytial virus (HRSV) N, P and M proteins – Armando Morais Ventura, University of São Paulo, São Paulo, SP, Brazil

Room ÁgataRound Table 11 - Immunobiologicals - Experimental animal models in virology

1. Experimental infection of Callithrix penicillata with dengue virus – Pedro Fernando Vasconcelos, Evandro Chagas Institute, Belém, PA, Brazil (Chair)2. Experimental infection of Mus musculus with Rocio vírus – Juliana Helena Chávez, Federal University of Mato Grosso, Cuiabá, MT, Brazil3. Experimental infection of Macaca fascicularis with human parvovirus B19 – Marcelo Alves Pinto, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil

Room TopázioRound Table 12 - Helio Gelli Pereira Award Oral Presentations

6:30 pm - 8:30 pm Poster Session 2 and Visit to Exhibits 10:00 pm Social mixer and dance


Wed

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8:00 am – 9:00 am

Mini-course 1: Introduction to viral replication / Room Ágata• Luciana Jesus da Costa, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Mini-course 2: Proteomic applications in virology / Room Turquesa • Alessandra Vidotto, Medical School of São José do Rio Preto, São José do Rio Preto, SP, Brazil

Mini-course 3: Molecular diagnosis in virology / Room Topázio• Roberta Bronzoni, Federal University of Mato Grosso, Sinop, MT, Brazil, and Luciano Kleber de Souza Luna, University of São Paulo, Ribeirão Preto, SP, Brazil

Mini-course 4: Viral phylogeny and sequence analysis / Room Ônix• Camila Romano, University of São Paulo, São Paulo, SP, Brazil

9:00 am - 10:00 am


• The cardiac innate response to viral infectionsBarbara Sherry, North Carolina State University, Raleigh, NC, USA

10:00 am - 10:30 am Coffee-break and Visit to Exhibits

10:30 am - 12:30 pm Room EsmeraldaSBV Business meeting and election of new board of directors

12:30 pm - 2:00 pm Lunch break and Visit to Exhibits

12:30 pm - 2:00 pm Room ÔnixThis week in virologyVincent Racaniello, Columbia University, New York, NY, USA

2:00 pm -3:00 pm


• The emergence of chikungunya virus in South AmericaXavier Nicolas de Lamballerie, Emerging Viruses Unit, University of Marseille, France


Room ÔnixRound Table 13 - Veterinary Virology - Antivirals for animal viruses

1. Viral activity of natural compounds and derivates against pet’s virus and animals herpesvirus – Márcia Rogéria Lamêgo Almeida, Federal University of Viçosa, Viçosa, MG, Brazil 2. In vitro and in vivo inhibition of rabies virus by RNAi – Paulo Eduardo Brandão, University of São Paulo, São Paulo, SP, Brazil (Chair) 3. Control of ruminant morbillivirus replication by small interfering RNA – Renata Servan de Almeida, Agricultural Research for Development (CIRAD), Montpellier, France 4. Antiherpes activity of Brazilian marine organisms - Claudia Maria Oliveira Simões, Federal University of Santa Catarina, Florianópolis, SC, Brazil

Room ÁgataRound Table 14 - Plant and Invertebrate Virology - Control of emerging plant viruses

1. Recent advances in the study of papaya sticky disease – Patricia Fernandes, Federal University of Espírito Santo, Vitória, ES, Brazil 2. Badnaviruses: Biology and management in tropical crops – Andrew Geering, University of Queensland, Brisbane, Australia 3. Epidemiology and management of viruses transmitted by Bemisia tabaci in solanaceous crops – Jorge Alberto Marques Rezende, University of São Paulo, Piracicaba, SP, Brazil (Chair)

Room TurquesaRound Table 15 - Immunobiologicals - Advances in biotechnology products with applications in virology

1. Parapoxviruses as vectors – Diego Diel, University of Illinois, Urbana-Champaign, USA 2. Expression of viral vaccines in plants – Tatsuya Nagata, National University of Brasília, Brasília, DF, Brazil (Chair)3. Advances in biotechnology products with applications in virology - Marcos Freire, Bio-Manguinhos/Fiocruz, Rio de Janeiro, RJ, Brazi4. Carbon nanotubes applied to point-of-care tesing for immunoassay – Rosa Amalia Fireman Dutra, Federal University of Pernambuco, Recife, PE, Brazil

Room TopázioRound Table 16 - Human Virology - Advances in HTLV research

1. Effects of HTLV-1 infection on human mesenchymal stromal cells - Simone Kashima Haddad, University of São Paulo, Ribeirão Preto, SP, Brazil (Chair)2. Leukotrienes in HTLV-1-related neuroinflammatory diseases - Lúcia Helena Faccioli, University of São Paulo, Ribeirão Preto, SP, Brazil 3. HTLV-1 reverse genetics and search for an intracellular carrier of ORF-1 gene product – Luiz Carlos Alcântara, Gonçalo Moniz Research Center, Fiocruz, Salvador, BA, Brazil


Hélio Gelli Pereira AwardThe evaluation of papers submitted for the Helio Gelli Pereira Award will take place on Tuesday, September 30, from 5:30 to 7:00 pm. Presenters will have 10 minutes for their presentation, followed by 5 minutes of questions from the examiners and, if time permits, from the audience.

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Hélio Gelli Pereira AwardA RESOURCEFUL GIANT: APMV IS ABLE TO INTERFERE WITH THE HUMAN TYPE I INTERFERON SYSTEMSilva, L.C.F.; de Almeida, G.M.; Oliveira, D.B.; Dornas, F.P.; Campos, R.K.; La Scola, B.; Kroon, E.G.; Ferreira, P.C.P.; Abrahão, J.S.

THE MOVEMENT PROTEINS (NSM) OF DISTINCT TOSPOVIRUSES SELF-INTERACT, ASSOCIATE WITH THE COGNATE NUCLEOCAPSID (NP) AND WITH HETEROLOGOUS NSM AND NP PROTEINS AND WITH THE CELLULAR MEMBRANE IN A NON-TRANSMEMBRANE FASHIONLeastro, M.O.; Pallás, V.; Resende, R.O.; Navarro, J.A.S.

HIV-1 TRANSCRIPTS USE IRES-INITIATION UNDER CONDITIONS WHERE CAP-DEPENDENT TRANSLATION IS RESTRICTED 1 BY POLIOVIRUS 2A. PROTEAS - Amorim, R.; da Costa, S.M.; Cavaleiro, N.; da Silva, E.E.; da Costa, L.J.

ACANTHAMOEBA POLYPHAGA MIMIVIRUS INHIBITS TRANSCRIPTION OF AMOEBAL 2 SUBTILISIN-LIKE SERINE PROTEINASE MRNA AND CIRCUNVENTS CELL ENCYSTMENTBoratto, P.V.M.; Almeida, G.M.F.; Botelho, L.; Lima, A.; Costa, A.O.; Santos, D.A.; La Scola, B.; Kroon, E.G.; Ferreira, P.C.P.; Abrahão, J.S.

HTLV-1 INFECTS HUMAN MESENCHYMAL STROMAL CELL IN VITRO AND MODIFIES THEIR PHENOTYPIC CHARACTERISTICSRodrigues, E.S.; de Macedo, M.D.; Pinto, M.T.; Orellana, M.D.; Rocha Junior, M.C.; de Magalhães, D.A.R.; Tanaka, Y.; Takayanagui, O.M.; Covas, D.T.; Kashima, S.


Oral Presentation

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SESSION 1 – Human Virology IHV33 - DETECTION OF IMPORTED CASES OF CHIKUNGUNYA VIRUS IN BRAZIL: VIRUS ISOLATION, MOLECULAR AND SEROLOGICAL ASSAYSCunha, M.S.; Maeda, A.Y.; Bisordi, I.; Rocco, I.M.; da Silva, F.G.; de Souza, R.P.; Coimbra, T.L.M.; Nogueira, J.S.; Kisielius, J.J.; Silveira, V.R.; Oliveira, A.L.R.; Suzuki, A.

HV125 - ORAL HUMAN PAPILLOMAVIRUS INFECTION IN HIV-POSITIVE PEOPLESilva, C.O.; Santos, L.S.; de Azevedo, K.M.L.; Pereira, O.M.D.; Oliveira, L. do H. dos S.

HV147 - NEW REAL TIME PCR ASSAY FOR RAPID DETECTION OF TRICHODYSPLASIA SPINULOSA-ASSOCIATED POLYOMAVIRUS IN BIOLOGICAL SAMPLESUrbano, P.R.; Nali, L.H. da S.; Pannuti, C.S.; Pierrotti, L.C.; Neto, E.D.; Romano, C.M.

HV213 - EXPRESSION PROFILE OF HUMAN ENDOGENOUS RETROVIRUS W (HERV-W) LOCI FROM MULTIPLE SCLEROSIS PATIENTS UNDER DISTINCT THERAPIES BY HIGH THROUGHPUT SEQUENCINGNali, L.H. da S.; Urbano, P.R.P.; Silva, D.F.; Olival, G.S. do; Penalva de Oliveira, A.C.; Romano, C.M.

HV240 - HIGH FREQUENCE OF SAFFOLD VIRUS IN TONSIL TISSUES FROM PATIENTS WITH CHRONIC TONSILLAR HYPERTROPHYLuna, L.K. de S.; Valera, F.C.P.; Tamashiro, E.; Lima, W.T.A.; Arruda, E.

HV247 - FIRST REPORT OF NOROVIRUSES OCCURRENCE AMONG ALLOGENEIC STEM CELL TRANSPLANT RECIPIENTS IN BRAZIL: EVIDENCE FOR PROLONGED VIRAL EXCRETION AND LONG-TERM VIRAL RNA PRESENCE IN THE BLOODSouza, M.; Lemes, L.N.; Correa, T. dos S.; Fiaccadori, F.S.; Souza, K.M.C. da Silva, L.P.; Arantes, A. de M.; Cardoso, D. das D. de P.; Souza, M.

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SESSION 2 – Plant and Invertebrate IPIV150 - TWO NOVEL BEGOMOVIRUS SPECIES FROM THE NEW WORLD WITH FEATURES RECALLING OLD WORLD BEGOMOVIRUSESGodinho, M.T.; Lima, A.T.M.; Xavier, C.A.D.; Zerbini, F.M.

PIV160 - COMPLETE GENOME SEQUENCE OF TWO NEW VIRUSES ISOLATES ASSOCIATED TO COTTON BLUE DISEASE BREAKING RESISTANCE IN BRAZILVaslin, M.F.S.; Fausto, A.K. da S.; Romanel, E.; Silva, T. da F.; Schrago, C.G.; Galbieri, R.; Bélot, J.L.; Vasli, M.F.S.

PIV210 - SYSTEMIC ACQUIRE RESISTANCE INDUCED BY CELL WALL PEPTIDOGALACTOMANNAN OF THE FUNGUS CLADOSPORIUM HERBARUM MEDIATES VIRUS PROTECTION IN TOBACCO PLANTSVaslin, M.F.S.; Montebianco, C.B.; Mattos, B.B.B.; Silva, T. da F.; Romanel, E.; Bergter, E.B.; Vaslin, M.F.S.

PIV221 - PROTEIN SYNTHESIS ANALYSIS OF INSECT CELL LINES INFECTED WITH SPODOPTERA FRUGIPERDA MULTIPLE NUCLEOPOLYHEDROVIRUS (SFMNPV)Marcio, M.S.; Sihler, W.; Souza, M.L.

PIV295 - CHARACTERIZATION OF A BACULOVIRUS HOST-ACQUIRED PROTEASE INHIBITOR AND ITS ABILITY TO AUGMENT VIRAL VIRULENCEArdisson, A.D.M.P.; Rohrmann, G.A.; Bergmann, M.R.; Rollie, J.C.

PIV357 - IDENTIFICATION OF A NEW DCL3 GENE IN COTTON GOSSYPIUM RAIMONDII (ULBRICH) D-GENOME AND STUDY OF THE ROLE OF NEW ISOFORMS OF GENE SILENCING RELATED GENES IN COTTON VIRUS INFECTED PLANTSVaslin, M.F.S.; Romanel, E.; Moura, M.; Lei, G.; Paterson, A.

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SESSION 3 – Veterinary Virology I VV27 - ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE APOBEC3H GENE OF DOMESTIC CATS (FELIS CATUS) AND THEIR ASSOCIATION WITH THE SUSCEPTIBILITY TO FELINE IMMUNODEFICIENCY VIURUS AND FELINE LEUKEMIA VIRUS INFECTIONSCostenaro, J.G.; de Castro, F.L.; Junqueira, D.M.; de Medeiros, R.M.; da Silva, T.R.; Knak, M.B.; Almeida, S.E. de M.; Campos, F.S.; Roehe, P.M.; Franco, A.C.

VV37 - PARTIAL CHARACTERIZATION OF AVIAN INFLUENZA VIRUS (H11N9) IN MIGRATORY BIRDS (ARENARIA INTERPRES) CAPTURED IN AMAZON REGION, BRAZILde Araujo, J.; Júnior, S.M. de A.; Gaidet, N.; Hurtado, R.F.; Walker, D.; Thomazelli, L.M.; Ometto, T.; Seixas, M.M.M.; Galindo, D.B.; Webby, R.J.; Webster, R.G.; Durigon, E.L.

VV96 - AVIAN CORONAVIRUS AND AVIAN BORNAVIRUS DETECTION IN FREE-RANGING PSITTACINESLima Neto, D.F.; Barnabé, A.C.S.; Caserta, L.; Martini, M.C.; Simas, P.V.M.; Nagel, N.E.; Lierz, M.; Hafez, H.M.; Felippe, P.A.N.; Arns, C.W.


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SESSION 3 – Veterinary Virology I VV113 - PRIMARY AND SECONDARY MUCOSAL IMMUNITY AFTER CHALLENGE WITH BRAZILIAN AVIAN INFECTIOUS BRONCHITIS VARIANTOkino, C.H.; Mores, M.A.Z.; Montassier, H.J.; Mattos, G.L.M.; Brentano, L.; Coldebella, A.; Ritterbusch, G.A.; Esteves, P.A.; Trevisol, I.M.

VV172 - ASTROVIRUS DETECTION IN THE BAT TADARIDA BRASILIENSIS (GEOFFROY, 1824: CHIROPTERA, MOLOSSIDAE) FROM RIO GRANDE DO SUL STATE, BRASILDuppont, Priscilla Medeiros - Pacheco, Susi M. - Rosa, Júlio Cesar A. - Streck, André Felipe-Alves, Christian D. B. T. - Da Silva, Mariana S. - Budaszewski, Renata F. - Weber, Matheus N.-Canal, Cláudio W.

VV207 - DETECTION OF AVIAN GROUP F ROTAVIRUS IN FECAL SAMPLES OF BROILER CHICKENS IN PARA STATE, BRAZILBezerra, D.A.M.; Silva, M.J.M.; Bezerra, D.A.M.; Silva, R.R.; Soares, L.S.; Mascarenhas, J.D.P.

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SESSION 4 – Environmental VirologyEV90 - PATHOGENS INACTIVATION BY UNIONIZED AMMONIA FOR THE PRODUCTION OF SAFE BIOFERTILIZER FROM SWINE EFFLUENT AND SLUDGEFongaro, G.; Kunz, A.; Magri, M.E.; Zeredo, A.C.B.; Schissi, C.D.; Zaguini, J.; Barardi, C.R.M.

EV140 - ANALYSIS OF MARINE VIRAL FAMILIES ASSOCIATED WITH SIDERASTREA STELLATA FESTIVAL IN ARRAIAL DO CABO AND BÚZIOS, RJ, BRAZILSantana-Pereira, P.; Giongo, V.; Pedrosa, L.G.; Paula, J.E.F.B.; Amorim, L.; Paixão, I.C.N.P.

EV255 - DETECTION OF HUMAN ADENOVIRUSES IN SEDIMENTS FROM STREAMS IN URBAN AREAS FROM RIO DOS SINOS WATERSHEDStaggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; Soliman, M.C.; Souza, F.G. de; Fabres, R.B.; Luz, R.B.; Zirbes, G.; Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, S.E. de M.

EV306 - ENVIRONMENTAL POLIOVIRUS SURVEILLANCE: DETECTION OF WILD AND VACCINE - DERIVED POLIOVIRUSES IN SÃO PAULO SEWAGEHachich, E.M.; Barbosa, M.RF.; Garcia, S.C.; Bonanno, V.M.S.; Eduardo, M.B.P.; Burlandy, F.M.; Costa, E.V.; da Silva, E.E.; Sato, M.I.Z.

EV430 - DIFFERENT PHAGES ACTION PATHWAYS IN REDUCING BIOFILMS FORMED BY OIL REFINERIES ASSOCIATED BACTERIADias, R.S.; Fonseca, L.A.B.V.; Albanese, J.M.; Silva, L.C.F.; Suhette, R.; Torres, A.P.R.; Sousa, M.P.; Silva, C.C.; Oliveira, V.M.; de Paula, S.O.

EV457 - FIRST IDENTIFICATION OF ACANTHAMOEBA POLYPHAGA MIMIVIRUS IN LIMNOPERNA FORTUNEI FROM GUAIBA LAKE, RIO GRANDE DO SUL, BRAZILdos Santos, R.N.; Arantes, T.; Correa, R.A.; de Albuquerque, N.R.M.; Kluge, M.; Santos, C.; dos Santos, C.P.; Campos, F.C.; Roehe, P.M.; Franco, A.C.

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SESSION 5 – Basic Virology IBV12 - OROPOUCHE ORTHOBUNYAVIRUS MINIGENOME SYSTEM REVEALS SIGNIFICANT ERRORS IN THE PUBLISHED GENOME SEQUENCESAcrani, G.O.; Lunel, N.L.T.; Spiegel, M.; Weidmann, D.M.D.S.; Nunes, M.R.T.; Ellott, R.M.

BV86 - KROON VIRUS: A NEW AND HIGHLY RESISTANT GIANT VIRUS WITH EXTRA SHELL LAYERDornas, F.P.; Silva, L.C.F.; Boratto, P.V.M.; Rodrigues, R.A.; Freitas, G.M.; Assis, F.L.; Trindade, G.S.; Kroon, E.G.; Cortines, J.; La Scola, B.; Abrahão, J.S.

BV95 - DYNAMIC COVALENT BONDS IN VIRAL CAPSIDS: AUTO PROTEOLYSIS INVOLVED IN CAPSID MATURATION CAN BE REVERSED UNDER PHYSIOLOGICAL CONDITIONSDomitrovic, T.; Matsui, T.; Johnson, J.E.

BV105 - ANALYSIS OF DIFFERENTIAL METHYLATION PROFILE IN PATIENTS WITH DENGUE USING METHYLATION-SENSITIVE ARBITRARILY PRIMED POLYMERASE CHAIN REACTIONAlessandra, V.B.T.G.; Morais, S.M. de S.; Ferreira, J.M.S.; Malaquias, L.C.C.; Coelho, L.F.L.

BV171 - STUDY OF STOP CODONS RECURRENCE IN HEPATITIS C VIRUS NS5A PROTEINCampos, G.R.F.; Bittar, C.; Rahal, P.

BV175 - CHARACTERIZATION OF THE INTERACTION BETWEEN THE METHYLOSSOME AND THE HUMAN RESPIRATORY SYNCYTIAL VIRUS NUCLEOCAPSIDOgawa, J.; Oliveira, A.; Simabuco, F.; Éleouët, J.F.; Ventura, A.


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mSESSION 6 – Human Virology IIHV258 - DETECTION AND TYPING OF HUMAN PAPILLOMAVIRUS BY NESTED MULTIPLEX PCR (NMPCR) REVEALS NEW EPIDEMIOLOGICAL PROFILEFaria, M.G.; Matias, B.F.; Costa, E.P.; Julião, J.A.S.; Junior, P.C.F.; Goulart, L.R.

HV262 - USE OF CHIMERIC PROTEINS EXPRESSED IN PROKARYOTIC SYSTEM IN THE DEVELOPMENT OF A SCREENING METHOD FOR THE EVALUATION OF ANTI-HTLV-1 ANTIBODIESSantos, D.M. da S.; do Carmo, A.P.; Martins, M.L.; da Fonseca, F.G.; Stancioli, E.F.B.

HV311 - HEPATITIS C VIRUS QUASISPECIES ANALYSIS USING ULTRA-DEEP PYROSEQUENCINGYamasaki, L.H.T.; Khudyakov, Y.; Vaughan, G.; Raeva, L.M.G.; Diminitrova, Z.E.; Skums, P.; Rendon, D.S.C.; Jardim, A.C.G.; Bittar, C.; Mello, I.M.V.G.C.; Rahal, P.

HV340 - EFFECTS OF HTLV-1 INFECTION ON BONE MARROW CELLS FROM HTLV-1 INFECTED INDIVIDUALSRodrigues, E.S.; Favarin, M. do C.; Macedo, M.D. de; Otaguiri, K.K.; Orellana, M.D.; Takayanagui, O.M.; Slavov, S.N.; Covas, D.T.; Kashima, S.

HV346 - EVIDENCE OF HEPATITIS E VIRUS CIRCULATION IN RURAL AMAZONIAMerlone, M.P.; Vitral, C.L.; Oliveira, J.M.; Silva, J.P.; Nunes, M.S.; Ferreira, M.U.; Pinto, M.A.

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SESSION 7 – Plant and Invertebrate Virology IIPIV378 - THE FIRST COMPLETE GENOME SEQUENCE OF COFFEE RINGSPOT VIRUS; AN EMERGING THREAT TO COFFEE PRODUCTION AND QUALITYFigueira, A. dos R.; Ramalho, T.O.; Sotero, A. de J.; Duarte, P. de S.G.; Wang, R.; Farman, M.; Goodin, M.M.

PIV383 - SUBCELLULAR LOCALIZATION OF COFFEE RINGSPOT VIRUS PROTEINSRamalho, T.O.; Figueira, A.R.; Sotero, A.J.; Duarte, P.S.G.; Wang, R.; Farman, M.; Goodin, M.M.

PIV423 - COMPLETE GENOME SEQUENCE OF A TOBACCO-INFECTING TOMATO BLISTERING MOSAIC VIRUSFernandes, J.E.F.; Melo, F.L.; Ribeiro, B.M.; Ribeiro, G.S.

PIV452 - COMPLETE GENOME SEQUENCE OF A NOVEL IFLAVIRUS ISOLATED FROM OPSIPHANES INVIRAESilva, L.A.; Melo, F.L.; Tinôco, R.S.; Fernandes, O.A.

PIV509 - THE FUNCTIONAL ANALYSIS OF DISTINCT TOSPOVIRUS MOVEMENT PROTEINS (NSM) REVEALS DIFFERENT BEHAVIOR FROM THE ALFALFA MOSAIC VIRUS (AMV) MODEL SYSTEMLeastro, M.O.; Peiró, A.; Pallás, V.; Navarro, J.A.S.; Resende, R.O.

PIV510 - THE MOVEMENT PROTEINS (NSM) OF DISTINCT TOSPOVIRUSES SELF-INTERACT, ASSOCIATE WITH THE COGNATE NUCLEOCAPSID (NP) AND WITH HETEROLOGOUS NSM AND NP PROTEINS AND WITH THE CELLULAR MEMBRANE IN A NON-TRANSMEMBRANE FASHIONLeastro, M.O.; Pallás, V.; Resende, R. de O.; Navarro, J.S.

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SESSION 8 – Veterinary Virology IIVV229 - LONG-TERM CIRCULATION OF INFLUENZA A VIRUSES IN SWINE IN BRAZILSchaefer, R.; Nelson, M.; Gava, D.; Cantão, M.E.; Zanella, J.R.C.

VV260 - EVIDENCE OF ORTHOPOXVIRUS CIRCULATION AMONG URBAN DOMESTIC CATS, MINAS GERAIS, BRAZILMiranda, J.B.; Costa, G.B.; Almeida, G.G.; Figueiredo, P. de O.; Bonjardim, C.A.; Ferreira, P.C.P.; Abrahão, J.S.; Kroon, E.G.; Trindade, G. de S.

VV303 - DEVELOPMENT AND STANDARDIZATION OF A MULTIPLEX RT-PCR FOR THE DETECTION THE PRINCIPAL RESPIRATORY VIRUS IN BIRDSSakata, S.T.; Rosales, C.A.R.; Reischak, D.; Orsi, M.A.

VV316 - SEQUENCING AND PHYLOGENETIC ANALYSIS OF NUCLEOPROTEIN OF RABIES VIRUS ISOLATED FROM CATTLE IN THE STATE OF RIO GRANDE DO SULFernandes, M.E.S.; Pereira, P.M.C.; Achkar, S.M.; Oliveira, R.N.; Ferreira, J.C.; Rosa, J.C.A.; Castilho, J.G.; Almeida, L.L.; Carnieli, Jr. P - Roehe PM - Batista HBCR

VV321 - HERPESVIRUS SIMPLEX TYPE-1 OUTBREAK IN MARMOSETS (CALLITHRIX)Ullmann, L.S.; Kurissio, J.K.; Linhares, A.G.; Linhares, L.S.A.; Milanelo, L.; Araujo Jr, J.P.

VV333 - ABC OF CANINE PARVOVIRUSES GENOGROUPS AND ANTIGENIC TYPESStreck, A.F.; Borchardt, A.; Daudt, C.; Canal, C.W.


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mSESSION 9 – Human Virology IIIHV353 - IMPLEMENTATION OF NEXT GENERATION SEQUENCING TECHNIQUES FOR PATHOGEN DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS WITH ENCEPHALITIS AND MENINGITISNunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; Romano, C.M.

HV417 - CO-DETECTIONS OF RESPIRATORY VIRUSES BY QPCR IN THE ABSENCE OF SYMPTOMS OF ACUTE RESPIRATORY INFECTION (ARI): NEW INSIGHTS ON SOURCES OF VIRUS SHEDDINGCriado, M.F.; Modena, J.L.P.; de Paula, F.E.; de Jesus, B.L.S.; Pestana, N.F.; Prates, M.C.; Silva, M.L.; Saturno, T.; Tamashiro, E.; Valera, F.C.P.; Lima, W.T.A.; Arruda, E.

HV428 - ANALYSIS OF INFLUENZA A(H1N1)PDM09 VIRAL LOAD IN PATIENTS WITH DIFFERENT CLINICAL PRESENTATIONPerosa, A.H.; Bellei, N.

HV436 - HIV-1 ENV SUBTYPES AND DISEASE PROGRESSION IN A COHORT OF HIV-1 POSITIVE INDIVIDUALS FROM RIO DE JANEIRO, BRAZILLeite, T.; Campos, D.; Coelho, A.; Teixeira, S.; Veloso, V.; Guimarães, M.; Morgado, M.G.

HV491 - OPTIMIZATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR NAKED-EYE DETECTION OF DENGUE VIRUS INFECTIONMonteiro, D.C.S.; Carvalho, B.K.S.; Nascimento, V.A.; Souza, V.C.; Naveca, F.G.

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SESSION 10 – Basic Virology IIBV225 - HIV-1 TRANSLATION INITIATION IS INHIBITED BY POLIOVIRUS 2A PROTEASEAmorim, R.; Costa, L.J. da

BV280 - 2A PROTEASE FROM HUMAN RHINOVIRUS C15 DOES NOT PROVIDE INTERFERON RESISTANCE AND TRAFD1 HAS AN ANTIVIRAL ACTIVITY AGAINST HUMAN RHINOVIRUSES AND ENCEPHALOMYOCARDITIS VIRUSGagliardi, T.B.; Dabelic, R.; Racaniello, V.R.

BV345 - IDENTIFICATION OF THE CELLULAR AND MOLECULAR DETERMINANTS OF FLAVIVIRUS FUSIONEsposito, D.L.A.; Nguyen, J.B.; Modis, Y.

BV379 - DEVELOPMENT OF TWO POTENTIAL TOOLS AGAINST THE REPLICATION OF THE DENGUE VIRUSFujimura, P.T.; Vaz, E.R.; Cardoso Jr, C.A.M.; Carvalho, W.J.; Reis, C.F.; Santos Jr, C.D.; Freitas, G.R.O.; Yokosawa, J.; Carneiro, A.P.; Goulart, L.R.; Vecchi, L.; Ueira,C.U.V.

BV490 - IN VITRO MICROEVOLUTION OF DENGUE VIRUS 4 IN AEDES ALBOPICTUS CELLSNascimento, V.A.; Souza, V.C.; Naveca, F.G.

BV501 - ALIX IS A HOST CELL FACTOR REQUIRED FOR LYSOSOMAL TARGETING OF CD4 BY HIV-1 NEFJanuário, M.E. da S.; da Silva, E.M.L.; de Castro, R.O.; Amorim, N.A.; da Costa, L.J.; da Silva, L.L.P.

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SESSION 11 – Veterinary Virology IIIVV351 - GENETIC CHARACTERIZATION OF CANINE CIRCOVIRUS DETECTED IN STOOL SAMPLES FROM DOGS IN THE SOUTH REGION OF BRAZILCruz, T.F.; Batista, T.N.; Baccarin, A.M.; Gradiz, J.J.; Vieira, E.M.; Tozato, C.C.; Kurissio, J.K.; Araujo Jr, J.P.

VV365 - PHYLOGENETIC ANALYSIS OF AICHIVIRUS B IN FECAL SAMPLES FROM CALVES IN BRAZILRribeiro, J.; Lorenzetti, E.; Medeiros, T.N. da S.; Junior, J.C.R.; Bronkhorst, D.E.; Crespo, S.E.I.; Favero, L.M.; Alfieri, A.F.; Alfieri, A.A.

VV393 - VIABILITY OF VACCINIA VIRUS IN EXPERIMENTALLY CONTAMINATED CHEESES AT DIFFERENT TIMES OF THE MATURATION PROCESSRehfeld, I.S.; Fraiha, A.L.S.; Matos, A.C.D.; Guedes, M.I.M.C.; Costa, A.G.; de Souza, M.R.; Lobato, Z.I.P.

VV396 - MOLECULAR DETECTION AND PHYLOGENETIC RELATIONSHIPS OF AN AMPHIBIAN RANAVIRUS ASSOCIATED TO OUTBREAKS OF MORTALITY IN BRAZILIAN FISH FARMQueiroz, S.R. de A.; de Pádua, S.B.; Filho, R.N. de M.; Araújo, A.P. de; Maganha, S.R. de L.; Navarro, J. de O.; Lima, M.P.; de Alencar, A.L.F.; Arruda, E. de P.; Munin, F.S.; Fernandes, A.M.; de Sousa, R.L.M.

VV470 - VIRUCIDAL ACTIVITY OF A PURIFIED COMPOUND ISOLATED FROM PRASIOLA CRISPA AGAINST EQUINE HERPESVIRUS TYPE 1Marinho, R.S.S.; Ramos, C.J.B.; Leite, J.P.G.; Belo, C.A.D.; Pereira, A.B.; Teixeira, V.L.; Paixão, IC.N.P.; Pinto, A.M.V.

VV473 - EMERGING VIRUSES MONITORING IN BATS, RODENTS AND MARSUPIALS FROM BRAZILIAN RAIN FOREST REGION AND AMAZON REGION COLLECTED BETWEEN 2008 AND 2013Campos, A.C. de A.; Anthony, S.J.; de Araújo, J.; Ometto, T.; Hurtado, R.; Nardi, M.S.; Nava, A.; Solorio, M.R.; Souza, M.C.P.; Rostal, M.; Loh, E.; Torrelio, C.M.Z.; Aguirre, A.A.; Daszak, P.; Durigon, E.L.


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SESSION 12 – Immunobiologicals in VirologyIV100 - EVALUATION OF TETRAVALENT AND CONSERVED SYNTHETIC PEPTIDES VACCINES DERIVED FROM DENGUE VIRUS ENVELOPE DOMAIN I AND IIRocha, R.P.; Franco, I.R.; Livonesi, M.C.; Fumagalli, M.J.; Rodrigues, N.F.; da Costa, L.C.F.; dos Santos, M.C. da S.G.; Rocha, E.S. de O.; Kroon, E.G.; Malaquias, L.C.C.; Coelho, L.F.L.

IV183 - THE IMPACT OF ADJUVANT IN WHOLE INACTIVATED VIRUS (WIV) VACCINES FOR INFLUENZA A INFECTION IN PIGS WITH VACCINE-ASSOCIATED ENHANCED DISEASESouza, K.C.; Rajao, D.; Loving, L.C.; Gauger, C.P.; Vincent, L.A.

IV218 - SELECTION AND CHARACTERIZATION OF MIMOTOPES THROUGH PHAGE DISPLAY FOR IMMUNOGLOBULIN A DETECTION IN HPV DIAGNOSISMatias, B.F.; Lima, M.I.S.; Faria, M.G.; Costa, E.P.; Alves, P.T. Pereira, U.P.; Fernandes Jr, P.C.; Goulart, L.R.

IV477 - INFLUENZA: DEVELOPMENT OF A REVERSE GENETICS SYSTEM BY YEAST-BASED HOMOLOGOUS RECOMBINATION AND ITS APPLICATION IN THE IMPROVED VACCINE PRODUCTION IN CULTURE CELLSilva Jr, J.V.J.; Cruz, F. da S.P.; Bertani, G.R.; Machado, A. de M.V.; Gil, L.H.V.G.

IV482 - RECONSTRUCTION OF LATENT AND REACTIVATED QUASISPECIES OF SIVMM251 INFECTING RHESUS MONKEYS AFTER TREATMENT WITH THE LATENCY ANTAGONIST INGENOL-BGonçalves, G. dos S.; Abreu, C.M.; Price, S.L.; Gama, L.; Lewis, M.; Pianowski, L.F.; Tanuri, A.

HELIO GELLI PEREIRA AWARD

XXV Brazilian Congress of Virology & IX Mercosur Meeting of VirologySeptember/October, 28 - 01, 2014, Ribeirão Preto, São Paulo, Brazil

Helio Gelli Pereira Award 17

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Helio Gelli Pereira Award

THE MOVEMENT PROTEINS (NSM) OF DISTINCT TOSPOVIRUSES SELF-INTERACT, ASSOCIATE WITH THE COGNATE NUCLEOCAPSID (NP) AND WITH HETEROLOGOUS NSM AND NP PROTEINS AND WITH THE CELLULAR MEMBRANE IN A NON-TRANSMEMBRANE FASHIONLeastro, M.O.1; Pallás, V.2; Resende, R.O.1; Navarro, J.A.S.2

1. UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900

2. IBMCP/CISIC-UPV - Instituto de Biología Molecular y Celular de Plantas do Consejo Superior de Investigaciones Científicas de la Universidad Politécnica de Valencia, Ingeniero Fausto Elio, s/n 46022 Valencia - Espana.

Tospovirus most economically important plant viruses worldwide with significant losses in agronomic production. The M RNA genomic segment that encodes a non-structural protein (NSm) involved in cell-to-cell and systemic movement commonly associates with ribonucleoprotein complexes (RNP) located in the tubular structure of plasmodesmata and seems to interact with cell periphery forming aggregates on the cell surface. Membrane-proteins topology can be predicted based on amino acid sequence analysis or experimentally determined by biochemical techniques, such as alkaline enzymatic modifications, Tag modification by glycosylation and chemical modification. One of the aims of this study was to understand the in vivo membrane association of the movement proteins of the tospoviruses species BeNMV, CSNV, TCSV and TSWV. For this purpose, we used two different approaches in planta: the bimolecular fluorescence complementation (BiFC) assay and the chemical treatments addressed to identify the type of MP-membrane association. Was proposed that the four MPs analyzed are peripherally associated to the cytosolic face of the ER membranes in living plant cells. Finally, dimerization analysis of homologous and heterologous tospoviurus proteins (NSm and N) generated information that can facilitate a better interpretation of viral proteins interactions, strengthening the understanding of mixed infection issues.

A RESOURCEFUL GIANT: APMV IS ABLE TO INTERFERE WITH THE HUMAN TYPE I INTERFERON SYSTEMSilva, L.C.F.; de Almeida, G.M.; Oliveira, D.B.; Dornas, F.P.; Campos, R.K.; La Scola, B.; Kroon, E.G.; Ferreira, P.C.P.; Abrahão, J.S.

Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG.

Acanthamoeba polyphaga mimivirus (APMV) is a giant, double-stranded virus of the Mimiviridae family that was discovered in 2003. Recent studies have shown that this virus is able to replicate in murine and human phagocytes and might be considered a putative human pathogen that causes pneumonia. However, there is little data regarding APMV and its host defense relationship. In the present study, we investigated how some components of the interferon (IFN) system are stimulated by APMV in human peripheral blood mononuclear cells (PBMC) and how APMV replication is affected by IFN treatment. Our results demonstrated that APMV is able to replicate in human PBMC, inducing type I Interferons (IFN) but inhibiting interferon stimulated genes (ISG) induction by viroceptor and STAT-1 and STAT-2 dephosphorylation independent mechanisms. We also showed that APMV is resistant to the antiviral action of interferon-alpha2 (IFNA2) but is sensitive to the antiviral action of interferon-beta (IFNB1). Our results demonstrated the productive infection of professional phagocytes with APMVand showed that this virus is recognized by the immune system of vertebrates and inhibits it. It provides the first data regarding APMVand the IFN system interaction and raise new and relevant evolutional questions about the relationship between APMV and vertebrate hosts.




HIV-1 TRANSCRIPTS USE IRES-INITIATION UNDER CONDITIONS WHERE CAP-DEPENDENT TRANSLATION IS RESTRICTED 1 BY POLIOVIRUS 2A. PROTEAS - Amorim, R.1; da Costa, S.M.1; Cavaleiro, N.1; da Silva, E.E.2; da Costa, L.J.1

1. Instituto de Microbiologia, Departamento de Virologia. Universidade Federal do Rio de Janeiro. 5 Rio de Janeiro. RJ – Brazil.

2. Laboratório de Enterovírus. Instituto Oswaldo Cruz. Fundação Oswaldo Cruz. Rio de Janeiro. RJ – Brazil.

The 30 different species of mRNAs synthesized during the HIV-1 replication cycle are all capped and polyadenilated. Internal ribosome entry sites have been recognized in the 5’ untranslated region of some mRNA species of HIV-1, which would contribute to an alternative mechanism of initiation of mRNA translation. However, the Cap-dependent translation is assumed to be the main mechanism driving the initiation of HIV-1 protein synthesis. In this work, we describe a cell system in which lower to higher levels of transient expression of the poliovirus 2A protease strongly inhibited cellular Cap-dependent translation with no toxic effect to the cells during a 72-hour time frame. In this system, the synthesis of HIV-1 proteins was inhibited in a temporal dose-dependent way. Higher levels of 2A protease expression severely inhibited HIV-1 protein synthesis during the first 24 hours of infection consequently inhibiting viral production and infectivity. Intermediate to lower levels of 2A Protease expression caused the inhibition of viral protein synthesis only during the first 48 hours of viral replication. After this period both protein synthesis and viral release were recovered to the control levels. However, the infectivity of viral progeny was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation contribute to the synthesis of HIV-1 proteins; during the first 24-48 hours of viral replication HIV-1 protein synthesis is strongly dependent on Cap-initiation, while at later time points IRES-driven translation initiation is sufficient to produce high amounts of viral particles.

ACANTHAMOEBA POLYPHAGA MIMIVIRUS INHIBITS TRANSCRIPTION OF AMOEBAL 2 SUBTILISIN-LIKE SERINE PROTEINASE MRNA AND CIRCUNVENTS CELL ENCYSTMENTBoratto, P.V.M.1; Almeida, G.M.F.1; Botelho, L.1; Lima, A.2; Costa, A.O.1; Santos, D.A.2; La Scola, B.3; Kroon, E.G.1; Ferreira, P.C.P.1; Abrahão, J.S.1

1. Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Laboratório de Vírus, Av. Antonio Carlos, 6627, Pampulha, Belo 10 Horizonte, MG, Brazil.

2. Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Laboratório de Micologia, Av. Antonio Carlos, 6627, Pampulha, Belo Horizonte, MG, Brazil.

3. URMITE CNRS UMR 6236 – IRD 3R198, Aix Marseille Université, Marseille, France

Acanthamoeba is a genus of free-living amoebas distributed worldwide known as agents of public health concern for causing disease in humans. On natural environments, they can act as hosts for many microorganisms, including members of the family Mimiviridae, one of the largest and most complex group of viruses ever described. Many studies have investigated the interactions between these protozoa and some of its harbored microorganisms (mostly pathogenic bacteria) but few have explored the mechanisms involving their interaction with giant viruses. In the present study, we explored the behavior presented by Acanthamoeba spp. and Mimivirus (APMV) in response to different conditions of incubation and interaction. The results obtained suggest that amoeba encystment is a crucial phenomenon for avoiding viral infection success, and therefore it is the target of an arm wrestling between the virus and amoeba. We demonstrate that once amoeba encystment is triggered, trophozoites become significantly less permissive to APMV and, remarkably, APMV is able to interfere with the expression of the serine proteinase mRNA related to amoebal encystment. These results may represent one of the most ancient examples of fight for supremacy involving a virus and a eukaryotic cell.




HTLV-1 INFECTS HUMAN MESENCHYMAL STROMAL CELL IN VITRO AND MODIFIES THEIR PHENOTYPIC CHARACTERISTICSRODRIGUES, E.S.1,2; DE MACEDO, M.D.1,2; PINTO, M.T.1,2; ORELLANA, M.D.1,2; ROCHA JUNIOR, M.C.1,2; DE MAGALHÃES, D.A.R.1; TANAKA, Y.4; TAKAYANAGUI, O.M.3; COVAS, D.T.1,3; KASHIMA, S.1,2

1. Regional Blood Center of Ribeirão Preto, University of São Paulo, Brazil

2. School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil

3. School of Medicine of Ribeirão Preto, University of São Paulo, Brazil

4. University of the Ryukyus, Okinawa, Japan

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection

ORAL PRESENTATION


Oral Presentation 21

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Oral Presentation

958bp, with a longer 5’ UTR. The published UTR was not functional in the minigenome system, whereas a minigenome containing the recently discovered longer version was strongly active. Our results pave the way for the establishment of a reverse genetics system to generate infectious virus from cloned cDNA. Financial support: Wellcome Trust and Medical Research Council / FAPESP – Sao Paulo Research Foundation, grant 2013/02798-0.

BV86 - KROON VIRUS: A NEW AND HIGHLY RESISTANT GIANT VIRUS WITH EXTRA SHELL LAYERDornas, F.P.1; Silva, L.C.F.1; Boratto, P.V.M.1; Rodrigues, R.A.1; Freitas, G.M.1; Assis, F.L.1; Trindade, G.S.1; Kroon, E.G.1; Cortines, J.2; La Scola, B.3; Abrahão, J.S.1

1. UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901

2. UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901

3. UNIVERSITÉ DE LA MÉDITERRANÉE, Jardin du Pharo - 58, bd Charles Livon -13284 Marseille Cedex 07

The members of the Mimiviridae have been isolated from various environments due mainly the host ubiquity, although few studies have demonstrated biological and structural assays of them. Therefore, this work aimed to characterize a new virus of the Mimiviridae, named Kroon virus, isolated from a lake in Lagoa Santa city, MG, Brazil. For this, water samples were collected, were submitted to an enrichment process in rice medium and then were filtered afterwards (in 200nm filters) to retain the viruses. Samples were eluted in PBS and then inoculated in amoebae (Acanthamoeba castellanii) cultures where were isolated. In parallel, the samples were submitted to a Real-Time PCR assay, targeting the helicase viral gene to be phylogenetics analyzed. The virus was also purified for further characterization. Biological and structural analyses, including cytopathic effect assays, resistances tests, optical microscopy, SDS-PAGE and mass spectrometry analyses reveal unique features of Kroon virus. Electron microscopy imaging revealed particles with dimensions similar to that described for other mimiviruses and some multiplication phases similar to giant viruses. The helicase gene sequencing

BV12 - OROPOUCHE ORTHOBUNYAVIRUS MINIGENOME SYSTEM REVEALS SIGNIFICANT ERRORS IN THE PUBLISHED GENOME SEQUENCESAcrani, G.O.1; Lunel, N.L.T.2; Spiegel, M.3; Weidmann, D.M.D.S.3; da Silva, D.4; Nunes, M.R.T.4; Ellott, R.M.2

1. USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070

2. FACULTY OF VETERINARY, UNIVERSITY OF GLASGOW, 464 Bearsden Road, Glasgow, G61 1QH, Scotland

3. UNIVERSITY MEDICAL CENTER GOTTINGEN, Georg-August-Universität Robert-Koch-Straße 40, 37075 Göttingen Briefpostadresse 37099 Göttingen

4. IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000

Oropouche virus (OROV, Bunyaviridae) is the second most frequent arboviral febrile illness in Brazil. OROV occurs predominantly in the North Amazon region, but has been isolated in the Southeast region of the country and also in Peru, Venezuela and Panama. OROV is a negative sense RNA virus with three genome segments named S, M, and L. Complete sequences for the three genomic segments are available at the NCBI Database. The 3’ and 5’ untranslated regions (UTRs) of each viral segment function as important regulatory sequences for replication and transcription, are specific for each genus and are highly conserved among them. We report here 5’ and 3’ RACE results and a minigenome system using the UTR of each segment controlling a Renilla luciferase reporter gene. The analysis of the function of the UTRs of OROV strain BeAn19991 showed that the sequences published in the NCBI Database contain numerous errors. Our results show that the published UTRs are not functional, while changing the residue number 9 of the 3’ genomic UTR was necessary to regain its function. The viral 3’ and 5’ UTR form a “panhandle” structure that is necessary for proper replication and transcription of the viral genome, and the mismatch we generated at residue number 9 has been shown to be important in other bunyaviruses. We also show that the published L ORF contains some deletions, insertions and mutations that alter the translated protein and should be revised. Finally, we prove that the published sequences for the S segment are shorter than the real viral genome. The published sequences indicate that the viral S segment is 754bp long, but we prove that it has a genome of




and phylogenetic analyses indicated that Kroon virus belongs to group A mimivirus, clustering together with APMV and others. At the heat resistance test, the Kroon virus show 2 logs more resistant than APMV at the 75oC while at the resistance UV light assay the Kroon virus show approximately 1.5 logs more than Kroon virus when submitted at 10 minutes. Details of the transmission electron microscopy images revealed a remarkable extra shell layer, suggesting the first evidence of the high resistance of this virus. To evidence this propose, a SDS-PAGE were performed after the virus protein heat denaturation. The gel analyses show different proteins approximately the same weight of the capsid APMV protein. Mass spectrometry shows the homolog proteins of the APMV capsid and an extra putative capsid protein. One of this proteins show a deleted region indicating the possibility to answer the reason of the extra shell layer. The number of the shell layers virus might be involved as an environmental evolution resistance, as UV incidence of a tropical area and the adaptation with the high organic matters due the increase of the host multiplication. So, the Kroon virus structural discovery can show how complex giant viruses may be, opening ways to new research in this field.

BV95 - DYNAMIC COVALENT BONDS IN VIRAL CAPSIDS: AUTO PROTEOLYSIS INVOLVED IN CAPSID MATURATION CAN BE REVERSED UNDER PHYSIOLOGICAL CONDITIONSDomitrovic, T.1; Matsui, T.2; Johnson, J.E.3

1. INSTITUTO DE MICROBIOLOGIA PAULO DE GÓES/UFRJ - Centro de Ciências da Saúde - Bloco I, Cidade Universitária - Ilha do Fundão, Rio de Janeiro - RJ, 21941-590

2. STANFORD UNIVERSITY - 450 Serra Mall, Stanford, CA 94305, Estados Unidos

3. THE SCRIPPS RESEARCH INSTITUTE, 10550 N Torrey Pines Rd, La Jolla, CA 92037, Estados Unidos

Virtually all animal viruses transition from a procapsid noninfectious state to a mature infectious state. Auto proteolysis of the capsid protein is a common event during maturation of Picorna, Birna, Noda and Reoviruses and generally results in the generation of a membrane-active peptide that remains associated to the capsid by non-covalent interactions. In this work, we show that self-cleavage is reversible and that this

reaction may be involved in structural transitions important for infectivity. Nudaurelia capensis omega virus (NωV) is a non-enveloped (+)ssRNA virus from the alphatetraviridae family that infects insects. This virus is an accessible system for the studies of non-enveloped virus maturation and entry in animal cells. In neutral pH, 240 copies of NωV capsid protein assemble around RNA, forming a round procapsid. After acidification, the procapsid collapses into a compact capsid with distinctive quasi-equivalent contacts (T=4 capsid). This structural transition activates auto-proteolysis of the capsid protein generating lytic peptides that stay non-covalently associated to the capsid. The mature capsid presents lytic activity against membranes when exposed to alkaline pH, what correlates with the in-vivo environment of infection inside caterpillars mid-gut. We found that the structural transition of mature capsids on high pH not only induces the exposure of the lytic peptides to the environment, but also, induces an unexpected reconstitution of the covalent bond between the capsid protein and the peptides that were cleaved during maturation. This reverse-reaction is restricted to a subset of the peptides present in the capsid and does not affect the lytic activity against membranes. Possible functional implications of this reaction for capsid disassembly and RNA exposure will be discussed. FINANCIAL SUPPORT: CNPQ, PEW CHARITABLE TRUST AND NATIONAL INSTITUTE OF HEALTH (NIH)

BV105 - ANALYSIS OF DIFFERENTIAL METHYLATION PROFILE IN PATIENTS WITH DENGUE USING METHYLATION-SENSITIVE ARBITRARILY PRIMED POLYMERASE CHAIN REACTIONAlessandra, V.B.T.G.1; Morais, S.M. de S.1; Ferreira, J.M.S.2; Malaquias, L.C.C.1; Coelho, L.F.L.1

1. UNIFAL - Universidade Federal de Alfenas, R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, 37130-000

2. UFSJ - Universidade Federal de São João del-Rei, Praça Frei Orlando, 170, Centro, São João del-Rei, Minas Gerais, 36307-352

Dengue virus (DV) is an enveloped virus, positive single-stranded RNA, transmitted to humans through the bite of infected female Aedes aegypti mosquito. There are two main clinical manifestations caused by DV infection named Dengue Fever (DF) and Dengue Hemorrhagic




Fever (DHF). Several studies indicate that one of the most important factor involved in the DHF development is the presence of non-neutralizing antibodies. These antibodies can facilitate DV infection in susceptible cells in a secondary infection caused by a different serotype of DV. However, this hypothesis can not explain the cases of primary infection with DHF outcome, indicating that genetic or epigenetic host factors may be influence the predisposition and development of DHF. DNA methylation is the best characterized epigenetic modification exerting great importance in silencing and gene regulation. In eukaryotes, it is found predominantly in the 5 position carbon of cytosine followed by a guanine in the CpG dinucleotide. Therefore, the analysis of differential methylation between different clinical manifestations and the identification of hyper or hypomethylated regions may provide evidence to elucidate the pathogenesis of dengue. In this present study, the DNA from patients with DF and DF with hemorrhagic manifestations was extracted and used for analysis of differential methylation profile by using Methylation-Sensitive Arbitrarily Primed Polymerase Chain Reaction (MS-AP-PCR). After electrophoresis, one band showed a differential methylation pattern and this band was cutted, purified, re-amplified and cloned in pGEM-T-Easy vector. The recombinant plasmid were extracted and sequenced. The sequence data showed 98% homology to a region of chromosome 13 present in SMAD family member 9 gene, which transduces signals from TGF-β family members. Searching for the sequence with the BLAST tool, we found out that it is located next to a CpG island, according to the MethPrimer program. Further studies are needed to confirm the methylation status in SMAD region in other samples by another technique such as Methylation Specific PCR or Bisulfite Sequencing.FINANCIAL SUPPORT: FAPEMIG; CNPQ; CAPES.

BV171 - STUDY OF STOP CODONS RECURRENCE IN HEPATITIS C VIRUS NS5A PROTEINCampos, G.R.F.; Bittar, C.; Rahal, P.

UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José do Rio Preto - SP, 15054-000

The occurrence of mutations that introduce TGA stop codon in the genomic region of Hepatitis C Virus (HCV), recurrent in different patients, led to the search of alternative explanations for these mutations. In a conventional translation process, these stop codons would take premature stop of polyprotein translation, preventing the complete codification of NS5A and therefore de following protein, the viral RNA dependent RNA polymerase NS5B. In order to investigate the impact of this codon in the position 111 of NS5A protein, a site directed mutation was produced in the CMV_SGR_JFH1_GFP5A plasmid and in the subgenomic replicon SGR-JFH1-FEO, introducing the TGA codon in the position 111 of NS5A protein of these constructions. The expression of GFP protein, set after codon 111 of CMV_SGR_JFH1_GFP5A, wild type and mutated, was analyzed by fluorescence microscopy 48h after transfection in hepatocellular carcinoma strains (Huh 7 and HepG2). The results show the expression of GFP in both wild type and mutated plasmid, an indication of complete NS5A expression. A replication assay was carried out for the mutated and wild type replicons, by the quantification of luciferase protein expression, establishing the replication level of these constructions, in Huh 7. The results show that up to 12h after electroporation the mutated replicon has replication levels similar to wild type, corroborating previous result, but these levels reduce over time. The present study suggests that TGA codon in position 111 of Hepatitis C Virus is not determinant of translation termination. New studies are necessary to determinate which mechanism is responsible for the readthrough of this codon, as well as its influence in complete virus activity, in different steps of viral cycle and impact in host pathogenicity.FINANCIAL SUPPORT: FAPESP, PROCESSO Nº 2013/02856-0




BV175 - CHARACTERIZATION OF THE INTERACTION BETWEEN THE METHYLOSSOME AND THE HUMAN RESPIRATORY SYNCYTIAL VIRUS NUCLEOCAPSIDOgawa, J.; Oliveira, A.; Simabuco, F.; Éleouët, J.F.; Ventura, A.


2. UNICAMP - Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970

3. INRA - Institut National de la Recherche Agronomique, Siège; 147 rue de l’Université 75338 Paris Cedex 07

The Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract, causing respiratory illness particularly in newborns, babies, children and immunocompromised patients. The genome of HRSV encodes eleven proteins, being fundamental to understand its relationship with the host, to characterize the interactions between those proteins and cellular components. Viral nucleoprotein (N) associates with the viral RNA forming the basic structure of the HRSV helical nucleocapsid. In a previous work of our laboratory it was found that N interacts with cellular proteins Hsp70, PRMT5 and WDR77, utilizing a FLAG tagged N expression in HEK293 cells protocol and polyclonal mouse sera against N. Association of two PRMT5 and two WDR77 polypeptides form a methylosome core unit and its interaction with the nucleocapsid might have a role in virus replication. N has arginine residues exposed on the surface that are potential targets of methylation by PRMT5 and this modification could be modulating the interaction between N and the viral RNA, enabling access of viral RNA polymerase during the processes of transcription and replication. We used Hep2 cells infected by HRSV A2 strain extracts to confirm, by immunoprecipitation and western blot, N-methylosome interaction utilizing commercially available monoclonal antibodies against N. In addition a HRSV minigenome system, functional in BSRT7 cells transfected with a set of vectors expressing viral replication proteins and a reporter gene, was used to probe co-localization of N and PRMT5 during replication process in inclusion bodies. The results confirmed N-methylosome interaction by both strategies. We conclude that the inhibition of PRMT5 activity is a

potential target to block HRSV replication. FINANCIAL SUPPORT: FAPESP and CAPES

BV225 - HIV-1 TRANSLATION INITIATION IS INHIBITED BY POLIOVIRUS 2A PROTEASEAmorim, R.; Costa, L.J. da

UFRJ - Universidade Federal do Rio de Janeiro, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro - RJ, 21941-901

Translation initiation in eukaryotic cells is made mainly via two mechanisms: the recognition and association of several eukaryotic initiation factors (eIFs) to the 5’ Cap structure present at eukaryotic messenger RNAs (mRNAs); or the association of eIFs to specific regions of highly structured regions of mRNAs named internal ribosome entry sites (IRES). IRES-dependent translation occurs for some mammalian mRNAs under certain metabolic conditions and for RNAs of some viruses as poliovirus. Human Immunodeficiency Virus Type 1 (HIV-1) uses cellular factors to start its protein synthesis, which is done mainly by the cap structure of its mRNAs. However, there is still some controversy whether translation of retroviral mRNAs is strictly Cap-dependent, since some studies have demonstrated the existence of IRES elements on the 5′UTR of HIV-1, FIV and SIV. In this study, we used poliovirus 2A protease (2APro), that interrupts cap-dependent translation by the cleavage of initiation factor eIF4G, and analyzed its interference on the replication of HIV-1 in Hek293T cells. We made co-transfections by cationic liposomes of the infectious clone of HIV-1 or a reporter plasmid containing luciferase gene with an expression vector of 2Apro. The expression of luciferase was evaluated by luminometry, expression of viral proteins by western-blotting and production of HIV-1 infectious particles by the infection of TZM-bl cells indicated by β-galactosidase. We observed a strong reduction in the expression of luciferase in 2Apro co-transfected cells at 24 hour post-transfection, along with a significant decrease in eIF4GI levels and a mild decrease in eIF4GII levels. Expression of the viral proteins GagPol (and its cleaved products) and Nef was strongly reduced in cells co-transfected with 2APro and remained inhibited until 72 hours post-transfection. Moreover, in the presence of 2APro, the release of infectious viral particles was 90% reduced compared to control cells. This decrease in protein




synthesis does not seem to be related with mRNA traffic, as viral full-length mRNAs were detected in the cytoplasm of 2APro transfected cells. Together, our results indicate that 2Apro is able to reduce significantly the translation of viral structural and accessory proteins and therefore to inhibit the production of HIV-1 infectious particles. Studies are under way to precisely demonstrate that in Hek293T cells the synthesis of HIV-1 proteins is solely cap-dependent.FINANCIAL SUPPORT: CNPQ, CAPES/PROEX, FAPERJ.

BV280 - 2A PROTEASE FROM HUMAN RHINOVIRUS C15 DOES NOT PROVIDE INTERFERON RESISTANCE AND TRAFD1 HAS AN ANTIVIRAL ACTIVITY AGAINST HUMAN RHINOVIRUSES AND ENCEPHALOMYOCARDITIS VIRUSGagliardi, T.B.1; Dabelic, R.2; Racaniello, V.R.2


2. CU

Innate immune response is the first one to be activated when a virus infects the cell and that results in interferon (IFN) production following transcription of interferon-stimulated genes (ISG). This study evaluated the activity of 2A protease from human rhinoviruses (HRVs) against IFN and ISG responses. This study was developed using HRV1A, 2, 14 and16; encephalomyocarditis virus (EMCV); and five EMCV recombinants (E2AHRV) expressing 2A protease from HRV1A, 2, 14, 16 and C15. All experiments were done in H1Hela cells, using multiplicity of infection of 1 and the viruses were quantified by plaque assay. Cells were pre-treated with 0, 1000, 2000 and 5000u of IFN-α2a. After 24h, they were infected with a virus. Supernatant and cells were collected at 24h post-infection and the viruses were titrated. The presence of 2A protease from all HRV improved EMCV replication mainly at the end of cycle (exponential phase), turning it 2h faster. In addition, the E2AHRV infectiousness was also improved (replicative burst increased 3 to 5 orders of magnitude). As expected, HRVs were IFN-resistant. All E2AHRV showed to be IFN-resistant, except for E2AHRVC15, which was IFN-sensitive like the wild type. We also analyzed the antiviral response of 31 ISGs. Plasmids that express ISGs tagged with “red fluorescent protein” (RFP) were transfected into H1Hela cells and, after 48h, the cultures were infected with one of the

viruses. Supernatant was collected at 24h post-infection and viral titres were normalized by RFP-expression, quantified by flow cytometry. RFP-expression varied greatly between plasmids, i.e., pPARP12 was expressed in 19.3% of the cells while pTLR3 only in 0.33%. HRVs are resistant to most ISGs used in this study, specially HRV14 and HRV16, both members of the major group. EMCV was sensitive to most ISGs, and the titre decreased around 3 orders of magnitude (p<0.001). On the other hand, 2A protease from all HRV provided more resistance to all E2AHRV. “Tumor necrosis factor receptor-associated factors domain 1” (TRAFD1) was the ISG with antiviral activity against all viruses used in this study. In conclusion, 2A protease from HRV improved EMCV replication and also resistance to IFN-treatment, except to E2AHRVC15 that was IFN-sensitive as the wild type. 2A protease from all HRVs improved EMCV resistance to the ISGs used in this study. TRAFD1 was the ISG with greatest antiviral activity against HRVs and other viruses.

BV345 - IDENTIFICATION OF THE CELLULAR AND MOLECULAR DETERMINANTS OF FLAVIVIRUS FUSIONEsposito, D.L.A.; Nguyen, J.B.; Modis, Y.

YALE UNIVERSITY, New Haven, CT 06520, Estados Unidos

Flaviviruses are major pathogens that are responsible for a number of important mosquito-borne diseases of both humans and animals. Flavivirus infection begins with cellular entry of the virions, which occurs by a membrane fusion reaction. It is unclear whether virus-like particles (VLPs - noninfectious ordered structures that resemble whole virions in terms of their structural organization, cellular processing, and capacity for membrane fusion, and therefore serve as useful models for investigating viral entry) have the same requirements for fusion and the same cell entry pathways as full-sized virions. In this study, we examined the functional requirements for productive membrane fusion of West Nile (WN) and Japanese encephalitis (JE) VLPs to liposomal target membranes by monitoring membrane fusion using a lipid-mixing assay. WN and JE VLPs were produced in mammalian cells with the prM-E region of the WN/JE virus genome and, after purification, were labeled with the hydrophobic fluorescent indocarbocyanine




dye DiI (Invitrogen). A fluorescent plate assay was performed by mixing the labeled VLPs with 100-nm liposomes, and fusion was observed after acidifying the reaction (0.1 M sodium acetate pH 5.2). Fusion of JE and WN VLPs to liposomes occurred on the time scale of minutes with optimal fusion efficiencies observed at the human physiological temperature of 37 oC. Fusion was also enhanced with increasing acidity, with the threshold for fusion between pH 5.5-5.7. Importantly, the presence of anionic lipids, namely BMP (LBPA) and phosphatidylinositol 3-phosphate (PI3P), in the liposome target membranes significantly enhanced the fusion potential of VLPs, consistent with an emerging model of flavivirus fusion to mid-to-late endosomal compartments. Financial Support: * Subcontract within Contract No. W81XWH-12-C-0028 (L2 Diagnostics) 07/22/13 – 07/21/15 US Department of Defense Small Molecule Antiviral Agents Against Flaviviruses * P01 GM022778-37 (Steitz, Project Director; Modis, Co-PI) 04/01/09 – 06/30/14 NIH/NIGMS Program Project Grant Structural bases of the functions of RNA-protein machines

BV379 - DEVELOPMENT OF TWO POTENTIAL TOOLS AGAINST THE REPLICATION OF THE DENGUE VIRUSFujimura, P.T.1; Vaz, E.R.1; Cardoso Jr, C.A.M.1; Carvalho, W.J.1; Reis, C.F.2; Santos Jr, C.D.3; Freitas, G.R.O.1; Yokosawa, J.1; Carneiro, A.P.4; Goulart, L.R.1; Vecchi, L.1; Ueira,C.U.V.1

1. UFU - Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, 38408-100


3. UFSCAR - Universidade Federal de São Carlos, Rodovia Washington Luís, Km 235 - SP 310 - Jardim Guanabara, São Carlos - SP, 13565-905


Dengue is the most important mosquito-borne viral disease in tropical and subtropical areas and has become a global threat affecting around 100 countries in the world. Dengue virus infection can be manifested by the self-limited febrile illness known as dengue fever (DF) or by a severe complication caused by the hemorrhagic

fever (DHF) or dengue shock syndrome (DSS). Although, much research has been done in Dengue, currently, there is no licensed antiviral agent to defend against the dengue. Thus, development of therapeutic strategies is needed to fight against the virus. In this study we selected by Phage Display an antibody fragment capable to recognize the viral structural protein NS1. NS1 is a versatile nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. The intracellular NS1 has been associated with early steps of viral replication, since it can be found co-localized together whith dsRNA and other nonstructural proteins like NS3 and NS5. Futhermore, we development a mechanism to degrade the NS1 when scFv is fused to the SEL1L molecule (scFv viral-degradin). SEL1L is a component of HRD1 complex, which is involved on the Quality Control protein process of the endoplasmatic reticulum (ER). This molecule (SEL1L) is able to recognize and to translocate protein with misfolded luminal domains to proteasome. After three round of selection, selected cDNAs encoding antibody fragments (scFv) were subcloned into an expression vector in mammalian cells to produce scFv in soluble form or to produce scFv fused to the SEL1L molecule (scFv viral-degradin). In our study, we performed immunofluorescence to co-localize the soluble scFv with NS1 in the infected cell. In addition, we also performed Western blot to verify the specific degradation and qPCR to quantify the new production of viral particle after inabation with our soluble scFv and scFv viral-degradin. Our results showed that the soluble antibody scFv co-localizes with NS1 inside the cell, probably in the viral replication complex, and it is able to block the viral replication in more than 90%. Moreover, the scFv viral-degradin degraded the specific molecule (NS1) and it was also able to inhibit the new viral particle production in more than 90%. Therefore, this soluble scFv and scFv viral-degradin may be the potential tools to combat dengue virus replication. Financial Support: Capes, CNPq, FAPEMIG, UFU.




BV490 - IN VITRO MICROEVOLUTION OF DENGUE VIRUS 4 IN AEDES ALBOPICTUS CELLSNascimento, V.A.; Souza, V.C.; Naveca, F.G.

ILMD/Fiocruz - Instituto Leônidas e Maria Deane - Fiocruz Amazônia, Rua Terezina, 476 - Adrianópolis, Manaus - AM, 69057-070

The Dengue virus (DENV) belongs to the Flaviviridae family, genus Flavivirus and four distinct serotypes are recognized. Like other RNA viruses, DENV displays genome variations due to error-prone RNA polymerase. These variants may be observed not only when samples from different individuals are analyzed, but also within the same host. Considering the need for a better understanding of DENV evolutionary process, and the possible emergence of highly virulent lineages, this study aimed to evaluate the in vitro microevolution of DENV serotype 4, genotype II, during the adaptation process in insect cells culture. For this purpose, a sample of DENV-4 collected in May 2011 (BrAM005/11) was inoculated into C6/36 cells (P1), followed by serial passages in the same cell line until the passage 25 (P25). Subsequently, viral RNA was extracted and cDNA was generated using a specific primer targeting the last 21 nucleotides at 3’ UTR. The full-length genome of the BrAM005/2011 sample, as well P1, P5, P10, P15, P20 and P25 were amplified in eight overlapping amplicons, which were sequenced by Sanger’s based method. The same samples were also submitted to Next-Generation Sequencing (NGS) using the Ion Torrent technology. The files generated after sequencing were further analyzed using Geneious software version 7.1.7. The BrAM005/11 sequence was initially aligned with the reference sequence available in GenBank (NC_002640.1) for annotation. Furthermore, the insect cells passages (P1 to P25) were aligned for the nucleotide variation comparison using annotated BrAM005/11 as reference. The data analysis provided by the Sanger’s sequencing showed two nucleotide mutations that occurred during the viral adaptation. One of these mutations occurred at position 1602 and results in a residue substitution (T222P) in the domain II of the viral envelope protein; whereas a silent mutation was observed in position 3365 (NS1 region). The NGS results corroborate those obtained by Sanger’s sequencing and showed two additional mutations: at nucleotide position 1810, which results in a residue substitution K291R in the viral envelope and a silent mutation at position

4248 (NS2B region). Further experiments are being performed in order to verify the significance of these mutations for viral fitness. The results of this work may provide data that can help to a better understanding of the mechanisms of viral adaptation in vitro, thus contributing to evolution studies with RNA viruses.FINANCIAL SUPPORT: FIOCRUZ/CNPQ PAPES VI; POM - FIOCRUZ; CAPES ÁREA

BV501 - ALIX IS A HOST CELL FACTOR REQUIRED FOR LYSOSOMAL TARGETING OF CD4 BY HIV-1 NEFJanuário, M.E. da S.1; da Silva, E.M.L.1; de Castro, R.O.1; Amorim, N.A.1; da Costa, L.J.2; da Silva, L.L.P.1

1. FMRP/USP - Faculdade de Medicina de Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900

2. IMPG/UFRJ - Instituto de Microbiologia Paulo de Góes da Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - Cidade Universitária, Rio de Janeiro, Ilha do Fundão Rio de Janeiro - RJ, 21941-590

Nef is an accessory protein of HIV-1 that enhances production of infectious viral particles and promotes progression to AIDS. The most studied function of Nef is the ability to downregulate the expression of specific cell surface proteins, such as CD4. CD4 is a type I transmembrane glycoprotein expressed on the surface of helper T-cells and cells of the macrophage/monocyte lineage that plays important roles in adaptive immune responses, and serves as the primary receptor for HIV-1, HIV-2 and SIV. Downregulation of CD4 by Nef is thought to prevent deleterious superinfection and to facilitate release of newly produced viral particles. Nef induces CD4 endocytosis via clathrin-coated vesicles (CCVs) through interaction with AP-2. CD4 that is internalized by Nef is then targeted to late endosomes/multivesicular bodies (MVBs) and subsequently to lysosomes for degradation, but how this is achieved is mostly unknown. Our previous work showed that Nef interacts with the middle (V) and the Bro1 domains of Alix, a protein involved in MVB biogenesis. Moreover, overexpression of truncated Alix V-domain impairs CD4 degradation induced by Nef. Here we show that Nef-Alix interaction takes place in late endosomes that contain internalized CD4 and that depletion of Alix impairs delivery of CD4 to lysosomes induced by Nef. Alix depletion or V-domain overexpression does




not compromise transmembrane protein degradation through the canonical MVB-pathway, suggesting that Nef uses a alternative mechanism, mediated by Alix, to deliver cargo to MBVs. Together our results show that Nef interacts with Alix to facilitate delivery of CD4 to the MVB pathway, thus promoting sustained depletion of this surface receptor in infected cells.

EV90 - PATHOGENS INACTIVATION BY UNIONIZED AMMONIA FOR THE PRODUCTION OF SAFE BIOFERTILIZER FROM SWINE EFFLUENT AND SLUDGEFongaro, G.1; Kunz, A.2; Magri, M.E.1; Zeredo, A.C.B.1; Schissi, C.D.1; Zaguini, J.1; Barardi, C.R.M.1

1. UFSC – Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima - Trindade, Florianópolis - SC, 88040-900

2. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901

The biomass derived from swine manure has good potential to be used as biofertilizer due to its high nutrient concentration. However, the land application of manure should be based on safety parameters. This work used ammonia for hygienization as an additional method to produce a safe biofertilizer from swine effluent and sludge after treatment in anaerobic bioreactor. The microorganisms used as models were: somatic coliphage ɸX-174, Human Adenovirus (HAdV-2) and Salmonella typhimurium. The enumerations of the HAdV, ɸX-174 and S. tyfimurium were respectively performed by integrated cell culture assay–preceded by reverse transcription (ICC-RT-qPCR), double layer agar method and by ISO 6579 (2002). The following final concentrations of urea were used: 186 mM (T1), 379 mM (T2) and 754 mM (T3) reaching pH 9.8 at 23oC, providing, in this condition, unionized ammonia (NH3), as inactivating agent. All experiments were performed in triplicate and with negative controls. The results revealed that i) S. typhimurium either in swine effluent or in sludge, had a 4 logs10 reduction in up to 3 days for all treatments; ii) HAdV had a 4 logs10 reduction in swine effluent at 27, 20 and 15 days with the respective treatments T1, T2 and T3, and in swine sludge the inactivation occurred at 15, 9 and 3 days with the respective treatments T1, T2 and T3; iii) ɸX-174 had

3 logs10 reduction in swine effluent at 80 and 45 days of treatments (T2 and T3, respectively). T1 was able to reduce 2 logs10 of ɸX-174 after 80 days of treatment. Considering the relative time of treatment to inactivate 99.9% (3log10) of all the studied pathogens, considering the amount of urea added and efficiency of inactivation, the T2 treatment was the most promising for swine effluent and T3 treatment was more indicated for sludge. Hygienization by NH3 provides new possibilities for alternative treatments of different types of waste, liquid as well as solid, at both small and large scale, being a viable method for reuse of agricultural waste as biofertilizer, considering the animal and human health quality. FINANCIAL SUPPORT: CNPQ 472804/2013-8.FINANCIAL SUPPORT: CNPQ 472804/2013-8.

EV140 - ANALYSIS OF MARINE VIRAL FAMILIES ASSOCIATED WITH SIDERASTREA STELLATA FESTIVAL IN ARRAIAL DO CABO AND BÚZIOS, RJ, BRAZILSantana-Pereira, P.; Giongo, V.; Pedrosa, L.G.; Paula, J.E.F.B.; Amorim, L.; Paixão, I.C.N.P.

UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900

The reef ecosystem is considered as one of the oldest, stable and biodiverse on the planet. A complex network of interactions and interrelationships due to the high diversity of organisms in this environment is sustained by primary productivity, which is characterized as one of the largest ocean. Studies have shown that viruses are highly dynamic components in the aquatic ecosystem, because they have a fundamental role in the chain to be significant microbial agents in controlling the abundance and diversity of bacteria and phytoplankton, which are symbionts of corals and are important in controlling availability of nutrients. In this study we performed the molecular and morphological analysis for identification of viral groups in a scleractinian coralreef that didn’t have been described yet, being the first work in Brazil studying the presence of viruses associated with marine organisms. For this we used TEM and PCR for determination of marine virus families as widely tools applied in marine virology studies. The samples were collected in two áreas (Arraial do cabo and Búzios) with the aid of chisel and hammer, kept on ice until the time of processing. After processed were




stored at -80 ° C ultrafreezer until the analysis. We perform the transmission electron microscopy using concentrated phage prepared in glutaraldehyde (2.5%) with addition of uranyl acetate (2% final concentration) for performing negative staining in grids of Cu + + 300 mesh coated collodion. After incubation the grids were examined in a transmission electron microscope (JEOL, JEM1011 - Electron Microscopy) at 80 Kv. The images were observed in increases 18000-300000 times. The PCR analyses were performed using the DNA extraction kit (ROCHE) and specific sequences of oligonucleotides belonging to Podoviridae and Myoviridae viral families (CPS1, CPS2, P60, T3, T7 and T4 primers). Through these analyzes it was possible determine the presence of the viral groups belonging to Podoviridae, Myoviridae and Nodaviridae viral families by TEM. In addition, it was possible to identify fragments belonging to the groups Myoviridae and Podoviridae by PCR. Also, some amplified fragments, having non expected size, were observed suggesting the presence of viral particles with different sequences of the target sequence. These results confirm the interaction between viruses and cnidarians and thus more robust studies is required to determine the possible consequences of this association.FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPI AGIR

EV255 - DETECTION OF HUMAN ADENOVIRUSES IN SEDIMENTS FROM STREAMS IN URBAN AREAS FROM RIO DOS SINOS WATERSHEDStaggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; Soliman, M.C.; Souza, F.G. de; Fabres, R.B.; Luz, R.B.; Zirbes, G.; Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, S.E. de M.

FEEVALE - Universidade Feelave, Av. Dr. Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, 93510-250

Soil can act as an important reservoir of natural resources, however, it may also allow the permanence of many potentially dangerous microorganisms. Enteric viruses in the soil may migrate through the successive adsorption-desorption phenomena, thus providing risk of contamination of groundwater. Among the enteric viruses, adenoviruses (AdV) have been the focus of many studies, because of their persistence in the environment and their great impact on public health. The aim of this study is the detection of HAdV

in sediment samples from streams located in urban areas of EstânciaVelha/Portão (EstânciaVelha and Portão), Schmidt (Campo Bom), Pampa and Luiz Rau (Novo Hamburgo), RS, Brazil. Samples (n=102) were collected from 17 different points, bimonthly, from September/12 to July/13. Samples were eluted 10% v./v. in E-MEM (pH 10,5). Viral DNA was extracted by a commercial kit and HAdV detection was performed by qPCR using primers that targeted a conserved region (hexon gene) of the virus genome. Overall, 42.16% (43/102) samples were positive for the presence of HAdV: Arroio Schmidt (45.8%; 11/24), Arroios Pampa and Luis Rau (both 37.5%; 9/24), and EstânciaVelha/Portão (36.7%; 11/30). There was a variation in viral load among 4.52 x 102 a 1.30 x 105 genome copies per gram of sediment. These viral agents detected in these streams are related to various pathologies, such as gastroenteritis, respiratory infections and conjunctivitis. The results suggest significant anthropic contamination. FINANCIAL SUPPORT: CAPES, FAPERGS, CNPQ, PROJETO MAIS ÁGUA, UNIVERSIDADE FEEVALE.

EV306 - ENVIRONMENTAL POLIOVIRUS SURVEILLANCE: DETECTION OF WILD AND VACCINE - DERIVED POLIOVIRUSES IN SÃO PAULO SEWAGEHachich, E.M.1; Barbosa, M.RF.1; Garcia, S.C.1; Bonanno, V.M.S.1; Eduardo, M.B.P.2; Burlandy, F.M.3; Costa, E.V.3; da Silva, E.E.3; Sato, M.I.Z.1

1. CETESB - Companhia Ambiental do Estado de São Paulo, Av. Prof. Frederico Hermann Jr.,345, Av. Prof. Frederico Hermann Júnior, 345 - Alto de Pinheiros São Paulo - SP, 05459-010

2. CVE/SES-SP - Centro de Vigilância Epidemiológica da Secretaria de Saúde do Estado de São Paulo, Av. Dr. Arnaldo, 351 - 6º andar — Pacaembu, São Paulo - SP, 01246-000

3. FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900

The environmental poliovirus surveillance (ENV) is an important complementary tool to the investigation of acute flaccid paralysis (standard approach for polio surveillance) for the Global Polio Eradication Initiative (GPEI) of WHO. CETESB, the Environmental Company of Sao Paulo State, works in the field of Environmental Virology for about 40 years, and since 1999 develops a Program of ENV in collaboration with the Center of




Epidemiological Surveillance of Sao Paulo State. The surveillance is based on the examination of sewage and seawater samples from potential points of foreign people entrance, such as international airport and seaports, and from wastewater treatment plants (WWTP). Sampling is performed bimonthly through gauze pad (Moore swab) or grab sampling. Moore’s swab is transported to the laboratory in 3% dessecated beef extract and concentrated by organic flocculation. The grab samples are concentrated using the PEG 8000. Samples are treated with chloroform to clarification and decontamination and tested for sterility. Poliovirus is isolated according to the WHO alternative test algorithm (S1 Supplement to the WHO Polio Laboratory Manual), a specific approach comprising of several simultaneous inoculation into L20B and RD cell. Virus isolates are submitted to intratypic differentiation by RT-PCR. All poliovirus isolated are sent to the Enterovirus Laboratory of FIOCRUZ (WHO Reference Laboratory for Poliomyelitis) for sequencing, aiming to diagnose the presence of vaccine derived poliovirus (VDPV) and confirm the wild poliovirus (WPV) isolation. From August 1999 to April 2014, 1,274 samples of sewage and seawater were tested. A WPV sorotype1 with nucleotide identity of 99,6% to the Equatorial Guinea recent isolates was detected in a sewage sample from the influent of the Viracopos Airport WWTP, Campinas, collected on 5th March, 2014. The first passage of the sample in L20B rendered 50% CPE (5 days) and the second passage in RD rendered 100% CPE (48 h). On January 20th, at the Petrobras Pier in São Sebastião Coast, a VDPV was also isolated from a seawater sample collected after a first passage in L20B (50% CPE, 5 days) and a second one in RD (100% CPE, 4 days). RT-PCR showed the presence of a Poliovirus Sabin 2. The sequencing of VP1 gene (903 nucleotides) of this isolate, showed a nucleotide identity of 91% with the reference PV Sabin 2. These findings reinforce the importance of the environmental monitoring as a preventive action for the GPEI.

EV430 - DIFFERENT PHAGES ACTION PATHWAYS IN REDUCING BIOFILMS FORMED BY OIL REFINERIES ASSOCIATED BACTERIADias, R.S.1; Fonseca, L.A.B.V.1; Albanese, J.M.1; Silva, L.C.F.1; Suhette, R.2; Torres, A.P.R.2; Sousa, M.P.2; Silva, C.C.1; Oliveira, V.M.3; de Paula, S.O.1

1. UFV - Universidade Federal de Viçosa, Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570-000

2. CENPES3. UNICAMP - Universidade Estadual de

Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970

Biofilms occur spontaneously in different environments, whether biotic or abiotic, is an important survival strategy. Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this work, the influence of non-specific bacteriophage was investigated in the biofilm formed by isolated bacteria. Bacteria isolated from feed water sampled from a RO system that showed greater ability to form biofilm, i.e. higher biomass, were selected to be used in the biocontrol assay employing phages against biofilm formation. A phage suspension of a multiplicity of infection (MOI) of 0.01 was added into 96-well microtiter plate containing bacterial suspension, and biofilm formation was measured using biomass quantification with crystal violet staining (CV). Statistically significant reductions were observed in 7 of 11 tested bacteria, some showed dose-dependent reduction and others, increasing the MOI used did not cause further reduction in biofilm. After the CV analysis, two bacteria which responded differently with MOI increasing were selected and analyzed in MBEC® devices by scanning electron microscopy (SEM) in order to evaluate the influence of phages on biofilm structure. By SEM is possible to observe that phage interferes with the adherence of one tested bacteria and in the stability of biofilm formed by other bacteria. The results suggest that the phage vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation by two strategies, by action of enzymes or phage infection, without bacterial lysis. This approach may represent a good alternative in biofilm control on membranes in reverse osmosis systems.FINANCIAL SUPPORT: PETROBRAS




EV457 - FIRST IDENTIFICATION OF ACANTHAMOEBA POLYPHAGA MIMIVIRUS IN LIMNOPERNA FORTUNEI FROM GUAIBA LAKE, RIO GRANDE DO SUL, BRAZILdos Santos, R.N.; Arantes, T.; Correa, R.A.; de Albuquerque, N.R.M.; Kluge, M.; Santos, C.; dos Santos, C.P.; Campos, F.C.; Roehe, P.M.; Franco, A.C.

UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060

The discovery of a complex group of viruses, known as giant viruses, has aroused the interest of many environmental virologists. The first representative of this group, Acanthamoeba polyphaga mimivirus, was isolated from samples collected in a cooling tower of an air conditioning system in a hospital in England in 2003. This virus has a large size (750 nm in diameter), with a 1.2 mb genome and 911 protein-coding genes. This study aims the isolation and molecular identification of mimiviruses in samples collected from golden mussel (Limnoperma fortunei) at Guaiba Lake, in Rio Grande do Sul. A total of 300 samples were collected from grids submerged in the lake, and transported to the laboratory under refrigeration. Next, these samples were incubated with rice enrichment medium for 30 days at 25 °C and submitted to DNA extraction. Real-time PCR was performed using primers to the helicase gene of mimiviruses. So far, in the 25 samples examined, mimivirus genome segments were identified in all of them. Next, we will perform a subsequent cloning and molecular characterization of the helicase amplicons. In addition, the samples will be submitted to isolation in cultures of Acanthamoeba castellani T4 to observe the citopatic effects. Although preliminary, these results indicate that the golden mussel can host and may play a role in mimiviruses maintenance in nature. FINANCIAL SUPPORT: CAPES.

HV33 - DETECTION OF IMPORTED CASES OF CHIKUNGUNYA VIRUS IN BRAZIL: VIRUS ISOLATION, MOLECULAR AND SEROLOGICAL ASSAYSCunha, M.S.; Maeda, A.Y.; Bisordi, I.; Rocco, I.M.; da Silva, F.G.; de Souza, R.P.; Coimbra, T.L.M.; Nogueira, J.S.; Kisielius, J.J.; Silveira, V.R.; Oliveira, A.L.R.; Suzuki, A.

IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 - Cerqueira César, São Paulo - SP, 01246-000

Chikungunya virus (CHIKV) is a mosquito-borne emerging pathogen that has a major health impact in humans causing a dengue-like illness characterized by high fever, headaches, rash, nausea, vomiting, myalgia, and arthralgia. Recent outbreaks have been reported in many parts of the world since 2004 after an outbreak at the coast of Kenya. More recently, the virus has reached the Americas, where there is an ongoing outbreak at the Caribbean Countries, and positive cases have been confirmed in the United States in travelers returning from these areas. Viremic travelers are a risk for spreading CHIKV to non-endemic regions, specially in Brazil, where there is a high vector incidence and a great number of immunologically naïve people. Sera from 13 Brazilian soldiers and one female missionary, suspected of Chikungunya fever, all coming from Haiti, were sent to Núcleo de Doenças de Transmissão Vetorial, Centro de Virologia at the Instituto Adolfo Lutz, São Paulo, Brazil, for diagnosis. Blood samples were collected between days 2 and 16 after onset of symptoms, and so the choice of each laboratory method was based upon the time of sample collection relative to symptom onset. Samples varying from 2 and 7 days were innoculated in Vero and C6/36 cell lines and in mice by intracerebral and subcutaneous routes, while samples with 4 days or more were tested for Chikungunya IgM antibody capture enzyme-linked immunsorbent assay (MAC-ELISA). All serum samples as well as the supernatants of cells were tested by Real-time RT-PCR (RT-qPCR) for CHIKV detection. Viral RNA was amplified in 10 serum samples tested by RT-qPCR. Four infected Vero cells showed cytophatic effect after 4 and 5 days post-innoculation. All these supernatants were confirmed by RT-qPCR, as well as other two without cytophatic effect. Plus, electron microscopy also confirmed viral particles. One serum sample negative for RT-qPCR was positive after one passage in C6/36 cells. CHIKV was also isolated in three mice groups inoculated subcutaneously that showed intense signs of alopecia at the inoculation site, while two animal groups inoculated only by intracerebral route presented alopecia on the head and occular inflamation. MAC-ELISA detected specific immunoglobulin M for CHIKV in all four samples tested. Here we report the first isolation of Chikungunya virus in Brazil, confirmed by real-time RT-PCR, as well as IgM detection, in samples from patients returning from Haiti.




HV125 - ORAL HUMAN PAPILLOMAVIRUS INFECTION IN HIV-POSITIVE PEOPLESilva, C.O.; Santos, L.S.; de Azevedo, K.M.L.; Pereira, O.M.D.; Oliveira, L. do H. dos S.


Human papillomavirus are associated with a variety of oral lesions and they have been detected in healthy oral mucosa of HIV-infected individuals. The aim of this study was to survey information concerning HPV infection from randomly selected HIV infected individuals and to compare the results obtained with a control group. Methods: This cross-sectional study included oral samples from 75 HIV positive and 120 HIV negative individuals (control group) who were asymptomatic for oral mucosa lesions. To detect human papillomavirus status, polymerase chain reaction amplification was performed. HPV typing was determined by restriction fragment length polymorphism analysis. Demographic data, life style and tobacco history was obtained through a questionnaire. Results: The HIV patients (n=75) aged from 21 to 75 years, mean 41, 96 years and standard deviation 10,313. None of the variables studied was significantly associated with increased odds of HPV detection in HIV individuals. Of the total, 54,7% were women and 25,3 % smokers. They presented a greater frequency (70.7%) of HPV infection compared with the control group (45.83%). It was also observed a higher rate of multiple HPV infection (18.9% vs 8, 82%) than in health people. The most commonly type detected in HIV individuals was HPV-53 (38.88%), whereas in control group it was HPV-6 (31.58%). Indeterminate types were largely detected in both groups. Conclusions: The HIV positive population in this study had a high HPV oral infection. The prevalence of indeterminate types can be due to the presence of non-genital infection by α-HPV not detected by methodology utilized, or they can belong to other genera as β- HPV and γ-HPV types. FINANCIAL SUPPORT: CNPQ, PROPPI - UFF.

HV147 - NEW REAL TIME PCR ASSAY FOR RAPID DETECTION OF TRICHODYSPLASIA SPINULOSA-ASSOCIATED POLYOMAVIRUS IN BIOLOGICAL SAMPLESUrbano, P.R.1; Nali, L.H. da S.1; Pannuti, C.S.1; Pierrotti, L.C.1; Neto, E.D.2; Romano, C.M.1

1. IMT/FAMUSP - Intituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, 01246903

2. DIVISÃO DE UROLOGIA DO SERVIÇO DE TRANSPLANTE RENAL/HC/FMUSP - Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 155 - Cerqueira César, São Paulo - SP, 05403-000

A new polyomavirus, TSV was recently described in a solid organ transplant recipient with rare and serious skin disease, initially described as side-effect of cyclosporine treatment. The disease, Trichodysplasia spinulosa (TS) is characterized by the development of follicular papules and keratin spines known as spicules, which usually manifests on the face of the patient. TS has a similar pattern among affected patients, demonstrating hair follicle dilatation and keratotic plugging of the infundibulum and keratin spicules. Usually occurs proliferation of abnormal enlarged and eosinophilic cells, containing trichohyaline irregular granules. Although there is a strong association between TS disease and the virus, data regarding the mechanisms of pathogenesis and transmission as well as viral diversity are still unknown. Recently, we identified TSV on a skin cancer biopsy from an immunosuppressed patient using metagenomic approaches (GenBank ID KM007161). In this work, we developed a Real Time PCR for TSV detection and investigated the virus in spicules and serial sampled urine obtained from the same patient six months before and after trichodysplasia spinulosa diagnostic. We developed a novel Real Time PCR assay directed to AgT gene using SyBR® Green chemistry. Our method was very specific since do not detect any other polyomaviruses but TSV. Using plasmid as a control, the limit of detection was 1000 copies per microliter in water and 500 copies per microliter in urine. Intra-laboratory repeatability and reproducibility was also conducted. According to our test, TSV was detected in the spicules, biopsy and all urine samples tested. The viral load ranged from 10E3 to more than 10E8 copies varying according to the time of sampling. In conclusion, we presented a new and sensitive test to detect TSV, responsible for a rare and aggressive skin cancer. We also show for the first time that virus is shed in urine at least 6 months before the tumorigenesis and remains detectable even after the treatment. We suggest that early detection of TSV




in urine can be used to monitor immunocompromised patients that are at risk to develop the disease.

HV213 - EXPRESSION PROFILE OF HUMAN ENDOGENOUS RETROVIRUS W (HERV-W) LOCI FROM MULTIPLE SCLEROSIS PATIENTS UNDER DISTINCT THERAPIES BY HIGH THROUGHPUT SEQUENCINGNali, L.H. da S.1; Urbano, P.R.P.1; Silva, D.F.4; Olival, G.S. do3; Penalva de Oliveira, A.C.2; Romano, C.M.1

1. IMT/FAMUSP - Intituto de Medicina Tropical da Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, 01246903

2. Instituto de Infectologia Emílio Ribas, Av. Dr. Arnaldo, 165 - Cerqueira Cesar, São Paulo - SP, 01246-900

3. Centro de Atendimento e Tratamento de Esclerose Múltipla - Santa Casa De São Paulo

4. Instituto de Ciências Biomédicas

HERVs are remaining viral elements, once exogenous, that infected germ line cells and became fixed in host genome through the generations. HERVs sequences compose 8% of human genome and are found in many sites throughout the genome. Several findings indicate that HERVs from the W family (HERV-W) may play a role in Multiple Sclerosis (MS) pathogenesis. Such findings vary from higher HERV-W expression levels in MS patients than in healthy controls to ENV HERV-W protein detection in MS brain lesions. Considering the wide distribution of HERV-W sequences within the human genome some studies have determined the origin of the of HERVW transcript in MS patients through cloning and Sanger sequencing. Here we aimed to determine the HERV-W expression level of these patients and the origin of these transcripts using Next Generation Sequencing (NGS). Blood samples were obtained from MS patients, three from patients under Natalizumab therapy (monoclonal antibody) and three from patients under other therapies. RNA was obtained using Trizol and before cDNA synthesis it was treated with DNAse. A 730bp amplicon from envelope gene was then obtained from each sample and sequenced through Ion Torrent PGM plataform. Reads representing the transcripts from different sources were then mapped to putative chromosomes using a databank containing the most active HERV-W (28 loci) using CLC7 WorkBench. Our workflow indicated that all MS patients presented HERV-W expression from at least 4 different

chromosomes. All patients presented transcripts from different sources, however chromosomes with highest read count frequently found in all patients were: chromosomes 1, 6, 9, 14 and X, suggesting that these are the most frequently sources of ENV-HERVW expression in MS patients. Patients receiving Natalizumab presented transcripts from average 16 different chromosomes in contrast to those treating with βInterferon, which presented transcripts from average 8 different sources. This finding might suggest that patients under Natalizumab therapy, usually with higher Expanded Disability Status Scale, are expressing HERV-W from more chromosomes than patients under βInterferon therapy. Although the small sampling of this study we showed that our method is useful to determine the source of HERV-W expression in biological samples. Increasing the number of samples in this study may clarify if there is actually a distinct HERV-W expression profile in MS patients under different therapies.

HV240 - HIGH FREQUENCE OF SAFFOLD VIRUS IN TONSIL TISSUES FROM PATIENTS WITH CHRONIC TONSILLAR HYPERTROPHYLuna, L.K. de S.1; Valera, F.C.P.2; Tamashiro, E.2; Lima, W.T.A.2; Arruda, E.1

1. Cell Biology Department And Virology Research Center, University Of Sao Paulo School Of Medicine

2. Department Of Otorhinolaryngology And Head And Neck Surgery, University Of Sao Paulo School Of Medicine

Chronic tonsillar hypertrophy (CTH) is a persistent hypertrophy of palatine and pharyngeal tonsils of unknown etiology. Respiratory viruses have been detected in high frequencies in tonsils from patients with CTH, but their role in CTH pathogenesis is unclear. In this study, Saffold virus (SafV), an emerging human cardiovirus which has been found mainly in patients with respiratory or gastrointestinal diseases, was detected by real-time RT-PCR aimed to the conserved 5’UTR in patients with CTH. Samples were comprised of nasal swabs (NS), nasopharyngeal washes (NW) and tissue fragments of palatine (PaT) and pharyngeal (PhT) tonsils obtained from patients (ages: 1-25 years; mean±SD: 6.18±3.23; median: 5.00) with CTH who underwent tonsillectomy at the University of Sao Paulo Hospital in Ribeirao Preto, Brazil. Samples were




collected from November 2011 to March 2013 and were previously tested by real-time PCR for a panel of common respiratory viruses. A total of 709 samples from 210 individuals were tested, of which 643 (179 NS, 168 NW, 146 PaT and 150 PhT) were from 190 patients with CTH, and 56 samples (18 NS, 17 NW; 16 PaT and 15 PhT) were from a control group (CTRL) of 20 individuals without CTH. SafV was detected in 45 of 190 (23.68%) patients with CTH, in at least one sample type (7 NS, 3 NW, 23 PaT and 23 PhT), and in 8 of 20 (40.0%) CTRL patients (1 NS, 0 NW; 4 PaT and 4 PhT). The SafV overall detection frequencies were not significantly different between CTH and CTRL (p = 0.09). In addition, SafV specific detection frequencies in respiratory secretions and tissue samples were also not significantly different between CTH and CTRL (p = 0.7335 and p = 0.1158, respectively for secretions and tissues). Both in CTH and CTRL SafV was more frequently detected in tonsillar tissues (15.54% for CTH and 25.81% for CTRL) than in secretions (2.88% for CTH and 2.86% for CTRL), suggesting that tonsils could be sites of SafV persistence and viral shedding. To the best of our knowledge, this is the first study of SafV infection in tonsils, and further studies including screening of blood samples of CTH patients, fluorescent in situ hybridization in tonsillar tissues, sequencing and phylogenetic analysis of these SafV strains are underway. Financial support: FAPESP, CNPq.

HV247 - FIRST REPORT OF NOROVIRUSES OCCURRENCE AMONG ALLOGENEIC STEM CELL TRANSPLANT RECIPIENTS IN BRAZIL: EVIDENCE FOR PROLONGED VIRAL EXCRETION AND LONG-TERM VIRAL RNA PRESENCE IN THE BLOODSouza, M.1; Lemes, L.N.1; Correa, T. dos S.1; Fiaccadori, F.S.1; Souza, K.M.C.1; da Silva, L.P.2; Arantes, A. de M.2; Cardoso, D. das D. de P.1;

1. LABORATÓRIO DE VIROLOGIA/IPTSP/UFG - Laboratório de Virologia do Instituto de Patologia Tropical e Saúde Pública da Universidade Federal de Goiás, Rua 235 s/n st. Universitário, Goiânia - Goiás, 74605-050

2. HAJ/ACCG - Hospital Araújo Jorge da Associação de Combate ao Câncer em Goiás, R. 0239, 181 - St Universitário, Goiânia - GO, 74605-070

Human caliciviruses (Norovirus and Sapovirus) are important acute gastroenteritis agents. Recent studies

show that in immunocompromised patients, such as transplanted patients, norovirus infection can lead to worsening of symptoms and be confused with clinical symptoms of graft versus host disease (GVHD). However, routine human caliciviruses (HuCV) screening is not performed. In this prospective study we have monitored allogeneic stem cell transplant (ASCT) patients for HuCV infection to investigate the occurrence of HuCV infection, to evaluate prolonged viral excretion in feces and long term viral RNA presence in sera. Fecal samples were collected weekly, and blood samples were obtained every two weeks from ten patients who underwent ASCT, for a minimum period of five months and a maximum of one year. The secretor status was determined by enzyme immunoassay and the detection of HuCV was performed by RT-PCR using primers targeting region C of NoV genogroup I and II (GI and GII) and SaV capsid genes. Genomic sequencing and phylogenetic analysis were also performed for all NoV-positive samples. The results showed that 6/10 patients (60%) had positive samples for NoV, and all of them had a secretor phenotype. SaV were not detected in any of the samples. The main symptoms presented were vomiting and diarrhea. The duration of NoV excretion in feces ranged from five to 143 days, and long term presence of viral RNA in serum ranged from 29 to 36 days in the patients infected with NoV. Three of the six patients had acute intestinal GVHD. All NoV-positive samples were characterized as genotype GI.3. The data highlight the urgent need of the inclusion of HuCV screening in the routine testing performed before transplantation and during follow-up of these patients. This is the first report of NoV occurrence in patients undergoing ASCT in Brazil. FINANCIAL SUPPORT: CNPq, FAPEG AND PPGBRPH/UFG

HV258 - DETECTION AND TYPING OF HUMAN PAPILLOMAVIRUS BY NESTED MULTIPLEX PCR (NMPCR) REVEALS NEW EPIDEMIOLOGICAL PROFILEFaria, M.G.; Matias, B.F.; Costa, E.P.; Julião, J.A.S.; Junior, P.C.F.; Goulart, L.R.

UFU - Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, 38408-100

Epidemiological distribution and genotypic prevalence of the human papillomavirus (HPV), globally, remains troubling due to the strong association between HPV




infection and neoplasia; present in up to 90% of cases of cervical cancer. In order to increase the detection and molecular typing of HPV in gynecologic samples, we have developed a system nested multiplex polymerase chain reaction (NMPCR) for simultaneous amplification of 40 HPV genotypes, detected by capillary gel electrophoresis. Cervical secretion samples were collected from 72 women, randomly chosen at Gynecology Ambulatory of Clinical Hospital of Federal University of Uberlândia (UFU), for HPV genotyping through NMPCR technique. In addition, epidemiological analyses were performed on database of cytological reports (Pap smears) released by pathology service of UFU. For genotypic assessment, it was designed a modified version of the original MY09/11 primer system to cover all 40 sequences of HPV genotype. To identify genotypes of high-, intermediate- and low-risk, 40 specific primers were labeled with fluorophores. The results were classified as positive in the presence of amplification for one (single infection) or more HPV types (multiple infection) (74 to 338 bp) and negative when only the β-globin gene (366 bp) was amplified. Among the 72 women studied, 45.8% were positive for HPV genotyping. In cytological evaluation, 8.3% patients were observed with cell disorders suggested of HPV infection and 8.3% showed atypical squamous cells of undetermined significance (ASCUS). Among the positive patients, only 6.1% were compatible with the cytological analysis. A total of 44.4% of the women studied were negative for both genotyping and cytology, while 34.7% showed positive genotype and negative cytology. Among the patients positive for HPV by NMPCR, 39.4% were detected with single infection, while 60.6% had multiple infections. The most prevalent type in simple viral infection was HPV 52 (38.5%) and multiple infections were HPV 06 (30%). Notably, the discrepancy between genotyping and cytology can be attributed to frequent false-negative smears, which may be due to inadequate sampling or results misinterpretation. On the other hand, false-positive Pap smears may be related to other cervical diseases unrelated to HPV. The NMPCR presented in this study is a very promising and effective tool for HPV diagnostics, not only for population screenings, but also for clinical follow-up of patients. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ.

HV262 - USE OF CHIMERIC PROTEINS EXPRESSED IN PROKARYOTIC SYSTEM IN THE DEVELOPMENT OF A SCREENING METHOD FOR THE EVALUATION OF ANTI-HTLV-1 ANTIBODIESSantos, D.M. da S.1; do Carmo, A.P.3; Martins, M.L.2; da Fonseca, F.G.1; Stancioli, E.F.B.1


2. HEMOMINAS, Rua Grão Pará, 882 - Santa Efigênia - Belo Horizonte - MG, 30622-020

3. IF-ES

Human T-Lymphotropic Virus (HTLV) was the first retrovirus isolated from human beings and presents a wide world distribution. It might be transmitted by breast feeding, sexual contact and blood transfusion. The most important practice to fight this virus consists in prevention, once there is no cure nor efficient treatment against it and, in most of the cases, the carrier stays asymptomatic. Regarding the cross-reaction problems linked to the currently available diagnostic methods, it is necessary to create new tests, which would be more efficient and accurate. A recombinant HTLV-1 multiepitope protein was tested by immunological tests in order to verify its accuracy and efficiency when used in a diagnostic test. A recombinant gene containing highly immunoreactive epitopes was cloned in Qiagen pQE30 expression vector and then transformed into Escherichia coli SG13009 cells. After different induction processes, the proteins were purified using affinity chromatography columns. The proteins were tested by Western blot using infected and non infected patients sera as primary antibodies. The purified protein which showed the best result was used in an Enzyme Linked Immunosorbent Assay (ELISA). In this last test, 80 different sera were used, being 40 from seronegative donors, 20 from asymptomatic seropositve patients and 20 from seropositive patients presenting HAM/TSP. The results were analyzed using the GraphPad Prism 5 software. The statistical tests showed that the means of the HTLV seropositive and seronegative sera differed significantly between themselves according to the paired samples t-test. These results suggest that this protein may be used in clinical serological tests due to its capacity of being recognized by infected patients and not by uninfected patients. Individually, both symptomatic




and asymptomatic HTLV-1 seropositive patients showed absorbance values which differed significantly from the seronegative ones according to one-way ANOVA followed by Tukey test (p=0.0001). This result indicates that the protein could be used to identify the presence of HTLV-1 virus even in situations, such as blood donations, in which the symptoms absence suggests no disease.FINANCIAL SUPPORT: CAPES, FAPEMIG AND CNPQ

HV311 - HEPATITIS C VIRUS QUASISPECIES ANALYSIS USING ULTRA-DEEP PYROSEQUENCINGYamasaki, L.H.T.; Khudyakov, Y.; Vaughan, G.; Raeva, L.M.G.; Diminitrova, Z.E.; Skums, P.; Rendon, D.S.C.; Jardim, A.C.G.; Bittar, C.; Mello, I.M.V.G.C.; Rahal, P.

1. UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José do Rio Preto - SP, 15054-000

2. CDC/HEPATITIS DIVISION - Centers for Disease Control and Prevention 1600 Clifton Road Atlanta, GA 30329-4027, USA


Hepatitis C is a major public health problem. New HCV antiviral drugs were released on market on 2010; however, excluding for genotype 1, the most used therapy used currently is still based on Interferon (IFN) and Ribavirin. Nowadays, genotype 3 is the one with the highest rate of treatment failure. Viral genome variability is one of the factors that lead in therapy failure. HCV presents a high mutability during replication course, implicating in arising of intra-host variants called quasispecies. The hypervariable region 1 (HVR1) from envelope protein presents as quasispecies and may be related to IFN therapy resistance. Resistant quasispecies may not represent majority of variants population in the host, therefore, in these cases traditional sequencing techniques are unable to detect. For detection of minority quasispecies, ultra-deep pyrosequencing (UPDS) is a reliable and efficient tool, being able to detect even variants with frequency <1% in the population. Regarding this issue, we determined HVR1 quasispecies from 14 patients infected with HCV genotype 3 using the UPDS approach. In total, 64,400 HVR1 sequences were obtained from pre-therapy sample. From these

sequences, 27,398 ones with high quality were filtered. Genetic distance and Shannon entropy values were not related to therapy outcome. These sequences were analyzed using median-joining networks and Bayesian population structural analysis. These analysis identified samples with different structures, from high conserved (one sub-population) to high stratified ones (6 sub-populations). Networks analysis also confirmed this result. Mutations exclusive for a type of response of therapy were identified along HVR1. Amino acid sequences indicated that this region presents conserved structure, even if sequence and physical and chemicals properties seem flexible. Especially in turns and coils positions, this conservation seems notable. Potential epitopes positions are concentrated in carboxiterminal and can vary of number and size between quasispecies These results will contribute to the understanding of HCV quasispecies dynamics and therapy and how a high resolution tool as UPDS is essential to it.

HV340 - EFFECTS OF HTLV-1 INFECTION ON BONE MARROW CELLS FROM HTLV-1 INFECTED INDIVIDUALSRodrigues, E.S.1; Favarin, M. do C.1; Macedo, M.D. de1; Otaguiri, K.K.1; Orellana, M.D.1; Takayanagui, O.M.2; Slavov, S.N.1; Covas, D.T.1; Kashima, S.1

1. Regional Blood Center of Ribeirão Preto, Faculty of Medicine of Ribeirão Preto, University of São Paulo (Usp); Faculty of Pharmaceutical Sciences of Ribeirão Preto

2. Faculty of Medicine of Ribeirão Preto, Usp, Brazil

The infection of mesenchymal stromal cell (MSC) by the human T lymphotropic virus (HTLV-1) in vitro can induce alterations in the biological characteristics of these cells. However, little is known about the effects of this infection on the bone marrow (BM) cells of HTLV-1 infected individuals and the MSC biological functions in these patients. Objective: Our objective was to evaluate the HTLV-1 infection in BM cells isolated from HTLV-1 asymptomatic carriers (HAC) and symptomatic patients with tropical spastic paraparesis/HTLV-associated myelopathy-1 (HAM/TSP). Methods: BM mononuclear cells were isolated and characterized by flow cytometry (CD3, CD4, CD8, CD14, CD19, CD34, CD45, CD73 and CD105). From these cells, CD4+ T cells and mesenchymal cells were separated. In both populations, we evaluated




the presence of HTLV-1 by PCR, confocal analysis of the p19 specific protein (Gag) and by EIA (p19 protein). Results: Initially, we observed an infiltration of CD4+ T lymphocytes in BM from HTLV-1 infected individuals when compared to the group of non-infected individuals. Additionally, the detection of proviral DNA by PCR revealed the presence of integrated provirus in the tested CD4+ T cells. The number of fibroblast progenitor cells colonies (CFU-F) was lower in HTLV-1 infected individuals (CFU-F/5 x 105 cells in HAM/TSP (2,11,6), HAC (7,52,1), when compared to non-infected subjects (10,41,1). MSCs isolated from HTLV-1 infected patients showed the expression of typical surface molecules, and differentiation potential into adipocytes and osteocytes similar to the control MSCs. Moreover, proviral DNA and p19 viral protein were detected in MSC in the majority of HTLV-1 patients. However, no p19 antigen was detected in MSC supernatant obtained from HTLV-1 patients Conclusion: These results suggest that MSCs are potential reservoirs for HTLV-1 infection and can contribute to viral dissemination and persistence in the host. These results also can elucidate the pathogenesis of HTLV-1 and the related diseases. FINANCIAL SUPPORT: CTC, INCTC, FAPESP, FUNDHERP and CNPq.

HV346 - EVIDENCE OF HEPATITIS E VIRUS CIRCULATION IN RURAL AMAZONIAMerlone, M.P.1; Vitral, C.L.1; Oliveira, J.M.3; Silva, J.P.1; Nunes, M.S.2; Ferreira, M.U.2; Pinto, M.A.3

1. UFF - Universidade Federal Fluminense, Rua Miguel de Frias, 9, Icaraí, Niterói - RJ, 24220-900



Hepatitis E virus (HEV) is transmitted by the faecal-oral route, and represent a common cause of acute hepatitis in developing countries. Human cases of HEV infection seem to be rare in Brazil, although the virus has been detected in swine livestock and effluents of slaughterhouses. This study was to determine the epidemiology of hepatitis E in one of the largest agricultural settlements in the Amazon Basin of Brazil. Serum samples collected from 397 individuals aged between 5 and 90 years during a population-based cross-sectional survey were tested for anti-HEV antibodies using commercial enzyme

immunoassays kits. Associated risk factors and spatial clustering of HEV seropositivity were also analyzed. Anti-HEV IgG were detected in 50 out of 388 settlers (12.9%, 95% CI, 9.5-16.2%), a HEV seropositivity rate substantially higher than those previously found across the Amazon Basin and other regions in Brazil. HEV IgM antibodies were detected in 7/43 (16.3%) anti-IgG positive samples, and 4 of them had a confirmed result by immunoblot. Increasing age was the only significant determinant of HEV seropositivity detected by multilevel logistic regression analysis (OR, 1.033; 95% CI, 1.016-1.050; P < 0.001). No significant spatial clustering of HEV seropositivity was detected in the area. This study provides data on the prevalence of HEV in a rural setting of the Brazilian Amazon. Anti-HEV prevalence observed in the present study was considerably higher than those previously reported in Brazil. Moreover, the detection of HEV- specific IgM antibodies in four asymptomatic individuals is highly suggestive of the circulation of HEV in this rural population. FINANCIAL SUPPORT: FAPERJ, FAPESP, CNPQ

HV353 - IMPLEMENTATION OF NEXT GENERATION SEQUENCING TECHNIQUES FOR PATHOGEN DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS WITH ENCEPHALITIS AND MENINGITISNunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; Romano, C.M.

IMT/USP - Instituto de Medicina Tropical de São Paulo da Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, Jardim América, São Paulo - SP, 05403-000

The central nervous system (CNS) may be affected by several agents, including viral bacterial or fungal. CNS infections can trigger severe symptoms and, according to the site of infection, maybe designated as encephalitis or meningitis. Viruses are the most common cause of these diseases, followed by bacteria and fungus. Agents such as polyomavirus, Herpesvirus (Simplex, 6 and varicella-zoster) influenza A, enterovirus, mumps, flavivirus, M. tuberculosis and C. neoformans, are responsible for most of the CNS infections, and have a high incidence worldwide. However, prevalence of different agents varies according to population, individual’s immune status, age and region of study. In fact, the prevalence of these infectious agents is well established in many countries. However, there is a lack of information




regarding the prevalence of these agents in the Brazilian population. Although there are diagnostic methods available for identifying most of the etiologic agents that cause encephalitis and meningitis (EM), to obtain results in short period is essential for targeting the most appropriate treatment of the disease. Here we use next generation technology (Ion Torrent) to investigate putative microbial agents in CNS infections through metagenomic approaches in liquor. The advantage of this technique over traditional ones is the capability to detect not only known agents, but also identify pathogens not commonly associated to these pathologies in a fast way. Samples (n=4) from patients with suspected viral EM is were spiked before extraction with baculovirus and bovine viral diarrhea viruses as a control for acid nucleic extraction. After a set of centrifugation steps to exclude human cells, genomic DNA/RNA was obtained using Macherey Nagel commercial kit. Reverse transcription was performed with random primers using High Capacity (Applied Biosystems) kit. Double-stranded DNA was synthesized using Klenow enzyme Fragment (3’>5 ‘exo-) (NEB), followed by NGS sequencing. Filtering, trimming and assemble of the reads were performed in CLC genomic workbench software. Our methods appear to be efficient since small reads of viruses used to spike the samples as internal control were recovered during analysis. In one sample investigated, around 150 contigs of Burkolderia (bacterial agent) were assembled. In conclusion, we demonstrated that the metagenomic methods implemented in the present work are capable to detect viral and bacterial agents affecting the CNS.

HV417 - CO-DETECTIONS OF RESPIRATORY VIRUSES BY QPCR IN THE ABSENCE OF SYMPTOMS OF ACUTE RESPIRATORY INFECTION (ARI): NEW INSIGHTS ON SOURCES OF VIRUS SHEDDINGCriado, M.F.1; Modena, J.L.P.1; de Paula, F.E.1; de Jesus, B.L.S.1; Pestana, N.F.1; Prates, M.C.1; Silva, M.L.1; Saturno, T.1; Tamashiro, E.2; Valera, F.C.P.2; Lima, W.T.A.2; Arruda, E.1

1. University of Sao Paulo, School of Medicine, Virology Research Center

2. University of Sao Paulo, School of Medicine

Viral acute respiratory infections (ARI) are the most frequent illnesses of mankind. Diagnosis of respiratory viruses is useful in medicine and public health, and

the advent of PCR has greatly increased the sensitivity of genome detection and, as a consequence, the simultaneous detection of multiple viruses has become very frequent, challenging the establishment of disease causality. Chronic tonsillar hypertrophy (CTH) is a very frequent ailment and genomes of multiple respiratory viruses are detectable by qPCR in nasopharyngeal washes (NW) and tissues from CTH patients without ARI symptoms. This prompted a search for in situ evidence of productive viral infections in adenoid tissues from patients who shed two or more viruses in respiratory secretions. The patients were 179 children with CTH without ARI symptoms who underwent tonsillectomy at the Otorhinolaryngology Clinic, University of Sao Paulo Hospital in Ribeirao Preto. Remarkably, 47.47% (85/179) had at least 2 viruses, and 21.78% (39/179) of patients had 3 or more viruses detected in NW by qPCR. In addition, one quarter of them (45/179; 25.13%) had 3 or more viruses detected directly in the adenoid tissue by qPCR. Selected formalin fixed, paraffin-embedded adenoid sections from patients with 2 or more viruses detected in NW were stained by immunofluorescence (IF) for major human respiratory viruses: respiratory syncytial virus (HRSV), metapneumovirus (HMPV), parainfluenza virus (HPIV), enterovirus (HEV), rhinovirus (HRV), adenovirus (HAdV) and Epstein–Barr virus (EBV). Slides were analyzed by confocal and multiphoton microscopy. Results showed that in most cases (15 patients tested were positive by IF at least for 2 viruses) structural proteins of the same viruses co-detected by qPCR in NW were also present in situ by IF in hypertrophic adenoid sections. Importantly, these findings strongly indicate that children with CTH without ARI symptoms may shed multiple viruses in secretions, due to ongoing viral activity within a hypertrophic adenoid. Financial support: FAPESP, CNPq, CAPES

HV428 - ANALYSIS OF INFLUENZA A(H1N1)PDM09 VIRAL LOAD IN PATIENTS WITH DIFFERENT CLINICAL PRESENTATIONPerosa, A.H.; Bellei, N.

UNIFESP - Universidade Federal de São Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001

The kinetics of viral load has not been sufficiently studied in patients with pandemic 2009 influenza A(H1N1) and the optimal timing of therapy with antiviral drugs




for critically ill patients is not well established. We performed viral load analysis in samples from patients (children and adults) with confirmed influenza A(H1N1)pdm09 admitted to Sao Paulo Hospital from July 2009 to August 2013. We also studied consecutive samples from hospitalized patients which received oseltamivir treatment. A standard curve for viral RNA quantification was constructed with the target region of M gene from influenza A and viral loads were normalized according to RNaseP to adjust the quantity of virus detected for the quality of the samples. We studied 153 nasal swabs collected from 76 hospitalized patients (mean age ± standard deviation, 31.3 ± 21.3), 65 ambulatory patients (11.7 ± 14.9) and 12 asymptomatic individuals (36.7 ± 6.1). The mean viral load in the 153 collected samples was 6.57 (±1.78) log copies/mL. Viral load in symptomatic patients was significantly higher than asymptomatic (mean ± standard deviation, 6.74 ± 1.69 vs 4.77 ± 1.78 log copies/mL, p<0.001). Ten hospitalized patients with severe acute respiratory illness collected during 2013 influenza season and treated with oseltamivir were followed up and samples were collected until clinical improvement and/or reduction in viral load. Maximum duration of treatment was 30 days. The mean viral load for these patients was 6.64 ± 1.82 log copies/mL. Viral RNA concentration decreased with treatment and prolonged viral shedding (> 7 days after treatment onset) was observed in 70% (7/10) of these patients. Slower viral clearance was observed in patients with major comorbidities like chronic liver disease, lupus, cardiac disease and leukemia, even under prolonged treatment. These data showed that quantification of influenza viral load would be useful for antiviral treatment management.FINANCIAL SUPPORT: FAPESP (2013/00715-0), CNPQ

HV436 - HIV-1 ENV SUBTYPES AND DISEASE PROGRESSION IN A COHORT OF HIV-1 POSITIVE INDIVIDUALS FROM RIO DE JANEIRO, BRAZILLeite, T.1; Campos, D.2; Coelho, A.2; Teixeira, S.1; Veloso, V.2; Guimarães, M.1; Morgado, M.G.1

1. IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - RJ, 21040-360

2. INI/FIOCRUZ - Instituto Nacional de Infectologia Evandro Chagas da Fundação Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-360

Viral and host factors have been described to play a role on the different patterns of AIDS progression. The co-circulation of HIV-1 subtype B, F, variant B” of B subtype and BF1 recombinants has been fully described in Rio de Janeiro, Brazil. Thus, the aim of this study was to evaluate the potential association of HIV-1 subtypes circulating in Rio de Janeiro with the distinct profiles of disease progression. For this purpose, 246 HIV-1 patients under clinical and laboratory follow-up at INI/FIOCRUZ from 1986 to 2011 were classified according to their progression to AIDS in typical progressors (133; TP), rapid progressors (95; RP) and long term non-progressor (18; LTNP). HIV-1 proviral DNA were amplified by env-gp120 nested PCR and then sequenced. Neighbor Joining phylogenetic inferences were performed in Mega 5 program. Chi-square test or Fisher\’s exact test were used to compare the groups. Kaplan Meier method and Cox modeling were performed to analyze the time until AIDS progression according to the HIV subtypes/variants. The classification of the HIV-1 subtypes according among the progression profiles were as follows: TP (63.1% B, 24.8% B”, 8.3% F1, 2.2% C, 0.8% BF1 and 0.8% BD); RP (53.7% B, 24.2% B”, 11.5% F1, 4.2% C, 3.2% D, 2.1% BF1 and 1.1% CRF01_AE) and LTNP (66.7% B, 27.7% B” and 5.6% F1). Similar distribution of HIV-1B and HIV-1B” was observed for the three studied groups. A trend for a higher frequency of HIV-1F1 was observed for the RP group compared to either TP or LTNP groups, even though no statistically significant differences were made evident in these comparisons. In the survival analysis, no differences in progression rate were observed for subtypes B, B” and F1. On the other hand, the group including other less prevalent subtypes (C, D) and recombinants presented a significant risk of progression to AIDS (HR 1.99 [IC95% 1.16; 3.40] P<0.02). Despite the fact of being a heterogeneous group, probably the presence of HIV-1D strongly contributed to this association, since this subtype was found here only in RP group and has rapid evolution profile as previously described. However, in the multivariate analysis this association lost its statistical power. Our results contrast with previous ones that described a slowly disease progression in B” group in comparison with HIV-1 subtype B. Taken together, these data try to contribute to the discussion of the possible role of HIV-1 subtypes in determining the HIV-1 infection outcomes.




HV491 - OPTIMIZATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR NAKED-EYE DETECTION OF DENGUE VIRUS INFECTIONMonteiro, D.C.S.; Carvalho, B.K.S.; Nascimento, V.A.; Souza, V.C.; Naveca, F.G.

ILMD/Fiocruz - Instituto Leônidas e Maria Deane - Fiocruz Amazônia, Rua Terezina, 476 - Adrianópolis, Manaus - AM, 69057-070

The development of a simplified system for nucleic acid amplification, with the possibility of using at minimum equipped laboratories, may improve access to molecular diagnosis of infectious diseases, especially at resource-poor settings. The Loop-Mediated Isothermal Amplification (LAMP) is an example of a very sensitive technique which amplifies DNA at very high levels. Since its development, LAMP has been used to detect various pathogens including bacterias, fungi and viruses. In the present study, a previously published protocol for dengue virus (DENV) detection was reevaluated for the best work conditions and naked-eye visualization using a Brazilian DENV isolate. Firstly, a laboratory sample of DENV serotype 4 virus was propagated in Aedes albopictus C6/36 cells for ten days at 28°C. Subsequently, RNA was extracted from cell supernatant with a commercial kit and converted to cDNA with GoScript (Promega). The positivity to DENV infection was further confirmed by a semi-nested PCR protocol described elsewhere. Variables such as temperature (62°C, 63°C, 64°C, 65°C, 66°C and 67°C) and incubation time (30, 45, 60, 90 and 120 minutes), as well as reagents concentrations were evaluated. Moreover, the addition of hydroxynaphthol blue (HNB), a reagent that may allow to the visualization of LAMP results by naked-eye, was also tested. All conditions were evaluated in triplicate with positive and no-template controls. Finally, all reactions were visually analyzed for color changes and further subjected to electrophoresis on 2% agarose gel to confirm amplification. The best results were achieved when the reaction was conducted at 65°C over a one-hour period, with eight units of Bst DNA polymerase; 8mM of MgSO4; 1,4mM of dNTPs; 1M of betaine; 1,6uM/0,2uM/0,8uM of FIP/BIP, F3/B3 and Loop primers, respectively. The color change due a positive reaction was clearly observed when 120uM of HNB was included. Further studies, including samples representing all four serotypes, are been conducted in

order to confirm the efficiency of the LAMP protocol with Brazilian samples. FINANCIAL SUPPORT: FAPEAM - PAIC; FAPEAM - PPSUS REDE; POM - FIOCRUZ

IV100 - EVALUATION OF TETRAVALENT AND CONSERVED SYNTHETIC PEPTIDES VACCINES DERIVED FROM DENGUE VIRUS ENVELOPE DOMAIN I AND IIRocha, R.P.1; Franco, I.R.1; Livonesi, M.C.1; Fumagalli, M.J.1; Rodrigues, N.F.1; da Costa, L.C.F.1; dos Santos, M.C. da S.G.1; Rocha, E.S. de O.2; Kroon, E.G.2; Malaquias, L.C.C.1; Coelho, L.F.L.1



Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progress to the severe form of the disease named dengue hemorrhagic fever (DHF). In this study, the vaccine potential of a three tetravalent and conserved synthetic peptides derived from Dengue virus envelope domain I (named Pep01) and II (named Pep02 and Pep03) was evaluated. A panel of Dengue IgM/IgG positive human serum was used to determine their ability to recognize the synthetic peptides using an indirect ELISA assay. Of a total of 16 dengue IgM/IgG positive serum, only 3 (18.75%) showed IgM against Pep01; 15 (93.75%) against Pep02 and 16 (100%) against Pep03. The presence of IgG against the peptides was evaluated and14 sera (87.5%) reacted against Pep01; 11 (68.75%) against Pep 02 and 15 (93.75%) against Pep03. Mice immunization experiments showed that these peptides were able to induce a humoral response with an absence or a low titer of neutralizing activity. The IgM mean titers were 37 ± 17 for Pep01 and 300 ± 141 for Pep03. Sera from Pep02 immunized animals showed absence of IgM. The mean total IgG titers of Pep01, Pep02and Pep03 were 96 ± 56; 133 ± 46 and 4800 ± 2262, respectively. The spleen cells derived from mice immunized with




the peptides and infected with a low viral dose of non-adapted DENV-1 showed a significant cytotoxic activity (only for Pep02 and Pep03), a high expression of IL-10 (P<0.01) as well as reduced expression of TNF-α and IFN-g (P<0.001) compared to DENV-1 derived spleen cells. Thus these peptides, and specially the Pep03, can induce a humoral response characterized by antibodies with low neutralizing activities and probably a T cell response that could be beneficial to induce an effective immune response against all DENV serotypes and do not contributed to the immunopathogenesis. However, further studies in peptide sequence will be required to induce the production of neutralizing antibodies against all four DENV serotypes and also to improve immunogenicity of these peptides. FINANCIAL SUPPORT: FAPEMIG; CNPQ; CAPES.

IV183 - THE IMPACT OF ADJUVANT IN WHOLE INACTIVATED VIRUS (WIV) VACCINES FOR INFLUENZA A INFECTION IN PIGS WITH VACCINE-ASSOCIATED ENHANCED DISEASESouza, K.C.1; Rajao, D.2; Loving, L.C.2; Gauger, C.P.3; Vincent, L.A.2

1. UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060

2. NATIONAL ANIMAL DISEASE CENTER/UNITED STATES DEPARTMENT OF AGRICULTURE, Department of Animal Science 1221 Kildee HallIowa State University Ames, Iowa 50011-3150

3. IASTATE - Iowa State University, Ames, Iowa 50011

Adjuvants can improve vaccines by modulating and/or magnifying the immune response. Whole inactivated virus (WIV) vaccines can reduce clinical disease against homologous influenza A virus (IAV) infection; however, WIV vaccines using oil-in-water adjuvants have been associated with enhanced respiratory disease in swine when challenged with heterologous IAV of the same hemagglutinin subtype. We evaluated the immune response of WIV with different adjuvants in a swine model of vaccine-associated enhanced respiratory disease (VAERD) to test the hypothesis that different types of adjuvants similarly predispose to VAERD. Pigs were vaccinated with WIV containing swine δ-cluster H1N2 (human-like, MN08) IAV formulated with five different

adjuvants. The groups given WIV (WIV-MVP, WIV-AddaV, WIV-Gel, WIV-ISA) were vaccinated at 4 weeks of age and were boosted at 7 weeks of age by an intramuscular route. The 5th group, WIV-IMS, was vaccinated at the same time points by the intranasal route. The control groups included non-vaccinated and challenged pigs (NV/C) and non-vaccinated, non-challenged pigs (NV/NC). WIV-MVP (22.1%) and WIV-ISA (21.7%) groups had significantly higher percentage of macroscopic lung lesions consistent with VAERD compared with WIV-AddaV (11.3%), WIV-Gel (9.3%), WIV-IMS (8.1%) and controls NV/C (6.1%) and NV/NC (0.3%). At the time of challenge, WIV-MVP and WIV-ISA groups showed the greatest geometric mean reciprocal hemagglutination inhibition antibody (HI) titers of 640±13 and 1371±12, respectively, against the homologous vaccine virus. The WIV-AddaV and WIV-Gel groups had mean reciprocal titers of 149±13 and 184±13, respectively. The WIV-IMS and non-vaccinated control groups had HI mean titers below the positive cut-off (20). The WIV-IMS did not result in VAERD following heterologous challenge, but also failed to elicit HI antibodies to vaccine antigen. None of the groups had cross-reactive HI antibodies against the challenge strain (pH1N1). The WIV-MVP and WIV-ISA groups had significantly higher levels of IgG antibody in serum against homologous and heterologous virus; however, the WIV-AddaV and WIV-Gel groups also had serum IgG antibody against both viruses. These data show that adjuvant plays a significant role in WIV immunogenicity and subsequently, a role in VAERD. FINANCIAL SUPPORT: USDA-ARS, CAPES AND CNPQ.

IV218 - SELECTION AND CHARACTERIZATION OF MIMOTOPES THROUGH PHAGE DISPLAY FOR IMMUNOGLOBULIN A DETECTION IN HPV DIAGNOSISMatias, B.F.; Lima, M.I.S.; Faria, M.G.; Costa, E.P.; Alves, P.T. Pereira, U.P.; Fernandes Jr, P.C.; Goulart, L.R.


Screening programs based on Pap smear in most developing countries are not well established and often fail in HPV detection and prevention of cervical cancer due to its lack of sensitivity and specificity. Our aim was to develop a new and simple strategy based on IgA detection during HPV infection. The strategy was




to identify epitopes or mimotopes by phage display that could bind to IgA antibodies in saliva and cervical secretion samples from patients with HPV infection. We have selected mimetic peptides to the capsid regions of HPV through a commercial library of random 7-mer peptide library (Ph-D.C7C) against purified IgA of cervical secretion samples on 15 women that were positive to high-risk HPV, confirmed by both Pap smears and DNA genotyping. After three rounds of selection, 96 clones were isolated and sequenced, generating 31 valid sequences. In silico analyses (BLAST, ClustalW2 and I-Tasser programs) revealed 4 sequences that presented partial alignment to antigenic regions of HPV, such as L1 and L2 viral capsid proteins. Clones were submitted to ELISA assay to assess their immunoreactivity against IgA from cervical secretions and saliva of healthy and HPV-infected women. Statistical analysis (ANOVA with Bonferroni post-test) showed that clones differed significantly in both positive and negative samples and also against the control (Irrelevant phage). Our clones are potential biomarkers for HPV screening and therapeutic monitoring, distinguishing infected patients from healthy individuals. These results provide new perspectives for HPV detection with a fast and non-invasive screening strategy. FINANCIAL SUPPORT: FAPEMIG; CAPES; CNPQ.

IV477 - INFLUENZA: DEVELOPMENT OF A REVERSE GENETICS SYSTEM BY YEAST-BASED HOMOLOGOUS RECOMBINATION AND ITS APPLICATION IN THE IMPROVED VACCINE PRODUCTION IN CULTURE CELLSilva Jr, J.V.J.1; Cruz, F. da S.P.1; Bertani, G.R.2; Machado, A. de M.V.3; Gil, L.H.V.G.1

1. CPQAM/FIOCRUZ - Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, Recife - PE, 50740-465

2. UFPE - Universidade Federal de Pernambuco, Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - PE, 50670-901

3. CPqRR/FIOCRUZ MINAS - Centro de Pesquisas René Rachou/ Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002

Recombinant influenza vaccines are produced using genetic reverse system and grown in embryonated chicken eggs. The viruses are generated by cell co-

transfection with cloned cDNA of hemagglutinin (HA) and neuraminidase segments from wild-type strain plus six segments from influenza A/PR/8/34 (PR8) strain. However, theses clones are obtained by use of specific restriction sites and in vitro ligation, which sometimes are difficult to perform. In advantage, the homologous recombination in yeast (HRY) cloning technique is an efficient and simple process, where DNA fragments containing homologous ends with the vector can be directly cloned using in vivo recombination. The currently influenza vaccine also is limited by capacity of the egg supply in pandemic situation, e.g. as occurred in 2009 (pnd2009). Additionally, the WHO recommends Vero cells as an alternative substrate for recombinant influenza vaccine production, however some viruses grow sub optimally in this cell line. Recently, were reported the enhanced growth of H1N1 in Vero cell by changing an amino acid residue in HA (117N>D). Thus, to overcome the difficulties of producing recombinant pandemic influenza vaccine, the objective this work was the development of the first reverse genetics system of influenza made by HRY and the insertion of the HA/117N>D mutation (by HRY) in a chimeric virus expressing HA from isolated pandemic (2009) capable of improved replication in Vero cells. For this, the pJG-HW2000-2013 vector was constructed and the mutation effect was first evaluated in prototype influenza PR8 strain. The PR8 genome was subcloned by HRY from pJG-HW2000 to pJG-HW2000-2013. The pJG-HW2000-2013/PR8 was transfected in HEK 293T/MDCK co-culture and recombinant PR8 (IC-PR8) was generated. After, the HA/117N>D mutation was inserted by HRY into pJG-HW2000-2013/PR8 and mutant virus (IC-PR8/HA-117N>D) was generated. Plaque assay performed in Vero cell showed replication larger in IC-PR8/HA-117N>D than in IC-PR8. After, the HA segment of 2009 influenza isolate was cloned by HRY into pJG-HW2000-2013 and the clone was co-transfected with pJG-HW2000-2013/PR8 and chimeric virus IC-PR8/HApnd2009 was generated. The mutation HA/117N>D was inserted by HRY in IC-PR8/HApnd2009 and mutant virus (IC-PR8/HApnd2009-117N>D) was recovered. Comparison of the plaque assay and viral growth kinetics between IC-PR8/HApnd2009-117N>D and IC-PR8/HApnd2009 in Vero cell is in progress. FINANCIAL SUPPORT: CAPES, CNPQ, FIOCRUZ




IV482 - RECONSTRUCTION OF LATENT AND REACTIVATED QUASISPECIES OF SIVMM251 INFECTING RHESUS MONKEYS AFTER TREATMENT WITH THE LATENCY ANTAGONIST INGENOL-BGonçalves, G. dos S.1; Abreu, C.M.1; Price, S.L.2; Gama, L.2; Lewis, M.3; Pianowski, L.F.4; Tanuri, A.1


2. JHU - The Johns Hopkins University, Baltimore, MD 21218, Estados Unidos

3. BIOQUAL4. KYOLAB, Rua Isaura Ap. Oliveira Barbosa

Terini n° 231,Jd. Itapuã, Valinhos - SP, 13273-105

Viral latency is one the major obstacles for obtaining a cure for HIV infection. Recently, it was proposed a new strategy (“shock-and-kill”) using drugs that reactivate latent virus in combination with antiretrovirals. It is important to evaluate the potential of latency antagonist in reaching viral reservoir, in order to develop an efficient strategy. Ingenol-B is a promising PKC agonist that reactivates integrated HIV-1 in J-Lat cells and in resting CD4 T cells (from humans and monkeys). Rhesus monkeys infected with SIV is an important model to study HIV infection. We have analysed the hypervariable region V1-V2 of gp120 from SIVmm251 infecting two rhesus monkeys treated orally with Ingenol-B throughout 9 weeks, in a dose escalating protocol (1.0 , 2.5, and 5.0 mg BID), with 7 days cycles on and off drugs. Samples from PBMC and plasma from on and off drug cycles, as well as organs autopsies were used in the study. An amplicon sequencing was performed in Illumina MiSeq sequencer. The data generated for each sample was submitted to quasispecies reconstruction using QuRe software, producing different haplotypes (subpopulations) and their related percentage. The gp120 region of reconstructed quasispecies showed a significant diversity between samples and even between subpopulations inside the same sample. The Shannon entropy analysis of the subpopulation diversity in each sample showed a direct relation with the viral load and the diversity circulating in plasma, both fluctuating in response to Ingenol-B. A compartmentalization of quasispecies in reservoir organs was also observed. New subpopulations circulating in plasma emerged during the treatment and the comparison of these subpopulations

with the ones present in reservoirs , demonstrated their relation with different organs. These findings show the potential of deep sequencing for the characterization of quasispecies and Ingenol-B as potent latency antagonist in vivo.

PIV150 - TWO NOVEL BEGOMOVIRUS SPECIES FROM THE NEW WORLD WITH FEATURES RECALLING OLD WORLD BEGOMOVIRUSESGodinho, M.T.; Lima, A.T.M.; Xavier, C.A.D.; Zerbini, F.M.

DFP/UFV - Departamento de Fitopatologia da Universidade Federal de Viçosa, Campus Universitário, Viçosa - MG, 36570-000

Begomoviruses (family Geminiviridae) have a circular, ssDNA genome encapsidated in twinned icosahedral particles. In Brazil, a number of begomoviruses have been described infecting weeds. Here, we describe two novel begomovirus species infecting Sida acuta plants collected from a small area (about 10,000 m2) at Viçosa, state of Minas Gerais in December 2011. Total DNA was extracted from S. acuta samples and the viral genome was amplified by RCA, cloned and sequenced. A total of 12 full-length DNA-A component were obtained from four samples, and the ICTV-established 89% DNA-A identity threshold was used for taxonomic placement. This analysis indicated that the cloned components correspond to two novel species, for which the names Sida golden yellow mosaic virus and Sida yellow spot virus (SiGYMV and SiYSV, respectively) are proposed. The DNA-A components exhibited a highly divergent 5´ half , including part of the intergenic region, the putative CP gene and an AV2-like ORF (present only in Old Word begomoviruses). The deduced amino acid sequence of the CP had very low identity with other begomoviruses, but the presence of conserved motifs in the CP and Rep coding regions, characteristic of OW begomoviruses, was detected. Although New World-like begomoviruses have been found in the OW, this is the first time that OW-like begomoviruses are found naturally in the NW. FINANCIAL SUPPORT: FAPEMIG, CAPES AND CNPQ




PIV160 - COMPLETE GENOME SEQUENCE OF TWO NEW VIRUSES ISOLATES ASSOCIATED TO COTTON BLUE DISEASE BREAKING RESISTANCE IN BRAZILVaslin, M.F.S.1; Fausto, A.K. da S.1; Romanel, E.1; Silva, T. da F.1; Schrago, C.G.1; Galbieri, R.2; Bélot, J.L.2; Vasli, M.F.S.1


2. IMAMT - Instituto Mato-Grossense do Algodão, Av. Rubens de Mendonça, 157. Sala 100, Ed. Mestre Ignácio. Baú, Cuiabá - MT, 78008-000

Cotton blue disease (CBD) is an important disease that affects cotton crops in Asia, South America and Africa. In Brazil it is present in almost all the cotton crop areas, leading to important productivity losses by up to 80% in cotton production. The symptoms include leaf rolling, intense green foliage and a severe to moderate stunting. The disease is transmitted by the Aphis gossypii (Glover) and associated to Cotton leafroll dwarf virus (CLRDV) from the family Luteoviridae, genus Polerovirus. In order to by pass productivity losses associated, Brazilian cotton fields are nowce days almost only planted with CBD-resistant cotton cultivars. Interesting, since 2006, some CBD-resistant cotton fields started to show individual groups of plants presenting CBD-like symptoms. This new disease was called atypical vein mosaic disease and infected plants show CBD mild symptoms as leaf rolling at the plant top and small or null internode shortening associated with reddish and some withered shape of the leaves of the central part of the plants. In the subsequent years this phenomenon spread around all the Cerrado and Southeast cotton planting area and it is nowce days an important disease widely distributed in cotton crops areas in Brazil. Sequencing of the POL-CP block of 13 viruses isolated from symptomatic resistant cotton collected in the field showed that a virus extremely close to CLRDV was associated to it. However, until now any, other part of its genome was met known. In order to better characterize the virus associated with this new disease we sequenced the complete genome of two distinct viral isolates, Acr3 and IMA2, recovered from CBD resistant host plants in 2006 and in 2011, respectively. Infected plants total small RNAs were sequenced by Fasteris Co., Switzerland and the libraries were analyzed at UFRJ using a new software develop by

us for the assembly of viral genomes using small RNA data sets, the SearchSmallRNA, freely available at http://www.microbiologia.ufrj.br/ssrna/. Genomes sequences with 99% and 99,3% of coverage were obtained for Acr3 and IMA2, respectively, using CLRDV-PV1 Brazilian isolate (HQ827780.2) as reference genome. After that new analysis were performed by the mapping software using the reconstructed genomes as reference genome. Complete putative genome sequences were obtained for both isolates. RT-PCR assays were performed to validate the reconstruct genomes and the resulting amplicons were sequenced by Sanger. Here we describe for the first time the complete genome of these two viruses isolates. Identities analysis showed that the new isolates share high nucleotide and amino acid identities with Cotton leafroll dwarf virus, causal agent of Cotton blue disease. Although, theirs P0 protein are 86.1% identical, indicating that these new isolates represent a new polerovirus species. We propose the name Cotton atypical red leaf virus (CARLV) to this new virus. FINANCIAL SUPPORT: CAPES AND FAPERJ

PIV210 - SYSTEMIC ACQUIRE RESISTANCE INDUCED BY CELL WALL PEPTIDOGALACTOMANNAN OF THE FUNGUS CLADOSPORIUM HERBARUM MEDIATES VIRUS PROTECTION IN TOBACCO PLANTSVaslin, M.F.S.; Montebianco, C.B.; Mattos, B.B.B.; Silva, T. da F.; Romanel, E.; Bergter, E.B.; Vaslin, M.F.S.


Virus protection of susceptible crops is an important goal in the agriculture all over the world. Bioactive agents that can mediate this kind of response minimizing damages associated with usual vector control by chemical insecticides are each day more receiving importance in plant applied research. Here we test the ability of a fungal cellular wall glycoprotein from the Cladosporium herbarum to induce systemic acquire resistance against virus infection. C. herbarum is the most prominent mold in air spores. It grows over a wide range of temperatures, and has frequently been reported causing spoilage of meat in cold storage and associated with the development of respiratory allergic disease in humans. Nevertheless, it is also found as a plant pathogen often associated with scab of passion




fruit, besides having been reported to cause disease in others crops as onion, wheat, oats, peanuts, potatoes, tobacco, grapes and coffee. Until now, only one study of the interaction of C. herbarum and plants at molecular level was reported (Mattos et al., in preparation). Here, we checked if the most abundant glycoprotein of the fungus cell wall, the peptidogalactomannan (pGM), may be use for viral protection. Cladosporium herbarum pGM was extracted in sodium phosphate buffer 0.05 M pH 7.0 at 100°C for 2h under reflux, after growth on potato dextrose medium (PDB) for seven days. Suspensions of the pGM, in the concentration of 600μg/mL, were sprayed on plants of Nicotiana tabacum growing in green house conditions using a high pressure apparatus. After 24 hours, the plants were infected mechanically with the Tobacco Mosaic Virus (TMV) with potassium phosphate buffer 0.01M pH 7.2. Control plants, sprayed with pGM or water only, showed no sign or symptoms showing that the pGM spray by itself do not affect plant wellness. The same was observed for plants sprayed with pGM and mock inoculated 24 hours later. N. tabacum SR1 cv. plants sprayed with the pGM before TMV inoculation exhibited milder or no symptoms of the viral disease. Besides that a reduction of approximately 38% of the necrotic lesions was observed when N. tabacum Xanthi cv, where mechanically infected 24 hours after pGM spray. These results show an important role of pGM in the induction of TMV protection in Nicotiana tabacum. To understand the molecular response involved, seven defense-related genes are being evaluated by qRT-PCR assays. They are the pathogenesis-related genes PR-1a (unknown function), PR-2 (β1-3 endoglucanase), PR-3 (chitinase) and PR-5 (thaumatin-like protein); the phenylpropanoid pathway gene PAL (phenylalanine ammonia-lyase); and genes involved in plant stress responses and innate immunity, such as LOX (lipoxygenase) and Prx (peroxidase). FINANCIAL SUPPORT: CNPQ, FAPERJ , CAPES, PROEX AND UFRJ

PIV221 - PROTEIN SYNTHESIS ANALYSIS OF INSECT CELL LINES INFECTED WITH SPODOPTERA FRUGIPERDA MULTIPLE NUCLEOPOLYHEDROVIRUS (SFMNPV)Marcio, M.S.; Sihler, W.; Souza, M.L.

Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901

Baculovirus have been widely used as successful biopesticides for lepidopteran control. The fall armyworm, Spodoptera frugiperda, is an important pest in South America damaging several different crops. A baculovirus pathogenic to this insect, Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNV), has a great potential to be used for controlling this pest. In the present work kinetics of viral protein synthesis was carried out in order to confirm previous results in which two Spodoptera frugiperda cell lines showed to be highly susceptible to this virus (IPLB-SF-21AE and Sf9). Six different lepidopteran cell lines were assayed: Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286) and the two S. frugiperda cells (SF21 and Sf9). Initially, cells seeded at a density of 1X106 per 60mm2 dish were incubated with the SfMNPV I-19 isolate for 1h adsorption time and kept in TNMFH complete medium at 27ºC. At different times post infection (0h, 24h and 72h pi) the medium was replaced by phosphate buffered saline pH 6.2. After a 30min starvation period, a total of 50 Ci of [35S]methionine was added in a 1h pulse. In order to detect the presence of radioactively labeled proteins, a polyacrylamide gel electrophoresis (SDS–PAGE) was carried out and the gel treated for autoradiography. In parallel, morphological analysis was monitored by light microscopy during five days. Typical cytopathic effects began to be visualized after 48h p.i. in the S. frugiperda cell lines. Although both cells showed to be very productive, polyhedra formation was even more intense in SF21 cells than in Sf9 cells. No morphological changes were observed in UFL-AG-286 and BTI-Tn-5B1-4 cells. The LD-625Y and BM-5 cells became highly vacuolated with some visible changes in the cell membrane surface, but none polyhedra production could be observed. Analysis of the kinects of radiolabed proteins showed that the cell protein synthesis was shut off while an intense band of aprox. 30 kD was synthesized in SF21 and Sf9 cells.




This peptide is regarded to be the polyhedrin, the main protein component of the occlusion body (polyhedra). No similar band was observed in the other cell lines. This was predictable since only these two cell lines showed occluded virus particles formation. These results confirm the susceptibility to both Spodoptera frugiperda cells to SfMNPV and point out their use for further in vitro production studies. FINANCIAL SUPPORT: EMBRAPA

PIV295 - CHARACTERIZATION OF A BACULOVIRUS HOST-ACQUIRED PROTEASE INHIBITOR AND ITS ABILITY TO AUGMENT VIRAL VIRULENCEArdisson, A.D.M.P.1; Rohrmann, G.A.2; Bergmann, M.R.1; Rollie, J.C.3

1. Dept. of Cell Biology, University of Brasília2. Dept. of Microbiology, Oregon State

University3. Molecular, Cellular and Developmental

Biology Program, Division of Biology, Kansas State University

The insect immune system responds innately against many invading pathogens. Defensive cells neutralize pathogens by engulfing or trapping them into nodules which become melanized through the action of phenoloxidase (PO). POs are in the insect plasma in an inactive form (proPO) and are activated upon pathogen invasion by a cascade of serine proteases and serine protease-inhibitors (Serpins). Although serpins have been reported in poxviruses, recently the first baculovirus serpin ortholog gene was described in the genome of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV). Baculoviruses are insect viruses with circular dsDNA infective mainly to moth and butterfly larvae. While baculoviruses are known to manipulate many intracellular host processes such as apoptosis and mitosis, as well as host physiology and metabolism, it is not clear whether they can directly control host innate immune responses. Therefore, in this work, we investigated the functionality of the HespNPV serpin and whether it could alter the fitness of a prototype baculovirus. We found evidence that the HespNpV serpin gene is functional and could efficiently inhibit both lepidopteran PO activity and a set of serine proteinases. The gene is most similar to lepidopteran serpins and presents signatures previously described in other insect serpins. When expressed in the prototype baculovirus Autographa californica MNPV, the protein

was secreted, and slightly increased virus titers in vitro. No significant differences in lethal time were observed in infections of two lepidopteran host species, but the virus lethal concentration was 4-fold lower in Trichoplusia ni. During virus infections, the serpin increased cathepsin activity and partially reduced caspase activity. Based on our observations and previous work, we hypothesize that acquisition of this serpin gene by HespNPV ancestor may have resulted in increased viral virulence, and its expression may be advantageous in pest control strategies.

PIV357 - IDENTIFICATION OF A NEW DCL3 GENE IN COTTON GOSSYPIUM RAIMONDII (ULBRICH) D-GENOME AND STUDY OF THE ROLE OF NEW ISOFORMS OF GENE SILENCING RELATED GENES IN COTTON VIRUS INFECTED PLANTSVaslin, M.F.S.1; Romanel, E.1,2; Moura, M.1; Lei, G.3; Paterson, A.4



3. IASTATE - Iowa State University, Ames, Iowa 50011

4. UGA - University Of Georgia, Athens, GA 30602, Estados Unidos

The genus Gossypium is composed by ~50 species ranging from 885 Mb per haploid nucleus in D-genome (G.raimondii) to 2572 Mb in K-genome. Genes involved in plant small RNA biogenesis pathway, keys component of RNA-silencing mechanism, are essential for eukaryote development, nutritional and stress responses, chromatin regulation and viral defense. Searching for Dicer-like (DCL), Argonaute (AGO), RNA-dependent RNA polymerase (RDR) and nuclear RNA polymerase IV/V (PolIV/V) gene sequence in G. raimondii genome (98.3%), we surprisingly find duplication gene event for DCL3, AGO1, AGO4, AGO7, AGO10, RDR1 and Pol IVa. Beyond these extra DCL3, AGO1, AGO4, AGO10 and Pol IVa-2 proteins, G. raimondii genome showed a higher number of isoforms of these genes compared to the others plants species studied here. All these paralogous cotton duplicated genes showed a KS value which fit with the unique cotton whole genome duplication event.




It is the first report of an extra-DCL3 and Pol IVa protein existence in eudicots. Most of these genes (DLC3, AGO4 and Pol IVa) are involved in 24-nt siRNA production and secondary steps DNA methylation and chromatin remodeling at their target loci. The increase number of genes and its isoforms points out a possible role of them in the control the large amount of retrotransposons (53%) and repetitive DNA elements present in cotton species. In a previous study, we find that the DCL2 is downregulated as well DCL4 are unregulated in cotton cultivate plants (from G. hirsutum specie) during Cotton leafroll dwarf virus infection. Now, the expression of some of these new genes and isoforms during virus infection are been assayed by qRT-PCR. Primers for DCL2a, DCL2ab, DCL3a, DCL3b, DCL4; AGO1a, AGo1b, AGO1c, AGO2; PollVa1; PollVa2, PollVb were design and are been tested in order to try to understand theirs possible role during virus:plant interaction. FINANCIAL SUPORT: FAOERJ, CNPQ AND PIBIC/UFRJ

PIV378 - THE FIRST COMPLETE GENOME SEQUENCE OF COFFEE RINGSPOT VIRUS; AN EMERGING THREAT TO COFFEE PRODUCTION AND QUALITYFigueira, A. dos R.1; Ramalho, T.O.1; Sotero, A. de J.1; Duarte, P. de S.G.1; Wang, R.2; Farman, M.2; Goodin, M.M.2

1. UFLA - Universidade Federal de Lavras, Câmpus Universitário, Lavras - MG, 37200-000

2. UKY - University of Kentucky, Lexington, Kentucky 40506

Coffee ringspot virus (CoRSV) was first described in 1938 in Sao Paulo state, Brazil, and considered a minor disease for decades thereafter. Symptoms of coffee ringspot disease include chlorotic or necrotic spots, usually forming concentric rings, which can also be observed in fruits and young branches. CoRSV infected leaves usually fall from infected plants within four to five weeks after symptom appearance, resulting in both loss of photosynthetic capacity and source of shade for developing coffee cherries. In addition, infection of fruits results in their accumulation of compounds that contribute to depreciate the coffee brew. CoRSV is transmitted by the false spider mite Brevipalpus phoenicis and was tentatively classified as a member of the Rhabdoviridae family, genus Nucleorhabdovirus, based on the site of replication. In this study the genome of

CoRSV was sequenced by the first time, and the sequence was analyzed and compared with other rhabdoviruses available in the GenBank. The CoRSV genome is bipartite, containing negative ssRNA, with 6,522 nucleotides in the RNA1and 5,945 nucleotides in the RNA2. The RNA1 is composed by five open reading frames (ORFs): ORF1 likely encodes the CoRSV nucleocapsid protein, with a predicted molecular weight of 49 kDa; ORF 2 is likely to encode the phosphoprotein with a molecular weight of 27 kDa; ORF 3 encodes a 36 kDa protein with homology to viral cell-to-cell movement proteins; ORF 4 encodes a 20 kDa protein with unknown function and the ORF5 encodes a 60 kDa glycoprotein. Each ORF is separated by a conserved tri-modular intergenic spacer. The RNA2 encodes a single protein (ORF6), which has all the hallmarks required for assignment as a RNA dependent RNA polymerase. The leader and trailer regions of RNAs 1 and 2 are separated from their nearest ORF by truncated gene junctions containing only the transcription start site or polyadenylation signal, respectively. Phyogenetic analysis of L protein sequences firmly establishes CoRSV as a member of the proposed Dichorhavirus genus, which has an Orchid fleck virus as type member, and shares 70% similarity with CoRSV. In this study a wealth of information and resources were generated, serving as a foundation of future studies for the control of this emerging virus that is an increasing threat to coffee production and quality. FINANCIAL SUPPORT: CNPQ, FAPEMIG, CAPES AND CNP&D-EMBRAPA CAFÉ.

PIV383 - SUBCELLULAR LOCALIZATION OF COFFEE RINGSPOT VIRUS PROTEINSRamalho, T.O.1; Figueira, A.R.1; Sotero, A.J.1; Duarte, P.S.G.1; Wang, R.2; Farman, M.2; Goodin, M.M.2


2. UKY - University of Kentucky, Lexington, Kentucky 40506

Coffee (Coffea arabica) is the second most valuable traded commodity after petroleum. Although grown commercially in most tropical and subtropical countries, the world supply of coffee is dominated by Brazil, which produces 35% of the global exports of green coffee beans. As such, the emergence of Coffee ringspot virus (CoRSV), which is transmitted by the false spider mite Brevipalpus phoenicis, is of critical concern. CoRSV




was first described in Sao Paulo state in 1938 and is tentatively classified as a member of Rhabdoviridae family, genus Nucleorhabdovirus. The CoRSV genome consists of bipartite RNAs with negative-sense polarity. The RNA1 is composed by five Open Reading Frames: ORF1, nucleocapsid protein; ORF 2, phosprotein; ORF 3, viral cell-to-cell movement protein; ORF 4 a protein of unknown function and ORF 5, glycoprotein. The RNA2 encodes a single protein, RNA dependent RNA polymerase. Subcellular localization of viral proteins is frequently difficult to determine strictly from in silico methods. Therefore, in this study an established strategy for protein localization was employed, using an Agrobacterium-mediated transient expression of fluorescent protein fusions in transgenic Nicotiana benthamiana lines that express subcellular markers. The five proteins encoded by the ORFS of RNA 1 were expressed as carboxy-terminal fusions to GFP in transgenic plants expressing a red nuclear marker. The analysis at confocal microscope showed that GFP-P1 fusion was detected at steady state in both the nucleus and the cytoplasm. In marked contrast, the GFP-P2 fusion was localized exclusively in the nucleus. Consistent with a predicted cell-to-cell movement protein function, the GFP-P3 fusion accumulated predominantly at the cell periphery. GFP-P4 accumulated into the nucleus but could also be found as cytoplasmic aggregates. GFP-P5 was targeted to the nuclear envelope and, to a lesser extent, at perinuclear membranes. Taken together, the nucleophillic character of the proteins encoded by ORFs 1, 2, 4 and 5 are consistent with their predicted roles in viroplasm formation and viral morphogenesis. This study gives important information and provides resources to a function prediction and localization of CoRSV proteins.

PIV423 - COMPLETE GENOME SEQUENCE OF A TOBACCO-INFECTING TOMATO BLISTERING MOSAIC VIRUSFernandes, J.E.F.1; Melo, F.L.1; Ribeiro, B.M.1; Ribeiro, G.S.2


2. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901

The tymoviruses (family Tymoviridae, genus Tymovirus) are icosahedric, non-enveloped, single-stranded positive sense RNA viruses. Only five tymoviruses infecting different plant species were reported in Brazil. Most of these viruses were identified as distinct tymoviruses based on biological, biochemical and serological properties, and no complete genomic RNA sequences are available for these viruses, except for the recently described Tomato Blistering Mosaic Virus (ToBMV). ToBMV was isolated from tomato, and was serologically related to Eggplant mosaic virus (EMV), suggesting a cryptic diversity within Brazilian tymoviruses. Therefore, we revisited a previously described tobacco-infecting EMV to confirm its taxonomic status. Frozen viral stock was used mechanically inoculate N. tabacum ‘TNN’ plantlets. The virus was purified from symptomatic tobacco leaves and the viral RNA was extracted, gel purified and sequenced using an Illumina HiSeq 2000 platform. The paired-end reads were assembled using CLC Genomics Workbench version 6.0.3. The assembled contigs were submitted to blastx search against a viral genome database. A contig of 6,256 nucleotides was found to be similar to tomato blistering mosaic virus. This contig was annotated and tree intact ORFs were identified. The ORF1 (5427 nt) encodes the replication polyprotein with 1809 amino acids. The ORF2 (1956 nt) encodes the movement protein and the ORF3 (573 nt) encodes the capsid protein, with 652 and 191 amino acids, respectively. The 5′ and 3′ untranslated regions (UTRs) are 129 and 117 nt long. A phylogenetic analysis using 24 complete genomes available in GenBank confirmed that the prior identified EMV-tobacco isolate is in fact, an isolate of ToBMV, with 88% nucleotide identity over the entire genome. Interestingly these two ToBMV isolates differ in the ability to infect N. tabacum ‘TNN’ plants. The comparison of the coding region of both isolates revealed that most of the nucleotide differences among the two genomes are synonymous, except for those occurring in the ORF2, which presented a high degree of non-synonymous substitution.




PIV452 - COMPLETE GENOME SEQUENCE OF A NOVEL IFLAVIRUS ISOLATED FROM OPSIPHANES INVIRAESilva, L.A.1; Melo, F.L.1; Tinôco, R.S.2; Fernandes, O.A.3


2. FCAV/UNESP - Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, SP, 14884-900

3. PLANT PROTECTION AND RESEARCH MANAGER OF GROUP AGROPALMA S/A, Alameda Santos, 466 10. andar Cerqueira Cesar, São Paulo, SP, 01418-000

A new Iflavirus was isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae) and its complete genome sequenced. O. inviridae, in its larval stage, is considered a pest, causing defoliation in oil palm, coconut and some native palms in northern part of South America. This new virus was isolated from O invirae larvae collected in Tailândia, Pará State - Brazil, which showed a brown color and discoloration of the posterior and middle parts of the insect body. The virus particles were purified by ultracentrifugation in a sucrose cushion and the viral RNA was extracted according to TRIzol® Reagent (Invitrogen) protocol. The viral RNA was sequenced at Macrogen (South Korea) using an Illumina HiSeq 2000 platform. The paired-end reads were assembled using CLC Genomics Workbench version 6.0.3. The assembled contigs were submitted to blastx search against a viral genome database. A contig of 10,083 nucleotides was found to be similar to Iflavirus, which have a single-stranded RNA genome of positive polarity that possess a single Open Reading Frame (ORF) of 9,558 nucleotides (nt), encoding a polyprotein, which is post-translationally processed into viral proteins essential for its replication, packaging and transmission. The 5’UTR (254 nt) includes an internal ribosome entry site (IRES). The single large ORF encodes both structural (5’terminus) and non-structural (3’ terminus) proteins. The ORF is followed by a 3’UTR (271 nt). The phylogenetic analysis revealed that this new iflavirus is related to Spodoptera exigua iflavirus 1. FINANCIAL SUPPORT: CNPQ (PROC. 147765/2012-9)

PIV509 - THE FUNCTIONAL ANALYSIS OF DISTINCT TOSPOVIRUS MOVEMENT PROTEINS (NSM) REVEALS DIFFERENT BEHAVIOR FROM THE ALFALFA MOSAIC VIRUS (AMV) MODEL SYSTEMLeastro, M.O.1; Peiró, A.2; Pallás, V.2; Navarro, J.A.S.2; Resende, R.O.1


2. IBMCP/CISIC-UPV - Instituto de Biología Molecular y Celular de Plantas do Consejo Superior de Investigaciones Científicas de la Universidad Politécnica de Valencia, Ingeniero Fausto Elio, s/n 46022 Valencia

Plant viruses have developed a class of proteins responsible for ensuring viral infection and dissemination denoted movement proteins (MPs). The MPs interact with the plasmodesma enhancing its size exclusion limit and, in this way, allowing the virus passage. For tospoviruses, it was demonstrated that virus movement requires tubules formation driven by its non-structural movement protein (NSm). Although, it is expected that this mechanism would be conserved among the 28 tospovirus species reported so far within the genus, comparison among them reveals significant differences in amino acid sequences of viral proteins and in their biological features such as host range. Some tospovirus species present a narrow spectrum of host plants, e.g BeNMV, while others such as TSWV, TCSV and CSNV display a broad host range infecting plants from a large number of different botanical families. Due to its main function, the NSm proteins are often assigned as potential determinants of host specificity and/or adaptation. The aim of this study was to evaluate the role and the efficiency of four tospovirus MPs on cell-to-cell and systemic movements using the heterologous Alfalfa mosaic virus AMV system, that allows the functional exchangeability of viral movement proteins (MPs) assigned to the “30K family”. Here, differences in the efficiency in cell-to-cell and systemic movement were observed based on the average sizes of the infected areas supported by the distinct tospovirus MPs tested. Also, we observed that all MPs analyzed were not competent to support the transport of an AMV RNA 3 carrying a CP mutant (CP206) defective in virus particles, indicating the incapacity of the MPs to transport other complexes different than virions in the AMV context. It was also demonstrated that the MPs of the four tospovirus




species were similarly efficient to form tubules. However, C-terminal deletion of the MPs revealed that C-terminus of the MP of BeNMV (the only tospovirus here showing a narrow host range) was shown not to be essential for virus movement, in contrast to CSNV, TCSV and TSWV-MPs which required the entire NSm proteins to guarantee the cell-to-cell movement. These results confirmed different features and behaviors among the tospovirus movement proteins. We suggest that the NSm protein plays a role in the determination of the viral infection spectrum of host plants based on the differences observed in cell-to-cell and systemic movement patterns among the tospovirus MPs. FINANCIAL SUPPORT: UNIVERSIDADE DE BRASÍLIA (PPG BIOLOGIA MOLECULAR), CNPQ, CAPES, FAP-DF, UPV-CISIC, UNIÓN EUROPEA, GOBIERNO DE ESPAÑA

VV27 - ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE APOBEC3H GENE OF DOMESTIC CATS (FELIS CATUS) AND THEIR ASSOCIATION WITH THE SUSCEPTIBILITY TO FELINE IMMUNODEFICIENCY VIURUS AND FELINE LEUKEMIA VIRUS INFECTIONSCostenaro, J.G.; de Castro, F.L.; Junqueira, D.M.; de Medeiros, R.M.; da Silva, T.R.; Knak, M.B.; Almeida, S.E. de M.; Campos, F.S.; Roehe, P.M.; Franco, A.C.


The Feline Immunodeficiency Virus (FIV) and the Feline Leukemia Virus (FeLV) belong to the Retroviridae family, are widely distributed and induce a significant immunosuppressive effect in infected domestic cats (Felis catus). Proteins with activity against retroviruses are conserved between mammals and determined as restriction factors. Cytidine deaminases of the APOBEC3 gene family are the most studied class of restriction factors and the APOBEC3H gene encodes two proteins (APOBEC3H and APOBEC3CH) that are the most responsible for this restriction among cats, through hypermutations in provirus DNA during reverse transcription. Changes on the gene sequence can alter the stability and cellular localization of homologous proteins to APOBEC3H in mammals. Considering the importance of APOBEC3H gene in cats, the investigation of its variability is relevant. Fifty DNA samples of cats

FIV and/or FeLV positives and fifty-nine of negative cats to both viruses were used as template to amplify two different regions of the gene, with subsequent sequencing and analysis. The first investigated region was conserved among all samples. On the second one it was possible to identify six single nucleotide variation points, and of them, the A65S (A65I) was significantly correlated with the susceptibility to FIV and/or FeLV infection. On the other hand, the haplotype analysis showed that the combination “GGGGCC” was significantly correlated with the lack of retroviral infection, maybe indicating a protective effect. Whereas a polymorphism at position 65 have been found in Indochinese Tiger and given the correlation found in this research, more studies about the effect on the activity of restriction factors encoded by the A3H gene should be performed. Similarly, research about the effects of the combination “GGGGCC” should be carried so that we can confirm a possible protective effect shown in this work. FINANCIAL SUPPORT: CNPQ, FINEP

VV37 - PARTIAL CHARACTERIZATION OF AVIAN INFLUENZA VIRUS (H11N9) IN MIGRATORY BIRDS (ARENARIA INTERPRES) CAPTURED IN AMAZON REGION, BRAZILde Araujo, J.1; Júnior, S.M. de A.2; Gaidet, N.5; Hurtado, R.F.1; Walker, D.4; Thomazelli, L.M.1; Ometto, T.1; Seixas, M.M.M.1; Galindo, D.B.3; Webby, R.J.4; Webster, R.G.4; Durigon, E.L.1

1. ICB/USP - Instituto de Ciências Biomédicas da Universidade de São Paulo, Edifício III USP - Administração - Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, 05508-900

2. DB/UFRPE - Departamento de Biologia da Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife - PE, 52171-900

3. Agência de Defesa Agropecuária do Estado do Pará- ADEPARA, Pa-Brazil

4. Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tn, USA

5. CIRAD-ES, Ur Agirs, Montpellier, France

The prevalence of Avian influenza viruses in North America and Eurasia is known a long time, while their prevalence in wild birds in South America is largely unknown. Aquatic birds are considered natural reservoir for avian influenza viruses (AIV) and Brazil is the second country with the highest biodiversity in species of




migratory birds. So, the current study was conducted to characterize of the avian influenza virus detected in four samples of migratory birds (Arenaria interpres). Samples were collected in Ilha de Canelas, located in Amazon region, Pará State, in November 2008. Mist nets were strategically placed near the beach and the mangrove to capture of birds. In this occasion, a total of 80 samples were collected by orotracheal/cloacal swabs, and were put in the VTM medium and immediately stored in liquid nitrogen. Blood was collected and sera were separated on field. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) to identify influenza viruses in wild birds were used with AIV-M TaqMan Reagents kit (ABI). Four samples were Influenza A virus positive by molecular test. Then were submitted to viral isolation, inoculated in embryonated chicken eggs for viral replication. The result was confirmed per Avian Influenza Virus Type A Test Kit (Synbiotics). From these samples, three viruses were isolated, sequenced and characterized by Haemagglutination-inhibition (HI) test using full panel of HI sera standard. All positive samples showed similarity with subtype H11N9 groups. Analyze of sequences confirmed the HI results. Our results from surveillance of migratory birds have shown great importance, because these birds have not nationality defined, and are in constant migration between the Northern and Southern Hemispheres. Therefore, these movements deserve special attention, particularly after the incidence of recent outbreaks of avian influenza in others continents. To our knowledge, this is the first isolation of H11N9 in the Arenaria interpres in South America.

VV96 - AVIAN CORONAVIRUS AND AVIAN BORNAVIRUS DETECTION IN FREE-RANGING PSITTACINESLima Neto, D.F.1; Barnabé, A.C.S.1; Caserta, L.1; Martini, M.C.1; Simas, P.V.M.1; Nagel, N.E.2; Lierz, M.2; Hafez, H.M.2; Felippe, P.A.N.1; Arns, C.W.1


2. FREIE UNIVERSITÄT BERLIN, Kaiserswerther Straße 16-18, 14195 Berlin, Alemanha

A key issue in understanding the dynamics of multi-host pathogens in an ecosystem is the Identification of the pathogen reservoir. Coronaviruses (CoV) are

enveloped, positive sense, single-stranded RNA virus. CoV has been recognized as a major cause of respiratory disease in several species with important outcomes such as the SARS pandemic event. Infectious bronchitis virus (IBV), a group 3 coronavirus, causes a costly viral disease of chickens that is found worldwide. It can cause respiratory disease in chickens of all ages and a loss of production and egg quality in mature hens. Some strains are nephropathogenic, resulting in renal-induced mortality rates of up to 25% for susceptible flocks. PDD Proventricular Dilatation Disease (PDD) is a worldwide known fatal disease in birds, mainly affecting psittacines but also other species. This disease is characterized by a gastrointestinal dysfunction with or without neurological symptoms. However, bornavirusABV was also found in healthy birds, indicating that additional factors may be required for ABV to cause the clinical disease. To date, 9 different genotypes of ABV including non-psittacine birds have been detected from birds in Africa, Europe, North America, Japan and Australia. All recent studies involved psittacines originating from captivity. No further sampling or diagnose was possible with these birds. According to the available literature, no PDD or ABV cases have been reported in free-ranging psittacines and few studies were published on the detection of Coronaviruses from parrots. In this study we report about the presence of both Coronavirus and ABV infection in apparently healthy, free ranging psittacines in Brazil for the first time. 142 tracheal and cloacal swabes were collected between 2009 and 2010 of 7 species of psitacideos São Paulo and Mato Grosso do Sul states, Brazilian Southeast and Midwest. By RT-PCR, 33.8% ABV infections, 15.49% Pancoronavirus infections and 6:33% had coinfection ABV-Pancoronavirus. This work was supported by CAPES/CNPq and FAPESP, grant number 2011/50919-5.




VV113 - PRIMARY AND SECONDARY MUCOSAL IMMUNITY AFTER CHALLENGE WITH BRAZILIAN AVIAN INFECTIOUS BRONCHITIS VARIANTOkino, C.H.1; Mores, M.A.Z.1; Montassier, H.J.3; Mattos, G.L.M.1; Brentano, L.1; Coldebella, A.1; Ritterbusch, G.A.2; Esteves, P.A.1; Trevisol, I.M.1

1. EMBRAPA SUÍNOS E AVES, Parque Estação Biológica - PqEB s/nº, Brasília, DF, 70770-901

2. UFPEL - Universidade Federal de Pelotas, Capão do Leão - RS, 96160-000

3. UNESP - Universidade Estadual Paulista, Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010

Avian infectious bronchitis virus (IBV) is one of the most important poultry industry pathogens. IBV is a gammacoronavirus, characterized for high genomic mutations, with frequent emergence of new variants and incomplete vaccine protection. The aim of this study was to evaluate humoral and cell-mediated immune responses in birds vaccinated with the H120 attenuated strain and challenged with Brazilian IBV field isolates genotyped as IBV variants. Twenty seven SPF chickens were equally distributed in three groups placed in positive pressure isolators. At one-day old group A birds received full-dose H120 vaccine. At 28 days of age, all birds from vaccinated group A and from not vaccinated group B were challenged with 104.0 EID50/bird of F3735 Brazilian field strain of IBV. Group C was mock infected. All birds were euthanized at 5dpi, tracheas were removed and evaluated for ciliary activity, microscopic lesions and viral load to determine degree of vaccine protection, and for RT-qPCR quantification of immune response genes (innate:TLR3, TLR7, MyD88, IFNα; inflammatory: IL6; cell-mediated: CD8β, CD3ε, CD4, IFNγ and Granzyme homolog A). Tears and serum were analyzed for anti-IBV IgG by ELISA. Scores of lesions observed by ciliary activity and histopathology, and levels of viral load, were significantly reduced on vaccinated group A compared to group B. All immune related genes tested were significantly up-regulated for the primary immune response in group B. Although lower levels of mRNA for TLR7, MyD88, IFNα, IFN γ, IL6, CD8β, Granzyme homolog A and CD3 were found on tracheal samples of birds from group A (secondary response) compared to group B, the differences were not significant, what in part may be due to the presence of two birds that resulted as unprotected in the vaccinated

group A. Transcripts for CD4 were significantly lower on vaccinated group A compared to group B. Levels of local and systemic IgG were significantly higher in group A compared to unvaccinated/challenged group B. These results indicate an exacerbated local cell-mediated primary immune response to the IBV field variant strain, and a lower level secondary response to vaccination and variant IBV challenge for the evaluated interval post-infection. Otherwise, both local and systemic humoral responses were boosted during secondary response. FINANCIAL SUPPORT: PROJETO EMBRAPA 03.12.03.012.0.00

VV172 - ASTROVIRUS DETECTION IN THE BAT TADARIDA BRASILIENSIS (GEOFFROY, 1824: CHIROPTERA, MOLOSSIDAE) FROM RIO GRANDE DO SUL STATE, BRASILDuppont, P.M.1; Pacheco, S.M.2; Rosa, J.C.A.3; Streck, A.F.1; Alves, C.D.B.T.1; da Silva, M.S.1; Budaszewski, R.F.1; Weber, M.N.1; Canal, C.W.1


2. ISAUVER - Instituto Sauver, Av. Pernambuco, 2623 sala 404 Bairro Floresta,Porto Alegre - RS, 90240-005

3. IPVDF/FEPAGRO - Instituto de Pesquisas Veterinárias Desidério Finamor da Fundação Estadual de Pesquisa Agropecuária, Estrada do Conde, 6000, Eldorado do Sul - RS, 92990-000

Bats are the second largest group of mammals on the planet, with about 1,200 species and 174 are found in Brazil. Due to changes and fragmentation of natural habitats, these animals seek shelter alternatives and thus become increasingly exposed to anthropic enviroments. Some bat species are recognized as natural reservoirs of several viral families and this feature gives them an important role in the transmission and maintenance of these microorganisms. This work aims to detect the presence of astrovirus genome fragments in bat organ samples from Rio Grande do Sul State, Brazil. The bats were caught with mist nets at roost or foraging sites in natural enviroments and entomological network, gloves and long tweezers were used for the urban areas. The animals were euthanized and sent to the Instituto de Pesquisas Veterinárias Desidério Finamor/FEPAGRO for rabies diagnosis. Negative rabies samples were further




analysed to detect the presence of astrovirus using RT-PCR. In total, 38 bats samples belonging to thirteen species were studied. The samples were submitted to RNA extraction followed by RT-PCR. As results, three positive samples (11%) were found in only one species (Tadarida brasiliensis). Those positive samples were collected in urban areas, while the presence of astrovirus was not identified in samples from natural enviroment. Possibly, the anthropic enviroment may contribute to the presence of the virus, since previous studies showed that bat astroviruses may be related to human astrovirus, but these possibility will be further studied. FINANCIAL SUPPORT: CAPES, CNPq, FAPERGS and Propesq/UFRGS.

VV207 - DETECTION OF AVIAN GROUP F ROTAVIRUS IN FECAL SAMPLES OF BROILER CHICKENS IN PARA STATE, BRAZILBezerra, D.A.M.1; Silva, M.J.M.1; Bezerra, D.A.M.1; Silva, R.R.2; Soares, L.S.1; Mascarenhas, J.D.P.1


2. Laboratório Nacional Agropecuário

Rotavirus (RV) is a major viral agent of enteric disease in humans and animals. They are non-enveloped viruses with genome divided into eleven segments of double-stranded RNA that encodes 12 proteins. The VP6 protein is conserved and an excellent target for laboratory diagnosis as well as being determinant of species/groups of RV these are classified into eight (A-H). The group F rotaviruses (RVF) infect only birds causing damage to health and consequently losses to the poultry market. Presently, there are few data in the literature on the RVF particularly using molecular methodologies such as the reverse transcriptase-polymerase chain reaction (RT-PCR). In this context, this study aimed to detect RVF in broilers in Para state, Brazil, during 2008 to 2011. A total of 85 pools of fecal specimens of broilers were collected in 37 farms located in eight different municipalities in the metropolitan region of Belém (Belém, Ananindeua, Benevides, Castanhal, Santa Isabel do Pará, Inhagapi, Santa Bárbara do Pará e Santo Antônio do Tauá). Viral genome was extracted from stool suspensions and subjected to RT-PCR using a specific primer pair designed for the gene encoding VP6 protein of RVF (RF6F / RF6R). The positive samples were sequenced using the same primers in RT-PCR. RVF positivity was detected in 9.4%

(8/85) of pools. Samples from five of eight municipalities were positive for RVF with infections spread in 7 of 37 (18.9%) farms. The higher rate of infection was related to the age of 16-30 days. This study is one of the pioneers to detect RVF by RT-PCR, as well as adding information about the occurrence of RVF in Brazil, providing more representative data about this group of RV. FINANCIAL SUPPORT: FAPESPA; CNPQ.

VV229 - LONG-TERM CIRCULATION OF INFLUENZA A VIRUSES IN SWINE IN BRAZILSchaefer, R.1; Nelson, M.2; Gava, D.1; Cantão, M.E.2; Zanella, J.R.C.1


2. FIC/NIH - Fogarty International Center - National Institutes of Health, 31 Center Drive, MSC 2220 Bethesda, MD 20892-2220 USA

Influenza A viruses (IAVs) circulating in swine present important economic concerns for the swine industry and a pandemic threat for humans. Although Brazil hosts one of the largest swine populations in the world, there has been little evidence prior to 2009 of IAV circulation in Brazilian swine herds. Following the detection of pandemic H1N1 (H1N1pdm) IAVs in pigs in Brazil in 2009, surveillance efforts increased. Screening of 1440 nasal swab samples was carried out by RT-qPCR assay. Thirty-nine positive samples were submitted to virus isolation. Genetic sequencing was performed by ABI 3130xl and Illumina MiSeq. Five H1N2, four H3N2 and seven H1N1pdm IAVs, collected from swine in different Brazilian states during 2009-2012, were sequenced and analyzed. Nucleotide alignments were generated for five data sets: ‘H1s’ (human seasonal virus-like), ‘H1p’ (pandemic virus-like), H3, N1p (pandemic virus-like) and N2, including other related human and swine viruses, collected globally, as background. The four H3N2 viruses from Brazilian swine are monophyletic (100% bootstrap) and closely related to human seasonal viruses from the late 1990s. The five H1N2 viruses are also monophyletic (64% bootstrap) on the H1 tree, and are closely related to seasonal H1N2 viruses that circulated in humans during 2001-2003. The lower support for this clade appears to be driven by the early divergence of the Brazilian H1 viruses into two distinct sub-clades. The Brazilian swine IAVs are not monophyletic on the N2




phylogeny. Five Brazilian swine viruses of the H1N2 and H3N2 subtypes belong to one N2 clade (97% bootstrap) and two H1N2 belong to a second N2 clade (100% bootstrap). Both clades are closely related to human seasonal H3N2 viruses from the late 1990s. Therefore, Brazilian swine viruses of the H1N2 subtype that contain H1 segments related to human seasonal H1N2 viruses have acquired a different N2 of human H3N2 origin via two different reassortment events. The seven viruses of human pandemic H1N1 origin also were not monophyletic on the H1 or N1 tree, indicating that these viruses are the result of multiple separate human-to-swine introductions of the H1N1pdm virus, rather than clonal expansion of a single introduced lineage. The co-circulation of multiple antigenically diverse influenza virus lineages of the H1N1, H1N2, and H3N2 subtypes introduces new challenges for the control of influenza in Brazil’s swine herds, including design of cross-protective vaccines. FINANCIAL SUPPORT: EMBRAPA (PROCESS Nº. 02.11.10600-01)

VV260 - EVIDENCE OF ORTHOPOXVIRUS CIRCULATION AMONG URBAN DOMESTIC CATS, MINAS GERAIS, BRAZILMiranda, J.B.; Costa, G.B.; Almeida, G.G.; Figueiredo, P. de O.; Bonjardim, C.A.; Ferreira, P.C.P.; Abrahão, J.S.; Kroon, E.G.; Trindade, G. de S.

UFMG - Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901

Cessation of smallpox vaccination occurred 36 years ago in reason of its eradication. Because of this, several outbreaks involving other orthopoxviruses have been reported worldwide, such as Monkeypox virus which is endemic in Africa and Cowpox virus associated to human and animal cases in Europe, specially having cats as hosts. In Brazil, Vaccinia virus is a causative agent of an exanthematous disease, always affecting dairy cattle and humans and related to rural environment. Furthermore, other mammalian species are known to be infected, such as equids, monkeys and wild rodents. Taking into account that cats are well known to transmit Cowpox virus in European urban areas, our goal was to investigate Orthopoxvirus circulation among cats in a Brazilian urban environment and its implication for epidemiological studies. Whole blood,

plasma and serum samples were analyzed from 78 domestic cats that live in an urban area from state of Minas Gerais. Plaque reduction neutralizing test was applied in serum samples to check anti-Orthopoxvirus neutralizing antibodies. Viral DNA was accessed using phenol, chloroform and isoamyl alcohol method and real time PCR targeting vgf and ha genes were also applied. Most animals are female (55.1%) and age range from 5 months to 11 years. It was found 13 seropositive samples (16.7%), with antibodies titers ranging from 100 to 1600 neutralizing units per ml. Molecular analysis of seropositive cats showed 7 positives for vgf gene and 4 for ha. Poxviruses are ubiquitous among mammals and the host spectrum is wide. Although in Brazil occurrence of poxviruses are, until now, restricted to rural environment involving dairy cattle and humans, investigations conducted in urban areas highlights the importance to clarify epidemiological chain of this emerging infectious disease. Since smallpox eradication, vaccination is discontinued worldwide increasing the number of immunologically unprotected people. These data also reinforce the impact of viral spread to urban environments affecting vulnerable populations.FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG, PRPQ UFMG, PPG-MICROBIOLOGIA UFMG.

VV303 - DEVELOPMENT AND STANDARDIZATION OF A MULTIPLEX RT-PCR FOR THE DETECTION THE PRINCIPAL RESPIRATORY VIRUS IN BIRDSSakata, S.T.1; Rosales, C.A.R.1; Reischak, D.2; Orsi, M.A.2

1. Bolsista Do Projeto Sagres- MAPA/CNPQ2. Laboratório Nacional Agropecuário -

LANAGRO-SP

The avian Metapneumovirus (aMPV), the Newcastle disease virus (NDV), Influenza A virus (AIV) and infectious bronchitis virus (IBV) are the most important respiratory virus pathogens that affect breeding hens and broilers, with high importance to the poultry farms, both in terms of economic losses, impact in the exportation as the possibility of transmission between birds species. The aim of the present paper proposes the development, evaluation and standardization of a Multiplex RT-PCR, in order to assess the purity of avian vaccines. Reference virus: aMPV subtypes A and B; NDV- La Sota; AIV subtype H5N2; IBV-M41 were used for standardization of the assay and commercial vaccines




from different manufactures of biological products were screened. For IBV has been applied to RT-PCR with primers directed ORF 1B; for the presence of aMPV, we used an RT-PCR directed to the gene encoding the G protein subtypes A and B; with respect to the NDV, the region of the gene encoding the F protein of virulent and avirulent strains was performed, and to virus AIV, a pair of oligonucleotides encoding the protein of M1 and M2 RNA virus matrix gene was used. The pairs of primers used in this work confirmed to be specific for each target sequence. From reference virus and commercial vaccines was possible to shown the amplification products 444pb (aMPV), 310bp (NDV), 244pb (AIV) and 179pb (IBV), evidencing the differentiation of sequences targets of each virus concerned. The best results were obtained from the multiplex RT-PCR identified as “49”, referring to the annealing temperature of the reaction. The multiplex RT-PCR is a fast and easy technique to be carried out in any laboratory equipped to perform the PCR. This screening test can be considered as a first step for further studies of purity of vaccines used in Brazil. It is one method that can assist with greater clarity and certainty the identification of aMPV, NDV, AIV and IBV viruses, both in quality control of biological products (avian vaccines) and for diagnosis of these infectious agents in birds. Financial Support: MAPA/CNPq Cesar

VV316 - SEQUENCING AND PHYLOGENETIC ANALYSIS OF NUCLEOPROTEIN OF RABIES VIRUS ISOLATED FROM CATTLE IN THE STATE OF RIO GRANDE DO SULFernandes, M.E.S.1; Pereira, P.M.C.1; Achkar, S.M.1; Oliveira, R.N.1; Ferreira, J.C.1; Rosa, J.C.A.2; Castilho, J.G.1; Almeida, L.L.2; Carnieli, Jr. P.1; Roehe P.M.2,3; Batista H.B.C.R.1

1. INSTITUTO PASTEUR, Av. Paulista, 393 - Cerqueira César, São Paulo - SP, 01311-000

2. FEPAGRO SAÚDE ANIMAL/IPVDF - Instituto de Pesquisas Veterinárias Desidério Finamor, Estrada Municipal do Conde, 6000. Eldorado do Sul - RS, 92990-000


Rabies is a viral infection that reaches the Central Nervous System (CNS), causing fatal encephalitis in all mammals. The etiological agent of this zoonosis is Rabies Virus (RABV), a RNA virus member of Rhabdoviridae family,

Lyssavirus genus. Bats and canids are main reservoirs of the disease and are responsible by the maintenance of the rural and urban cycles of rabies. Different species of herbivores could be affected by rabies in rural cycle but cattle are the most important host of rabies at the cycle. In the State of Rio Grande do Sul (RS), southern Brazil the urban cycle of rabies was controlled but rural rabies is still endemic. Recently it has been registered an increase of rabies in cattle in the State causing losses at livestock. This work was carried out with the aim to identify genetic lineages of RABV circulating in cattle in the RS. For this 14 samples of RABV isolated from cattle were submitted to RT-PCR and sequencing of nucleoprotein gene (N) of virus. For the edition of sequences was used CHROMAS and BIOEDIT software and for the phylogenetic analysis was used MEGA 5 software. After sequence edition, 684 nucleotides of the protein were aligned and phylogenetically analyzed. In the 14 samples isolated RABV was clustered with the genetic lineage from haematophagous bat Desmodus rotundus. To generate the phylogenetic tree genetic sequences of RABV samples from cattle isolated in different regions of Brazil (26) and in Uruguai (4) were recovered from GenBank and included in the work. Based on the topology of the phylogenetic tree of studied RABV, three main clusters (1, 2 and 3) could be identified. Cluster 1 consists by 4 samples from Uruguai and 18 samples from different regions of Brazil (Rio Grande do sul- 4, Goias- 1, São Paulo- 5, Rio de Janeiro- 1, Mato Grosso- 2 e Pará- 5). Cluster 2 consists by 15 samples from south and southeast regions of the country (Rio de Janeiro- 5 and Rio Grande do sul- 10). The cluster 3 was compound by 7 samples from midwest and southeast regions (Tocantins- 3, Rio de Janeiro- 1 and São Paulo- 3). Samples from RS were included in clusters 1 and 2, with these results could be affirm that at least two sublineages of RABV from haematophagous bat D. rotundus are current in RS. Another sample of RABV isolated from cattle in RS will be analyzed to better understand rabies in the region.




VV321 - HERPESVIRUS SIMPLEX TYPE-1 OUTBREAK IN MARMOSETS (CALLITHRIX)Ullmann, L.S.1; Kurissio, J.K.1; Linhares, A.G.2; Linhares, L.S.A.1; Milanelo, L.2; Araujo Jr, J.P.1


2. PARQUE ECOLOGICO TIETE, Centro de Lazer Engenheiro Goulart Rua Guira Acangatara, 70, Engenheiro Goulart, São Paulo - SP. 03719-000

The increasing number of monkeys as pets can bring two different situations: monkeys as hosts to important human zoonoses and the opposite, man transmitting pathogens to the monkeys. One of these pathogens is human herpesvirus 1 (HHV-1). Different susceptibility levels are observed among the wide variety of Neotropical non-human primates and members of the Callitrichidae family are considered high susceptible to human herpesviruses. We report here an outbreak of HHV-1 in seven healthy marmosets: 3 white-tufted-ear (C. jacchus), 3 black-tufted-ear (C. penicilatta), and one hybrid (C. sp.), received at the reception center for animals (RCA), Tietê Ecological Park, a municipal park located in São Paulo. Marmosets were sheltered near the howley monkeys’ (Alouatta guariba) enclosure. In a period of a month howley monkeys presented clinical signs similar to herpesvirus with oral lesions that disappeared. During the following 24 days all seven marmosets died acutely. At necropsy, only one had oral lesions similar to hematoma, and all had encephalitis. One week before the onset on the monkeys, one person with herpes symptoms was seen at the park. The clinical suspicion included herpesvirosis and brain tissue was sent for diagnosis. As the animals did not show any signs but encephalitis, rabies was also included on the differential. DNA purification was performed with ReliaPrepTM gDNA Tissue Miniprep System kit and a nested PCR with degenerated primers was used to diagnose herpesvirus on the samples. Degenerated primers used on the first PCR were: DFA (5`-GAYTTYGCNAGYYTNTAYCC-3´), ILK (TCCTGGACAAGCAGCARNYSGCNMTNAA-3´), and KG1 (5´-GTCTTGCTCACCAGNTCNACNCCYTT-3´); and on the second PCR: TGV (5´-TGTAACTCGGTGTAYGGNTTYACNGGNGT-3´) and IYG (5´-CACAGAGTCCGTRTCNCCRTADAT-3´). The PCR product was purified and sequenced by Sanger methodology, ABI 3500 platform with BigDye Terminator

3500. Sequences were analyzed with MEGA 6.0, resulting in 100% of identity with sequences available on GenBank KF498959, JQ352184, and JQ780693, confirming Human Herpesvirus 1. The restrict access of HHV-1 people is essential due to the lethality of the disease in marmosets. Emphasis should be given to the importance of differential diagnoses mainly with wild animals whereas few epidemiological studies are available. FINANCIAL SUPPORT: FOUNDATION OF THE BIOSCIENCES INSTITUTE (FUNDIBIO).

VV333 - ABC OF CANINE PARVOVIRUSES GENOGROUPS AND ANTIGENIC TYPESStreck, A.F.; Borchardt, A.; Daudt, C.; Canal, C.W.

LABORATÓRIO DE VIROLOGIA VETERINÁRIA/UFRGS - Laboratório de Virologia Veterinária da Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090, Agronomia, Porto Alegre - RS, 91540-000

Canine parvovirus type 2 (CPV-2) is a member of the autonomous replicating Parvoviridae family that has a single-stranded DNA genome which encodes two capsid proteins (VP1 and VP2) and two non-structural proteins (NS1 and NS2). The CPV-2 emerged from the feline parvovirus (FPV) or a closely related virus as a novel pathogen in the late 1970s and rapidly spread worldwide in the canine population. Within a few years, the virus underwent a rapid evolution and, new “antigenic” types, termed CPV-2a, CPV-2b and CPV-2c, replaced the original CPV-2. Notably, it has been observed that canine parvoviruses display high evolutionary rates, and for this reason, nucleotide substitutions are often found. In order to evaluate if the CPV-2a, CPV-2b and CPV-2c represent different antigenic types, hypothetical viral proteins were generated and analyzed. Additionally, all genomes from CPV-2 were retrieved from GenBank and phylogenetically analyzed. Six distinct genogroups were observed. For each genogroup, hypothetical viral proteins were also generated based on the consensus sequences and compared. According to a structural, electrostatic and hydrophobical analysis and total polar/apolar charges caused by the amino acids modifications, those six genogroups resulted in four main prevalent structural types. It was observed that modifications in the VP2 apical structure involved in the virus binding domain represents more expressive electrostatic and hydrophobical changes than the formerly sites used for




CPV typing. Therefore, it is suggested that CPV-2 typing need to be revised and that vaccine strains should be based on those main prevalent structural types to be more effective. FINANCIAL SUPPORT: CNPQ, FAPERGS, CAPES.

VV351 - GENETIC CHARACTERIZATION OF CANINE CIRCOVIRUS DETECTED IN STOOL SAMPLES FROM DOGS IN THE SOUTH REGION OF BRAZILCruz, T.F.1; Batista, T.N.2; Baccarin, A.M.2; Gradiz, J.J.2; Vieira, E.M.2; Tozato, C.C.1; Kurissio, J.K.1; Araujo Jr, J.P.1


2. FURB - Universidade Regional de Blumenau, R. Iguaçu, 404 - Itoupava Seca, Blumenau - SC, 89030-030

Circoviruses are non-enveloped, icosahedral viruses with single-stranded circular genome DNA that belong to the family Circoviridae. Circoviruses infect birds and mammals, and porcine circoviruses were reported as only species in the genus Circovirus that infect mammals. Currently, a specie nonporcine circovirus (canine circovirus) was detected in samples from dogs. Thus, the objective of this study was to detect canine circovirus in stool samples by quantitative PCR (qPCR) and perform the genetic characterization of the virus. DNA was extracted from 45 feces samples collected from dogs with hemorrhagic gastroenteritis (n=5), hematochezia (n=10), normal stool (n=12), and without information about feces (n=18). The dogs were one months-old to 10 years-old. The animals were from Santa Catarina (n=44) and Paraná (n=1) states. The samples were tested for canine parvovirus (CPV) and canine circovirus by qPCR. The results determined by qPCR were analyzed qualitatively. Twenty-three samples were positive for CPV and two were positive for canine circovirus. A six months-old female dog (D11) from Paraná was co-infected with CPV and canine circovirus. The other female dog (D10) infected with canine circovirus was 10 months-old and was from Santa Catarina. The two dogs had hemorrahagic gastroenteritis. The complete genome of canine circovirus was amplified by rolling-circle amplification (RCA). The digestion reaction was performed after RCA. The formation of fragments larger than 2000 bp confirmed the detection of canine circovirus, which has genome DNA of 2,063 kb. The

purified products were sequenced using Illumina Miseq platform. A consensus tree based on nucleotide sequences from complete genome was generated by the neighbor-joining method (MEGA5), using maximum composite likelihood model and 1,000 bootstrap replicates. The complete genome of canine circovirus D11 shared 85.5%-98.0% nucleotide identity with others canine circovirus. The capsid and replicase nucleotide sequences of D11 share 86.7%-98.3% and 82.2%-98.5% identities, respectively, with the known canine circovirus. The capsid and replicase proteins of D11 showed 86.7%-98.3% and 82.2%-98.5% amino acid identity, respectively, with others canine circovirus. The phylogenetic tree showed that D11 grouped with others canine circovirus. These group forming a distinct clade with porcine circovirus, wherever avian circovirus clustered separately.

VV365 - PHYLOGENETIC ANALYSIS OF AICHIVIRUS B IN FECAL SAMPLES FROM CALVES IN BRAZILRribeiro, J.1; Lorenzetti, E.1; Medeiros, T.N. da S.1; Junior, J.C.R.1; Bronkhorst, D.E.2; Crespo, S.E.I.1; Favero, L.M.1; Alfieri, A.F.1; Alfieri, A.A.1

1. UEL - Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, Londrina - PR, 86057-970

2. UNOPAR - Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140

The genus Kobuvirus belongs to the Picornaviridae family, and the virions are non-enveloped, with icosahedral symmetry and a diameter of 27-30 nm. Currently, the Kobuvirus genus includes three species, designated as Aichivirus A, Aichivirus B, and Aichivirus C, which can cause infections in humans, cattle, and pigs, respectively. The presence of Aichivirus B has been detected worldwide. However, there are few studies about the genetic heterogeneity of these viruses. A total of 30 fecal samples diarrheic from calves (<30 days) with diarrhea were analyzed from a dairy production farm in the state of Paraná, Brazil. Fecal suspensions were prepared at 10-20% (w/v) in PBS buffer and centrifuged at 3,000 x g for 5 min. The RNA extraction was performed with 400 µL of fecal suspensions. The presence of segment double-stranded RNA (dsRNA) of rotavirus in the faecal samples was evaluated by polyacrylamide gel electrophoresis (PAGE) technique. The SN-PCR assay for the bovine




coronavirus was performed to amplify a sequence based on the highly conserved region N gene with a predicted product of 251 bp. All fecal samples were submitted to RT-PCR assay to detect the 3D region of the Aichivirus B using the primers UNIV-F/R, which target a 216 bp. Two positive samples to Aichivirus B was performed VP1-F/R, which target a 1,159 bp region of the complete VP1 capsid gene. Two Aichivirus B region VP1 products were purified, quantified, sequenced in an ABI3500 Genetic Analyser and submitted for nucleotide sequence analysis. Eleven out of 30 (36.7%) fecal samples tested were positive for Aichivirus B form 3D region, two fecal samples were positive for rotavirus, and none fecal sample was positive for bovine coronavirus. The Aichivirus B had already been described in Brazil and also found high infection (38%) in calves with diarrhea. The phylogenetic analysis of the VP1 region of Aichivirus B showed that the Brazilian strains clustered in the same branch of the genotype A prototype (U-1 strain). Further studies should be performed to characterize the Aichivirus B strains circulating in all regions of Brazil. FINANCIAL SUPPORT: FINEP, CAPES, CNPq, and Fundação Araucária/PR.

VV393 - VIABILITY OF VACCINIA VIRUS IN EXPERIMENTALLY CONTAMINATED CHEESES AT DIFFERENT TIMES OF THE MATURATION PROCESSRehfeld, I.S.; Fraiha, A.L.S.; Matos, A.C.D.; Guedes, M.I.M.C.; Costa, A.G.; de Souza, M.R.; Lobato, Z.I.P.


Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing major economic impact in the Brazilian dairy production. Previous studies have described the presence of viable viral particles in milk samples of cows experimentally and naturally infected with VACV. Furthermore, others studies showed that VACV remains viable in fresh artisan cheese, made with VACV contaminated raw milk. However, it is not known if the maturation process used during the production of artisan cheese is able to inactivate VACV particles. The artisan cheese produced in Minas Gerais was declared a Brazilian cultural heritage in 2008, and its sanitary quality is a very important Public Health issue. Therefore, the aim of this study was

to produce artisan cheese using raw milk experimentally contaminated with VACV and to analyze the viral viability at different times of the maturation process. The raw milk used for the cheese production was tested by PCR for Orthopoxvirus, and it was negative. The milk was contaminated with 105 PFU/ml of VACV-GP2. Control cheeses were produced separately with raw milk not contaminated with VACV. Natural yoghurt and commercial rennet were used to produce the cheeses. The control and contaminated cheeses were submitted to the maturation process during 1, 7, 14, 21, 45 and 60 days. Cheeses production was repeated four times in different moments. They were stored in a B.O.D. incubator at 25ºC. In each previously established maturation day, one control and one contaminated cheese were collected and macerated. For viral isolation, aliquots of macerated cheese were inoculated into BSC-40 cells. VACV viable particles were detected in contaminated cheeses until 60 days of maturation. These results suggest that artisan cheese produced with milk contaminated with VACV might be a risk for Public Health. FINANCIAL SUPPORT: FAPEMIG, CNPQ AND CAPES

VV396 - MOLECULAR DETECTION AND PHYLOGENETIC RELATIONSHIPS OF AN AMPHIBIAN RANAVIRUS ASSOCIATED TO OUTBREAKS OF MORTALITY IN BRAZILIAN FISH FARMQueiroz, S.R. de A.1; de Pádua, S.B.2; Filho, R.N. de M.2; Araújo, A.P. de3; Maganha, S.R. de L.1; Navarro, J. de O.1; Lima, M.P.1; de Alencar, A.L.F.1; Arruda, E. de P.1; Munin, F.S.1; Fernandes, A.M.1; de Sousa, R.L.M.1

1. FZEA/USP - Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Av. Duque de Caxias Norte, 225 - Campus da USP, Pirassununga - SP, 13635-900

2. AQUIVET - Aquivet Saúde Aquática, Rua Antonio Ernesto Célico, número 359, quadra 8, lote 16, Residencial Marcia Damha III, São José do Rio Preto - SP, 15061810

3. ACQUAPISCIS - Acquapiscis Consultoria e Medicina Veterinária em Aquicultura, Rua Vieira de Morais, 1201, Campo Belo, São Paulo - SP, 04617-014

Ranavirus (RV) are emerging pathogens responsible for epizooties of great ecologic and economic impact in fishes, amphibians and reptiles around the world. Disease caused by these viruses was detected in




amphibians from Brazil, but until now there is no data about RV infection of Brazilian fishes. Currently, tilapia of Nile (Oreochromis niloticus) is the fish farm of the most economically rentable farming activities in Brazil. Due the economic importance of tilapia farming, the present study was aimed to investigate mortality of tilapias from states of Ceará (CE) and Bahia (BA). For this purpose, 30 moribund and 15 apparently healthy specimens were collected during mortality events in tilapia farms installed around Castanhão Dam, Jaguaribara (CE), and from nursery farms in Horizonte (CE) and Paulo Afonso (BA). Darkening skin and hemorrhagic foci in the base of the pectoral fins and perianal region, exophthalmos, corneal opacity and bleeding ulcers on the skin with loss of scales were observed. Concurrent bacterial infections were also detected. Fragments of liver, pancreas, spleen, kidney, heart, intestine, stomach, gills, brain and eye were sampled and stored in buffered formalin solution 10%. Histopathological analysis revealed impairment of liver tissue at various levels with the presence of vacuolar degeneration associated with necrotizing hepatitis. Basophilic inclusions bodies in acinar cells were detected in all samples. Some tissue samples showed necrosis of the kidney, depletion of hematopoietic tissue, and degeneration of cardiac muscle cells and endothelial cells. Fragments of fixed tissues were submitted to PCR (polymerase chain reaction) for sequential amplification of 321bp- and 625bp-products, corresponding to the MCP (Major Capsid Protein) gene of Ranavirus. All samples tested positive for ranavirus. The amplimer sequences revealed high percentage of similarity (>96%) with other homolog amphibian ranavirus sequences deposited in GenBank. In phylogenetic reconstructions, the samples were grouped together in the same clade with other amphibian ranavirus. The results indicate, in an unprecedented manner, the circulation of frog virus-like, an amphian ranavirus, in fish farm in Brazil with economic implications for tilapia farming from Brazilian northeast region. This work was financially supported by FAPESP (Proc. nº 2011/19002-8; 2012/08846-3).

VV470 - VIRUCIDAL ACTIVITY OF A PURIFIED COMPOUND ISOLATED FROM PRASIOLA CRISPA AGAINST EQUINE HERPESVIRUS TYPE 1Marinho, R.S.S.1; Ramos, C.J.B.1; Leite, J.P.G.2; Belo, C.A.D.3; Pereira, A.B.3; Teixeira, V.L.1; Paixão, IC.N.P.1; Pinto, A.M.V.1



3. UNIPAMPA - Universidade Federal do Pampa, Av. General Osório, 900, Bagé - RS, 96400-100

Equine herpesvirus type 1 (EHV-1) is a major equine pathogen. It is etiologic agent for a triad of clinical signs: abortion, neurological and respiratory disease. It is enzootic throughout the world and almost all horses older than 2 years of age have been exposed. Following initial exposure, EHV-1 has the ability to develop into an unapparent, latent infection. It is this ability to reside as a silent and persistent infection in horses which provides a reservoir of virus for continual transmission. The terrestrial seaweed Prasiola crispa (Lightfoot) Kützing as collected in ice-free areas, the region near the station Polish Arctowski, Admiralty Bay, King George Island (61° 50’ - 62° 15’ S and 57° 30’- 59° 00’ W), in Antarctica. Seaweeds were dried in a dark chamber with air circulation at 40°C in dark bags, and stored in a freezer and after a new screening was performed for the removal of waste and other materials getting dried seaweed. The compound C3F4 was isolated from crude extract in dichloromethane in silica gel 70-230 chromatography column and characterized in Thin-Layer Chromatography (TLC) and identified by H- and C-Nuclear Magnetic Resonance (RMN) spectras. This study aimed to evaluate the in vitro cytotoxic effect and virucidal properties of C3F4 compound purified from Prasiola crispa in EHV-1 replication. The cytotoxic effect of C3F4 has been measured in RK-13 cell treated with different drug concentrations. Cytotoxic concentrations (CC50) values have been determined for acyclovir 1010 ± 2.0 and C3F4 compound C3F4 1884 µM ± 9.0. As a first approach to characterize the action of C3F4 on EHV-1 a virucidal assay activity was performed. A virus suspension containing 1x106 PFU EHV-1 were mixed with C3F4 (50 µM) and acyclovir (200 µM) were kept at room temperature (24 ºC) for 1 h. Meanwhile,




control of untreated infected virus was performed in the same conditions. Then, treated virus suspension and untreated control were diluted and percentage of inhibition of EHV-1 infectivity on RK-13 cell was determined by plaque assay. The result of virucidal activity for dichloromethanic fraction C3F4 was 90% ± 8.5. Acyclovir had slightly virucidal activity on viral particle. For better understanding about mechanisms of the antiviral activity by C3F4 further investigations are underway.

VV473 - EMERGING VIRUSES MONITORING IN BATS, RODENTS AND MARSUPIALS FROM BRAZILIAN RAIN FOREST REGION AND AMAZON REGION COLLECTED BETWEEN 2008 AND 2013Campos, A.C. de A.1; Anthony, S.J.2; de Araújo, J.3; Ometto, T.3; Hurtado, R.3; Nardi, M.S.4; Nava, A.5; Solorio, M.R.6; Souza, M.C.P.7; Rostal, M.8; Loh, E.8; Torrelio, C.M.Z.8; Aguirre, A.A.9; Daszak, P.8; Durigon, E.L.1

1. Laboratório de Virologia Clínica e Molecular, Depto Microbiologia, ICB-II, Universidade de São Paulo And Ecohealthalliance-EHA

2. The Center for Infection and Immunity, Columbia University and Ecohealthalliance-EHA

3. Laboratório de Virologia Clínica e Molecular, Depto Microbiologia, ICB-II, Universidade De São Paulo

4. DEPAVE 3, Secretaria do Verde e do Meio Ambiente da Prefeitura Municipal de São Paulo and Laboratório de Virologia Clínica e Molecular, Depto Microbiologia, ICB-II, Universidade De São Paulo

5. Ecohealthalliance-EHA and Depto de Medicina Veterinária Preventiva e Saúde Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo

6. Depto de Medicina Veterinária Preventiva e Saúde Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo and Ecohealthalliance-EHA

7. Laboratório de Virologia Clínica e Molecular, Depto Microbiologia, ICB-II, Universidade de São Paulo and Universidade Santo Amaro

8. Ecohealthalliance-EHA9. George Mason University. Environmental

Science and Policy

In the last decades some viruses has been concern among researchers and health professionals because

they can emerge silently or aggressively. Wildlife it is the principle source of these viruses and constitutes a large and often unknown reservoir of zoonotic diseases that can be responsible for epidemics or outbreaks events. The University of Sao Paulo in a partnership with EcoHealthAlliance-EHA and Columbia University (NY) developed a project that aim the identification of different virus families circulating in animals from wild and urban areas with potential epidemic risk. Samples were collected from 758 animals during 2008 to 2013 in Rain Forest region and Amazon Forest in sites close and inside city of Manaus-AM characterized as Pristine, Urban and Intermediate depending of the level of conservation. Samples from722 animals (620 bats, 64 rodents and 38 marsupials) were analyzed in the Clinical and Molecular Virology Laboratory-USP and 216 animals (179 bats and 37 rodents) were analyzed in duplicate by Columbia University-USA. After the capture oral and rectal swabs, feces, urine and blood were collected, directly transferred to liquid nitrogen and stored at -70ºC in laboratory. Total nucleic acids were extracted using NucliSens Lysis Buffer (BioMerieux) and the randomic cDNA synthesis was made with SuperScript III kit. The presence of thirteen different viruses and/or families (Alphaviruses, Arenaviruses, Astroviruses Bocaviruses, Coronaviruses, Enteroviruses, Filoviruses, Flaviviruses, Hantaviruses, Herpes virus, Nipah virus, Paramyxoviruses and Seadornaviruses) were analyzed in samples by conventional PCR or Real-Time PCR assays. All amplicons were sequenced by Sanger’s method to results confirmation. One rodent sample was positive to Paramyxovirus. Twelve bats were positive to Coronaviruses, seven to Hantaviruses, one for Paramyxoviruses and ten to Herpes virus. Only one marsupial was positive to Hantavirus. This data showed the presence of virus with emergent potential in animals from Rain Forest and Amazon region. Financial support: EcoHealthAlliance - EHA

BASIC VIROLOGY -BV


Basic Virology: BV 62

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Basic Virology: BV

BV9 - IN VITRO EVALUATION OF THE ANTIVIRAL ACTIVITY OF BOTHROPS ALTERNATUS VENOM ON THE REPLICATION OF RESPIRATORY SYNCYTIAL VIRUS (RSV)Polloni, L.C.; Barros, H.L.S.; De Oliveira, F.; Yokosawa, J.


Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is responsible for high morbidity and mortality in children throughout the world. This fact runs partly due to the limited treatment options available for this infection, which makes the development of antiviral agents economically feasible a priority in healthcare. This study aimed to evaluate the antiviral activity of crude venom of the snake Bothrops alternatus against RSV. Through the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, the concentration of crude venom that reduced in 50% the viability (CC50) of Hep-2 cells was determined as 23,1 ng/µL. The evaluation of the antiviral activity was also performed by using the MTT assay. Treating Hep-2 cells with 23,1 ng/µL crude venom for 3 h, exposing them with RSV for 1 h, and then incubating them for six days showed antiviral activity at MOI that ranged from 1,0x10-4 to 9,5x10-4. Thus, the antiviral activity exerted by poison seemed to be dependent on the low amount of virus presented to the cells (low MOI). On the other hand, treating cells with crude venom after exposing them with RSV (post-treatment) did not seem to affect viral activity. These results may indicate that the antiviral activity of the crude venom on RSV in vitro is apparently preventive and not therapeutic. Reduced incubating period may help elucidate the effect of the venom after limited rounds of viral replication. FINANCIAL SUPPORT: UFU, FAPEMIG, CNPQ, CAPES, INCT NANO BIOFAR

BV7 - EVALUATION OF NITAZOXANIDE ANTIVIRAL ACTIVITY AGAINST ROCIO AND SAINT LOUIS ENCEPHALITIS VIRUSESBarros, H.L.S.1; Polloni, L.C.1; Oliveira, T.F. de M.S.1; Yokosawa, J.1; Chávez, J.H.2


2. UFMT - Universidade Federal de Mato Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT, 78060-624

Nitazoxanide (NTZ), a thiazolide anti-infective, presents activity against anaerobic bacteria, protozoa, and a range of viruses in cell culture models, including influenza A, hepatitis C and B, rotavirus and Japanese encephalitis virus. Originally developed as a treatment of intestinal protozoan infections, the antiviral properties of NTZ were discovered during the course of its development for treating cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS). The mechanism of antiviral activity of NTZ has not been fully elucidated, although some studies indicate that NTZ blocks viral infection via modulation of host cell processes. Rocio virus (ROCV) and Saint Louis encephalitis virus (SLEV) belong to the Flavivirus genus of the Flaviviridae family. Both viruses may cause important disease, such as human encephalitis. No vaccine or antiviral drug is currently available to prevent or treat such viruses. Thus, the aim of this study was to evaluate the in vitro antiviral activity of Nitazoxanide (NTZ) against ROCV and SLEV. This evaluation was performed in infected VERO cells. The cytotoxicity (CC50) in VERO cells and antiviral activity of NTZ against ROCV and SLEV was assayed by the MTT method. In the NTZ CC50% assay, was obtained the value of 3245,6 µM. Furthermore, in the NTZ antiviral effect assay was found the CE50% varying from 630µM to 1254,2 µM. NTZ showed antiviral activity against SLEV, with selectivity indexes ranging from 2,59 to 5,14, depending on multiplicity of infection (MOI) that was used. No antiviral activity was observed against ROCV. Additional studies should be carried out to investigate in vitro and in vivo NTZ action against other viruses, since antiviral activity against SLEV was demonstrated. FINANCIAL SUPPORT: UNIVERSIDADE FEDERAL DE UBERLÂNDIA




BV19 - ASSESSMENT PROINFLAMMATORY CYTOKINES AFTER TREATMENT WITH 17B-ESTRADIOL CELL LINE (MT-2) CONTINUOUSLY INFECTED WITH HTLV-1Carvalho, L.D.1; Martins, C.P.S.2; Martins, M.L.3; Souza, J.G.2; Stancioli, E.F.B.2; Fagundes, E.M.S.2; Gomes, R.A.3

1. UESC - Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900



HTLV-1 was the first retrovirus to be isolated in humans. Diseases associated with this infection include Adult T-cell leukemia (ATL) and myelopathy (TSP / HAM). HTLV-1 can be transmitted through infected lymphocytes present in breast milk, during sex and through blood transfusions or blood products. Transmission of HTLV-1 is more effective from men to women and infected women are more likely to develop the disease. This different pattern is gender-related worldwide and remains to be clarified. Recent studies point to a possible hormonal cause. In this context, this study aims to evaluate proinflammatory cytokines levels in the human lymphocyte culture supernatant permanently infected with HTLV-1 (MT-2) treated with 17β-estradiol at different concentrations (1 uM to 0.0001 uM). Cytokines were measured in the MT-2 cell culture supernatant after stimulation with PMA and after treatment with 17β-estradiol at different concentrations (1 uM to 0.0001 uM), using the Human Inflammatory Cytokine CBA Kit, and the result points to a change in IL-6, IL-10 and IL-12 p70 levels in cells treated with 17β-estradiol, in a dose-dependent manner, compared to the untreated control. The greatest change occurred in IL-6 levels compared to untreated control (elevation around 5-fold compared to control cell). These data suggest that fluctuations in the levels of estradiol in the HTLV-1 carrier may alter the pro-inflammatory cytokines profile and, consequently, influence the outcome of HTLV-1 pathogenesis. This is the first study showing the modulation of proinflammatory cytokines by HTLV-1 hormone 17β-estradiol. FINANCIAL SUPPORT: CNPQ, FAPEMIG E FUNDAÇÃO HEMOMINAS

BV20 - FITNESS EVALUATION OF A RECOMBINANT MURINE NOROVIRUS DURING SERIAL PASSAGES IN CELL CULTUREFilho, E.F. de O.; Di Felice, E.; Toffoli, B.; Zonta, W.; Mathijs, E.; Mauroy, A.; Thiry, E.

Université de Liège - Place du 20 Août 7, 4000 Liège, Bélgica

Noroviruses are single stranded positive sense RNA viruses which can infect human and different animal species. Human norovirus (NoV) infections are among the most important causes of gastroenteritis in both children and adults. Infections often occur as outbreaks which may be foodborne. Due to the lack of an efficient cell culture system as well as a workable animal model, many aspects of the NoV infection in human are still poorly understood. The murine norovirus (MuNoV) grows easily in cell culture in contrast to the Human NoV, and constitutes an excellent animal model. Recombination can dramatically change virulence properties of the viruses and has been evidenced in silico for different human NoV strains isolated from clinical cases. Recently, after in vitro coinfection of RAW264.7 cells with parental MuNoV strains CW1 and Wu20, we obtained a recombinant Wu20/CW1 strain. This recombinant strain showed reduced plaque size compared to the parental strains. The aim of the study was to observe and molecularly characterize the natural genetic evolution of the recombinant MuNoV strain across in vitro replications. Viral fitness is a complex concept. Here we defined this fitness as the ability of a viral population to adapt to the cell culture system. Thus, the recombinant strain was serially replicated in vitro in RAW264.7 cells (up to 14 passages). Viral plaque sizes of early and late progenies were compared with the Image J software. A significant difference was shown between them with the Mann and Whitney non parametric statistical test. Afterwards, viruses from different cell passages were cloned and sequenced. The average plaque size increased from the earlier to the later progenies (from 0.1 mm2 to around 0.5 mm2). Molecular investigations are currently performed in order to specify in which genetic region mutations occur and whether or not this could explain fitness modifications during in vitro evolution. In addition, two other parameters of in vitro virulence modification will be investigated: (i) virus production and (ii) one step growth kinetics. The data




should provide interesting information about genetic evolution in the genus Norovirus, especially regarding recombination events and explain how a recombinant strain, first disadvantaged compared to its parental strains, could regain fitness by genetic evolution.

BV23 - EVALUATION OF AN AMINOMETHYLNAPHTHOQUINONE (R425) IN ACUTE TOXICITY AND ANTI-HSV-1 ACTIVITY IN BALB/C MICEGarrido, V.; Barros, C.S.; Chagas, C.; Melchiades, V.; Ribeiro, C.; Cavalcante, M.; Bittetti, M.; Silva, A.; Vargas, M.D.; Neves, A.P.; Teixeira, G.; Paixão, I.C.P.N.


Herpes Simplex-1 virus (HSV-1) is a worldwide endemic infection and one of the most prevalent pathogenic microorganisms in Brazil. The drugs employed to treat HSV-1 have several side effects including resistance, latency, and toxicity. The research for new effective drugs against these viruses therefore remains crucial. Preliminary in vitro results with an aminomethylnaphthoquinone (R425), synthesized by The Organic Chemistry Department of UFF have shown that R425 has low cytotoxicity (CC50= 1722 ±175.9 µM) and effective anti-HSV-1 activity (EC50=2.13±0.11 μM), compared with reference drug, acyclovir (CC50= 960 μM; EC50= 1.00 μM). In vitro assays were performed in Vero cells infected with HSV-1 KOS strain (MOI-1) and then treated with different concentrations (12.5, 25 and 50 µM) of the substance R425 for 24 h. Each drug has a degree of systemic toxicity, so that research aims at finding out the acute toxicity level of a single dose of R425 in BALB/c mice weighing 18-25g and 60-120 days old. First, for the LD50 test, two groups of mice (n= 5/group) were treated: G1= R425 (2000 mg/kg) in 10% DMSO as the vehicle, and G2= the vehicle only control. Four groups were also employed for the 14-day tests (n= 5/group): G1 and G2 were treated with 550 mg/Kg and 175 mg/Kg doses of R425, respectively; G3= acyclovir control (50mg/Kg) and G4= 10% DMSO only. Blood samples were drawn before (D0) and after each experiment (D7, D14) for haematological and biochemical evaluation (urea, creatinine, uric acid, proteins, albumin, ALT, AST, and alkaline phosphatase), and some organs were removed for histological analysis.

The weight and behavior of the animals showed no significant changes. For the LD50>2000 mg/kg group no changes in animal behavior or times of death were recorded within 48 hours. Platelets showed a significant increase in numbers (p<0.001) while urea values decreased significantly (p<0.001) at the 550 and 175 mg/kg R425 concentrations. Although there have been significant changes, the 14-day parameters returned to normal the physiological levels. In histological sections, there was reversible inflammatory responses in the major organs (liver, kidney, stomach and duodenum), as already reported with other drugs. According to the OECD 423 guidelines we concluded that R425 has low toxicity in BALB/c mice. Thus, the maintenance of the anti-HSV validity tests is mandatory in experimental animals. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, UFF (PROPPI)

BV39 - PROSPECTIVE STUDY OF FLAVIVIRUSES IN SMALL MAMMALS, IN MINAS GERAIS, BRAZILRezende, I.M.de1; Amaral, C.D.2; Sacchetto, L.1; Miranda, J.B.2; Borges, I.A.2; Vieira, F.N.2; Alves, P.A.2; Paglia, A.P.2; Trindade, G. de S.2; Drumond, B.P.1

1. UFJF - Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036-330


The wildlife is a reservoir of microorganisms that, once transferred to humans, may emerge as public health threats. Most reservoirs animals are mammals, mainly of the orders Rodentia, Chiroptera, Primates and Carnivora. The great diversity of rodents, its remarkable reproductive potential and their ability to adapt to different niches are factors that explain, in part, the emergence and reemergence of some viral diseases. The genus Flavivirus belongs to the group of arboviruses of greatest global importance, responsible for serious diseases such as encephalitis, fever, hemorrhagic fever and hepatitis in humans. The members of the genus Flavivirus of major importance to public health are the Dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), Japonese encephalitis virus (JEV) and Saint Louis encephalitis virus (SLEV). The aim of this study was to do a prospective study of flaviviruses in




small mammals in a rural area of Minas Gerais, Brazil. To detect flaviviruses, a collection of viscera of small mammals from Laboratório de Vírus/UFMG was used. These viscera were collected at Rio Pomba city, from October 2012 to August 2013 and kept in RNAlater at -70 °C. Of the 112 samples present in the collection, the liver of 49 animals was initially tested for the presence of DENV, YFV, SLEV, WNV and JEV. The liver was macerated with liquid nitrogen and subsequently, the total RNA extraction was performed using Viral RNA Midkit (QIAGEN, USA). cDNA synthesis was performed using random primers and then used in a real time PCR (qPCR) assay, which aimed to amplify a fragment of the NS5 region of the flaviviruses genome. None of the 49 liver samples tested presented flaviviruses RNA. Some had indeterminate result and qPCR assays will be repeated and amplicons will be sequenced. The serum of 109 animals are also going to be tested. All positive samples are going to be sequenced to confirm the results. The presence of mammals infected with zoonotic viruses in areas occupied by humans may increase the risk of re-emergence of zoonotic diseases, since these animals may be close to humans, which may favors the transmission of the infectious agents. FINANCIAL SUPPORT: FAPEMIG, CNPq, CAPES, UFJF, PROPESQ/UFJF.

BV46 - HUMORAL IMMUNE RESPONSE ELICITED BY A DNA BASED IMMUNIZATION ENCODING AN ARTIFICIAL ANTIGEN CONTAINING SEGMENTS OF E1 AND E2 HCV GLYCOPROTEINSFreitas, G.R.O.; Froelich, L.; Oliveira, M.M.; Oliveira, T.F.M.S.; Yokosawa, J.

Laboratório de Virologia, UFU - Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121 - Santa Mônica, Uberlândia - MG, 38408-100

Approximately 130 million people are infected chronically with hepatitis C virus (HCV). The development of vaccines has been hampered by the high genetic variability of the virus as well as by the lack of suitable animal models. In this study, we describe the construction of an artificial antigen containing segments of the HCV glycoproteins E1 and E2 that have been reported to elicit neutralizing antibodies against HCV. The immunogenicity of the E1E2 antigen was assessed in mice by DNA immunization given in three doses. Furthermore, another group of mice was immunized with two doses of DNA and one

additional dose of bacteria-expressed E1E2 protein. The sera of immunized mice were tested by ELISA and reacted against the recombinant E1E2 protein as well as four synthetic peptides (I - IV) covering parts of the E1E2 sequence. In addition, sera of mice of the two immunized groups reacted against viral proteins in Huh7.5 cells producing JFH1 strain HCV in immunofluorescence assay. Our results demonstrate the ability of an artificial antigen, administered as DNA vaccine, to elicit specific antibodies against HCV glycoprotein(s). Further investigations are still needed to explore the neutralizing capability of these antibodies, and to investigate the cellular immune response elicited by this antigen.

BV56 - EVALUATION OF MITOCHONDRIAL ACTIVITY IN CELLS INFECTED BY ADENOVIRUS SEROTYPE 40Guissoni, A.C.; Parente, J.; Badr, K.; Soares, C.; Cardoso, D.

UFG - Universidade Federal de Goiás, Av. Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970

The mitochondria are involved in a variety of metabolic processes including cell ATP production, calcium homeostasis, cell proliferation, apoptosis, and synthesis of amino acid, nucleotide and lipid. The distribution, shape and operation of this organelle are regulated by intrinsic and extrinsic stimuli which may be a virus. These may induce or inhibit various mitochondrial processes in highly specific manner. When a cell infected with adenovirus, usually undergoes metabolic changes that can trigger apoptosis through mitochondrial pathway. These changes occur not only as a result of viral replication, but also by the interaction between transcripts and / or viral proteins with factors of cellular regulation and immune response. Thus, some viral proteins interact with mitochondrial proteins which regulate cellular responses. In this study we evaluated the activity of the respiratory chain in A549 cells (human lung carcinoma) infected with adenovirus serotype 40 (HAdV-40). The procedure included with the cultivating of A549 cells followed by inoculating of HAdV-40 and observation of cytopathic effect after 30 hours. After this, the infected and control cells were treated with MitoTracker ® Green FM, where the probe Mitotracker (green) marks all mitochondria, and rosamina (Red) selectively marks just the active mitochondria. The visualization of these changes was done by A1 Axioscope




fluorescence microscope (Carl Zeiss) at wavelength 579/599 for rosamina and 490/516 for Mitotracker. The images were generated in AxioCamMR photographic system (Carl Zeiss). From the results, it was possible to observe the mitochondria in the cytoplasm as an increase in fluorescence intensity zones. Mitochondrial activity was quantified based on the evaluation of the fluorescence intensity of rosamina. It was observed that the mitochondria of infected cells HAdV-40 had higher mitochondrial activity, suggesting that adenovirus infection in A549 cells up-regulates electron transport chain. FINANCIAL SUPPORT: FAPEG / CNPQ

BV61 - FIBRILS OF THE GIANT ACANTHAMOEBA POLYPHAGA MIMIVIRUS – INITIAL STUDIES OF PROBABLE FUNCTIONS OF THIS PECULIAR STRUCTURERodrigues, R.A.L.1; Silva, L.K. dos S.1; Dornas, F.P.1; Boratto, P.V. de M.1; Arantes, T.S.1; La Scola, B.2; Kroon, E.G.1; Abrahão, J.S.1


2. Université de la Méditerranée, Jardin du Pharo - 58, bd Charles Livon -13284 Marseille Cedex 07

Mimiviridae family comprises giant viruses with huge genomic and structural complexity, with Acantamoeba polyphaga mimivirus (APMV) as its type virus. It was firstly isolated in 1992, but only in 2003 it was identified as a virus, being included in the group of the Nucleo-Cytoplasmic Large DNA Viruses. During the following years many singular characteristics was described, such as several genes related to translational apparatus, besides some very distinct morphological characteristics, such as two separate portals related to the packaging and delivering of the genome and glycoprotein fibrils surrounding almost all viral particle. It remains unknown which would be the functions of these fibrils. Thus, the aim of this work is to evaluate probable roles for this structure. In that purpose, APMV particles were submitted to a sequential enzymatic treatment, with lysozyme and bromelain, to remove the fibrils. Then, it was realized analysis of those particles by transmission electronic microscopy and SDS-Page, comparing the eletrophoretic protein profile from treated and untreated APMV and M4 lineage, the one

which has many deleted genes, resulting in the absence of fibrils. Bioinformatic analysis was also done in order to predict the mass of the fibrils. In addition, it was verified the viral replication by evaluation of cytopathic effect of the virus in acanthamoeba cells displayed in agar plates with PAS buffer. A fluctuability assay was performed using a discontinuous glucose gradient, where treated and untreated APMV were submitted to ultracentrifugation. Electronic microscopy revealed that the enzymatic treatment removed the fibrils and it was corroborated by SDS-Page. The protein profile of treated APMV and M4 was quite similar, showing the absence of two proteins, one of approximately 45 kDa and other of about 80 kDa. These proteins refer to ORFs R135 and L829 respectively, both related to fibrils’ synthesis. The absence of the fibrils has not led to changes in viral replication and cytopathic effect was the same. The absence of fibrils showed no difference in viral fluctuability but the assay is being done with different centrifugation conditions. The evaluation of fibrils in viral particle’s resistance when exposed to high temperatures and UV light is underway. The discovery of the functions of this mimiviruses’ peculiar structure will bring insights about its biology and may help discussions about its evolutionary origin and environmental roles. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, MAPA, PPG-MICROBIOLOGIA UFMG

BV85 - ANTIVIRAL ACTIVITY OF A POLYSACCHARIDE OF DIMORPHANDRA GARDNERIANA ASSOCIATED WITH MANGIFERIN AGAINST POLIOVIRUS TYPE 1 (PV-1)Rechenchoski, D.Z.1; Espada, S.F.1; Lopes, N.1; Godoi, A.M.1; de Faccin-Galhardi, L.C.1; Nozawa, C.1; Ricardo, N.M.P.S.2; Linhares, R.E.C.1


2. UFC - Universidade Federal do Ceará, Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, 60020-181

Poliovirus (PV), member of the Picornaviridae family, is a non-enveloped positive single-stranded RNA virus, causative agent of poliomyelitis, an acute disease of the central nervous system that may result in paralysis and death. Currently, poliomyelitis is under control in




most part of the world, however, the disease remains endemic in Afghanistan, Nigeria and Pakistan and, therefore, represents a major threat for the world. Considering the importance of viral diseases, the search for natural products for their control has been encouraged. Dimorphandra gardneriana is a Brazilian native leguminous tree, popularly known as faveira or fava-d anta and belongs to the Fabaceae family. The plant is widely distributed in the Brazilian Cerrado and its fruits are exploited to obtain rutin and quercetin, both bioflavonoids. Mangiferin is the major constituent obtained from Mangifera indica and has multiple pharmacological activities such as antitumor, immunomodulatory, antidiabetic, antioxidant, antiviral, antibacterial, anthelminthic, antiallergic and anti-inflammatory. This study aimed to evaluated the antiviral activity of a polysaccharide of D. gardneriana associated with mangiferin against poliovirus type 1 (PV-1). The antiviral action was investigated by plaque reduction assay (PRA), using different protocols: the time-of-addition (-2, -1, 0, +1, +2, +4, +8), inhibition of adsorption and virucidal activity. The 50% cytotoxic concentration (CC50) of the compound, evaluated by MTT assay, was >2000 μg/mL and the 50% inhibitory concentration (IC50) 206.25 μg/mL, with selectivity index (SI) >9.69. The compound was tested at 500, 250, 125 and 62.5 μg/mL. Preliminary, we showed the percent of viral inhibition (%IV) of 77.8%, 45.1%, 54.1%, 70.6% and 65.2%, at the time 0h (treatment simultaneously with infection), evaluated 1h, 2h, 4h and 8h thereafter, respectively, at the highest concentration (500 μg/mL). At the same concentration, the compound inhibited the PV-1 in approximately 46.8% and 61.5% when added 1h and 2h before the infection, respectively. For virucidal assay, the compound demonstrated an inhibition around 26.2% and 30%, while the inhibition of adsorption showed a %IV of 65% and 27.5%, at the concentrations of 500 μg/mL and 250 μg/mL, respectively. These results suggested that the compound interfered inhibiting PV-1 replication. Therefore, polysaccharide of D. gardneriana may be a potential candidate for the control of PV infection. FINANCIAL SUPPORT: CAPES, CNPQ, FUNDAÇÃO ARAUCÁRIA, PROPPG/UEL

BV88 - ANTIHERPETIC ACTIVITY OF NONI (MORINDA CITRIFOLIA) PECTINSRechenchoski, D.Z.1; Lopes, N.1; de Faccin-Galhardi, L.C.1; Espada, S.F.1; Godoi, A.M. de1; Bomfim, W.A.1; Ricardo, N.M.P.S.2; Linhares, R.E.C.1; Nozawa, C.1



The natural substances capable of presenting alternative mechanisms, unlike synthetic antiviral action, may be potential candidates for the control of viral infections. These substances have been shown to interact with many viral targets ranging from adsorption of the virus to the host cell to release the new viral particles into the extracellular medium, which may result in additional mechanisms of action of existing antiviral drugs. In this context and considering the high prevalence of herpes virus (HSV) infections and the incidence of severe disease in immunocompromised, the search for new antiherpetic compounds, particularly, makes it extremely challenging. The aim of this study is to investigate the antiviral activity of four pectins of noni (Morinda citrifolia), PDNA, PNDB, PDNN and PDNS to HSV-1 in HEp-2 cell cultures. The 50% cytotoxic concentration (CC50) for PDNA, PNDB, PDNN and PDNS were 605, 845, 420 e 44 µg/ml, respectively, by MTT assay. The antiviral action was assayed by plaque reduction assay by the following protocols: the time-of-addition assay (-2h, 0h, +1h, +2h) and the virucidal effect. The four compounds showed the 50% inhibitory concentration (IC50) of approximately 45 µg/ml. For the PDNS, this concentration was close to the CC50 and, therefore, excluded. For the others, we observed significant antiviral activity when they were used concomitantly with viral infection, reaching 100% of viral inhibition at the concentration of 100 µg/ml. In post-infection treatment, only PDNA maintained the activity. No inhibition was observed in the pre-infection treatment and virucidal assays for any of the three substances. Thus, the viral inhibition caused by these substances most likely occurs in the initial stages of viral replication. Further studies will be conducted in order to better understanding at what step(s) of HSV-




1 replication M. citrifolia pectins may act. FINANCIAL SUPPORT: CAPES/CNPQ/FUNDAÇÃO ARAUCÁRIA/UEL

BV92 - EVALUATION OF THE INFLUENCE EXERCISED BY A MICROORGANISM PROBIOTIC DURING VACCINIA VIRUS INFECTION BALB/C MICEAndrade, A.C. dos S.P.1; Oliveira, G.P.1; Lima, M.T.1; Calixto, R.S.1; Martins, F. dos S.1; Ferreira, J.M.S.2; Kroon, E.G.1; Abrahão, J.S.1



The circulation of Orthopoxvirus in the world and the prevalence of bovine vaccinia in Brazil have generated significant economic and social impacts. Thus, the establishment of methods for control and prevention of poxvirus infections is important for society. In view of this, new strategies to combat these viruses have been studied. Recently, the use of probiotic microorganisms has been proposed as a novel therapeutic approach for the control of various viral diseases. Therefore, the aim of this study is to understand what are the possible effects caused by probiotics in Balb/c mice infected with Vaccinia virus Western Reserve (VACV-WR), in relation to letality and differential leukocyte count. The VACV-WR was multiplied and titrated in Vero cells, purified on sucrose cushion and used at a concentration of 106 pfu in intranasal infection of Balb/c mice. The probiotic X was obtained from an industrial product and grown in broth De Man, Rogosa & Sharpe (MRS). The animals were divided into four groups: animals that received (i) only phosphate buffered saline 0.9%, (ii) only the probiotic X (iii) only VACV-WR, (iv) probiotics X for 10 days after infection (d.a.i) with VACV-WR. The differential count of leukocytes in whole blood of mice was made. Infected groups showed clinical signals such as ruffling fur, arching back, balanopostitis and weight loss. Uninfected groups showed no signal of infection. This experiment showed that letality in the probiotic-treated group was 50% lower than the group infected with VACV-WR. The differential leukocyte count showed that the reduction in the proportion of lymphocytes in the group treated with probiotic occurred only between the fourth and

the eighth d.a.i and the group that received only VACV-WR the reduction of lymphocytes occurred between the first and fourth d.a.i. The mechanism through which the treatment with the probiotic was able to reduce letality and delay the reduction of lymphocytes in infected animals probably involves a immunomodulation as previously described for other viral infections. However, this mechanism will be further investigated by other experiments. The tested probiotic was able to reduce the letality of mice and delay the decline of lymphocytes, showing its potential use as a method of controlling infection with VACV. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, MAPA, PPG-MICROBIOLOGIA UFMG

BV94 - CLONING AND EXPRESSION OF TRUNCATED HEPATITIS C VIRUS E2 PROTEINS IN BACTERIAL SYSTEMMoreira-Vieira, F. de F.; Freitas, G.R.O.; Yokosawa, J.


The hepatitis C virus (HCV) belongs to the Flaviviridae family and to the Hepacivirus genus. This virus has an enveloped icosahedral capsid, and its genome consists of a segment of simple-stranded RNA that has a single open read frame (ORF). This ORF is translated to produce a single protein, which is processed to produce seven structural proteins and three non-structural proteins. There are seven genotypes and several subtypes. The HCV causes hepatitis C in humans and it is one of the main causes of chronic liver disease. It is a blood-borne virus that is transmitted mainly through the reuse of needles by injecting drug users and accidents in health care settings. In acute phase, the infection is asymptomatic in 80% of cases, and chronic infection may envolve to cirrhosis, hepatic carcinoma and cause death. Around the world more than 350 thousand people die each year due to diseases associated with hepatitis C. Early HCV diagnosis isn’t common and treatment is expensive, with many collateral effects and it isn’t always successful, although it can decrease the number of deaths and severe disease. There is no preventive vaccine for HCV but researchers around the world are working in search for an effective vaccine. In these studies, the envelope glycoproteins E1 and E2 are the main proteins of interest because of their position in the virus surface, turning them into




targets for the immune system. The objective of this study was to express two truncated proteins containing selected segments of HCV envelope glycoprotein E2 that induces the production of neutralizing antibodies. The gene sequences were cloned into vector pGS-21a for expression in E. coli of a protein fused with 6xHis-tag. Plasmids from selected colonies were sequenced and used in transformation of bacteria BL21-CodonPlus (DE3)-RIPL. The products of transformation were used for protein expression by induction with IPTG (isopropyl β-D-1-thiogalactopyranoside). Bacteria cells were lysed and submitted to electrophoresis in SDS-polyacrylamide gel and Western Blotting with anti-6xHis monoclonal antibody. The expression of both truncated proteins was successfully observed.

BV101 - EVALUATION OF INFLUENCE FROM NORMAL MICROBIOTA ON VACCINIA VIRUS INFECTION IN GNOTOBIOTICS AND CONVENTIONALS SWISS MICE BY TWO DIFFERENT INOCULATION FORMSLima, M.T.; Andrade, A.C. dos S.P.; Calixto, R.S.; Oliveira, G.P.; Alvim, L.B.; Martins, F. dos S.; Kroon, E.G.; Abrahão, J.S.


Normal microbiota has important role in the control and development of some kinds of autoimmune diseases, cancers, allergies and also in the formation of immune response against pathogens. Deficient microbiota is a potential risk factor for the development of animal diseases with high frequency and severity and also enables ineffective immunization in cases of vaccines using live attenuated pathogens, such as smallpox vaccines using Vaccinia virus strains. Vaccinia virus is a member of Poxviridae family and is the type virus of genus Orthopoxvirus. It is currently responsible for zoonotic human infections, affecting workers from dairy cattle, with outbreaks being observed in several regions of Brazil. To determine the possible influence of normal microbiota in the development of disease caused by Vaccinia virus infection, a comparative study using murine model was performed. Germ-free Swiss NIH mice and conventional Swiss mice of 6-7 weeks-old were infected with Vaccinia virus Western Reserve (VACV-WR) by two different pathways of inoculation,

intranasal and tail scarification. Control groups for both routes were inoculated only with phosphate buffered saline 0.9% (PBS). The infections were done using 106 pfu (intranasal) and 105 pfu (scarification) of VACV-WR per animal, which was multiplied and titrated in Vero cells and purified on sucrose cushion. Four groups, each one containing five animals, received intranasal doses. In this experiment, the Germ-free animals inoculated with VACV-WR showed clinical signals such as periocular alopecy, ruffling fur, arching of back and weight loss. The infection also resulted in the death of two animals. Conventional mice inoculated with VACV-WR and control groups did not show any clinical signs. Other four groups with five animals in each were submitted to tail scarification. In groups where the virus had been inoculated, a lesion has appeared 4-6 days post scarification and developed into a scab. In these groups, all mice survived and did not show any signs of morbidity. All living animals of the experiments were sacrificed 10 days after infection and several samples were collected for future assays. The experiments were approved by the Ethics Committee of UFMG. Our study showed that, there is an influence of the normal microbiota in pathogenicity and virulence in gnotobiotic and conventional Swiss mice, inoculated with VACV-WR applied intranasally. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, PPG-MICROBIOLOGIA UFMG

BV109 - DEVELOPMENT OF CELL SYSTEM FOR IN VITRO DETECTION OF NON-NEUTRALIZING ANTIBODIES AGAINST THE DENGUE VIRUSGomes, A.V.B.T.1; Rodrigues, N.F.1; Prado, A.A.O.1; Carneiro, A.C.A.2; Silva, B.M.1; Malaquias, L.C.C.2; Coelho, L.F.L.1


2. UFOP - Universidade Federal de Ouro Preto, Campos Morros do Cruzeiro, s/n - Bauxita, Ouro Preto - MG, 35400-000

Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different




serotype progresses to the severe forms of the disease, dengue hemorrhagic fever/dengue shock syndrome. Secondary infection with a different serotype from that in primary infection increases the risk of development of severe complications. In these cases, cross-reactive, non-neutralizing antibodies can bind to DENV surface and this DENV-antibody complexes are taken up more efficiently by Fcγ receptor (FcγRIIA)-expressing cells. Thus, in the present study we developed a cell system able to identify the presence of non-neutralizing antibodies against the DENV. The plasmid encoding the FcγRIIA receptor (pTCN- FcγRIIA) was used to transfect Baby Hamster Kidney cells (BHK-21). Since the vector has a neomycin resistance, we treated the cells with the G418 antibiotic to select the transfected clones. The FcγRIIA expression was assayed by Real Time PCR and immunofluoresnce staning. The clone that has the high expression of FcγRIIA was selected to be used in vitro assays to determine the presence of non-neutralizing DENV antibodies in human serum. FINANCIAL SUPPORT: FAPEMIG; CNPQ; CAPES.

BV115 - STUDIES ON THE BIOLOGICAL AND GENETIC DIVERSITY OF CLINICAL SPECIMENS OF CANTAGALO VIRUS ISOLATED FROM DAIRY CATTLE IN RONDÔNIARezende, B.; Damaso, C.


Cantagalo virus (CTGV) is a vaccinia virus (VACV) strain isolated in 1999 in Rio de Janeiro state from lesions in dairy cattle. The disease has caused substantial economic loss in different states of Brazil, and recently in Rondônia (RO). No antiviral therapy is currently available for poxvirus-related disease. Therefore, the search for potential antiviral drugs is important. In addition, the genetic diversity of the different clinical isolates should be investigated since this feature could contribute to different responses to antiviral drugs. In this work, we evaluated different clinical isolates from RO collected during outbreaks in dairy cattle after 2009. The samples were positive for CTGV by PCR and sequencing of the HA gene. We evaluated some biological features in cell culture and the viral response to three antivirals, i.e. sulfated galactan (SG), cidofovir (CDV) and ST-246. The size of poxvirus viral plaques reflects the virus capacity to spread to neighboring cells. Therefore,

we measured the diameter of 20 viral plaques after 48 h of infection with the clinical isolates. We observed that CTGV reference isolate CM-01, isolated in RJ in 1999, had viral plaques with mean diameter of 307.3 µm. On the other hand, isolates collected in Urupá (URU-07) in 2009 and in Jaru, Governador Jorge Teixeira, Espigão D’Oeste, Campo Novo de Rondônia and Ji-Paraná in 2010 presented mean diameters of 249.1 µm, 279.0 µm, 281.0 µm, 241.8 µm, 240.0 µm and 300.5 µm respectively. In contrast, isolates from Governador Jorge Teixeira in 2012 (GJT-05) presented plaques with mean diameter of 613.5 µm. To analyze the effect of SG on the formation of viral plaques, we infected cells with 300 PFU of CTGV CM-01 or isolates from RO, in the presence of different concentrations of SG during viral adsorption. After 48h, we verified an inhibition of 50.2% for CTGV CM-01, 60.8% for URU-07 and 64.6% for GJT-05 at 0.1 µg/ml. To evaluate the replication of CTGV isolates in the presence of CDV and ST-246, we infected BSC-40 cells with 300 PFU and added different concentrations of these drugs after adsorption for 48h. Our results showed an inhibition of 53.8% for CTGV, 83.6% for the URU-07 and 37.9% for GJT-05 at 0.01 µM of ST246. For CDV, we detected an inhibition of 56.2% for CTGV CM-01, 91.1% for URU-07 and 73.8% for GJT-05 at 30µM of CDV. Therefore, these results suggest that may be interesting differences in virus circulating in RO that should be better investigated. FINANCIAL SUPPORT: CAPES, CNPQ, FAPERJ AND INPETAM

BV122 - ANTIHERPETIC ACITVITY OF ANNONA SQUAMOSA LSilva, Í.T.S.S.1; Araújo, S.B.2; Fernandes, M.J.B.2; Oliveira, R.A.1; Rebouças, A.S.J.3; Conceição, A.O.1

1. UESC - Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900

2. Instituto Biologico SP, Avenida Conselheiro Rodrigues Alves, 1.252 - Vila Mariana, São Paulo - SP, 04014-002

3. UESB - Universidade Estadual do Sudoeste da Bahia, Estr. do Bem-Querer, Km 4, Vitória da Conquista - BA, 45083-900

Annona squamosa L. (Annonaceae) is a tree or shrub cultivated in tropical and subtropical regions of Central America that bears edible fruits known as sugar-




apples, ‘ata’, ‘pinha’, or ‘fruta-do-conde’. In vitro and in vivo studies revealed the biological activity potential of A. squamosa extracts and special attention has been done to acetogenins isolated from the seeds, which have antitumoral activity. Despite several biological activities reported for Annona species, there is a lack of information about antiviral activity of this plant against herpesvirus. Then, for this study, we evaluated the effect of seeds and leaves extract from commercial specimens of A. squamosa L. using Vero (ATCC-CL81) cells and animal herpesviruses. Aqueous, ethanolic, and hexanic extracts were obtained by conventional techniques and, after solubilization, they were brought to concentrations from 0.03 mg.mL-1 to 4 mg.mL-1. Cell viability was established using trypan blue exclusion technique and cell morphological evaluation. The antiviral assays were based on viral titer reduction assay against suid herpesvirus type 1 (SuHV-1) and equine herpesvirus type 1 (EHV-1) and the results were expressed as inhibition percentage (IP). Leaves and seeds ethanolic extracts were less toxic compared to aqueous and hexanic extracts and all of them were suitable to antiviral test in concentrations below 0.5 mg.mL-1. The ethanolic extract from seeds showed IP of 82.22 against EHV-1; while hexane extract from seeds showed IP of 94.24 for SuHV-1. These are the first report of activity of A. squamosa L. extracts against animal herpesviruses indicating the presence of hydrophobic compounds in seeds with promising antiviral activity.

BV137 - ISOLATION AND THERAPEUTIC EFFICACY IN BALB/C MICE OF DITERPENE ISOLATED FROM MARINE ALGA CANISTROCARPUS CERVICORNIS AGAINST HERPES SIMPLEX VIRUS TYPE 1Barros, C.; Garrido, V.; Melchiades, V.; Cavalcante, M.; Ribeiro, M.; Figueiredo, C.; Souza, K.S.; Pinto, A.M.; Giongo, V.; Teixeira, V.; Paixão, I.


Herpes Simplex Virus (HSV) infection is a worldwide endemic and is one of the most prevalent infections in Brazil. Although several antiviral substances are available for the treatment of HSV-1, increased viral resistance to usual antiherpetic drugs and the occurrence of severe cases of the disease, mainly in immunocompromised individuals, have highlighted the importance of finding

new effective therapeutics against herpes. In this context, an in vitro study with the diterpene from Canistrocarpus cervicornis used in this work showed the antiviral potential of the diterpene in controlling the replication of HSV-1 and maintaining low cytotoxicity. Hence, the aim of this work was the isolation of diterpene (4R, 9S, 14S)-4α-acetoxy-9β,14α-dihidroxydolast-1(15),7-diene and the evaluation of its antiherpetic efficacy in vivo. To this end, the air material alga was exhaustively extracted with CH2Cl2. The solvent was evaporated under reduced pressure, yielding a brownish residue. An aliquot from the crude extract was dissolved in pyridine and was treated at room temperature with acetic anhydride for 65 h. Extraction of the reaction mixture in the usual way afforded (addition using H2O Mili-Q® and extraction by CHCl3) and was subjected to silica gel chromatography elution by increasing the polarity of pure n-hexane/ethyl acetate mixtures, resulting in 44 fractions. Fractions 26, 27 and 28 yielded the diterpene, confirmed by 1H NMR spectra data, and new cycles of isolation procedures were repeated to obtain a sufficient concentration for the experiments. For the antiherpetic efficacy in vivo, three groups of 5 5-month-old BALB/c mice were used: 1 - diterpene (15mg/Kg); 2 - acyclovir (15mg/Kg); and 3 - DMSO 1% (vehicle). The right midflank of each mouse was clipped and depilated with a chemical depilatory. After two days, the skin was scratched using a sterile 27-gauge needle, and 10 μL of HSV-1 KOS (1 X 109 PFU) in DMEM with 2% bovine serum was applied to the scarified area. All mice were orally gavaged with 200 μL solution of the substances twice daily over 18 days, beginning 1 hour after virus inoculation. The cutaneous herpes infection was scored throughout the experimental period. Animals gavaged with diterpene (score = 5,0) and acyclovir (score = 3,8) had their disease scores reduced after day 10, compared with group 3 (vehicle) (score = 7,0). These results suggest that diterpene may be useful as an oral agent to reduce the severity of HSV-1 cutaneous lesions. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, UFF (PROPPI)




BV144 - ANTIVIRAL ACTIVITIES OF PHOSPHOLIPASES A2 FROM SNAKE VENOM AGAINST DENGUE VIRUSCaldas, S.; Cecílio, A.B.; Silva, F. de O.; Sanchez, E.F.

FUNED - Fundação Ezequiel Dias, R. Conde Pereira Carneiro, 80 - Gameleira, Belo Horizonte - MG, 30510-010

Introduction - The economic and human burden of dengue is substantial in endemic countries. High numbers of cases, absence of specific treatment or vaccine demonstrate the need for the development of drugs against Dengue virus. Recently we have observed the antiviral activity of phospholipase A2 from Bothrops leucurus against Dengue virus. Considering several studies of antimicrobial activity with animal venoms worldwide and the limited number of studies with derivatives of snake venom from Brazilian cerrado, this work aimed to assess the anti-Dengue potential of phospholipase A2 (pool) and it´s isoforms (K49 and D49) purified from B. leucurus venom. Material and methods - First, the maximum non-cytotoxic concentration of phospholipase A2 and it´s isoforms was determined by MTT assay in LLC-MK2 cells. Then, the cells were treated with the maximum non-cytotoxic concentration of those purified molecules and were incubated at 37°C with Dengue virus-1, 2 and 3 at a multiplicity of infection of 0.05 in a 5% CO2 humidified incubator. After 48 hours of incubation, the quantification of viral RNA load of treated cells compared to untreated controls was performed by one-Step qRT-PCR. Subsequently, the antiviral assays were performed with both isoforms inactivated by Diethyl Pyrocarbonate (an inhibitor of catalytic activity) to suggest a possible mechanism of action. Results and discussion - The maximum non-cytotoxic concentrations of phospholipase A2 were 40 µg / mL (Pool) and 20 µg/mL for isoforms. Antiviral assays showed significant reduction of viral RNA in the treated cells, with similar reductions for phospholipase A2 (Pool) and isoforms. The trials with inactivated isoforms suggest that the catalytic action is not important for antiviral activity. However, more specific enzyme inhibitors will be tested to more conclusive results. Moreover, our tests after viral adsorption showed no significant inhibition of viral replication in the treated cells, suggesting an activity at cell membrane level, whereas after cell penetration, no decrease was observed in their viral load. Conclusion - The data show that phospholipases A2 from B. leucurus are able to inhibit the growth of Dengue virus, probably

acting at the entrance into the cell, suggesting its applicability as a tool diagnostic or prototype for drug development. FINANCIAL SUPPORT: Fapemig (BIP-0056-14, BIP-00168-14, BIP-00139-13), CNPq (Proc. 482502/2012-6), FUNED.

BV154 - HSP27 PROTEIN HAS ANTIVIRAL ACTIVITY AGAINST HCVAkinaga, M.M.; Braga, A.C.S.; Carneiro, B.M.; Batista, M.N.; Rahal, P.

UNESP/IBILCE - Universidade Estadual Paulista/ Instituto de Biociências, Letras e Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José do Rio Preto - SP, 15054-000

Several cellular proteins are known to interact with the HCV or being necessary to the viral replication process, such as the family of heat shock proteins (HSP). The Hsp are usually synthesized in response to cellular stress such as heat shock, nutrient deprivation and bacterial or viral infections. Among Hsps, Hsp27 belongs to a family of small heat shock proteins and has shown antiviral activity in hepatitis B virus (HBV) and human immunodeficiency virus (HIV). Other studies have observed an increased expression of Hsp27 in cells containing an HCV subgenomic replicon compared to the cells without replicon and in patients with hepatocellular carcinoma, high expression of Hsp27 is associated with HCV presence. Thus, the present study aimed to evaluate in vitro whether HSP27 has antiviral activity for Hepatitis C. For this, human hepatoma Huh7.5 cells containing the subgenomic HCV replicon (SGR-JFH1 FEO) were transfected with siRNA HSP27 and viral replication was assessed 12, 24, 48 and 72 hours after transfection. At all times HCV replication was greater in cells treated with siRNA Hsp27 (135%, 117%, 115% and 122% respectively) to cells treated with siRNA control (100%, 101%, 94% and 98% respectively). These data suggest that Hsp27 protein represents a cellular defense mechanism, playing an antiviral activity in presence of HCV. FINANCIAL SUPPORT: FAPESP




BV170 - REGULATION OF HEAT SHOCK PROTEINS EXPRESSION BY HEPATITIS C VIRUSBraga, A.C.S.; Carneiro, B.M.; Batista, M.N.; Akinaga, M.M.;-Rahal, P.

UNESP - Universidade Estadual Paulista, Rua Quirino de Andrade, 215, São Paulo - SP, 01049-010

Hepatitis C is a disease caused by hepatitis C virus (HCV), and it is estimated that about 170 million people are infected with the virus worldwide. During infection HCV interacts with several cellular proteins to promote viral replication. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. This increase or decrease in HSPs expression may assist in understanding the mechanisms involved in viral replication and provide an alternative in viral replication reduction. Thus the present study aimed to evaluate in vitro the expression of heat shock proteins in the presence and absence of HCV. For this human hepatoma Huh7.5 cells were electroporated with the replicon HCV JFH-1 and maintained until approximately 90% of the cells were infected by the virus. These cells were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array comparing uninfected and infected cells with HCV. The results demonstrate that five genes showed increased expression (over Log2 2), while four others had reduced expression. These results may help in understanding the mechanisms involved in HCV replication. FINANCIAL SUPPORT: FAPESP

BV177 - CLONING AND EXPRESSION OF THE NONSTRUCTURAL PROTEIN 3 (NSP3) OF MAYARO VIRUSPinhatti, A.K.; Pacca, C.C.; Ribeiro, M.R.; Mota, M.T. de O.; Vedovello,D.; Nogueira, M.L.

FAMERP - Faculdade de Medicina de São José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São Pedro, São José do Rio Preto - SP, 15090-000

The Mayaro virus (MAYV) is an arbovirus of the Togaviridae family, Alphavirus genus enzootic in tropical South America and endemic in rural areas. This virus causes Mayaro Fever and its first outbreak in Brazil was reported in 1957, in the state of Pará. It is maintained in the sylvatic cycle involving vertebrates like monkeys of

Cebidae, Callithricidae, Saguinus e Alouatta family, and birds, that can act like secondary hosts. It is transmitted by blood-sucking arthropods, typically the mosquitoes, specially the Haemagogus janthinomys. Mayaro is an enveloped virus composed of a single strand of positive-sense RNA approximately 11.5 kb in length. The genome encodes nonstructural and structural proteins, forming a complex of replication that is required for the starter genomic RNA. The nonstructural protein NSP3 have an essential function at the replication of the virus and maturation of proteins, and its formed by tree domains: “macro” domain or X-domain, N-terminal domain and C-terminal domain. In order to understand the replication cycle of virus we decided to clone, express and purify the nSP3 protein of Mayaro virus. A DNA fragment equivalent of the coding region of nSP3 was obtained by RT-PCR and was cloned in pET21a vector. The plasmid built was amplified by Escherichia coli (DH5α) and the confirmation of the cloning was done by PCR. Several clones were then transferred to Escherichia coli (BL21) and he expression of this protein was be detected by SDS-PAGE. We will now take this production to a high volume and the protein will be purified and characterized. FINANCIAL SUPPORT: CNPQ / FAPESP

BV182 - EFFECT OF EXTRACTS SCHINUS TEREBINTHIFOLIUS AND PUNICA GRANATUM ON MAYARO VIRUS REPLICATIONFerreira, F.D.1; Salles, S.T.1,3; Meneses, M.D.F.1; Guimarães, T.E.1; Caldas, L.A.4; Meira, G.L.S.1; Yamamoto, K.I.1,3; Kuster, R.M.2; Campos, R.M.1; da Silva, M.R.S.3

1. Laboratório de Interação Vírus Célula, Departamento de Virologia, Instituto Microbiologia Prof. Paulo De Góes. Centro de Ciência E Saúde, Universidade Federal Do Rio De Janeiro.

2. Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro

3. LAMMP, Departamento de Bioquímica, Instituto de Química, Centro Tecnológico, Universidade Federal do Rio de Janeiro.

4. Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho. Universidade Federal do Rio de Janeiro.

Mayaro virus (MAYV) is an arbovirus that belongs to the genus Alphavirus, Togaviridae family which causes




a disease known as Mayaro Fever, with symptoms similar to the classic dengue fever. The anti-inflamatory properties of substances extracted from the plants Schinus terebinthifolius (mastic) and Punica granatum (Pomegranate) has already been explored by the Brazilian popular medicine. In this work we tested the antiviral effect of these substances against MAYV. The toxicity of these substances in VERO cells was tested by the incorporation of neutral red after 24h of treatment. The CC50 of each substance was determined. The CC50 of each substance was: 242, 315, 102 and 5000 µg/mL in acetate, flavonoids 1 and 2, oil of Schinus, respectively, and 590 and 442 µg/mL for Crude Extract and Acetate of Punica, respectively. After the determination of non-toxic concentrations of these substances, the antiviral effect was tested. Cells were infected for 1 h with Mayaro virus using a multiplicity of infection of 0,1. Treatment of cells was carried out for 24 hours with increasing concentrations. We observed that the substances exhibit antiviral activity. The IC50 of each was determined by a dose response graph, and were 4,3; 4,5; 14 and 830 µg/mL µg/mL in acetate, flavonoids 1 and 2, oil of Schinus, respectively, and 12 and 30 µg/mL for Crude Extract and Acetate of Punica, respectively. The selectivity index (ratio CC50/IC50), was determined and the values were greater than 7 for all substances. We also tested the substances for virucide properties, adsorption impairment and pretreatment. Our results showed more than 95% virucidal effect for the partitions acetate, flavonoids mastic and Crude Extract of pomegranate. Mastic oil, and acetate of pomegranate did not present virucidal effect. We also observed by transmission electron microscopy the damages caused upon the virus particles by the virucide substances. Proteomic analysis showed differences in the expression of secreted proteins during infection and treatment with the tetrahyamentoflavone substance. We conclude that some of the partitions in our substances act directly on the virus particle. FINANCIAL SUPORT: CAPES, FAPERJ AND INBEB

BV185 - SUSCEPTIBILITY OF BRAZILIAN VACCINIA VIRUS STRAINS TO THE ANTIVIRAL COMPOUND ST-246Rodrigues, N.F.S.; Pires, M.A.; Almeida, G.M. de F.; Ferreira, P.C.P.; Bonjardim, C.A.; Trindade, G. de S.; Abrahão, J.S.; - Kroon, E.G.; Mota, B.E.F.


The Vaccinia virus (VACV), which belongs to the Poxviridae family, is the etiological agent of a disease that affects bovines, humans and other hosts, being characterized by the appearance of exanthematous lesions at the virus entry site. The transmission occurs mainly by direct contact with lesions and it happens among wild hosts and bovines, and among bovines and humans. Recently several outbreaks have been described in rural properties across the Brazilian territory. In previous studies, our group observed genotypical and phenotypical differences among the viruses that were isolated from these outbreaks. This disease has been considered an important public and animal health problem, besides compromising local economy. The drug ST-246 (Tecovirimat®) has demonstrated potency and specificity for the treatment of poxvirus infections. However, so far only one isolate of Brazilian VACV (BRZ-VACV) has been tested for its susceptibility to this drug (Cantagalo virus, EC50 of 0.0086 µM). Therefore, due to great diversity of BRZ-VACV, it is necessary to evaluate further the in vitro susceptibility of these viruses to different ST-246 concentrations. The susceptibility test for eight BRZ-VACV isolates was done by using a viral suspension of 150 PFU, which was inoculated into monolayers of BSC-40 cells. The F13L gene in the samples was amplified by conventional PCR and then sequenced to assess the existence of polymorphisms related to drug resistance. The comet assay was performed to evaluate qualitatively the susceptibility of the isolates. EC50 values for the isolates showed noteworthy variation (from 0.0054 to 0.051 µM), which is within the range of values described in scientific literature for other VACV strains. The CC50 value in BSC-40 cells was higher than 50 µM, which provides a selectivity index greater than 1,000. Sequencing of F13L gene showed that the isolates did not exhibit the previously described polymorphism associated to ST-246 resistance (Gly277Cys). There




has been drastic reduction in comet formation in ST-246 concentrations as low as 0.0125 µM for all the isolates. In conclusion, the results show that EC50 varies significantly amongst Brazilian VACV isolates and that ST-246 may be useful in the treatment of human infections by BRZ-VACV.

BV187 - STUDY OF THE PHOTODYNAMIC THERAPY EFFECTS OF CURCUMIN-NANOPARTICLES IN HPV-16 POSITIVE CERVICAL CARCINOMA CELLSMatos, R.P.A. de1; Primo, F.L.2; Tedesco, A.C.2; Villa, L.L.3; Calmon, M. de F.1; Rahal, P.1



3. INCT-HPV - Instituto Nacional de Ciência e Tecnologia das Doenças do Papilomavirus Humano, R. Dr. Cesário Mota Júnior, 61 - Vila Buarque São Paulo - SP 01221-020

Human Papillomavirus (HPV) are a DNA virus family with more than 190 types identified, and can be classified in low and high risk HPVs. The most important high risk HPVs are the HPV-16 and -18, that together are responsible for more than 70% cervical carcinoma cases. The current available treatments are surgery, cryogenics, drugs and antiviral therapies. However, these treatment options have side effects such as pain, redness, itching, burning and hypersensitivity. So, due to the large number of people infected by HPV, the need of noninvasive antiviral treatment, with few or no side effects, has been increasing. Thereby, the nanotechnology associated with natural compounds may be a future treatment. Curcumin is a very versatile bioactive hydrophobic compound which has anti-inflammatory, anti-hyperlipidaemic, anti-angiogenic, anti-neoplastic and chemoprotective properties, and also has the ability to decrease the expression of HPV oncogenes. Furthermore, curcumin may be used as a photosensitizing agent in Photodynamic Therapy (PDT), as it was shown that several of the effects can be potentiated by the combination of curcumin with light. However, curcumin application as an active agent has been limited by low water solubility and bioavailability. The use of nanoparticles, where the curcumin can be encapsuled and delivered into cells, could allow

an improved pharmacological performance of this compound in antiviral therapies. Until now we evaluated the action of curcumin associated with nanoemulsion (NE-CUR) as photosensitizing drug on PDT system in cervical carcinoma cell lines HPV-16 positives (CasKi and SiHa). The fluorescence microscopy images showed that the nanoemulsions were able to internalize cells and were observed in the intracellular environment for up 36 hours after incubation in SiHa cells and for 48 hours in CasKi cells. The cellular viability assay showed biocompatibility of nanoemulsions, evidenced by the absence of cytotoxicity of empty nanoemulsions. Besides, we observed high phototoxic effect of the NE-CUR formulation with less than 5% of viable cells after irradiation for both cells lines. Additionally, photodynamic therapy using NE-CUR as photosensitizing drug was able to increase the P53 expression in 2.43 fold change for CasKi cell line. These results show that the developed formulation NE-CUR, in combination with photodynamic therapy, has a great therapeutic potential to be used in the treatment of lesions caused by HPV. FINANCIAL SUPPORT: FAPESP

BV192 - ANALYSIS OF THE ONCOLYTIC POTENTIAL OF AVIAN GYROVIRUS VP3 PROTEINCosta, C.S.; Knak, M.B.; Costenaro, J.G.; Mees, L.; Campos, F.S.; Franco, A.C.; Roehe, P.M.


Our group has recently identified a gyrovirus, named avian gyrovirus 2 (AGV2). The genome of AGV2 encodes three proteins, one of which is VP3 (AGV2-VP3), a protein homologous to apoptin of chicken anemia virus (CAV), for which several studies show a pro-apoptotic potential in tumor cells. In view of the similarity between these two proteins, it became of interest to investigate AGV2-VP3 potential to induce apoptosis in human tumor cells, since this property might be relevant for tumor suppression. Recombinant adenoviral vectors expressing three variants of VP3-AGV2 ( pAd, pAd and pAd), as well as a control vector pAd expressing the lacZ gene (pAd), were used for transformation of E. coli and subsequent cloning. Plasmid DNA was extracted and the nucleotide sequences of the recombinant adenovirus variants confirmed. Subsequently, the




vectors were transfected in 293A cells and cultured until observation of cytopathic effect, when new subcultures were performed in attempting to increase viral loads, as determined by titration. The transcription of mRNAs of VP3-AGV2 variants was confirmed by RT-PCR. The recombinants adenovirus were inoculated at different multiplicities of infection in normal MRC-5 cells (lineage of human lung fibroblasts) and in human tumor A549 cells (lineage of human lung carcinoma); cell viability was evaluated by MTT assay (3-bromide (4.5-dimetiltiazol-2-yl) -2.5-difeniltetrazolium). Statistical analysis of the results evidenced that the three recombinant adenovirus expressing variants of AGV2-VP3 inhibit cell growth of both tumor A549 cells and normal MRC-5 cells. However, the inhibition observed in A549 cells was significantly higher than in MRC-5 cells. These findings suggest that AGV2-VP3 is a potential candidate for a tumor suppressing protein. Future studies will be conducted to examine its potential as an oncolytic protein in more detail.

BV193 - PRELIMINARY EVALUATION OF ANTIHERPES ACTIVITY OF STRYCHNOS PSEUDOQUINA ST. HILSilva, I.T.2; Farias, L.M.3; Xavier, A.A.3; Pádua, R.M.1; Leite, J.P.V.3; Simões, C.M.O.2




Herpes Simplex Viruses (HSV) are responsible for many infections of oral, ocular and genital regions, and efforts have been made to find new drugs for their treatment, mainly due to their resistance to the most common available treatment (e.g. acyclovir). Natural products are an inexhaustible source of bioactive molecules, and considerable attention has been given to secondary compounds purified from plants in an attempt to search for new antiviral drugs. Strychnos pseudoquina St. Hil. is a native cinchona-like tree of the Brazilian savanna, popularly known as “quina” and used in the folk medicine to treat hepatic and stomach diseases, fever, and malaria.

Phytochemical studies employing S. pseudoquina have demonstrated the presence of some alkaloids and flavonoids, classes of compounds which are well known for their inhibitory effects of viral infections. In this study, we evaluated the cytotoxicity and the antiherpes activity of three extracts obtained by serial percolation of the stem bark (hexane, ethyl acetate and ethanol extracts), and one pure biflavone (strychnobiflavone) isolated from S. pseudoquina. Cytotoxicity was evaluated on Vero cells by using MTT assay, and antiviral activity was tested against HSV-1 (KOS strain) by viral plaque number reduction assay. Results were expressed as 50% cytotoxic concentrations (CC50) and 50% viral replication inhibitory concentrations (IC50), respectively, in order to calculate the selectivity index (SI=CC50/IC50) of each tested sample. Among the tested samples, the ethyl acetate extract and the pure compound SPEA2, inhibited HSV-1 replication showing SI values of 14.1 and 13.7, respectively. The detected antiherpes activity of S. pseudoquina studied extract seems to be linked to its high content of flavonoids, especially strychnobiflavone. FINANCIAL SUPPORT: CNPQ, CAPES, FAPEMIG

BV195 - POLICLONAL ANTIBODIES ANTI-P2, A CAPSID SUBDOMAIN FROM A SAPOVIRUS GI.2Fagundes, J.M.1; dos Anjos, K.; Lucinda, N.2; Machado, A.M.V.2; Nagata, T.1


2. FIOCRUZ MINAS - Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002

Sapovirus is one of the five genus within Caliciviridae family, it has, at the moment, only one specie member, Sapporo virus. Sapovirus genome is linear, positive-sense, single-stranded RNA, of approximately 7.3 to 7.5 kb. It has a linked protein at the 5’ terminus (VPg) and is polyadenylated at 3’ terminus. Sapporo virus is divided into, not officially, 14 genogroups, whereas GI, GII, GIV and GV are humans infective, GIII, GVI - GXI are porcine infective, GXII, GXIII and GIV are mink, dog and bat infective respectively. These viruses are worldwide known because their prevalence among human caliciviruses, that is very represented by Norovirus, which is the most important virus agent that causes acute gastroenteritis. Many epidemiological studies




are published every month about outbreaks caused by sapovirus, all of them uses RT-PCR or even RT-qPCR as detection methods. It is known that in Brazil although Norovirus is not included into the surveillance system for acute diarrhea it has been found positive samples all over the country; this means that if an effective and cheaper method to detect sapovirus was available these viruses would be also identified. It is known that Sapovirus GI.2 is worldwide prevalent, to detect this specific genogroup the subdomain P2 from the capsid protrude was expressed using E. coli system. The sequence of P2 from SapoBR01 was cloned using gateway system to pDEST17 and it was expressed into BL21AI E. coli. The protein was found into insoluble fraction; even when low temperature such as 15˚C was used for 24h of induction. The P2 was purified using MagneHis (Promega), 30ug/ml was used to immunize mice. A total of 6 injections using the purified protein plus aluminum were done through subcutaneous back of the animal. After 25 days after the first injection the blood was collected intraocularly. The serum was separated and then used as a primary antibody. A total of 4 animals were immunized and all the serum collected present positive reaction against the protein expressed. We expect to have human feces sample positive to Sapovirus GI.2 to evaluate these antibodies against human samples. FINANCIAL SUPPORT: CNPq.

BV202 - TREATMENT WITH MARINE ALGA PRODUCT AFTER INFECTION WITH HERPES SIMPLEX VIRUS 1 IN BALB/C MICEGarrido, V.; Barros, C.S.; Melchiades, V.; Cavalcante, M.; Teixeira, V.L.; Ribeiro, M.; Teixeira, G.; Giongo, V.; Paixão, I.C.N.P.


The search for new substances against herpes simplex virus 1 has advanced in the last decades mainly due to the resistance of existing drugs. Often HSV-1 is not lethal, but it becomes severe when causes encephalitis in children and immunocompromised patients. Research on marine alga shows a high potential bioactive in their secondary metabolites which could be developed into new drugs against pathogens. The aim of our study is to evaluate the efficacy of dolabellanodienetriol product (DBD3) isolated from marine brown alga Dictyota pfaffii

in BALB/c mice infected with a strain of HSV-1 mice. For the experiment three groups were used: G1-1% DMSO - vehicle (n=2); G2-Acyclovir-15 mg/kg (n= 5) and G3-DBD3- 20 mg/kg (n= 5). The right midflank of each mouse was clipped and depilated with a chemical depilatory. Two days later, 10μL of HSV-1 KOS (1 X 109 PFU) were inoculated by scratching the skin with a sterile 27-gauge needle. One hour after inoculation, the substances were administered by gavage twice daily for 18 days. The animals were monitored and photographed throughout the period to determine the score. The results showed the first signs of infection on the 4th day of inoculation in all groups. The disease has natural evolution in G1 (1% DMSO) group while most animals from G2 (ACV) and G3 (DBD3) group started to show signs of healing on the 10th day according to score done. Our results showed that concentration of 20 mg/Kg of DBD3 could inhibit the skin lesions caused by HSV-1 without animal death. Our conclusion based on in vitro and in vivo data, DBD3 seems to be a promising molecule since it has been showing low toxicity and antiviral efficacy. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, UFF (PROPPI)

BV203 - GENE EXPRESSION OF INTRACELLULAR FACTORS POTENTIALLY INVOLVED IN NATURAL RESISTANCE TO HIV IN SERODISCORDANT COUPLESColtro, V.P.; Santos, I.M.; Pinto, A.R.; da Rosa, R.D.

UFSC – Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima - Trindade, Florianópolis - SC, 88040-900

Background: Human immunodeficiency virus (HIV) infection is responsible for causing acquired immunodeficiency syndrome (AIDS). It is mainly transmitted through sexual intercourse, presents complex and variable natural history and shows different patterns of disease progression. Currently, there are many reports of individuals who were exposed to HIV and were not infected, suggesting the existence of mechanisms that lead to natural resistance to this virus. Some of these individuals are seronegative partners of serodiscordant couples. HIV interacts with many cellular host proteins during its replication cycle. Some of these proteins are required for HIV replication while others exhibit antiviral activity. In this way, HIV replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. This study aims




to evaluate gene expression of antiviral (APOBEC3G, TRIM5alpha, BST-2, SAMHD1, and cFLIP) and proviral factors (LEDGF/p75 and TREX1) in T CD4+ lymphocytes and monocytes from individuals exposed to HIV and uninfected, HIV positive and a control group composed by healthy individuals who have not been exposed to the virus. Methodology: This study was conducted on a cohort of heterosexual and homosexual couples, composed of 9 HIV-exposed seronegative subjects (HESN) and 9 HIV-infected patients in HIV (serodiscordant couples), and 12 healthy controls (HC) in HIV-negative seroconcordant monogamous couples. Serodiscordant couples were recruited at reference centers, and HC were recruited at HEMOSC, both in Florianópolis, SC, Brazil. Whole blood samples were collected and CD4 + T lymphocytes and monocytes were purified and total RNA isolated. Reverse transcription was performed and gene expression was carried out through qPCR using SYBR Green PCR Master Mix, and B2M, RPL13A and UBC as reference genes. Results: We observed significantly increase of cFLIP mRNA levels in CD4+ lymphocytes of HIV-exposed seronegative subjects compared to healthy controls and HIV-infected patients, whereas we found no differences in other genes expression. In monocytes, no significant differences were observed. Conclusions: Our data suggest that increase cFLIP levels may play a role in resistance to HIV-1 infection. FINANCIAL SUPPORT: CNPq.

BV204 - USING CARBON NANOTUBES AS A NANOVECTOR FOR A TETRAVALENT VACCINE CANDIDATE AGAINST DENGUE VIRUSCalegari, L.P.; Pessoa, C.R.; Monteiro, J.M. de C.; de Paula, S.O.

UFV - Universidade Federal de Viçosa, Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570-000

Dengue fever is the main disease caused by Arbovirus in the world and it is an issue of big concern in Brazil. The virus is represented by four serotypes DENV-1, DENV-2, DENV-3, DENV-4 and the control of them is based on the elimination of the mosquitos of genus Aedes, which transmit the virus. Antibodies produced in a first infection may worsen the disease in a secondary infection by a heterologous serotype, a phenomenon known as antibody-dependent enhancement (ADE)

of infection. Therefore, vaccines against dengue should be tetravalent to prevent the occurrence of this phenomenon. In our laboratory, we developed a tetravalent vaccine candidate which was made by successive insertions of the genes coding for the domain III of the E protein from each serotype of dengue virus in the expression vector pVAX1. In attempt to improve the efficacy of this vaccine candidate, in this work we used multi-wall carbon nanotubes (MWCNTs) as a nanovector to deliver the plasmid into cultured cells. For this goal, we made the conjugation of the plasmid DNA with MWCNTs using an ultrasonic sonicator bath. Then we used this solution of plasmid DNA+MWCNTs to transfect Vero cells, just adding this to the culture medium. To analyze the expression of the protein encoded by our vaccine candidate, we compared the level of expression between the plasmid mixed with MWCNTs and the plasmid pure, which was transfected to the cells by the method of calcium phosphate transfection. The analysis of the conjugation between plasmid DNA and MWCNTs was made by Raman spectroscopy, transmission electron microscopy and agarose gel electrophoresis. Protein expression was analyzed by SDS-PAGE, western blotting and immunofluorescence. The results showed that the plasmid DNA and the MWCNTs conjugated with each other, and the analysis of protein expression showed differences between the plasmid DNA transfected alone and when conjugated with MWCNTs. For the next steps we are going to test this vaccine candidate in mice and evaluate the immune response when it is administered with or without the MWCNTs. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPq.

BV209 - PRODUCTION OF INFECTIOUS RNAS FROM CDNA OF CLONES OF DENGUE VIRUS TYPE 3 (DENV-3) AND CHIMERIC CLONES DENV-3/VÍRUS ROCIOAmarilla, A.A.1; Alfonso, H.L.1; Muller, V.1; dos Santos-Junior, N.N.1; Russo, R.R.1; Soares, A.M.1; Oliveira, A.S.1; Rios, W.M.2; Silva, C.L.2; Trabuco, A.C.1; Figueiredo, L.T.2; Aquino, V.H.1

1. FCFRP/USP - Faculdade de Ciências Farmacêuticas de Ribeirão Preto/ Universidade de São Paulo, Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903

2. FMRP/USP - Faculdade de Medicina de Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900, Monte Alegre, Ribeirão Preto - SP, 14049-900




Dengue is an infectious disease caused by dengue virus (genus Flavivirus, family Flaviviridae), and transmitted by the bite of mosquitoes of the Aedes genus, mainly Aedes aegypti and this disease is an important problem of public health worldwide. In the 70s, the emergence of a new arbovirus caused a major outbreak of encephalitis in Brazil in 1975, in the Ribeira Valley, located in the southern of São Paulo state. This new arbovirus was named of Rocio virus (ROCV), which is a member of Japanese encephalitis virus seracomplex. Recently, an animal model to evaluate the pathogenesis of the disease, showed that the ROCV causes an acute inflammation in the NCS and that this virus has a neuroinvasive capacity. However, the mechanisms involved in neurovirulence and neuroinvasiveness of the ROCV have not been investigated. The objective of this work was the construction of infectious clones of DENV-3 and chimeric DENV-3/ROCV. Brazilian DENV-3 (D3BR/SL3/2002) and ROCV (SPH34675) isolates were used to prepare the infectious clones. A strategy of amplification of four DNA fragments with ends homologous to the adjacent fragments was used. Complementary DNAs (cDNAs) representing the entire viral genome were assembled using a recombination system into S. cerevisiae cells. The promoter sequence of T7 RNA polymerase was introduced in the viral cDNAs to allow in vitro transcription of the viral RNA. Restriction enzyme digestion, PCR, and nucleotide sequencing confirmed the assemblies of the cDNAs. In the chimeric clones, the E protein from D3BR/SL3/2002 isolated was replaced by E protein of the ROCV. These cDNAs were used as template to generate RNAs, which subsequently were introduced in eukaryotic cells. The viral particles have been recovered after several passages for all constructs, suggesting the infective capacity of RNA generated. This study describes the construction of cDNAs representing the entire viral genome from Brazilian isolated of DENV-3 and chimeric cDNAs between ROCV and DENV-3. These constructions could serve as an import tool for studying different aspects related to pathogenesis of the disease and could also provide a basis for future vaccines. FINANCIAL SUPPORT: FAPESP and CNPQ.

BV216 - NATURAL VARIANTS OF HUMAN PAPILLOMAVIRUS TYPE 6 EXHIBIT DIFFERENT PROMOTER ACTIVITYBonfim, C.M. do1; Sobrinho, J.S.2; Villa, L.L.2; Sichero, L.2; Rahal, P.1

1. UNESP/IBILCE - Universidade Estadual Paulista - Instituto de Biociências, Letras e Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro Jardim Nazareth, São José do Rio Preto - SP, 15054-000

2. ICESP - Instituto do Câncer do Estado de São Paulo, Av. Dr. Arnaldo, 251 - Sumaré, São Paulo - SP, 01255-000

Recurrent respiratory papillomatosis and anogenital warts are diseases associated with infection by Human papillomavirus (HPV) type 6. The promoter region of human papillomavirus, called of long control region (LCR), contains sequences important for the regulation of viral replication and transcription of early genes. This region can be used to determine HPV molecular variants whereas nucleotide variability in the LCR can be as high as 5%. Some transcription factors (TFs) have been shown to bind to the LCR and modulate viral transcription. The aim of this work was to compare the promoter activity of the HPV-6 variants and search for cellular transcription factors that could explain the differences observed. Were amplified and cloned LCR region of 5 different HPV-6 variants in the pGL3 vector upstream the luciferase gene. Transient luciferase expression experiments were performed in C33 cells. pCMV-β-Gal plasmid was used as an internal control for transfection efficiency. The TRANSFAC database was used to search for putative differences in TFs binding among the different variants. Then C33 cells were co-transfected with increasing amounts of selected TFs expression plasmids in addition to LCR-Luciferase vectors of different molecular variants of HPV-6 to analyze the impact of these proteins upon HPV-6 transcription.We observed a 10 fold enhanced promoter activity in the HPV-6vc reference variant as compared to the HPV-6a variant. A transition in nt 16 (G toA) detected in a HPV-6a related variant led to an increase in promoter activity similar to the HPV-6vc reference sequence, whereas a transversion in nt 7630 in a HPV-6vc related variant abolished the increased activity. Putative binding sites for FOXA1, ELF1 and GATA1 overlap variable nucleotide positions among molecular variants. Among these, only ELF1 superexpression was




shown not to impact upon HPV-6 transcription. CHIP experiments are being performed to analyze the binding of these TFs to the LCR. In this work, were identified TFs not previously implicated in the regulation of HPV early gene expression, and additional studies are needed since many of these transcriptional factors are mutated in cancer or are putative cancer biomarkers.

BV219 - MODULATION OF FORMYL PEPTIDE RECEPTORS EXPRESSION IN HIV INFECTIONRodrigues, C.M.1; Almeida, J.F.1; Morais, L.D. da S.1; de Sena, A.A.S.1; Borges, A.S.2; Goulart, L.R.1

1. INGEB/UFU - Instituto de Genética e Bioquímica da Universidade Federal de Uberlândia, Av. Pará, 1720, Uberlândia - MG, 38400-902

2. ICBIM/UFU - Instituto de Ciências Biomédicas da Universidade Federal de Uberlândia, Av. Pará 1720 - Bloco 2B - Sala 2B221, Uberlândia - MG, 38400-902

Human Immunodeficiency Virus (HIV) infection leads to a severe and rapid depletion of CD4+ T-cells and systemic activation, providing opportunities for secondary infections and chronic inflammation. The infection process is established with the virus entry through the interaction of the viral envelope proteins (gp120, gp41) with surface cell receptors (CD4 and CCR5) of the host. However, the Formyl Peptide Receptor 2 (FPR2/ALX) has also been described as an alternative co-receptor for HIV, a promiscuous G-protein coupled receptor that belongs to the FPR receptor family, which also includes FPR1 and FPR3 that are abundantly found in several cells of the human immune system. These receptors control several processes that support or prevent the inflammatory response according to the available ligand. Besides the specific HIV-gp120 recognition of FPR2, a recent demonstration of dimer formation among FPRs during their specific activation led us to investigate their role during HIV infection. We have focused on their expression pattern in HIV-1 positive subjects, in order to assess the involvement of all three FPRs in HIV pathogenesis. Samples of peripheral blood of 47 HIV-1 positive patients in chronic phase and 18 healthy control subjects were collected at the Clinical Hospital of Uberlandia. Total RNA samples were extracted and reverse transcription-qPCR (RT-qPCR) assay was performed using Taqman probes to quantify FPR1, FPR2/ALX and FPR3 mRNAs. The GAPDH gene was

used as endogenous control. No significant difference between patients and controls was observed for FPR1 (p=0.41). However, FPR2 had significantly lower levels in HIV-positive subjects (p<0.001), while the FPR3 was 5.3-fold higher (p<0.0001) than controls. Interestingly, the increased FPR3 expression in patients was correlated with opportunistic diseases (R = 0.31, p <0.001). We hypothesized that the FPR2/FPR3 expression disequilibrium caused by the HIV infection may have contributed to the disruption of the FPR signaling pathway, which has led to molecular mechanisms that are still unknown, but with consequences that resulted in opportunistic infections. The fine-tuning of FPRs signaling during HIV infection still needs to be thoroughly evaluated, and it may represent a key event in the HIV pathogenesis. FINANCIAL SUPPORT: FAPEMIG, CNPQ, CAPES

BV226 - SYNTHETIC NAPHTHOQUINONES ARE POTENT INHIBITORS OF DENGUE VIRUS REPLICATIONAmorim, R.1; da Silva, F.C.2; Rocha, D.R.2; Ferreira, V.F.2; Costa, L.J. da1



Dengue is a mosquito-borne viral infection caused by four serotypes of the dengue virus (DENV 1, 2, 3 and 4), a member of the Flaviviridae family. It has become a major international public health problem in recent years, as there are about 2.5 billion people living in endemic areas. Clinical manifestations of Dengue infection range from a flu-like illness to fatal cases of hemorrhagic fever. Currently, there are no effective vaccines or antiviral drugs against DENV, and there is an increasing need to develop and study molecules with potential antiviral activity against DENV. The aim of this study was to screen compounds with potential activity against DENV from a library of 30 new synthetic naphthoquinones. First, we tested Vero cells viability in the presence of different concentrations of these molecules using neutral red uptake assay. Three molecules (named 118, 143 and 150) were non-toxic at 100 µM concentration, other three (120, 127 and 128) were non-toxic at 50




µM concentration, and four (119, 122, 132 and 146 and 152) were non-toxic at 25 µM concentration. Next, we evaluated the antiviral activity of these molecules against DENV-2 in Vero cells at non-toxic concentrations. Viral titers were determined by detection of viral RNA in the supernatants of infected cells by quantitative real-time PCR using a probe and a pair of primers specific for DENV-2. From the eleven tested molecules, five (119, 120, 127, 132 and 146) presented antiviral activity against DENV-2. The molecule named 120 led to the highest inhibition of viral replication (95%), followed by 132 (86 %), 127 (83%), 119 (79%) and 146 (57%). Finally, we determined the EC50 of the molecules 127 and 132 as 12.10 µM and 8.21 µM, respectively. These results indicate that naphtoquinones are a class of molecules with potential antiviral activity against DENV-2. FINANCIAL SUPPORT: CNPQ, CAPES/PROEX, FAPERJ.

BV228 - BRAZILIAN PLANTS FROM ASTERACEAE FAMILY WITH PROMISING IN VITRO ANTIHERPES ACTIVITYBoff, L.1; Silva, I.T.1; Kratz, J.M.1; Reginatto, F.H.1; Ferraz, A. de B.F.2; Simões, C.M.O.1


2. ULBRA - Universidade Luterana do Brasil, Rua Martinho Lutero - 301 - Universitário, Cachoeira do Sul - RS, 96501-595

Herpes virus type 1 virus (HSV-1) establishes life-long latent infections in human hosts, and can be reactivated throughout life. Although most clinical infections do not progress to severe cases, immunocompromised individuals are in special danger and resistant strains have emerged, highlighting the need for new antiherpetic drugs. Natural products remain an important source of biologically active substances. The contribution of natural products and natural-derived scaffolds in the development of antiviral agents is huge, accounting for 57% of all small molecules that reached the market. In the search for antiviral agents, our group has been evaluating the antiviral activity of Brazilian plants. In this study, we evaluated the cytotoxicity and the in vitro anti-HSV-1 activity of crude extracts and fractions of Eupatorium serratum (ES) and Calea phyllolepis (CP). These two plants from the Asteraceae family were selected based

on previous antiviral and phytochemical studies carried out by our group. Cytotoxicity of the extracts was evaluated on Vero cells by MTT and sulforhodamine B assays, and the anti-HSV-1 activity (KOS strain) was investigated by plaque reduction assay. Results were expressed as 50% cytotoxic concentrations (CC50) and 50% viral replication inhibitory concentrations (IC50), respectively. At 100 µg/mL, crude extracts showed no toxic effects on Vero cells in both cytotoxicity assays, and inhibited 100% of viral replication. Fractionation of crude extracts showed the n-butanol fractions concentrated toxic constituents. On the other hand, aqueous extracts of ES and CP presented CC50 values >1,000 µg/mL, along with IC50 values of 19.1 ± 0.1 and 10.7 ± 1.5 µg/mL, respectively. The preliminary results presented in this study encouraged our group to perform further bioguided fractionation and isolation of natural inhibitors, screening against acyclovir-resistant strains and mechanism of action elucidation. These experiments are currently being conducted at our laboratory. FINANCIAL SUPPORT: CNPq and CAPES.

BV248 - REVERSE TRANSCRIPTASE INHIBITION EFFECT BY NEW AMANTADINE DERIVATIVESFranco, G.M.1; Souza, J.G.1; Fagundes, E.M.S.1; de Fatima, A.1; Martins, M.L.2; Stancioli, E.F.B.1



The Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus that infects human and presents high prevalence in some regions of the Earth. The HTLV-1 is known to cause adult T-cell leukemia/lymphoma - ATL, HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP), besides other inflammatory conditions also important. Its transmission occurs through cell-to-cell contact, being that after the entry of virus in the cell, the reverse transcriptase (RT) performs an essential step to the retrovirus replication: reverse transcription. At this moment, there is no specific treatment to be used, this being empiric and symptomatic with corticosteroids, interferon-α and anti-retrovirals used in AIDS treatment. The aim of this study is to evaluate the anti-HTLV-1 reverse transcriptase




activity of three amantadine derivatives, ADA1, ADA2 and ADA5, moiety (C10H15), for which antiviral properties had already been described to Influenza A, Rubella virus and HIV. Preliminary data are being evaluated in MT2 and C91PL cells, both permanently infected with HTLV-1. The cytotoxicity of the derivatives was evaluated by MTT assay in concentrations that ranged between 1000µM to 0.0001µM for 24h. A colorimetric assay in vitro was performed to evaluate inhibitory activity of the three derivatives, in concentrations that ranged between 10µM to 0.0001µM, against reverse transcriptase. Azidovudine (AZT) and lamivudine (3TC) activities in the concentration of 10µM were also tested. The results showed that both derivatives did not present toxicity to MT2 and C91PL cells in the tested dilutions. In the reverse transcriptase assay ADA1 and ADA2 in the concentrations of 0.0001μM presented inhibitory activity 40% and 35% higher than AZT and 17% and 12% than 3TC, respectively. ADA5 in the concentration of 10μM presented inhibitory activity 39% higher than AZT and 16% than 3TC. Despite further research would be necessary, these preliminary data demonstrated that these new amantadine derivatives could be potential antiviral candidates. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ

BV267 - EXPERIMENTAL MODEL OF INFECTION BY OROPOUCHE VIRUS IN MICEMamani, P.R.; Souza, W.M.; Badra, S.J.; Figueiredo, L.T.M.

CPG/FMRP/USP - Centro de Pesquisa em Virologia da Faculdade de Medicina de Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900

Oropouche virus (OROV) belongs to serogroup Simbu of the genus Orthobunyavirus (Bunyaviridae). This virus is transmitted to mammals and birds by mosquitoes (Aedes serratus, Culex quinquefasciatus) and midges (Culicoides paraensis) that are its primary vectors in urban cycle. OROV is the etiological agent of Oropouche fever in humans. This is an acute febrile illness that is endemic in in the Amazon region. OROV is the second arbovirus in number of reported cases in Brazil, only supplanted by dengue. We show here our preliminary results with an experimental model of infection by OROV in Swiss albinic mice (Mus musculus). OROV strain

BeAn 1999 from C6/36 (Aedes albopictus) cells; after confirmation by immunofluorescent assay, was filtered through a 0.22 μm membrane and stored at -70oC. This viral stock had 1.5 x 107 plaque forming units (PFU) per ml as observed in Vero cells. An amount of 3 x 106 PFUs of OROV, in 200 µl, was inoculated intraperitonially into 24 females of Swiss albinic mice, 3 weeks old. The animals were observed for 14 days and showed signals of disease 5 days post-inoculation. The animals presented loss of equilibrium, circular march and seizures suggesting they had encephalitis. The fatality rate of the infected animals was 50%. This experimental model will be improved to kill 100% of the animals and will be used in studies on the pathogenesis of OROV. The experimental model will also be used to test the protection to OROV infection by previous immunization with a nucleocapsid recombinant protein of this virus.

BV268 - STUDY OF THE EFFECT OF HEPATITIS C VIRUS NS2 PROTEIN IN DNA DAMAGE PATHWAYBittar, C.; Campos, G.R.F.; Rahal, P.


Hepatitis c is a worldwide health problem with an estimated incidence of the infection by hcv of 2.2%, corresponding to 130 million people in the world. it is estimated that more than 75% of the infected persons develop chronic infection that could lead to cirrhosis and in some cases hepatocellular carcinoma (hcc). the hcc is the third major cause of death by cancer in adults. at first it was believed that the great incidence of hcc in hcv infected patients was an indirect consequence of the viral infection, however now it is believed that the viral proteins have a direct role in the hepatocarcinogenesis. although some viral proteins have already shown hepatocarcinogenic potential, there are still many questions and a lot to be studied to understand the role of hepatitis c virus in cancer development. the ns2 protein is usually not studied in the context of hepatocarcinogenesis, however preliminary data indicates that it has an inhibitory potential of p53 protein which has a well known role in tumoral suppression. in this work we propose to address the effect of the viral protein ns2 in the dna damage pathway. we used the rt2




profiler pcr array – dna damage signaling pathway from sa biosciences to evaluate the expression of dna damage related genes in huh 7.0 cells, huh 7.0 cells expressing ns2 and huh 7.0 cells infected with hcv jfh-1. we analyzed the results focusing on genes which presented similar behavior both in ns2 expressing cells and virus infected cells. results show 9 genes down regulated. using string v9.1 a network of interaction between these proteins was constructed showing that they relate either directly or indirectly. further analyses are required to understand what is causing the down regulation of these genes. in order to have a more complete understanding of the effect of ns2 in this pathway we will also investigate its profile in hepg2 cells and hepg2 cell expressing ns2. FINANCIAL SUPPORT: FAPESP

BV277 - CAFFEINE EFFECT ON DIFFERENT STEPS OF HCV REPLICATION CYCLEBatista, M.N.; Carneiro, B.M.; Braga, A.C.S.; Rahal, P.


Hepatitis C is the liver inflammation caused by hepatitis C virus (HCV) infection. It often evolves to a chronic disease and has been considered the major world cause of cirrhosis and hepatocellular carcinoma. Standard treatment using PEG-IFN and ribavirin has low efficacy against some HCV genotypes. Furthermore, regular treatment has high cost and severe side-effects. Therefore, new treatments are needed. Caffeine can directly delay fibrosis, as well as improve the function of liver cellular pathways and interfere with cell pathways used by the HCV replication cycle. In the current study, the direct relationship between caffeine and viral replication cycle was evaluated. To this research, we utilized the subgenomic replicon SGR-JFH1-FEO, the full-length replicons FL-J6/JFH-5′C19Rluc2AUbi and JFH-1; and Huh-7.5 cell line. Caffeine viability was determined on Huh-7.5 cells by MTT assay. The effect of caffeine on virus replication cycle was evaluated by luciferase assay, western blotting, indirect immunofluorescence and qPCR. We observed in samples treated with caffeine a dose-dependent reduction on virus replication of subgenomic and full-length replication systems. Caffeine inhibited viral replication on different steps,

demonstrating an IC50 value of 0.7263 mM for virus replication. Caffeine inhibited SGR-JFH1-FEO replication around 82 ± 10% and HCVcc replication 79 ± 12% at its maximum safe concentration. Also caffeine reduced 30 % of viral entry. This inhibition increased two fold when particles were exposed to caffeine 1 hour before infection of host cells. This data possibly, indicates an interaction between caffeine and viral glycoproteins. On the other hand, there is no significant influence of caffeine on viral secretion process. In conclusion, caffeine inhibits HCV replication and entry/attachment on host cells in vitro and has a potential as a new antiviral therapy against HCV alone or in association with conventional drug treatment. FINANCIAL SUPPORT: FAPESP/ CAPES

BV278 - EFFECT OF CAFFEINE METABOLITES ON HCV REPLICATIONBatista, M.N.; Andrade, S.T.Q. de; Perissini, L.H.; Batista, M.N.; Carneiro, B.M.; Braga, A.C.S.; Rahal, P.


Caffeine is a phytochemical related to beneficial effects in several liver disorders. It improves abnormal liver biochemistry, cirrhosis and hepatocellular carcinoma and our group recently demonstrated this drug capacity to inhibit HCV replication on a dose-response fashion. Caffeine is metabolized to three main compounds: theobromine, theophylline and paraxantine. These molecules differ from caffeine only by loss of a methyl group in different positions. Theobromine lose methyl group at position 1; while theophylline lose the methyl group at position 3 and paraxantine lose this group at position 7. Theobromine proved to be a good antitumoral at very low concentrations and to this purpose it keeps similar effects to those observed to caffeine. Theophylline, showed beyond the antitumoral effect, inhibition of ERK and JNK phosphorylation. These pathways have already been described to be essential to HCV replication process. Thus, in this study we evaluated theophylline and theobromine ability to inhibit HCV replication. Hepatocellular carcinoma cell lineage (Huh-7.5) were cultured and stably transfected with subgenomic replicon (SGR-JFH1-FEO). Initially the cells expressing the SGR-JFH1-FEO were transferred to 96




wells plates and after 24 hours different theophylline/theobromine concentration were added: 1 µM to 5 mM on 10-fold dilution series. Cells were incubated for 24 h, 48 h and 72 h and tested for drug cytotoxicity by MTT assay. Viral RNA expression was evaluated by luciferase reporter assay. Caffeine metabolites showed more toxicity on tumoral liver cell line (Huh7.5), what is compatible with literature, and IC50 of theophylline was higher than caffeine, 1.2 mM (caffeine 0.726 mM). When analyzed on 1 mM, caffeine inhibits HCV replication around 60 %, while theophylline inhibits HCV replication around 45 %. It was not possible to analyze theobromine effect at same concentrations of caffeine or theophylline. Theobromine has low saturation point and was tested at following concentrations: 1 – 10 µM. At analyzed concentrations of theobromine, there was no effect on HCV replication. In conclusion, theophylline inhibited HCV replication around 45 % at maximum safe concentration and theobromine did not affect the virus replication at the tested concentrations. Therefore, we concluded that caffeine has a more potent effect before metabolism and this should be considered on a possible utilization of caffeine as therapy. FINANCIAL SUPPORT: FAPESP

BV285 - TIZOXANIDE INHIBITS THE REPLICATION OF DENGUE VIRUS-2 IN VERO CELLSYamamoto, K.A.1; Salles, T.S.1; de Meneses, M.D.F.2; Campos, R.M.2; Brown, D.T.3; Vancini, R.3; da Silva, M.R.S.2; Ferreira, D.F.2

1. IQ/UFRJ - Instituto de Química da Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos (antiga Av. 6), 149 Bloco A - 7° andar, Cidade Universitária, Rio de Janeiro - RJ, 21941-909

2. IMPG/UFRJ - Instituto de Microbiologia Paulo de Góes da Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - Cidade Universitária, Rio de Janeiro, Ilha do Fundão Rio de Janeiro - RJ, 21.941-590

3. NC State Biochemistry - Department of Molecular and Structural Biochemistry, 120 Broughton Drive, Raleigh, NC 27607, Estados Unidos

Dengue virus (DENV), a mosquito-borne virus (family Flaviviridae, genus Flavivirus), is a leading cause of illness in the tropics and subtropics. More than one-third of the world’s population is in areas at risk for infection, and there is no available antiviral treatment

or vaccine to cure or prevent DENV infection. Therefore, new approaches are needed to control Dengue virus infection and disease. Tizoxanide (TIZ) is the active compound of Nitazoxanide (NTZ), a thiazolide licensed for the treatment of parasitic gastroenteritis. In this study, the anti-DENV-2 activity of TIZ was evaluated in cultured cells. DENV-infected Vero cells were treated with TIZ at different concentrations. The replication of DENV in the control and TIZ-treated cells was examined by virus titration. TIZ was also administered at different time points of DENV infection to determine the stage at which TIZ affected DENV replication. TIZ significantly inhibited the replication of DENV in cell culture in a dose-dependent manner with 50% inhibitory concentration value of 3.07 ug/ml, a non-toxic concentration in Vero cells (50% cytotoxic concentration = 7.58 ug/ml). The selective index calculated was 2.5. The viral yields when the cells were treated with TIZ after the adsorption period decreased by 90% at 5 ug/ml, compared to the control. TIZ showed little (up to 20%) or none antiviral effect in other stages of viral replication (virucidal; 1, 12, 24 h pre-treatment, adsoption, penetration, virus release).Our results corroborate with those found in Shi et al (2014) study which indicated that NTZ has anti-Japanese encephalitis virus activity, acting at the early-mid stage of viral replication. These results suggest the potential application of NTZ/TIZ in the treatment of flavivirus infection. At the moment, further analyses are being analyzed in order to elucidate the mechanism of action of TIZ. FINANCIAL SUPPORT: CNPQ, CAPES, FAPERJ, INBEB, FARMOQUIMICA S.A.

BV293 - ACTION OF BACCHARIS DRACUNCULIFOLIA D.C. EXTRACT AGAINST HERPESVIRUS TYPE ICecílio, A.B.1; Dutra, A.G.S.2; Silva, F. de O.1; Caldas, S.1; Filho, A.A. da S.1

1. FUNED - Fundação Ezequiel Dias, R. Conde Pereira Carneiro, 80 - Gameleira, Belo Horizonte - MG, 30510-010

2. PUC- Pontifícia Universidade Católica

Introduction: The Herpesvirus type 1 (HHV-1) has an overall prevalence of about 90% in the human population. This prevalence can be explained by the establishment of a persistent infection in the host. The infection has become a public health problem worldwide, where besides the usual manifestations of the disease including




latency may arise numerous complications, such as eye infections. Plant extracts are commonly used in research against diseases and secondary metabolites provide defense against pathogens and may have antiviral activity. The Baccharis dracunculifolia, known as alecrim-do-campo, belongs to Asteraceae family and is considered the most important source of green propolis. According to the literature, the plant is used in folk medicine and has antioxidant, antimicrobial and cytotoxic activity. Therefore, the evaluation of the antiviral activity of B. dracunculifolia against HHV-1 indicates an alternative for treatment and prevention of this disease. Material and Methods: Cytotoxicity assays for B. dracunculifolia extract were performed and tested in interval of 48 hours. Through the tetrazolium salt colorimetric assay - MTT cytotoxicity was determined and the 50% EC50 effective concentration was determined by sigmoidal regression. The EC50 determined was 195,3μg/mL in 48 hours. From this analysis the antiviral assay without adsorption was performed, testing the concentrations of 285 to 75 μg/mL of the crude extract. A MOI = 0.1 was used in the experiment of 48 hours. The supernatant was collected, subjected to DNA extraction and Real-Time PCR. Results: The antiviral activity was detected using the concentrations of 285 a 165 μg/mL. Our tests indicate that the antiviral activity is effective in an assay without previous adsorption of the virus. Conclusions: The crude extract of B. dracunculifolia is an important approach in order to study a promising compound to treat the infection of Herpesvirus. FINANCIAL SUPPORT: FAPEMIG, FUNDAÇÃO EZEQUIEL DIAS – FUNED AND PONTIFÍCIA UNIVERSIDADE CATÓLICA DE MINAS GERAIS - PUC / MG.

BV297 - THE ROLE OF BOVINE LACTOFERRIN/HEPARAN SULPHATE INTERACTION ON HUMAN RHINOVIRUS 14 INFECTIONDenani, C.B.2; Santos, R.2; Carvalho, C.A.M.1; Roxo, T.2; Real-Hohn, A.1; Barros, C.A.2; Silva, J.L.1; Oliveira, A.C.1; Gomes, A.M.O.1; Gonçalves, R.B.2


2. UNIRIO - Universidade Federal do Estado do Rio de Janeiro, Avenida Pasteur, 296 - Urca, Rio de Janeiro - RJ, 22290-240

Rhinovirus, the causative agent of the common cold, is responsible for enormous damages to the world economy and health. Lactoferrin (Lf), a glycoprotein present in mucosal secretions of mammals, is widely studied for its antiviral activity. The mechanisms of Lf antiviral activity already described include intracellular biochemical processes, interaction with cell surface and competition for virus receptors. Our aim is to study the interaction of lactoferrin with HeLa H1 cells and how it interferes with the HRV14 infection cycle, investigating the possible interaction of Lf with molecules present at the cell surface, such as glycosaminoglycans. We observed the effect, to the cells, of Lf and NaClO3 using MTT cytotoxicity assay, and used confocal microscopy to investigate Lf ability to bind nonspecifically to heparan sulfate (HS), performing, for this, the fluorescent labeling of Lf and sulfation inhibition of cells HS with NaClO3. Time-lapse microscopy experiments showed that bLf causes morphological changes to the cellular surface. Structures similar to transitional blebs are observed immediately after bLf addition. After 30 minutes, bLf is observed bound to the cell membrane, while after 60 minutes a perinuclear distribution of bLf could be observed. Cells treated with sodium chlorate for sulfation inhibition presented less bLf binding as compared to control cells, suggesting a role for heparan sulfate during bLf binding to the cell. Our data show that bLf binds to HeLa H1 cells surface and is slowly internalized (60 min) moving to the perinuclear region of the cell. Sulfation inhibition suggests that the initial interaction of bLf is dependent on negative molecules, such as heparan sulfate. The bLf binding to glycosaminoglycans may be the key for an unspecific antiviral mechanism of bLf. The slow internalization of the molecule suggests that it may interfere with intermediate to late stages of the viral infection cycle. FINANCIAL SUPPORT: FAPERJ, CNPq, FINEP, CAPES.




BV313 - INHIBITORY ACTIVITY OF SEVERAL FUNGI AND PLANT EXTRACTS AGAINST DENGUE VIRUS TYPE-2Barbosa, E. de C.1; Francisco, F.L. de M.1; Mota, G.C.1; Alves, T.M. de A.1; Carlos, L.Z.1; Rosa, L.H.2; Calzavara-Silva, C.E.1; Kroon, E.G.2; de Oliveira, J.G.1



Dengue virus (DENV) is an important human pathogen, which causes a wide spectrum of clinical illnesses ranging from a silent or mild febrile infection, self-limited dengue fever to a severe dengue hemorrhagic fever and dengue shock syndrome. In fact, Dengue fever is the most prevalent arthropod-born viral diseases in the world. Currently, there is neither specific treatment nor vaccine available for dengue. Indeed, the development of an antiviral drug is considered a reasonable alternative for dengue treatment. In this study, a total of 2940 fungi and plant extracts obtained from the Fiocruz collection of plant and fungi extracts (COLAB) were screened for antiviral activity against Dengue virus type 2 (DENV-2). Extracts of different anatomical parts of a wide range of plants and fungi isolates were tested in vitro using BHK-21 cells in the presence of DENV. The antiviral effect of those extracts was verified by the inhibition of the cytopathic effect (CPE) caused by DENV and also by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. In addition, the potency of the extracts (EC50) that showed antiviral activity was further examined. For that, BHK-21 cells were treated simultaneously with different concentrations of those extracts (ranging from 100 to 0.78 µg/mL) in the presence of DENV-2. A total of 114 out of the 2940 extracts tested showed antiviral activity. Thirty-two out of the those 114 extracts were obtained from plants belonging to 17 distinct families: Malpighiaceae (6), Erythroxylaceae (1), Melastomataceae (2), Myrtaceae (1), Rubiaceae (1), Fabaceae (1), Clusiaceae (1), Combretaceae (1), Leguminosae (2), Lythraceae (2), Asteraceae (4), Amaryllidaceae (3), Sapindaceae (3), Ochnaceae (1), Begoniaceae (1), Annonaceae (1), Primulaceae (1). Most

of the extracts with remarkable antiviral activity were obtained from cultures of endophytic fungi, which are in the process of taxonomic identification. Eight fungal extracts presented interesting effective concentration 50 (EC50) values, at doses between 3.1 µg/mL and 12.5 µg/mL, with no cytotoxicity at 100 µg/mL. The molecular and cellular mechanisms of antiviral activity of those extracts are under investigation by our group. FINANCIAL SUPPORT BY FIOCRUZ, CPQRR, CNPQ AND FAPEMIG

BV325 - HEPATITIS B VIRUS SURFACE ANTIGEN (HBSAG) QUANTIFICATION IN PATIENTS WITH CHRONIC KIDNEY DISEASE: RELATION TO VIRAL REPLICATION AND HISTOLOGICAL FINDINGSLima, A.S.N.2; de Castro, I.R.D.2; Bernardino, J. de S.T.2; Alves, R.2; de Carvalho, I.M.V.G.1; Feldner, A.C. de C.A.3; Silva, A.E.B.3; Carvalho-Filho, J.R.3; Ferraz, M.L.C.G.3

1. Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São Paulo - SP, 05503-900

2. Universidade Federal de São Paulo, Laboratório de Hepatologia Molecular Aplicada, Departamento de Gastroenterologia

3. Universidade Federal de São Paulo, Departamento de Gastroenterologia

Patients with chronic kidney disease (CKD) on hemodialysis are at higher risk of HBV infection and at increased risk of developing liver cirrhosis, especially after undergoing kidney transplantation (KT). Quantification of HBsAg serum levels (qHBsAg) can help differentiate between HBV inactive carriers and patients with HBeAg-negative chronic hepatitis B, as well as in identifying patients with a lower probability of response to antiviral therapy with interferon. The aim of this study was to evaluate potential correlations between qHBsAg, serum HBV DNA levels and histological findings in patients with CKD. Three groups of chronic HBV carriers were included: Group 1 – Normal renal function (controls); Group 2 – CKD patients under hemodialysis; and Group 3 – KT recipients. Serum qHBsAg and HBV DNA levels were quantified using the ARCHITECT HBsAg Quantitative and the Abbott RealTime HBV assays, respectively. HBV genotyping was performed by sequencing and phylogenetic analysis. Liver histology was assessed using the METAVIR scoring system. A total of 104 patients was included as follows: Group 1: n=50,




2: n=21; and 3: n=33. Mean age was 42.0±12.2 years, with 72% males. Significant liver fibrosis (F2/F3/F4) was observed in 46% of the patients. Median serum ALT level was 1.4 times the upper limit of normal. Fifty-two patients (50%) were genotyped, with genotypes D and A being the most prevalent (56% and 33%, respectively). HBV viral load was obtained for 92 patients (88%), with higher levels found in Group 3 (median of 8.90 log IU/mL; p<0.001). qHBsAg was available for 61 subjects (59%), with a mean of 4.0±0.7 log IU/mL. Mean qHBsAg levels were similar among groups (4.02±0.71, 4.34±0.41, and 3.80±0.90 log IU/mL for Groups 1, 2, and 3; p=0.231). HBeAg-positive patients exhibited higher levels of qHBsAg as compared to HBeAg-negative subjects (median 4.41 vs. 3.49 log IU/mL; p=0.020). Comparable levels of qHBsAg were observed in subjects with mild and significant liver fibrosis (F0/F1: 4.04±0.80 log IU/mL; F2/F3/F4: 3.94±0.78 log IU/mL; p=0.644). No significant correlation was seen between the qHBsAg and HBV viral load (rs=-0.061; p=0.658). This analysis confirms the association between KT and higher HBV viral load. qHBsAg levels seem to be influenced neither by renal function status, nor by liver fibrosis stage, but are higher in HBe-positive patients. Up to now, the correlation between qHBsAg and HBV DNA levels could not be confirmed in CKD subjects. FINANCIAL SUPPORT: 2012/11081-9 AND CAPES

BV331 - EFFECT OF Β LAPACHONE AND PLOCAMIUM BRASILIENSES MONOTERPENE ON HERPES VIRUS SIMPLES TYPE 1 (HSV-1)Souza, K.F.C.S.1; Carvalho, D.G.1; Brito, V. de L.3; Lima, G.M.3; Silva, T.C.3; Garcia, D.G.2; Martins, D. de L.1; Teixeira, V.L.1; Paixão, I.C.N. de P.1; Burth, P.1



3. IEPIC - Instituto de Educação Professor Ismael Coutinho, Travessa Manoel Continentino, 32, São Domingos, Niterói - RJ, 24210-150

Infection with herpes simplex type-1 virus (HSV-1) can cause various diseases such as mucocutaneous infections, encephalitis in immunocompromised patients and neonates, as well as genital infections and keratitis, being

one of the main cases of blindness in underdeveloped countries. Although there is little information about the early stages of infection, it is known that transmission occurs by direct contact with skin or mucosa with some type of abrasion, when in contact of person presenting virus secretion. The most used compound for the tratament of such infections is acyclovir. However, the emergence of HSV-1 strains resistant to antiviral drugs as acyclovir, makes the search for new molecules, especially with different mechanisms of action, a constant urgency. The aim of this study was to evaluate the possible antiviral effect, of synthetic substance β lapachone and monoterpene fraction extracted from algae Plocamium brasilienses on Vero cells infected with HSV-1 virus, checking the cytotoxicity and the virucidal effect. Results obtained in plaque reduction assay showed that synthetic substances β lapachone AN6 and 39-42 monoterpene fraction obtained from Plocamium brasilienses at a concentration of 50μM were able to significantly reduce viral plaque forming units. The β lapachone was not cytotoxic at any concentration used (50μM and 500 μM) while the fraction 39-42 Plocamium brasilienses showed high cytotoxic effect at concentrations above 100μM, but at 50μM concentration values approached the one found for control cells. The virucidal analysis demonstrated that β lapachone substance AN6 and fraction 39-42 of Plocamium brasiliensis were able to prevent viral penetration into Vero cells. Other experiments are underway to determine in which step of the viral replicative cycle the tested substances are interfering. FINANCIAL SUPPORT: CAPES; CNPQ; FAPERJ; PROPI-UFF

BV352 - ANTIVIRAL ACTIVITY OF SYNTHETIC PEPTIDES AGAINST HSV-1 AND AICHI VIRUSSilva, P.A.; Boas, L.V.; Migliolo, L.; Mendes, G.; de Lima, L.M.P.; Franco, O.L.

UCB - Universidade Católica de Brasília, W5 Norte, Brasília - DF, 70790-160

Viral infections affect all living organisms. For control and prevention of such infections agents, public health and vaccination programs have been developed and the antiviral appear as a valuable alternative for virus treatment. In this context, antiviral peptides have been characterized with different mechanisms of action that are specific for each viral type. However, in recent




years, studies on antimicrobial peptides have reported an additional antiviral against both enveloped and non-enveloped viruses, making them promise candidates for antiviral drugs.OBJECTIVES: The present study aimed to evaluate the antiviral activity of synthetics peptides, Pa-MAP 1 and variants, clavanin MO, Cn-AMP1, and LL-37 against human herpes virus 1 and Aichi virus.MATERIALS AND METHODS: The peptides were synthetized by the F-MOC stepwise solid-phase method, further purified by using C-18 reversed-phase HPLC, and molecular masses were determined by using mass spectrometry MALDI-ToF/ToF Ultraflex III. The cytotoxic effect of the peptide in Vero cells was evaluated by MTT method and antiviral activity was determined by reduction of the virus titers. RESULTS: The peptides clavanin MO, Pa-MAP 1.8 and Pa-MAP18br showed a higher cytotoxicity at concentrations over 12 µM. Moreover antiviral assays, showed that the peptide Pa-MAP 1 caused 90% of inhibition of HSV-1, in a virucidal manner, with a SI higher than 4,8, and 0% of Aichi virus. LL-37 showed 90 % of inhibition of Aichi virus replication with a SI of 3,4. Other peptides did not showed any antivirus effects. CONCLUSIONS: This study was the first report of an antiviral peptide with potential for Aichi virus treatment. In conclusion, the data suggest that Pa-MAP 1 could be utilized as a potent candidate for virus infections control.

BV362 - STRUCTUTRE AND FUNCTION OF THE RHESUS CYTOMEGALOVIRUS RH137 PROTEINSantos, E.S.; Sperança, M.A.; da Silva, M.C.C.

UFABC - Universidade Federal do ABC, Rua Catequese, 242 - Jardim, Santo André - SP, 09090-400

Cytomegaloviruses (CMV) are ubiquitous members of Betaherpesvirinae sub-family, associated with serious diseases in immunocomprised hosts. Due to the strict specie-specificity, the studies of CMV biology and pathogenicity in vivo are performed in animals, using the CMV specific of the target specie. Rhesus macaques (Macaca mulatta), susceptible to infection with Rhesus CMV (RhCMV), are considered the ideal research model for these studies, becuase their close relationship to humans. Studies of the functional characterization of viral proteins are important for the understanding of viral replication and pathogenesis in vivo. We have previously demonstrated using, a HCMV Bacterial Artificial Chromosome (BAC) system, that the Human

Cytomegalovirus pp28 tegument protein, encoded by the UL99 gene, is essential for viral replication and functions during the final acquisition of the viral envelope in the cytoplasm of infected cells. In this work we aim to determine the function of the RhCMV137 protein, the RhCMV pp28 homologue. In order to generate a RhCMV lacking the RhCMV137 gene, its flanking regions were cloned into the PGS284 shuttle vector, which will be used for homologous recombination and allelic exchange in bacteria, creating a RhCMV with substitution of the RhCMV137 for the kanamycin resistance gene. In addition, we aim to perform structural and biophysical studies of pp28 and RhCMV137 proteins, to better understand their biochemical roles and to study their structure–function relationship. For this, the RhCMV137 and UL99 genes were cloned into the pET28a expression vector and experiments of protein expression are currently underway. The studies of the RhCMV137 protein will be important for determination of its function as compared with the pp28 protein, and for validation of the RhCMV as an animal model for CMV studies. In addition, the RhCMV137 defective mutant could also be tested as a vaccine against CMV in Rhesus monkeys. FINANCIAL SUPPORT:FAPESP, CAPES.

BV392 - EVALUATION OF APOPTOSIS INDUCED BY OROPOUCHE VIRUS NSS PROTEINOliveira, A.S.; Acrani, G.O.; Criado, M.F.; Silva, M.L.; Arruda, E.

FMRP/USP - Faculdade de Medicina de Ribeirão Preto/ Universidade de São Paulo, Av. Bandeirantes, 3900 - Monte Alegre, Ribeirão Preto - SP, 14049-900

Oropouche virus (OROV) is the second most common arboviral disease in Brazil and a major cause of febrile infections in South and Central America. OROV belongs to the Bunyaviridae family, genus Orthobunyavirus. OROV genome is a tri-segmented single-stranded negative sense RNA, composed of large (L), medium (M) and small (S) segments. The L segment encodes the polymerase; the M segment encodes a poly-protein whose cleavage products are the surface glycoproteins (Gn and Gc) and nonstructural protein (NSm). The S segment encodes the nucleocapsid structural protein (N) and the non-structural protein NSs. It has been previously shown that the non-structural proteins (NS) of other Bunyaviruses are involved in metabolic




processes such as apoptosis and synthesis of type I IFN. We have previously demonstrated that OROV induces apoptosis of HeLa cells and this requires the synthesis of viral proteins. The pathogenesis of OROV infection is not completely understood, but apoptosis seems to play a central role. Based on that, this study was done to evaluate the occurrence of apoptosis induced by OROV infection, and by NSs transfection in HeLa, Glioblastoma and HCE (human corneal epithelium) cell lineages. Semi-confluent monolayers were seeded in the 24-well plates containing coverslips and maintained respectively with MEM, DMEM and SHEM, and then infected with OROV or transfected with plasmid containing the NSs gene (pcDNA3.2- NSs), or with empty vector (pcDNA3.2) as a negative control. Subsequently, the plates were incubated at 37°C, 5% of CO2 for 24 or 36 hours. Then monolayers were fixed with paraformaldehyde and subjected to immunofluorescence (IF) with polyclonal antibody direct to OROV and TUNEL assay for DNA fragmentation. The IF confirmed the infection and transfection by the NSs protein, and also intense progressive labeling of fragmented DNA characteristic of apoptosis was noted at the times of 24 and 36 h pi. These results demonstrate that OROV NSs protein is pro-apoptotic for different tumor cell lines, and may become an important experimental tool for that purpose.

BV395 - HUMAN RESPIRATORY SYNCYTIAL VIRUS (HRSV) EXPERIMENTAL INFECTION IN NEONATE BALB/C MICEde Jesus, B.L.S.; Criado, M.F.; de Andrade, M.A.A.M.; de Sousa, E.; Arruda, E.


HRSV is the single most important viral cause of lower respiratory tract infections and re-infections in children worldwide. Passive immunoprophylaxis reduces hospitalizations, but vaccines or effective therapies are not available. The neonatal mouse model of HRSV infection, which most closely mimics human infants, offers a possibility to study pathogenesis of HRSV-induced airway disease, mechanisms of HRSV counteraction of the host immunity, and for preclinical testing of interventions. We have successfully established an experimental HRSV neonate BALB/c mice model in

order to study acute and persistent HRSV infection. Neonate BALB/c mice (10 day-old), were infected intranasally with 104 PFU of HRSV A long strain (ATCC). On days 3, 7, 14, 30, 60 and 90 post infection (pi), animals were euthanized with ketamine and xylazine and tissues from the respiratory tract (nostrils/trachea and lungs), liver, spleen and mesenteric lymph nodes were harvested for quantitation of viral RNA by real-time RT-PCR, and by plaque assay. qPCR showed that HRSV RNA was detected both in lungs and lymph nodes on day 7 pi, but remained detectable in lymph nodes on days 30, 60 and 90 pi. HRSV RNA was detectable in nostrils/trachea on day 90 pi. Remarkably, viable HRSV was recovered in mesenteric lymph nodes on days 7, 30 and 60 pi. This study successfully established an experimental model for HRSV intranasal infection, with additional important evidence that the virus remains viable in mice for prolonged periods of time. FINANCIAL SUPPORT: FAPESP, CNPq, CAPES

BV404 - HCMV INDUCED CHEMOTERAPIC RESISTENCE IN MRC-5 AND U251 CELLSSantos, C.J.; Souza, A.C.S.; Silva, M.C.C.


Human Cytomegalovirus (HCMV) is a ubiquitous virus that leads to lifelong persistent infection and can cause serious diseases in immunocompromised individuals. In the past years accumulating evidences suggest an association between HCMV infection and cancer. The virus has been detected in many types of cancers, especially in glioblastoma multiforme (GBM), the most prevalent and malignant tumor of the central nervous system. Although there is no direct confirmation that HCMV can transform cells, previously studies suggest that it might increase tumor malignancy through the action of viral proteins that disrupt cellular pathways involved in the cell cycle, apoptosis, angiogenesis, cell invasion and cellular immune response. Interestingly, other studies also demonstrated that the virus is capable of induce degradation of nuclear complexes, leading to a increase of cell resistance to chemoterapy. In this study, we analyzed the effect of HCMV infection in cell resistance to the chemotherapic agent Temozolamide (TMZ) by MTT assay. Human lung fibroblasts (MRC-5) and human glioblastoma cell line (U251) were treated




with TMZ (100 µm) in presence and absence of virus at multiplicity of infection 1 and 10, respectively (M.O.I. 1 and 10). The results demonstrated that at 120 hours post treatment TMZ reduced the amount of cells in both cell lines, as compared to untreated cells. The viral presence itself also reduces the cellular amount in both cell lines. The presence of HCMV in treated cells increases cell survival in approximately 50% in MRC-5 and 40% in U251 cells when comparing with no infected cells. The data reinforce a possible association between HCMV infection and tumor resistance to chemotherapic treatment. Further experiments are been conducted in other glioblastoma tumor cell lines. In addition retroviruses expressing individual HCMV proteins were constructed in order to test the effect of single proteins in drug resistance. FINANCIAL SUPPORT: FAPESP/CAPES.

BV405 - THE ANTI-HIV-1 REVERSE TRANSCRIPTASE ACTIVITY OF THE GREEN ALGAE PRASIOLA CRISPAAmorim, L. dos S.C.1,3; Ribeiro, M. de S.R.2; Brito, C.J.B.2; Stephens, P.R.S.1,3; Oliveira, R.G. de O.1; Lyra, J.S.P.O.2,3; Teixeira, V.L.1; Paixão, I.C.N. de P.1; Pereira, H. de S.P.1




Since the human immunodeficiency virus (HIV) has been established as the etiological agent of the AIDS, an appreciable number of drugs have been developed and approved for the treatment of the HIV-infected patients. Viral enzymes and particularly the Reverse Transcriptase (RT), which is an essential catalytic activity in the retrovirus replication cycle, have been chosen as the target for drug development. Despite of the remarkable pharmacological profiles of the classical nucleoside and non-nucleoside reverse transcriptase inhibitors, virus mutations are induced by antiretroviral therapy and drug-resistance is usually reported. In this work the anti-HIV-1 RT activity of the hexane and dichloromethane-methanolic fractions of the extracts of the green algae Prasiola crispa were investigated by using the Molecular Probe EnzChek® Reverse

Transcriptase Assay Kit. Furthermore, the cytotoxicity of both extracts were evaluated on human MT2 cells. The IC50 (the concentration of a drug that is required for 50% inhibition) and the CC50 (the concentration of a drug that is required for 50% of the cytotoxic effect) values were estimated by using Sigma Plot program. Our results show that both the hexane and the dichloromethane-methanolic extracts of P. crispa reduced the HIV-1 RT catalytic activity in a dose-dependent manner. The IC50 values obtained for both hexane fractions, F14 and F15, were similar (50ug/mL), while de the IC50 for the F16 fraction was 70ug/mL. The dicloromethane fraction F11 showed an IC50 value of 25ug/mL. The CC50 values obtained for both hexane fractions, F14 and F16, as well as the dicloromethane F11 fractions were similar, 0,8mg/mL, while the hexane F15 fraction was considered to be toxic for the MT2 cells. Conclusion: the hexane and the dichloromethane fractions of the algae P. crispa reduced the HIV-1 reverse transcriptase activity with low cytotoxic effect indicating a promising role for these algae extracts in the AIDS-therapy research. FINANCIAL SUPPORT: Capes, CNPq, FOPESQ-UFF and FAPERJ

BV408 - THE ANTI-HIV ACTIVITY AND LOW CYTOTOXICITY OF OXOQUINOLINE DERIVATIVES ON MT-2 CELLAmorim, L. dos S.C.1,3; Stephens, P.R.S.1; Lyra, J.S.P.O.2,3; Pereira, H. de S.P.1; Ribeiro, M. de S.R.1; Neurauter, A.L. de A.3; de Souza, M.C.B.V.1; Sangrillo, F.S.S.1; Cunha, A.C.C.1; Ferreira, V.F.F.1; Paixão, I.C.N. de P.1




According to UNAIDS 2013, there were more than 34 million people living with HIV worldwide. Brazil has a population of approximately 198 million inhabitants and (from 1980 to 2013) more than 480.000 AIDS cases were diagnosed, with more than 35.000 new cases per year. Currently, there are no effective HIV/AIDS vaccine or cure, although the introductions of antiretroviral drugs significantly improve the prognosis of infected individuals with access to treatment. However, the




emergence of drug-resistant viral strains is on the increasing. Therefore numerous studies have been developed, such as preventive strategies in order to find some low-toxicity and low-cost anti-HIV substances. The aim of this study was to evaluate the cytotoxicity and reverse transcriptase (RT) activity of oxoquinoline derivatives on MT-2 cells infected with HIV-1. The cells in 96-multiwell plates were treated with various concentrations of the compounds for 72 h. Then, the solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma)was added to evaluate cell viability. The 50% cytotoxic concentration (CC50) was calculated by linear regression analysis of the dose response curves and measurement of ELISA p24 antigen in supernatants from MT-2 cell lines treated with oxoquinoline derivatives and infected by HIV-1. The anti-HIV-1 RT activity of oxoquinoline derivatives were investigated by using the Molecular Probe EnzChek® Reverse Transcriptase Assay Kit. The oxoquinoline derivatives studied by our group have important effects on HIV replication, as we have observed more than 60% of RT viral inhibition. Cytotoxicity levels lower than 30% were observed on substances. Further pre-clinical studies are needed to better evaluate these substances as potential candidates for microbicides or sistemic drugs. FINANCIAL SUPPORT: FIOCRUZ, CNPq, FAPERJ, CAPES, FOPESQ-UFF-PROPPI.

BV419 - INTRACELLULAR LOCALIZATION PATTERN DURING HUMAN RESPIRATORY SYNCYTIAL VIRUS (HRSV) INFECTION IN A549 CELLSCardoso, R.S.; Criado, M.F.; Silva, M.L.; da Silva, L.L.P.; Neto, E.A.

USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070

Human Respiratory Syncytial Virus (HRSV) is the single most important respiratory viral pathogen for children worldwide. Nearly 95% of kids have been infected by HRSV before 5 years of age. Despite its importance, little is known about cell biology of HRSV infection in respiratory cells. We performed indirect immunofluorescence (IF) analysis of A549 cells infected with HRSV A (strain long) at different time points post infection, using antibodies for HRSV and for protein markers of cellular organelles. Sub-confluent monolayers of A549 cells grown in 24-well plates were infected with HRSV (MOI=1), absorbed

at 4°C for 1 h, and fixed with 4% paraformaldehyde at 0, 4, 8, 12, and 24 h post infection (pi). Coverslips were then stained by IF and analyzed by confocal microscopy. Primary antibodies used for IF were directed to plasma membrane (transferrin receptor TrF), endoplasmatic reticulum (ER, calnexin alpha - CNX2), trans-Golgi network (giantin), early and late endossomes (SNX2 and CD63, respectively) and HRSV. At early times pi HRSV proteins appeared to concentrate near the nucleus, starting heavy accumulation at 4 h pi, in a region suggestive of ER and Golgi. Thereof, HRSV proteins homogeneously disperse over the cytoplasm, losing the early pattern of concentration in well-defined granules, and at 24 pi they tend to be localized at the cell periphery, consistent with virus budding from the plasma membrane. There was no significant co-localization of HRSV structural proteins with Golgi or ER all through the 24-h period. Importantly, there was striking disorganization and dispersion of giantin and CNX2 over time, indicating loss of integrity of ER and Golgi, likely due to HRSV inflicted cell damage. This study points to important targets of cell damage in HRSV infected cells, underscoring the need for experiments to assess the mechanisms for cell damage, including ER stress. FINANCIAL SUPPORT: CAPES and FAPESP

BV422 - BIOINFORMATICS ANALYSIS OF THE PRO-APOPTOTIC NSS PROTEIN OF OROPOCHEVIRUSSouza, M.M.; Oliveira, A.S.; Acrani, G.O.; Arruda, E.


Oropouche virus (OROV), of the family Bunyaviridae, is the second most common cause of arboviralfebrile illness in Brazil, but its pathogenesis is practically unknown. OROV causes apoptosis of infected cells, both in vitro and in vivo. Previous results have shown that the non-structural protein NSs of OROV induces apoptosis in eukaryotic cells. A bioinformatics analysis of NSs was done in order to predict structural and physico-chemical parameters of the protein, which can help to guide experiments, and to understand how its relationship to apoptosis happen. The physicochemical parameters were analyzed by the Protparam protocols: molecular weight: 10.6 kDa; theoretical pI: 10.7;amino acid composition: 4 cysteine residues, 4 negatively charged and 12 positively charged residues; extinction coefficient:




1.829 assuming all Cys residues are reduced; estimated half-life: 30 hours for mamilian cells; instability index: 66.49,indicating an unstable protein; hydropathicity (GRAVY): -0.232,indicating NSs to be hydrophilic). Secondary structure prectiction by Jpred showed seven beta strands. BLASTn search has not indentified proteins with sufficient similarity to be helpful as template for molecular homology modeling. However, 3 structurally similar proteins could be identified: Williopsismakrii killer toxin (which matches 42% of NSs sequence with 23% identity), and two human methylesterases (matching 36% of NSs sequence with 36% identity).Ab initio modelling was done by QUARK,validated using PROCHECK, and visualized by PyMol. After validation,the modeling confirmed presence of two beta sheets,one with 3 and other with 4 beta strands, and showed disulfide bonds in coil regions nearthe C-terminus. A search by Dali identified similarity with streptavidin. These analyses help guiding experiments to structural targets of NSs in host cell metabolic pathways,in order to make advances in the understanding of its function. FINANCIAL SUPPORT: CNPQ AND FAPESP

BV445 - STUDY OF THE ORGANIZATION AND STABILITY OF THE LIPID ENVELOPES OF MAYARO VIRUS PARTICLES PURIFIED FROM VERTEBRATE AND INVERTEBRATE CELLSFerreira, V.N. dos S.; Carvalho, C.A.M.; Ferreira, D.F. Silva, J.L.; Gomes, A.M. de O.


Mayaro virus (MAYV) is an enveloped alphavirus that belongs to the Togaviridae family. MAYV has a genome composed of a positive-sense ssRNA molecule measuring nearly 12 kb. The replication cycle of the virus involves vertebrates and invertebrates. Cholesterol is an important lipid component in animal cells and is primarily responsible for dynamic and structural maintenance of cell membranes, and forms, along with sphingomyelin, organized regions of membrane known as “lipid rafts”. Studies have suggested the involvement of cholesterol and lipid rafts at different moments of the replication cycle of several enveloped viruses. However, it is interesting to note that arboviruses have an alternate cycle that involves infection of mammals

and insects, organisms which are quite different for the presence of cholesterol. In both, infection is productive and leads to the formation of equally infectious viral particles. This work aims to evaluate the thermostability of the envelope of MAYV particles originated from mammalian and mosquito cells in order to evaluate the structural characteristics of these virus particles. To achieve that, virus particles purified from BHK-21 (baby hamster kidney) and C6/36 (Aedes albopictus larvae) cells were labeled with the fluorescent probe laurdan and analyzed by fluorescence spectroscopy. The results show that particles obtained from BHK-21 and C6/36 cells have similar membrane organization, despite the difference in their lipid compositions, suggesting that cholesterol may not be the only determining factor for the maintenance of the organizaton of the virus envelope. The effect of high temperature on the virus morphology was analyzed by light scattering and viral infectivity was measured by plaque assay. Both particles were able to retain infectivity even when exposed to temperatures of 45 and 60 °C, suggesting a high thermostability for MAYV derived from BHK-21 or C6/36 cells. FINANCIAL SUPPORT: FAPERJ, CNPQ, CAPES, FINEP, UFRJ, INBEB

BV450 - EVALUATION OF THE ANTIVIRAL POTENTIAL OF PLANT EXTRACTS ANNONA MURICATA, CAESALPINIA ECHINATA AND SPONDIAS MOMBIN OF NORTHEAST BRAZIL AGAINST DENGUE-2 VIRUS IN C6/36 AND VERO CELLSFarias, K.J.S.; Machado, P.R.L.; de Almeida Jr, R.F.; Lima, T.L.C.; Nascimento, Y.M.; de Araújo, J.M.G.

UFRN - Universidade Federal do Rio Grande do Norte, Campus Universitário Lagoa Nova, Natal - RN, 59078-970

The dengue virus is the most prevalent arthropod-borne virus causing morbidity and mortality in tropical and subtropical regions of the planet. As it is a disease with no vaccine or effective treatment, it is important to study the antiviral potential of medicinal plants to reduce the patient’s viremia, one of the factors that may cause the severe form of the disease. This study examined the antiviral potential of three plant extracts from Brazilian Northeast: Caesalpinia echinata, Annona muricata and Spondias mombin, against dengue-2 virus (DENV-2) in C6/36 and Vero cell cultures. The stock solutions were 68 mg/mL for C. echinata, 100 mg/mL for A. muricata and 100 mg/mL for S. mombin. In the cytotoxicity assay,




different concentrations of each extract were prepared from stock solutions and tested in Vero and C6/36 cells to determine the ideal concentration to experiments. Those cells were seeded in 96-well plates and after incubation periods of 1, 6, 12, 24, 48, 72 and 96 hours of contact with the extracts, cell viability was evaluated by MTT technique and the method of exclusion of Trypan blue 0.4%. Therefore, the concentration which was taken to be used in the assay of antiviral activity was 0.68 mg/mL, 1 mg/ml and 1mg/mL for C. echinata, A. muricata and S. mombin, respectively. In the assay of antiviral activity monolayers of Vero and C6/36 cells seeded onto 24-well plates were infected with DENV-2 at a multiplicity of infection (MOI) of 1. At 1h post-infection the inoculum was removed. Vero and C6/36 cells were incubated in the presence of defined concentrations of the extracts as described above at defined time intervals, in different experiments: (i) 1 h after infection, and at 24-hour intervals for 7 days; (ii) 1 hour after infection and at 12-hour intervals for 7 days. Infected cell supernatants were collected at 24, 48, 72, 96, 120, 144, and 168 hours post-infection, cleared by centrifugation for the quantification of DENV-2 RNA copies by Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) and plaque assays (PFU). Our results demonstrated that A. muricata induced a significant reduction in viral yield in Vero cell cultures at 24-hour and 12-hour intervals when compared to untreated cells, but not in C6/36 cells; contrary to the other two extracts, which did not show a reduction in viral yield. Based on these data, we conclude that A. muricata has an inhibitory effect against DENV-2 virus in Vero cells culture.

BV460 - EVALUATION OF POTENTIAL ANTIVIRAL ACTIVITY OF RED PROPOLIS AGAINST DENGUE VIRUS

Veras, R.M. de A.; Machado, D.; Rocha, M.A.L. Goes, T.; Sales, C. de B.P.M.; Queiroz, A.; Moura, F. de B.P.; Moreira, M.S.A.; Borges, A.A.; Muller, V.D.M.

UFAL - Universidade Federal de Alagoas, Av. Lourival Melo Mota, Ciudad Universitaria - AL, 57072-900

Dengue fever, the most important arthropod-borne viral disease, is caused by dengue viruses belonging to the Flavivirus genus, Flaviviridae family, and are classified into four antigenically related serotypes (DENV-1 to

DENV-4). DENV infection can be asymptomatic or a self-limited, acute febrile disease ranging in severity. It is estimate that there are 390 million dengue infections each year, of which 96 million manifest apparently. In 2013 were reported 4819 cases in the State of Alagoas, Brazil. At the present time, no specific intervention against the virus exists, the sole measure of control is limiting the Aedes mosquito vectors. Therefore, there is a great need to develop new compounds for the treatment of patients infected with DENV. Propolis is a resinous mixture collected by honey bees from plant sources. The composition of the propolis depends on the season, the vegetation, and the area of collection. The red propolis is founded in Brazil northeast and presents a diverse biological activities such as antimicrobial, antioxidant and antitumoral. The aim of this work was to investigate antiviral activity of the red propolis etanolic extract against dengue virus type-2 (DENV-2). Concentrations of red propolis, ranging from 200 to 1,56 µg/mL, were evaluated for cytotoxicity by MTT assay in VERO E6 cells. The effect of red propolis over DENV-2 (NGC strain) was evaluated by replication inhibition, which was measured by MTT assay using two different methodological strategies: pre-treatment and pos-treatment. The results were expressed as 50% cytotoxicity (CC50) and 50% effective (EC50) concentrations, respectively, in order to calculate the selectivity indices (SI=CC50/EC50). The etanolic extract of red propolis showed a CC50=164,22 µg/mL. In the pre-treatment 10% of viral replication was inhibited by 25 µg/mL of red propolis. In the post-treatment, the concentration that inhibit 50% of viral replication (EC50) was 38,67 µg/mL, and the selectivity index (SI) was 4,24. The results showed that red propolis have a promising antiviral activity in the post-treatment. More studies should be carried out to investigate their mechanism of action and the action against others serotipes of dengue vírus. FINANCIAL SUPPORT: The authors are grateful to the CAPES (Fellowship VDMM), FAPEAL (PPSUS: 60030 000740/2013, Pronem 20110722-006-0018-0010), MCT, FINEP, CNPQ (479822/2013-1 e 404344/2012-7), INCT-INOFAR/CNPq (573.564/2008-6),Ministerio da Saude/CNPQ/SESAU AlCAPES




BV463 - EVALUATION OF ANTIVIRAL ACTIVITY OF COPAIBA OIL AGAINST DENGUE VIRUS TYPE 2Melo, D.M.; Veras, R.M. de A.; Rocha, M.A.L.; Goes, T.; Muller, V.D.M.; Borges, A.A.

UFAL - Universidade Federal de Alagoas, Av. Lourival Melo Mota, Ciudad Universitaria - AL, 57072-900

Dengue viruses (DENVs) cause the most common arthropod-borne viral disease. Repeated reemergence of dengue in sudden explosive epidemics often cause public alarm and seriously stress healthcare systems. It is estimate that there are 390 million dengue infections each year, of which 96 million manifests apparently. The dengue severity is divided into dengue without warning signs, dengue with warning signs, and severe dengue. Dengue virus belongs to the Flaviviridae family, genus Flavivirus and is classified into four antigenically related serotypes (DENV-1 to DENV-4). Currently there are no licensed vaccines or specific therapeutics, and substantial vector control efforts have not stopped its rapid emergence and global spread. Copaiba oil, is extracted from the trunks of Copaifera trees species. It has been reported for anti-inflammatory activity, analgesic, antimicrobial, antifungal, antileishmanial, antiproliferative, antimutagenic, and others. However, antiviral properties have not been studied yet. The aim of this work was to investigate the antiviral activity of copaiba oil against dengue virus type-2 (DENV-2). To evaluate the cytotoxicity by MTT assay in VERO E6 cells, the monolayer was treated with concentrations ranging from 1,000 to 1.56 µg/mL. The effect of copaiba oil over DENV-2 (NGC strain) was evaluated by replication inhibition, which was measured by MTT assay using two different methodological strategies: pre-treatment and post-treatment. The results were expressed as 50% cytotoxicity (CC50) and 50% effective concentrations (EC50), in order to calculate the selectivity indices (SI=CC50/EC50). The copaiba oil did not show CC50 to VERO E6 cell monolayers up to a concentration of 1,000 µg/mL. When the monolayer was treated with copaiba oil after the viral infection (post-treatment), there was a high inhibition of viral replication, and the EC50 obtained was 19.4, and the selectivity index was >51.5. When the cells monolayer was treated with 25 µg/mL before viral infection (pre-treatment), it was possible to inhibit 43% of viral infection. These results showed that the copaíba oil have a promising antiviral effect against DENV-2.

Further experiments are been carried out to completely evaluate the antiviral activity and to investigate which compounds are involved in the detected anti dengue activity. FINANCIAL SUPPORT: CNPq/ FAPEAL/ CAPES

BV464 - VPG AMINO ACID SEQUENCE CONSERVATION AMONG DIFFERENT HUMAN RHINOVIRUS SPECIESWatanabe, A.S.A.; Moreira, L.; de Oliveira, P.S.L.; Granato, C.; Bellei, N.


Human rhinovirus (HRV) infections are the most frequent cause of common cold and accounting for approximately 50% of all upper respiratory tract infections described. Currently the HRVs are classified in 3 distinct species: HRV-A, B and C, with A and C species clinically associated with more severe disease. Several studies have demonstrated that host factors, as age, immune status and severe clinical outcome are not associated with HRV specific species. A better understand of HRV molecular epidemiology raised new concerns about the mechanisms involved in pathogenesis of the different species. In this scenario the importance of viral physiology is evident. The HRV replication mechanism is initiated through a small genome binding protein called VPg (Viral Protein of the genome). HRV replication is initiated by VPg uridylylation. The VPg serves as a primer for initiation of intermediate RNA negative strand synthesis, which will subsequently be transformed into a genomic positive RNA strand. Several studies have demonstrated that VPg plays a critical role in HRV replication. Structural evaluation of VPg, and its role in the different species replication could clarify the infections dynamics and clinical diversity presented by patients. The first aim of the pilot study was assessed the amino acid sequence conservation among the different HRV species, and the second objective was to perform the prediction of the VPg structure. Two hundred and fourteen VPg sequences were obtained from GeneBank, with the following distribution: HRV A – 131 sequences, HRV B – 49 sequences and HRV C – 34 sequences. The analysis of sequences conservation was performed using the Jalview 2.8.1 software. Molecular modeling of the HRV VPg structure was performed using the Yasara software. The VPg peptide was composed of 20 amino acid sequence, among different HRV species sequence




conservation (⎕80%) was: HRV A – 85%, HRV B – 65% and HRV C – 55%. We could successfully predict the structure of two species, A and B, and the molecule structure was very similar between both species. Unlike expected the amino acid sequence was high conserved only in HRV A species. In HRV C species the sequence conservation was relatively low. The differences in amino acid sequences may reflect a possible difference in the species replication rates. Further studies can elucidate the VPg role in HRV pathogenesis.

BV483 - RETROSPECTIVE STUDY OF SEXUALLY TRANSMITTED DISEASES (STDS) IN PREGNANT AND NON-PREGNANT WOMEN IN PEOPLE ATTENDED BY LACEN MACEIÓ-ALAntão, K.L.1; de Sá, J.P.O.1; Pinheiro, T.M.L.2; de Lima, M.C.2; Pinheiro, M. da S.1; Maria, F.H. de O.S.1

1. UNCISAL - Universidade Estadual de Ciências da Saúde de Alagoas, Rua Doutor Jorge de Lima, 113, Trapiche da Barra, Maceió - AL, 57010-300

2. LACEN

Our basic strategy for controlling and prevention of Sexually Transmitted Diseases (STDs) will be through constant information and educational activities that focus on: risk perception, changes in sexual behavior and promotion and adoption of preventive measures to emphasize condoms use. Our aim was to draw a profile in pregnant and non-pregnant women with characteristics such as: early sexual behavior, different socio-economic and cultural aspects and age groups, who spontaneously sought treatment at Central Laboratory (Lacen/AL) for routine examinations as pre-natal vaginal discharge and pap smear, followed by referral to the preventive examination or treatment and depending on the case The total sample of cases was 5326 women, from which 3885 non-pregnant and 1441 pregnant. Ages ranging from 11 to 61 and the data were collected from the medical records in LACEN-AL from January 2004 to December 2005. The correlation between the data obtained from microorganisms associated with STD prevalence in pregnant and non-pregnant women were: Candida sp, Gardnerella vaginalis, Monilia, Staphylococcus aureus, Streptococcus agalactiae, Condylomata acuminata (HPV). The results show that the rate of pregnant women with STDs is very high and it supports the importance of having a thorough prenatal checkup regarding STDs. It

also sustains the need for laboratory tests cheap, simple and quick to be adopted routinely in women. Such tests aim to reduce STDs and increase the correct and early diagnosis of infection thus avoiding complications in the course of illness and treatment. FINANCIAL SUPPORT: LACEN/AL - Laboratório Central de Saúde Pública de Alagoas UNCISAL - Universidade de ciências da saúde de alagoas PROBIC – UNCISAL - Programa Institucional de Bolsas de Iniciação Científica

BV488 - ANTI-HIV-1 ACTIVITY OF NEW DITERPENES DERIVED FROM THE BROWN SEAWEED DICTYOTA PFAFFIIOliveira, I.B.1; Pardo-Vargas, A.3; Cirne-Santos, C.C.2; Castellanos, L.3; Teixeira, V.L.1; Paixão, I.C.N.P.1



3. UNAL - Universidade Nacional da Colômbia, Avenida Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS), a clinical syndrome which causes profound immunosuppression. HIV infects CD4+ cells and, once inside a cell, integrates its genetic material into the DNA of the host. Thus, it gets efficient replication, with a large number of copies of infective viral particles, making the organism susceptible to the acquisition of opportunistic infections, leading to the development of AIDS. Since the introduction of the highly active anti-retroviral therapy (HAART), the life expectancy of HIV-infected individuals has increased, even in cases in which the virus remains active. However, the discovery and development of new drugs candidates is required, as current drugs do not completely eradicate HIV from infected tissues and the long-term use of these drugs is restricted by the emergence of drug-resistant viruses, metabolic disorders and toxicity. Marine natural products have demonstrated pharmacological activities against a great number of pathogens, including HIV-1. In the latest years, natural compounds capable of inhibiting somehow HIV-1 have been target of study. The aim of this study is to show the potential anti-HIV-1 activity of 3 dolabellane diterpenes derived from the brown seaweed Dictyota pfaffii. Were




used MT-2 cells, which were maintained at 37 °C, 5% of CO2 and used for cytotoxicity and antiviral assays. For the antiviral assay were used the viral isolated from HIV-1 IIIB. Cytotoxicity and antiviral assays was performed by MTT method. Results of cytotoxicity assay showed that the compounds were not cytotoxic and MT-2 viability was higher than 90% for concentrations up to 250μM, resulting in a CC50 between 1345μM ± 2 and 1456μM ± 3,4. The compound also were less toxic than the positive control, neviparine (325μM ± 1,4). The antiviral activity was tested at concentrations between 6,25μM and 25μM. At 6,25μM, the compounds 1, 2 and 4 inhibited the viral replication in 83%, 69% and 52% of cases, resulting in EC50 values of 2,9μM ± 0,2, 4,1μM ± 0,4 and 6,16μM ± 0,7, respectively. Thus, these results suggest that such diterpenes could be considered potential new agents for HIV-1 therapy. Therefore, further studies on the precise mechanism of activity and on the in vivo antiviral activity are needed. FINANCIAL SUPPORT: FAPERJ, CAPES, CNPQ, PROPPI-UFF

BV494 - STRUCTURAL INSIGHTS INTO TOSPOVIRUS NUCLEOPROTEINPaula, D.F.; Lima, R.N.; Lucas, F.; Resende, R.O.

UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900

Efficient viral infection requires appropriate interactions between viral and host proteins that alter the routine of the cell to promote the virus infection. Viral proteins can also interact to form oligomers, such as some viral proteins of tospoviruses. The genus Tospovirus belongs to the family Bunyaviridae. All members of this genus have three segmented single stranded RNA genome. The L segment encodes the viral RNA-dependent RNA polymerase (RdRp). The M segment encodes two glycoproteins (Gn/Gc) and the nonstructural protein NSm responsible for the viral movement. The S segment encodes the nonstructural RNA silencing suppressor protein (NSs) and the nucleocapsid protein (NP). Multiple copies of the NP form oligomers that interact with the viral genomic and antigenomic RNAs to build ribonucleoprotein complexes (RNPs) that are functional templates for RNA replication and transcription carried out by the RdRp. Moreover, the NP interacts with NSm for cell-to-cell transport of the RNPs. Previously, studies based on yeast two-hybrid system (2YHS) and

site-directed mutagenesis in the TSWV NP sequence, identified regions involved in RNA binding and protein multimerization, which supports a “head-to-tail” interaction model. However, the tospovirus RNPs is uncharacterized at the molecular level and the lack of structural information about NPs prevents a more detailed description of those interactions. Recently, the NPs tridimensional structure from viruses of the related viral families Arenaviridae, Orthomyxoviridae and Bunyaviridae have been elucidated providing structural basis for understanding the tospovirus NP behavior. Whereas no structural models are available for Tospoviruses NP, several studies made for Orthobunyaviruses NP could be used as reference because of their kinship. NP proteins have distinct size and quite distinct folds. Nevertheless there are several similarities in the architectural principles by which both proteins form NP-NP multimers and NP–RNA complexes. Using Homology Modeling technique we determined that GRSV NP structure possesses two interacting N- and C-arms and a globular positively charged groove domain into which RNA is deeply encompassed. Tospovirus NP proteins share a high level of amino acid identity, indicating that GRSV NP structure could be used as a model for all members of the genus. The results reported here provide for the first time new insights into tridimensional structure for Tospovirus NP protein.

BV495 - EXPRESSION PROFILE OF FOUR CELLULAR GENES DURING 36 HOURS OF HUMAN ADENOVIRUS TYPE 5 INFECTIONGiehl, I.C.; Rigotto, C.; Staggemeier, R.; Jesus, L.F. de; Henzel, A.; Spilki, F.R.


During an infection, human adenoviruses, such as serotype 5 (HAdV-5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as mRNA production and translation. It is known that a small number of viral gene products can induce massive reprogramming of cellular gene expression. In order to understand what might be the impacts of adenoviruses on cells of different organisms, studies evaluating the effect of infectious viral particles on expression of cellular genes




are frequent. Therefore, the aim of this study was to evaluate the expression of four cellular genes involved in immune response, cell proliferation and cell cycle progression, in several time points during the infectious cycle of HAdV-5. HAdV-5 was inoculated onto human lung carcinoma cell line (A549), at 1 m.o.i (multiplicity of infection). Negative control, mock inoculated cells, was performed in order to normalize the results. At different time intervals upon infection (1h, 2h, 4h, 6h, 12h, 18h, 24h, 30h and 36h), total RNA was extracted by a commercial kit (PureLink®), followed by cDNA synthesis (SuperScript® III). Quantitative real time PCR (qPCR) was performed using primers that targeted the following genes: TGFBR1, CCNDBP1, DHFR and Smurf2. 18S gene was used as the endogenous control. The comparative threshold cycle (Ct) method was used to quantify changes in gene expression in infected group when compared to negative control. Values were expressed as fold differences (FD) between test and control treatments. In order to quantify the HAdV-5 present in the infected group, qPCR was performed using primers designed to amplify the hexon protein gene of HAdV, namely VTB2. Our preliminary results showed two major peaks with increased expression of the four genes, one appearing early in infection (4h) – FD values between 1.80 and 3.15 - and the other standing out at 30h – FD values between 2.96 and 4.34. In other periods, it was observed an increased, decreased or unchanged gene expression, with no pattern followed by the four genes. VTB2 gene was detected only in infected group, in all time points from 1h to 30h, with values ranging from 2.01x104 to 2.74x109 genome copies/5µL, in an ascending pattern over time. These results represent the erratic behavior of cell gene expression during viral infection, alternating between overexpression and normoexpression of the studied molecules. FINANCIAL SUPPORT: FEEVALE, CAPES, FAPERGS, CNPq.

BV496 - THE ROLE OF INTERACTION BETWEEN VIRAL NEF AND ALIX/AIP1 IN INFECTIVITY OF HIV-1da Silva, G.P.D.; Costa, L.J.; Mendonça, L.M.


Nef is an accessory protein expressed early during the replication cycle of the primate lentiviruses HIV and SIV.

This protein plays an essential role in viral infectivity and progression to AIDS. It has been reported that Nef interacts with different cellular partners to perform several functions in the viral replicative cycle, but the function related to increased viral infectivity in primary lymphocytes and macrophages has not been described. Nef may mediate downregulation of surface expression of CD4 membrane molecules and may prevent apoptosis in T cells infected with HIV-1. It has been reported that the cellular protein Alix/AIP1 plays a central role in directing the ESCRT machinery and this is essential for the budding of certain enveloped viruses such as HIV-1. We are investigating the role of the interaction between Nef and cellular protein Alix/AIP1. Nef interaction with Alix/AIP1 have been previously mapped to the amino acid residues YPLTF present at the positions 135-139 on the C-terminal of the Nef protein from the HIV NL4-3 isolate. The objective of this study is to elucidate the interaction between these two proteins has an influence on the increase in viral infectivity. For this, siRNA knockdown assays were performed in Hek293T cell cultures. The knockdown was carried out from the transfection of siRNA against Alix/AIP1. The expression of Alix was monitored by Western blotting with specific antibodies against Alix showing a 96% of knockdown after 24h when it was used 50mM siRNA. So we started testing transfection of plasmids NL 4.3 and NL 4.3 ΔNef after 24h of knockdown. Interestingly, infectivity assays of viruses produced in these conditions showed an Alix dependence to increase viral infectivity using NL 4.3 and this increase was not observed when was used the NL 4.3 ΔNef. In these experiments an infectivity reduction of NL 4.3 in knockdown cells of 60% was observed as well as the reduction observed in NL 4.3 ΔNef virus. So, these results indicate that the Alix/AIP1 protein has a role in increased infectivity of HIV-1 in the presence of Nef protein.




BV502 - EVALUATION OF ANTIVIRAL ACTIVITY OF EXTRACTS OF BACTERIA ISOLATED FROM SOILS MOUNDS AGAINST HUMAN HERPES VIRUS TYPE 1, KOS STRAINPadilla, M.A.1; Kohn, L.K.1; de Moraes, A.P.1; Morandi, B.C.2; Garboggini, F.F.2; Martini, M.C.1; Arns, C.W.1


2. CPQBA

Human herpes virus type 1 (HHV-1) belongs to the Herpesviridae family and represents serious threats for public health and increasing in the severity of ilnesses in immunocompromised patients. In addition, there are mutant strains resistant to available drugs. Since the difficulties associated with prevention and treatment of HHV-1, the use of natural products as antivirals can be an alternative. The aim of this study was to evaluate in vitro 93 extracts of bacteria isolated from soils mounds for antiviral activity against HHV-1. Isolated microorganisms were incubated in culture medium for four weeks at 30°C and these inoculums were extracted by liquid-liquid extraction with ethyl acetate. Antiviral activity was measured by virus titration technique and the percentage of viral inhibition (PI) was calculated for virus treated with samples. The extracts were considered active when PI was higher than 97%. From all extracts tested, five of them were considered active, which is equivalent to 5% of them and six showed toxicity at the concentration tested (50µg/mL). The extracts CDPA10, CDPA22, CDPA26 and CDPI92 presented 99% of PI. These extracts were selected for an evaluation to identify the selectivity index (SI) and were considered promising the extracts that showed IS value greater than 3.0. Of all, CDPA10, CDPA22 and CDPA26 presented the best values of SI with 17.10, 11.37 and 3.84 respectively. Finally, these extracts were evaluated for their mechanism of action through the addition of the extract and/or viral dilution at cell monolayer at different times. As a result, the CDPA10 and CDPA22 were classified as viruses inactivators with 99% of activity; and CDPA26 was active as an inhibitor of replication with 99% of action. These initial results of this work were promising; however additional studies are necessary to evaluate the chemical compounds responsible for the activity. FINANCIAL SUPPORT: CNPq, FAEPEX and FAPESP

BV508 - DISCOVERY OF PERILLYL ALCOHOL AND PERILLIC ACID ANTI-HSV-1 ACTIVITY AND MECHANISM OF ACTIONRibeiro, C.P.1; de Amorim, L.M. da F.1; Santos, T.F.Q.1; da Silva, V.A.G.G.1; Bloom, D.C.2; Paixão, I.C.N.de P.1


2. UFL - Universidade da Flórida, Gainesville, FL 32611, Estados Unidos

Infection by herpes virus simplex type-1 (HSV-1) causes several diseases, ranging from cutaneous, oral and genital infections to fatal encephalitis. Acyclovir is the reference compound used in antiviral therapy. With the emergence of strains of HSV resistant to the current antiviral drugs, new antiviral agents, especially those with different modes of action, are urgently needed. In this study, we examined the mechanism of action of perillyl alcohol (POH) and perillic acid (PA) in inhibiting in vitro replication of HSV-1 KOS strain, on Vero cells, by plaque assay. The cytotoxicity of the compounds was measured by MTT assay. Our results demonstrate that these compounds have high anti-HSV-1 activities in a concentration range that was not cytotoxic to Vero cells. Interestingly, despite the high anti-HSV-1 activity in Vero cells, the compounds helped the virus to reactivate from Trigemal ganglia neurons culture. DNA fragmentation assay confirmed the low cytotoxicity of these compounds, indicating that probably they are not causing apoptosis in Vero cells (infected or not). However, the compounds did not show virucidal activity, and therefore do not inactivate the viral particle. In addition, PCR analyses showed that these substances are not inhibiting viral genome replication, suggesting that the high anti-HSV-1 activity is not due to inhibition of viral replication. Our western blot results suggest that any of these compounds are affecting the total HSV-1 protein production. Since we did not find any inhibition in viral genome replication and protein production, we performed a virion egress assay, which revealed that POH is probably preventing the virus of getting out of the cells. Work is currently underway to determine if these compounds block capsid assembly or virion morphogenesis. Perillyl alcohol and perillic acid have a promising profile for further in vitro and in vivo assays toward development of new strategies in anti-HSV-1 therapy. FINANCIAL SUPPORT: CAPES/FAPERJ/CNPQ/PROEX/ FOPESQ-UFF-PROPPI




BV512 - PATTERN OF BACTERIAL MICROBIOME IN THE RESPIRATORY TRACT OF INFLUENZA PATIENTSCorrea, R.A.1; Borges, L.G. dos A.2; Giongo, A.3; Correa, R.A.1; Gregianini, T.S.4; Franco, A.C.1; Roehe, P.M.1; Campos, F.S.1; Veiga, A.B.G.2


2. UFCSPA - Universidade Federal de Ciências da Saúde de Porto Alegre, R. Sarmento Leite, 245 - Centro Histórico, Porto Alegre - RS, 90050-170

3. PUCRS - Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681 - Partenon, Porto Alegre - RS, 90619-900

4 IPB/LACEM - Instituto de Pesquisas Biológicas/ Laboratório Central de Saúde Pública do Estado, Av. Ipiranga, 5.400, Jardim Botânico, Porto Alegre - RS, 90610-000

Pandemic strains of Influenza A virus appear to be clinically more aggressive, mainly due to an imbalance in host immune activity against the virus, while in post-pandemic periods the mortality is lower and probably there are many flu patients cured without medical intervention. Host immunological response and factors associated with virus strains may define different outcomes among infected patients in a population. Studies have shown that mortality rate by influenza A virus can be a result of an associative network between virus-bacteria toward the host. Thus, the aim of the present study was to show a pattern of bacterial diversity in the microbiome of upper respiratory tract of influenza patients with different infection outcomes. Ten nasopharingeal samples of patients infected by influenza A H1N1 2009 virus in the post pandemic period were selected in a double blind, randomized study approved by the Research Ethical Committee of UFCSPA. Samples were divided in two groups (cure and death) based on the patient prognostic. In order to identify the microbiome, DNA was extracted and amplicons from the V4 hyper-variable region 16S rRNA gene were generated and sequenced on an Ion Torrent PGM, using a 314 chip. A total of 703,585 usable reads were generated and analyzed using Prinseq and MG-RAST. The most prevalent phyla in the samples were Bacteroidetes (40% of the total reads in the outcome deaths and 24.5% of the total reads in the outcome cures) and Firmicutes (36.7% of the total reads in the outcome deaths and 29.5% of

the total reads in the outcome cures). Interestingly, unclassified sequences at phylum level were observed four times more in the outcome cures than in the outcome deaths. A higher number of Porphyromonas and Staphylococcus reads were observed in the outcome deaths. On the other hand, a higher number of Pseudomonas and Rhodococcus were identified in the outcome cures. Some genera such as Haemophilus, Prevotella and Streptococcus presented high number of reads in at least half of the samples analyzed. We have observed that different bacteria genera could be presented in the virus infection depending on the outcome. More analyses need to be performed in order to identify patterns in the community diversity for both death and cure outcomes aiming to correlate those bacteria to the influenza infection and host immunity.

ENVIRONMENTAL VIROLOGY: EV


Environmental Virology: EV 101

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Environmental Virology: EV

EV40 - EVALUATION OF VIROLOGICAL PARAMETERS DURING MIMIVIRUS PRODUCTIONSilva, L.C.F.1; Boratto, P.V.M.1; Dornas, F.P.1; Campos, R.K.1; Kroon, E.G.1; La Scola, B.2; Abrahão, J.S.1


2. Aix-Marseille Université, 58 Boulevard Charles Livon, 13284 Marseille, França

Acanthamoeba polyphaga mimivirus (APMV) was first isolated few years ago from a water cooler, in an English hospital during a pneumonia outbreak and belongs to Mimiviridae family. APMV prompted the creation of an open field of study about function of various genes never-before-seen in viruses and their roles in virus-host interactions. In recent years, several giant viruses have been isolated from different environments and specimens. Although the scientific community has experienced a remarkable advancement in the comprehension of the mimivirus replication cycle in the last years, few studies have been devoted to the investigation of the methodological features and conditions for mimivirus production. This work aimed investigate the conditions for cultivation of mimiviruses isolates to obtain informations about the production of infectious particles, total viral particles and viral DNA. Our results suggest that low viral doses are more efficient for the production of infectious particles, yielding up to 5000 TCID50 for each inoculated TCID50. Besides methodological information, these data also reveal, for the first time, the ratio between total and infectious particles (in TCID50) that are produced during mimivirus cultivation in laboratory conditions. Then, can help prompt mimivirological studies in different fields. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG; MAPA.

EV22 - DETECTION AND MOLECULAR CHARACTERIZATION OF AICHIVIRUS FROM WASTEWATER DIRECTLY DISCHARGED INTO URUGUAY RIVER, URUGUAYTort, L.F.L.1; Burutarán, L.1; Victoria, M.1; Lizasoain, A.1; García, M.1; Miagostovich, M.2; Leite, J.P.G.2; Colina, R.1

1. Laboratory of Molecular Virology, Sede Salto, Cenur Del Noroeste, Universidad de La República, Uruguay

2. Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute (FIOCRZ), Ministry Of Health

Aichivirus (AiV) belongs to the genera Kobuvirus, inside Picornaviridae family, and there are three AiV genotypes capable of infecting human: A, B and C. AiV is a human enteric virus with a global prevalence between 0,9 to 4,1% in gastroenteritis sporadic cases, and, these virus have also been detected in environmental superficial water. The aim of this study was to assess the viral contamination of AiV in sewage directly discharged into Uruguay River, and to characterize AiV genotypes circulating in Uruguay. For this purpose, sewage samples (n=96) were collected biweekly from March 2011 to February 2012 in four Uruguayan cities: Bella Unión, Salto, Paysandú and Fray Bentos. Each sample was concentrated by ultracentrifugation method. A Nested RT-PCR directed to 3CD region of AiV genome, was performed. Positive samples were sequenced and phylogenetic analysis were performed in order to determine the genotypes present in the samples. A wide dissemination of AiV was observed in the sewage samples analyzed with a 57.3% of positivity, being detected with a high prevalence in the four sampled cities. Beside a clear seasonality was not observed in the present study, a higher positivity was observed in the coolest seasons (autumn and winter). The phylogenetic analysis showed that all the positive samples belong to genotype B. This study represent the first detection of AiV in our country, and showed the potential risk of infection with this virus of the local populations through recreational activities or consumption of water from the Uruguay River, in which untreated sewage is directly discharged.




EV41 - ACANTHAMOEBA POLYPHAGA MIMIVIRUS INHIBITS TRANSCRIPTION OF AMOEBAL SUBTILISIN-LIKE SERINE PROTEINASE MRNA AND CIRCUNVENTS CELL ENCYSTMENTBoratto, P.V.M.1; Almeida, G.M.F.1; Botelho, L.1; Lima, A.1; Costa, A.O.1; Santos, D.A.1; La Scola, B.2; Kroon, E.G.1; Abrahão, J.S.1


2. Aix-Marseille Université, 58 Boulevard Charles Livon, 13284 Marseille, França

Acanthamoeba is a genus of free-living amoebas distributed worldwide known as agents of public health concern for causing disease in humans. On natural environments, they can act as hosts for many microorganisms, including members of the family Mimiviridae, one of the largest and most complex group of viruses ever described. Many studies have investigated the interactions between these protozoa and some of its harbored microorganisms (mostly pathogenic bacteria) but few have explored the mechanisms involving their interaction with giant viruses. In the present study, we explored the behavior presented by Acanthamoeba spp. and Mimivirus (APMV) in response to different conditions of incubation and interaction. The investigated scenarios were: (1) Acanthamoeba trophozoites infection, (2) Acanthamoeba mature cysts infection, (3) trophozoites infection shortly after being subcultured in encystment medium and (4) trophozoites infection shortly before being subcultured in encystment medium. The regulation of some genes involved with encystation in Acanthamoeba in response to APMV infection were also investigated. The results obtained suggest that amoeba encystment is a crucial phenomenon for avoiding viral infection success, and therefore it is the target of an arm wrestling between the virus and amoeba. We demonstrate that once amoeba encystment is triggered, trophozoites become significantly less permissive to APMV and, remarkably, APMV is able to interfere with the expression of the serine proteinase mRNA related to the amoebal encystment. This work suggests that amoebas can use encystment as a form to protect themselves from viral infections, while APMV can interfere with the encystment process by regulating an amoebal gene. These results may represent one of the most ancient

examples of fight for supremacy involving a virus and a eukaryotic cell.

EV51 - COMPARISON OF DIFFERENT METHODS FOR ISOLATION OF GIANT VIRUSESArantes, T.S.; Andrade, K.R.; Silva, L.C.F.; Dornas, F.P.; Silva, L.K. dos S.; Rodrigues, R.A.L.; Kroon, E.G.; Abrahão, J.S.


The giant viruses are an important and unique group of viruses whose first representative was discovered in the year 2003. The Acanthameba polyphaga mimivirus (AMPV), so named was isolated from water collected in a cooling tower for air conditioning in the British city of Bradford. The AMPV caused interest in the scientific community because of its unique size, bigger than some bacterial and also by to provide genes never before observed in other viruses. After the discovery of the AMPV, many other giant viruses have been isolated from different samples and localizations through various protocols of isolation. This study aims to compare two mimiviruses isolation techniques: the agar PAS isolation technique and the direct inoculation of samples in Acanthamoeba castellanii monolayers growth in PYG liquid medium. For these analyses, were initially used 10 oyster samples previously enriched in middle rice during 30 days. After the enrichment, the samples were subjected the tests in order to check the efficiency. The isolation in cultivation of A. castellanii was performed using 100µl of sample, that were subjected to three passages lasting three days each for the visualization of cytopathic effect. For the agar PAS isolation test the enriched sample were first inoculated in a fresh culture of A. castellanii. After three days, 10 µl of each sample were collected and inoculated on an agar PAS plate containing a monolayer of A. castellanii. The plate was observed during three days for viewing lysis plaques on agar. After theconductingthe test it was possible to observe that the technique of isolation in culture of A. castellanii presented a positivity of 40% of de isolation after the third pass. Already the agar PAS teste showed an isolation rate of 50%. Through the results obtained was observed that the agar PAS technique was more effective than isolation technique on cultivation of A. castellanii.




In addition, a sample which showed no cytopathic effect after three passage in culture of A. castellanii was positive in agar PAS, demonstrating greater sensitivity of the technique. The agar PAS technique allowed the isolation in a short time using a smaller sample volume. The agar PAS isolation system has proved to be an efficient, sensitive and less fastidious method allowing the isolation of giant viruses in less time compared to isolation in culture of A. castellanii and can be widely used. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, MAPA, PPG-MICROBIOLOGIA UFMG

EV52 - EVALUATION OF THE DISTRIBUTION OF MIMIVIRUS IN A HOSPITAL ENVIRONMENT AND CORRELATION WITH THE PRESENCE OF ACANTHAMOEBASilva, L.K. dos S.; Andrade, K.R.; Rodrigues, R.A.L.; Boratto, P.V.M.; Arantes, T.S.; Silva, L.C.F.; Dornas, F.P.; Kroon, E.G.; Clemente, W.T.; Abrahão, J.S.


In 1992, during a pneumonia outbreak, a giant virus was isolated from a cooling tower of a Hospital in Bradford, England and was called Acanthamoeba polyphaga mimivirus (APMV). APMV infects amoebae of Acanthamoeba genus and is the prototype of the Mimiviridae family, which is clustered with the Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs). Pneumonia is a major cause of death related to infection around the world; however, between 20 to 50% of cases have unknown etiology. Therefore, identifying new causative agents of pneumonia is a public health concern. Some studies have pointed out the APMV as potential respiratory tract pathogens and probable causative agents of pneumonia in humans. In order to better understand the role of mimiviruses as etiological agents of this disease and its association with the presence of amoebae, we evaluate the distribution of these viruses in different settings of Hospital das Clínicas/UFMG. Altogether, 153 samples were collected from reception, stairs, elevators, hallways, nursery and respiratory isolation areas. The samples were collected using swabs and after were eluted in PBS. After this procedure, total DNA was extracted from the samples by phenol-chloroform method and used as template

for Real-Time PCR assays, targeting the conserved helicase gene of mimivirus, and the 18s ribosomal gene of Acanthamoeba. Phylogenetic analyses were also performed to investigate the relationship of these new samples with Mimiviridae family. Our results showed a higher positivity of mimiviruses in respiratory isolation area (36%) compared to other settings: reception (15%), stairs (10%), elevators (4%), hallways (8%) and nursery (10,5%). In addition, a correlation was observed between the presence of amoebae and mimiviruses: 26 out of 29 positive samples for the helicase gene were also positive for the 18s gene. The phylogenetic analysis showed a close relationship among our samples to other mimiviruses. In conclusion, this study showed that the respiratory isolation area presents a greater positivity to mimiviruses, indicating a possible risk factor for the occurrence of pneumonia. Moreover, the presence of mimiviruses appears to be directly related to the presence of Acanthamoeba. More studies are needed to clarify this relationship and the role of mimiviruses as pathogens of the respiratory system. FINANCIAL SUPPORT: CNPQ, CAPES, FAPEMIG, MAPA, PPG-MICROBIOLOGIA UFMG.

EV60 - AUTOMATED COMPOSTING ALLOWS THE DISINFECTION OF DIFFERENT ADENOVIRUS IN SWINE MANUREHenzel, A.1; Soliman, M.C.1; Ziber, G.1; Staggemeier, R.1; Rigotto, C.; Sá, M.2; Pujol, S.2; Aita, C.2; Spilki, F.R.1

1. FEEVALE - Universidade Feelave, Av. Dr. Maurício Cardoso, 510 | Bairro Hamburgo Velho, Novo Hamburgo - RS, 93510-250

2. UFSM - Universidade Federal de Santa Maria, Avenida Roraima, 1000 - Camobi, Santa Maria - RS, 97105-900

The automated composting is a method for stabilization of organic wastes. This technique is considered effective for elimination of pathogens in manure swine. Among the potential pathogenic microorganisms present in manure are adenovirus (AdV), members of the Adenoviridae family, consisting of double-stranded DNA genome, are often found. However, little is known about the efficiency of this technique in removing viruses. The aim of this work was to evaluate the efficiency of two automated composting system in elimination of different AdV species (canine, CAV; avian, AvAdV; bovine, BAV; human, HAdV and swine, poAdV) in liquid swine




manure (LSM). For this, two automated composting units were developed. First (treatment 1), consisted in a mix of LSM, wood shavings and phosphoric acid; and the second (treatment 2), only LSM and wood shavings were used. The frequency of application of the manure in the two piles of composting and its revolving occurred every seven days and the temperature of the piles was measured daily throughout the process (156 days). Throughout this period, samples were collected of raw LSM, of the compost (LSM more shavings) before and after its revolving. In both treatments was done viral analysis. The samples were diluted with Minimum Essential Medium (MEM) for the extraction of viral DNA and the adenoviral species were characterized through the amplification by real-time PCR followed by a differential step of high resolution melting. From the raw LSM added the two piles of composting during the process were detected in all samples BAV (28/28). Other viral species such as HAdV and poAdV in 7.14% (01/14) in treatment 1 and CAV and poAdV 7.14% (1/14) in treatment 2. During the process of composting (with the addition of manure every 7 days), adenovirus loads remained constant in both treatments before and after its revolving, showing that the time between the applications of the manure was not sufficient for viral removal. On maturity phase of the compost, when it was no LSM was, there was no detection of adenovirus in treatment 1 demonstrating efficiency of the process; however, was continued adenovirus being detected until the end of composting in treatment 2. Further analysis will be conducted to measures of temperature, pH, dry matter and moisture to understand this result. FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS ÁGUA, FEEVALE

EV78 - PRESENCE OF HUMAN ADENOVIRUSES IN SURFACE WATER AND SEDIMENTS IN SANGRADOURO RIVER, SANTA CATARINA, BRAZILElmahdy, E.M.; Schissi; C.D.; Fongaro, G.; Nascimento, M.A.; Barardi, C.R.M.


Human enteric viruses are a common microbial which can reach the aquatic environment through the discharge of untreated sewage. Aquatic sediments can

act as reservoirs from which contaminant viruses can be resuspended and return to the water column by several natural or artificial phenomena. Among them, human adenoviruses (HAdV) have been found to be the most prevalent enteric virus in biosolids. In the city of Florianópolis, Santa Catarina (SC), Brazil, located in the protected area of the Peri Lagoon Municipal Park. The Sangradouro River (SR) receives the Peri Lagoon water during the rainy episodes and this water is drained to the ocean located in the neighborhood. This river also receives wastewater and sewage from residences. This study aimed to quantify HAdV either in surface waters or sediment, as well as to evaluate the integrity and infectivity of HAdV in these samples collected along six points of the SR. A sum of 96 samples were collected during the summer and winter seasons of 2013. Two liters of surface water samples were concentrated by negatively charged membranes and 20g of sediment samples were concentrated and clarified using glycine buffer followed by polyethylene glycol (PEG) precipitation. The nucleic acids were extracted from samples before and after DNAse treatment in order to evaluate genome copies of HAdV (by qPCR) derived from intact or undamaged viruses respectively. The samples positive for genome copies were used to infect A549 cell monolayers for plaque assay (PFU) for viral infectivity determination. For surface waters, the incidence of HAdV was 17/24 (70.8%) ranging from 105 to 108 gc/L contained 70.8% undamaged HAdV particles (UP), from which 14/17 (82.4%) contained infectious HAdV ranging from 103 to 104 PFU/L during the summer. In winter, this incidence was 15/24 (62.5%) ranging from 105 to 108 gc/L contained 58.3% UP, and no infectious viruses were detected by PFU. For sediment samples, the incidence was 9/24 (37.5%) ranging from 109 to 1010 gc/Kg contained 37.5% UP, from which 6/9 (66.7%) contain infectious HAdV ranging from 103 to 104 PFU/Kg during the summer. The results of winter collection revealed 9/24 (37.5%) ranging from 108 to 109 gc/Kg contained 37.5% UP and no infectious samples by PFU. We could conclude that HAdV genomes can be more concentrated in sediment but its infectivity can be lost due to physical and chemical composition of the samples. FINANCIAL SUPPORT: CNPq/TWAS and CNPq Universal 472804/2013-8.




EV89 - SEDIMENTATION AND SURVIVAL EVALUATION OF PATHOGENS IN SWINE EFLLUENT AND SLUDGE FOR SAFE REUSE PURPOSESFongaro, G.1; Kunz, A.2; Schissi, C.D.1; Magri, M.E.1; Zaguini, J.1; Barardi, C.R.M.1


2. EMBRAPA, Rodovia SP 340 - Km 127,5 - Tanquinho, Jaguariúna - SP, 13820-000

The swine effluent is composed by urine, feces, digested food and water, characterizing high contents of solids, organic matters, phosphorus and nitrogen. Solid-liquid separation and aerobic treatment are routinely used to reduce the suspended solid concentration in the liquid fraction. In this study, treated swine effluent after aerobic reactor (AR) before settling tank, were used to evaluate the survival rates of the following pathogens artificially seeded: somatic coliphage phiX-174 (phi-X), Human Adenovirus (HAdV-2) and Salmonella Typhimurium. Imhoff cones (v. 1L) were filled with the effluents and the settling experiments were performed until 120 h, in triplicate. The pathogens survival in liquid and solid fraction were determined after 0, 0.08, 0.16, 0.33, 0.75, 2.5, 5, 10, 24, 48, 72 and 120 h. In the sludge (solid-fraction) this parameter was measured after 24, 48, 72 and 120 h of settling time. The enumeration of the HAdV, phi-X and S. Thyfimurium were respectively performed by integrated cell culture assay–preceded by reverse transcription (ICC-RT-qPCR), double layer agar method and by ISO 6579 (2002). In effluent, the survival rate was: 12.2% for S. Thyfimurium, 64% for HAdV-2 and 76% for phi-X-174 after 120h; In sludge (solid-fraction) the survival rate after 120h was 1.6% for S. Thyfimurium, 64% for HAdV-2 and 75% for phi-X-174. The settling rate of the three pathogens was also evaluated by measuring their reduction in the liquid-fraction. S. Thyfimurium was significantly reduced from effluent at 45 min and 5h post sedimentation remaining constant in the other periods evaluated; HAdV-2 at 20 min, 2.5 and 24h, and phi-X at 10, 20 min, 2.5, 5 and 10h. The sedimentation of the HAdV and phi-X was positivity correlated with the sedimentation of solid particles. HAdV-2 and S. Typhimurum increased significantly after 72h of settling in the secondary sludge. This means that the pathogens are present mainly in the solid

particles. Solid-liquid separation and survival studies of these prevalent pathogens in effluents and sludge are viable methods that allow studying the disinfection of these matrices. This can predict the safe reuse of these secondary-products as biofertilizers in agriculture or back to the swine facilities. FINANCIAL SUPPORT: CNPQ 472804/2013-8.

EV108 - ADENOVIRUS AND ENTEROVIRUS IN SURFACE WATERS IN THE WATERSHED SINOS RIVER, RS, BRAZILRigotto, C.; Dalla Vecchia, A.; Staggemeier, R.; Soliman, M.C.; Souza, F.G. de; Giehl, I.; Henzel, A.; Rigotto, C.; Spilki, F.R.


Adenovirus (AdV) and enterovirus (EV), among other viral agents are responsible for different pathologies, mainly associated with gastroenteritis. These non-enveloped viruses are eliminated through the feces of symptomatic or asymptomatic individuals released in high concentrations in water bodies and remain for long periods due to its high resistance to environmental conditions. The Sinos River watershed serves 1.5 million people (94% urban) living with serious problem caused by intense contamination of its waters. The watershed is geographically divided into three stretches: upper (source, low population density and small farms), medium (increased urban density) and lower (mouth, strong population density and industrial concentration). The present study evaluated the occurrence of AdV and EV in water samples by qPCR in catchment points for public supply in Sinos River, main watercourse of watershed. Five-hundred mL of raw water were collected monthly in sterile bottles in eight catchments of water treatment plants (WTP) along the river for 24 months. Initially the samples were submitted to virus concentration by adsorption-elution method and sequence nucleic acids were extracted with a commercial kit (RTP DNA/RNA Virus Mini Kit). For EV detection an additional step (cDNA) was performed using the kit High Capacity cDNA Reverse Transcription. For both viruses, primers targeting conserved regions of the genome were used for qPCR reaction. All 178 samples were negative for EV. In the upper part of the river high rates of AdV




were observed, with prevalence and average loads of: human adenovirus (HAdV, 62% and 5,94x103 GC/L); canine adenovirus (CAV-1 an 2, 66% e 1,04x106 GC/L); avian adenovirus (AvAdV,16%) and bovine adenovirus (BAV,4%). In the middle section viruses were observed in the upper stretch, plus the presence of porcine adenovirus PAdV (4%). In the lower stretch lower rates of HAdV (48%) and CAV (39%) were observed when compared to other stretches. There was a gradual decline in the detection rates for HAdV, CAV and AvAdV along the three sections of extension, which may be associated with the presence of inhibitors in the samples and the increasing of urbanization, since the lower stretch of the watershed suffers intense anthropogenic pressure and represents great human demand by the water use. Continuous monitoring of viral contamination is necessary to reduce negative impacts on public health. FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS ÁGUA, FEEVALE

EV127 - ENTERIC VIRUSES IN BOTTLED MINERAL WATER COMMERCIALIZED IN BRAZILRigotto, C.; dos Santos, V.R.; Staggemeier, R.; Dalla Vecchia, A.; Henzel, A.; Rigotto, C.; Spilki, F.R.


Brazil is the 6th largely consumer of mineral bottled water worldwide, stemming from increasing living standards and the general perception of population that bottle mineral water would be the healthier and safer option for consumption, when compared to other sources. Although several studies suggest that bottled water may have the same or worse bacteriological and chemical quality as tap water. The presence of microbial contaminants depends on source characteristics, methods, asepsis of the packaging material and product storage. This scenario is being aggravated by the fact that 64% of mineral water in Brazil is distributed to final consumer in reusable containers of 20 L. At present, the Brazilian resolution ANVISA RDC N.54 (June of 2000) regulates the microbial quality of natural mineral water requiring the detection of fecal coliforms and thermotolerans bacteria. Among the enteric viruses three of the most studied, as environmental contaminants are the adenoviruses, enteroviruses and rotaviruses.

These agents are transmitted by fecal-oral route, being associated with a number of diseases, especially gastroenteritis, either in human beings or animals. Despite the health impacts caused by outbreaks of these viruses, the investigation of their presence in drinking water, sewage effluent and recreational waters is not mandatory in many countries, including Brazil. Studies in Brazil aiming the detection of enteric viruses in mineral water have not yet been carried out. In this context, this study aimed to detect the presence of enteric viruses; human adenovirus (HAdV), human enterovirus (EV) and human rotavirus genogrupe A (GARV), in mineral water and discuss the possible sources of transmission. Samples of 1.5 L and 500 mL were analysed for virus by PCR and total and fecal coliforms by AQUACULT® kit. The most prevalent virus was Adenovirus, 32.5% followed by rotavirus 25% and enterovirus 17.5%. HAdV was also present in 100% of mineral water containers (20 L) with viral loads ranging from 1.34x103 to 9.94x104 gc/L. For bacteriology, total and fecal coliforms were absent in all samples and only three samples out of 60 analysed presented contamination by thermotolerant bacteria. It is concluded that, following the example of what has been considered in relation to the public supply of drinking water, stricter measures for microbiological control should also be applied to mineral water, so that this becomes indeed a safer alternative. FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS ÁGUA, FEEVALE

EV134 - VIRAL RECOVERY RATES OF HADV, EV AND RV EVALUATED IN DIFFERENT STAGES OF CONCENTRATION BY ADSORPTION-ELUTION PROCESS IN NEGATIVELY CHARGED MEMBRANESGiehl, I.C.; Dalla Vecchia, A.; Rigotto, C.; Souza, F.G. de; Soliman, M.C.; Staggemeier, R.; Giehl, I.C.; Henzel, A.; Spilki, F.R.


Viral particles are often highly diluted in water and difficult to detect, thus virus concentration methods are applied combined with molecular techniques to achieve a higher analytical sensitivity virus detection from environmental samples. Currently, the virus concentration method by adsorption-elution using




negatively charged membrane is the most widely used technique to concentrate viruses in different environmental matrices, however there are few studies aiming the assessment of quantitative virus recovery from different types of enteric viruses under the same conditions. The aim of this study was to evaluate the efficiency of this method for virus concentration in small volume samples (500 mL) and determine the recovery rate in different stages of the process using human adenovirus (HAdV), enterovirus (EV) and rotavirus (RV) as prototype viruses. Inoculums containing previously known viral titers of these viruses were individually and separately spiked into 500 mL of previously autoclaved MilliQ water. One milliliter from each step of the concentration method was withdraw at the beginning of the process and viral genomes were extracted using RTP DNA/RNA Virus Mini Kit and the viral nucleic acids were analyzed by real-time quantitative PCR with Platinum Sybr Green qPCR SuperMix-UDG, Invitrogen kit. The results showed higher rates of viral recovery before concentrating (step 1) in relation to the final step (step 5), except for HAdV with highest recovery efficiency of 77% at the end of the concentration when combined with other viruses in the sample. EV and RV showed better recovery rates in the early stages ranging from 30% to 74% when evaluated separately, compared to rates of 14% to 39% when evaluated combined. Results obtained for HAdV when associated to other viruses in the sample and the reduction in recovery efficiency for EV and RV genome suggests a possible competition of HAdV when in the presence of other viruses. Overall, the data showed wide variation in recovery rates (4% RV to 77% HAdV) using RNA and DNA genome viruses at the final stage of concentration, as previous studies. This study showed better recovery rates before concentration and loss in the intermediate stages, suggesting that virus concentration protocol could be replaced or excluded without harming the efficiency of viral recovery, however, future comparative analyzes with environmental matrices are needed to assess the efficiency in the presence of inhibitors. FINANCIAL SUPPORT: CNPQ, FAPERGS, PROJETO MAIS ÁGUA, FEEVALE

EV146 - MORPHOLOGICAL AND QUANTITATIVE ANALYSIS OF MARINE VIRUSES IN ARRAIAL DO CABO AND GUANABARA BAY, RJ, BRAZILPedrosa-Macena, L.G.1; Paula, J.E.F.B.1; Santana-Pereira, P.1; Amorim, L.1; Ferreira, D.F.2; Silva, R.2; Damaso, C.R.A.2; Giongo, V.1; Paixão, I.C.N.P.1



Viruses are the most abundant biological agents in aquatic environments. Responsible for most of the genetic diversity and interference in marine ecological processes, viruses distributed in limnic and marine ecosystems, such as oceans, lakes, coral reefs, glaciers, mangroves, among others. Viral activities influence the structure and composition of microbial communities and biogeochemical flows of matter and energy. This study aimed to analyze marine viruses (cyanophages) morphologically and determine their abundance, spatially and seasonally in regions of Arraial do Cabo and Guanabara Bay located in the state of Rio de Janeiro. Thus techniques were performed as Transmission Electron Microscopy (contrasted with uranyl acetate 1%) and Flow Cytometry, which was used in three different concentrations of SYBR Green I dye in the proportion of 5x10 -3, 5x10 -4 and 5x10 -5 from the stock solution. It was evidenced phages of Order Caudovirales (Families Myoviridae, Podoviridae and Siphoviridae) in Praia dos Anjos (Arraial do Cabo), the Aterro da Praia Grande and Enseada de Jurujuba (Guanabara Bay). The seasonal flow cytometry results indicated that the summer (3.61 ± 2.74 x 10 8.part.mL-1) and spring (2.85 ± 1.8 x 10 8.part.mL-1) had higher viral abundances. Spatially at Arraial do Cabo the sampling area with highest abundance was Praia dos Anjos (4.44 ± 2.62 x 10 8.part.mL-1) followed by Praia do Forte (3.23 ± 1.87 x 10 8.part.mL-1), otherwise the Guanabara Bay the highest abundance was represented in the Aterro da Praia Grande (3.33 ± 1.17 x 10 8.part.mL-1) followed by the Enseada de Jurujuba (2.57 ± 1.37 x 10 8.part.mL-1). Being found the presence and abundance of marine viruses in the study areas, especially in the period with higher solar incidence (Spring and Summer), showing a bacterial viability of maintenance for viral replication. Thus it concluded that




this environment is promising for future analyzes of role of viruses, organisms and microorganisms involved in the dynamics of the marine ecosystem. FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPPI AGIR

EV164 - VIRAL ABUNDANCE AND MARINE ENVIRONMENT ON ENDEMIC SIDERASTREA STELLATA CORAL REEF IN ARRAIAL DO CABO AND BÚZIOS/RJSantana-Pereira, P.; Giongo, V.; Pedrosa-Macena, L.G.; Paula, J.E.F.B.; Voguel, V.R.; Nepomuceno, A.; Crapez, M.A.; Paixão, I.C.N.P.


Coral reefs are known to be holobiont and integrated assemblage of the coral animal, its symbiotic algae, protists, fungi, a diverse consortium of Bacteria and Archaeas and even viruses. The symbiosis can be understood through de evaluation of corals and your symbionts, the breakdown of which can result in disease and mortality. Little is known, however, about the consequences of viruses that infect them. Studies have shown that the virus highly dynamic aquatic ecosystem components have a central role in these environments acting as antimicrobial agents in the control of the quantity and diversity of coral symbionts such as bacteria and phytoplankton. Viruses collaborate for the redistribution of micro and macro nutrients throughout the water column and in food webs. In this work, total and dissolved inorganic nutrients in the water column nitrate, nitrite, phosphate and ammonium were quantified and analyzed by grouping data, estimating the trophic conditions of the environment and its limitations. Spatially higher concentrations of nutrients were found in Búzios (Bz) and seasonally during the summer and autumn. The EFM technique was used to count and determination of the abundance of bacteria and virus particles, samples of seawater and coral mucus were subjected to serial dilutions and labeled with 3 dyes (AO, SYBR Green I and DAPI). The highest bacterial abundances were found during the winter in the coral mucus samples in Bz (15.1 x 107 cels. / mL) and in seawater in Arraial do Cabo (AC) (7.23 x 107 cels. / mL). The abundance of viral particles was found higher in summer in Bz in coral mucus samples (2.70 x 107 part. / mL) and during autumn in AC samples from seawater (3.24 x 107 part.

/ mL). These data support the characterization of these environments such oligotrophic, which are those with a low supply of nutrients. Therefore it was possible to determine the presence of viral particles associated with Siderastrea stellata and the water column in both sites. The relationship between nutrient availability, content of Chl-a and bacterial abundance supported the feasibility of viral replication. Bacteriophages are therefore regulators of bacterial abundance, as well as responsible for nutrient cycling. FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPPI AGIR

EV200 - PASSION AT SECOND SIGHT: AN UPDATE OF GENOME SEQUENCING AND ANALYSIS OF SAMBA VIRUSAssis, F.L.1; La Scola, B.2; Kroon, E.G.1; Abrahão, J.S.1


2. Université de la Méditerranée, Jardin du Pharo - 58, bd Charles Livon -13284 Marseille Cedex 07

The Samba virus (SBV) was isolated in Brazilian Amazon forest in 2011. At that time, a previous genomic analysis was performed using a partially sequenced molecule. Nowadays, using the Illumina Deep Sequencing Technology, we obtained the complete genome sequence of SBV. For these proposes, the DNA of sucrose-gradient purified SBV was extracted, sequenced and the raw data were assembled using the CLC workbench 6 software. The final genome of SBV is a molecule of 1.181.380 base pairs (bp) with a C-G content of 28%. This new molecule is ~32kb shorter than the previous SBV genome announcement, but still longer than Achantamoeba poliphaga mimivirus (APMV), the first discovered mimivirus. The new genome of SBV was predicted to encode to 971 ORFs, (33 more ORFs than the first report), ranging in size from 113nt to 8834nt, with a mean size of 1068pb. The ORF’s density was 0.821 genes per kb (1216 bases per gene), with a coding percentage of 87.9%. Its arsenal was comprised by 142 metabolic-associated cellular protein and up to 180 general metabolic processes, besides some regulatory proteins. Among the predicted ORFs, 45% was comprised by hypothetical proteins, and 85% showed best hit against APMV. We identified four aminoacil-tRNA sintetase sequences and six tRNA sequences being three cognates




to leucine, besides one to histidine, cysteine and tryptophan. Curiously, the C-G% of the tRNA sequences was 48.6%, greater than that found in the entire genome. All the predicted ORFs matched orthologous into NVCOG database (average similarity of 93,67%), meaning that new ORFans were not predicted. A total of 58 clusters consisting of 179 paralogous proteins were identified in the SBV genome, which was similar to that detected in APMV and Hirudovirus sangsue. The reciprocal best hit analysis identified 917 bona fide orthologous proteins between SBV and APMV. The same analysis identified 339 bona fide orthologous proteins shared with Moumouvirus (group B) and 485 ones with Megavirus chiliensis (group C). The phylogeny based on DNApol sequence grouped the SBV into mimivius clade A. This updated analysis has a prominent relevance once it might be used as a guide to future genomic studies. Its broad protein arsenal, the great number of uncharacterized putative proteins and the current discussions regarding virus evolution encourages us to go deeper into genomic analysis of giant virus. FINANCIAL SUPPORT: CNPQ, FAPEMIG, CAPES, MAPA, PRPQ

EV208 - BIOACCUMULATION, STABILITY AND DEPURATION OF HUMAN ADENOVIRUS IN OYSTERS CRASSOSTEA GIGASPilotto, M.R.; Garcia, L.A.T.; Barardi, C.R.M.


The Brazilian government implemented, in 2012, the National Programme for Hygienic and Sanitary Control of Bivalve Mollusks (PNCMB). The PNCMB sets minimum requirements to ensure the quality of bivalve mollusks, as well as supervise the attendance of these requirements, based only on bacterial standards. Some areas of mollusks production need to depurate their animals before selling them, but it does not give any specification about the depuration method, neither includes viral detection. The aim of this study was to evaluate the stability and depuration of Human Adenovirus type 2 (HAdV-2), considered one of the most resistant and prevalent viruses in the environment, in oysters Crassostrea gigas. The focus of the work was to use only in vitro cell culture methodologies to determine the infectivity of the virus, once these techniques can

precisely infer the real risk of these pathogens to cause a disease. The methods applied were the plaque assay (PA) and the integrated-cell-culture real-time PCR (ICC-RT-qPCR). Firstly, a bioaccumulation experiment was conducted to determine the optimal intake period of virus by the oysters. They were allocated in an aquarium, and the water was artificially seeded with a known amount of HAdV-2. The times of 3h, 5h, 8h, 10h, 12h and 24h were evaluated, and the period of 8h of bioaccumulation was selected. Secondly, samples of oysters were kept at 4oC to mimic the market storage temperature during commercialization, so the viral stability could be measured. The animals remained alive until 96 hours, and during this period, no significant reduction in the infectious viral load was observed. Finally, the viral depuration rate was measured up to 120h using a depuration tank supplied with an UV light. A tank without UV light was used as a negative control. After 24h of depuration in the tank with UV, and 48h in the tank without UV, no infectious viruses were detected by plaque assay. The results of the ICC-RT-qPCR are being finalized, but once this technique has a higher detection limit when compared with plaque assay, it is possible that infectious viruses could be detected for a longer period. This work shows that the depuration tank was effective to inactivate HAdV-2 and that UV disinfection speeds the depuration. It also provides information to complement the current legislation regarding depuration and viral inactivation. FINANCIAL SUPPORT: CNPq Universal 2012-1013 471755/2011-7

EV243 - INFLUENCE OF TIME AND TEMPERATURE OF STORAGE ON HUMAN ROTAVIRUSES VACCINE STRAIN STABILITY IN WATER MATRICESDamazo, N.A.; Moresco, V.; Barardi, C.R.M.


Human rotaviruses (RVA) are involved with many diarrhea outbreaks, most in children under 5 years old and mainly due to the poor sanitary conditions. Nowadays, two vaccines are available and their positive impacts on public health improvement were already showed. However, there are no studies evaluating the impact of these vaccine strains in the aquatic environment. The goal of this study was to evaluate the




influence of the time and temperature on the stability of the RVA vaccine strain (RotaTeq) in environmental waters. The RVA was propagated in MA104 cells and the titre of the viral stock was measured by plaque assay (PFU) as previously standardized (2,2x107 PFU mL-1). A quantity of 3mL was seeded in 17mL of three different water matrices: (SWL) surface water lagoon (water supply); (SDW) surface drinking water (unchlorinated); and (BWL) backish water lagoon (recreational area). Aliquots of 1mL were stored at the temperatures of 22ºC and 4ºC to mimic respectively environmental and lab storage temperatures. The RVA stability was evaluated by PFU at time zero, and at 5, 10, 30, 60, 90 and 120 days of storage. The viral titre reduction was expressed as log reduction values (Log10 = Nt/N0). In all cases, the higher Log10 reduction values were observed in the water samples stored at room temperature (22ºC). For the water matrix SWL, the samples showed a Log10 reduction of 2.99 and 1.40 at 22ºC and 4ºC respectively and for the water matrix SDW the Log10 values were 2.75 and 0.78 respectively for 22ºC and 4ºC both at the end of 120 days of storage. Samples belonged to BWL stored at 22ºC showed a complete inactivation of the RVA after 90 days of storage (up to 4 Log10 reductions). These samples stored at 4ºC also showed the higher values of log reduction in this temperature (2.44) after 120 days. The presence of other different microorganisms, as well as the distinct physicochemical properties of the samples SWL, SDW and BWL can explain the difference on the Log10 reduction values observed. This can be due to predation and the water composition that can affect the virus survival and/or to promote the virus aggregation to solid particles in the water. Further experiments will involve the study of the stability of the RVA genome by RT-qPCR in order to check the influence of the temperature and time of storage also on the genome persistence. FINANCIAL SUPPORT: CNPQ UNIVERSAL 471755/2011-7; CAPES

EV251 - DISTRIBUTION AND MORPHOLOGIC DIVERSITY OF VIRIOPLANKTON IN ARRAIAL DO CABO, CABO FRIO UPWELLING REGION, RIO DE JANEIRO STATE, BRAZILPedrosa-Macena, L.G.1; Paula, J.E.F.B.1; Pereira, P.S.1; Vogel, V.A.R.1; Nepomuceno, A.M.1; Crapez, M.A.1; Ferreira, D.F.2; Amorim, L.M.F.1; Giongo, V.A.1; Paixão, I.C.P.1



Viruses has been described as the most abundant biological components and dynamic in different aquatic ecosystems, being its greatest reservoir the oceans, where the concentration ranges from 107 to 109 mL-1. However, few virus ecology studies have addressed the assessment of virioplankton community in Brazilian tropical aquatic ecosystems. These is due in large part, by the majority of studies carried out in the area of environmental virology, are focused on just the aspects of public health and disease-causing agents and not the ecological aspects. We carried out a study with the aim of assessing virioplankton distribution and morphological diversity and determining how their variation in the marine waters of Arraial do Cabo, influenced by upwelling events and anthropogenic activities. Subsurface seawater samples were collected from Anjos Bay (AC581) and Forno Bay (AC570), more influenced by anthropogenic activities and the three others sampling sites (AC571, AC582 and AC561) were located in more external portion of AC region, therefore receiving high influence of oceanic environment where upwelling occur and with lesser input of anthropogenic activities. Viral abundances were highest in site AC581 (4.44 ± 2.62 x 10 8.part.mL-1) and AC570 (3.23 ± 1.87 x 10 8.part.mL-1), which were located in the inner bay and more influenced by anthropogenic and less influenced by upwelling (means of 3.33 x 10 8.part.mL-1 and 2.51 x 10 8.part.mL-1, respectively). The lowest viral counts were found in most influenced oceanic seawater located in site AC571 (1.32 ± 0.4 x 10 8.part.mL-1). Sites AC582, AC561, which presented little wastewater input, showed viral abundance mean of 1.4 ± 0.5 x 10 8.part.mL-1 and 1.53 ± 0.28 x 10 8.part.mL-1 respectively. The




highest virioplankton abundances was during summer, ranged from 1.47 to 7.95 x 10 8.part.mL-1, followed by spring with minimum of 1.24 x10 8.part.mL-1 and maximum of 5.7 x 10 8.part.mL-1, coinciding with the seasons in which upwelling phenomenum occurs. Most morphotypes have tails, belonging to Myoviridae, Podoviridae and Siphoviridae. The majority of tailed phages in Arraial do Cabo region were found at AC581, at the most antropogenic influenced area. In addition this study showed that phage represented an important fraction of virioplankton community in Arraial do Cabo coastal region. FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPPI AGIR.

EV254 - ENTERIC VIRUS IN SURFACE WATERS FROM URBAN AREAS IN RIO DOS SINOS WATERSHED, RS, BRAZILStaggemeier, R.; Heck, T.M. da S.; Andriguetti, N.B.; Soliman, M.C.; Souza, F.G. de; Rocha, S.; Faria, N.A.; Henzel, A.; Rigotto, C.; Spilki, F.R.; Almeida, S.E. de M.


The increased population density in urban areas is often reflected in a higher contamination of water resources.The four stream targets of this work are situated in this highly urbanized and industrialized region, within the municipalities EstânciaVelha, Campo Bom, Novo Hamburgo, form the Rio dos Sinos watershed, southern Brazil. These streams are recipients of large amounts of urban and industrial sewage (mostly without any treatments). The aim of this study was the detection and quantification of human adenovirus (HAdV) genomes in water samples collected from the streams EstânciaVelha/Portão (EstânciaVelha and Portão cities), Schmidt (Campo Bom city), Pampa and Luiz Rau (Novo Hamburgo city). In total, 102 water samples (500 ml each) from 17 different sampling points, at were collected bimonthy from September/2012 to to July/13. After, concentration by adsorption / elution, extraction of viral DNA was performed. The virus detection was performed by qPCR using primers with the potential alignment of highly conserved regions of the genome of the virus. According to the results 83.33% (85/102) were positive for the presence of HAdV: Schmidt (91.7%; 22/24), EstânciaVelha/Portão (83.3%; 25/30), Luiz Rau

(83.3%; 20/24), and Pampa (75%; 18/24). Viral load ranged from 1.76 x 103 to 5.11 x 107 genome copies/L. The results suggest intense anthropic contamination of surface water in the region. FINANCIAL SUPPORT: CAPES, FAPERGS, CNPQ, PROJETO MAIS ÁGUA, UNIVERSIDADE FEEVALE.

EV287 - ASSESSMENT OF VIRIOPLANKTON ABUNDANCE AND ECOLOGICAL FACTORS IN GUANABARA BAY, RIO DE JANEIRO STATE, BRAZILSantana-Pereira, P.1; Barbosa, J.E.F.1; Pedrosa, L.G.M.1; Vogel, V.A.R.1; Nepomuceno, A.M.1; Crapez, M.A.1; Ferreira, D.F.2; Amorim, L.M.F.1; Giongo, V.A.1; Paixão, I.C.P.1



Little is known about the ecology of viruses living in seawater columns over tropical estuarine ecosystems, especially the influence of environmental factors controlling their distribution. The aim of this study is to assess for the first time an attempt to elucidate the distribution and abundance of viruses and their relationships with hosts and environmental variables in the eutrophic estuary of Guanabara Bay, Rio de Janeiro, Brazil. Virus abundance ranged from 1.09 to 4.51 x 108 particles•mL-1, being among the highest observed in any brazilian aquatic system examined so far. The highest virus abundance found in this study was in GB537 (average 3.33 x 108.part.mL-1) and GB 566 (2.57 x 108.part.mL-1), which were located in the inner bay and less influenced by marine seawater. In the other hand, the lowest viral counts were found in most influence oceanic seawater located in sites GB557 (average 1.39 x 108.part.mL-1) and GB546 (average 1.45 x 108.part.mL-1). Interestingly, virus and bacterial abundances were strongly correlated (r= 0.70; P <0.01) as well when virus abundance were plotted against chlorophyll-a (r= 0.85; P <0.001). Further correlation analysis showed that virioplankton abundance also revealed high fits to linear models when plotted against phosphate (r= 0.75; P <0.001); ammonia (r= 0.78; P <0.001) and temperature (r= 0.59; P <0.05) and showed an inverse correlation to salinity (r= -0.45; p> 0.05) and conductivity (r= -0.39;




P > 0.05). All investigated phages were tailed phages of the Myoviridae and Podoviridae families, both belonging to the Caudovirales order, which represent the most abundant in nature, and account for 96% of the phages described so far. Understanding the correlation among virus abundance and other biological and physical-chemical measurements consist in an important approach to elucidate the main factors influencing viral abundance and its hosts in tropical estuaries submitted to pollution. FINANCIAL SUPPORT: CNPQ, FAPERJ, PROPI-UFF.

EV328 - PARTIAL CHARACTERIZATION OF NOROVIRUS GI AND GII IN WATER AND SEWAGE SAMPLES FROM BELÉM CITY, PARÁ STATE, BRAZILTeixeira, D.M.1; Spada, P.K. de P.1; Bandeira, R. da S.1; Soares, L. da S.1; Gurjão, T.C.M.1; Morais, L.L.C. de S.1; Sousa, M.S. de2; Mascarenhas, J.D.P.1; Fumian, T.M.3; Gabbay, Y.B.1

1. IEC/SVS/MS - Seção de Virologia do Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000

2. NÚCLEO DE MEDICINA TROPICAL/ UFPA - Núcleo de Medicina Tropical da Universidade Federal do Pará, AV. Generalíssimo Deodoro, 92, Belém - Pará, 66055-240

3. Laboratório de Virologia Comparada e Ambiental/FIOCRUZ - Laboratório de Virologia Comparada e Ambiental da Fundação Oswaldo Cruz Pavilhão Hélio e Peggy Pereira, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - CEP: 21040-900

Enteric viruses excreted in feces from infected individuals dispersed in aquatic environments by sewage discharge. Among these viruses, the norovirus (NoV) is actually considered the main cause of gastroenteritis outbreaks worldwide, resulting from the ingestion of contaminated food and water as well as is also associated with hospitalizations. This research aimed partially characterize the NoV (GI/GII) in different water matrices and in untreated sewage from Belém city. Environmental samples (2 liters) collected in seven points were concentrated on filtering membranes to obtain a final volume of 2 mL. The nucleic acid was extracted by silica method and initially submitted to semi nested RT-PCR (RNA-dependent RNA polymerase) and TaqMan® real time PCR (ORF1/ORF2 junction). The positive samples for both molecular methods

(N=57) were reanalyzed for 5’end ORF2 region by others nested (for GI) and semi nested (for GII) in order to obtain amplicon for genotyping. After the samples were purified using a commercial kit and submitted to molecular characterization in an automated sequencer. The obtained sequences were edited, aligned and compared to others available in gene bank (NCBI) and in the site NoV genotyping tool. Of 57 positive samples by TaqMan® real time PCR and/or semi nested RT-PCR, 53 were retested for 5’end ORF2, since four samples showed insufficient quantity of material which allowed a new analyze, so, in 47.2% (25/53) the NoV genome was detected, of these 12% (3/25) belonging to GI, 24% (6/25) to GII and 64% (16/25) for both. The most frequent GI and GII genotypes were GI.8 (n=8) and GII.4 (n=12), respectively, but others genotypes were also observed with lower incidence as GII.6 (n=3), GII.9 (n=2), GII.12 (n=1), GII.14 (n=1), GI.1 (n=1) and GI.4 (n=2). Due to the low quality of sequences obtained, eight samples could not be genotyped for GI and three for GII. It was verified that in the less rainy period (July to November) the NoV positivity increased and, in the months of highest rainfall (December to June) it decreased. The results obtained in the present study indicate the circulation of NoV GI and GII in aquatic environments in Belem, revealing the degradation that these water bodies have suffered, as a result of poverty or lack of sanitation in our city, allowing input of pathogens in these ecosystems, along with its effluents. According to our knowledge it was the first detection of GI.1 and GI.8 in environmental samples in Brazil. FINANCIAL SUPPORT: FUNDAÇÃO AMAZÔNIA PARAENSE DE AMPARO À PESQUISA (FAPESPA); CNPQ; INSTITUTO EVANDRO CHAGAS/SECRETARIA DE VIGILÂNCIA EM SAÚDE/MINISTÉRIO DA SAÚDE.

EV347 - SEARCHING FOR CIRCO-LIKE VIRUS-BRAZIL (CLV-BR) IN CEREBROSPINAL FLUID AND ENVIRONMENTAL SAMPLESNagasse-Sugahara, T.K.1; Castrignano, S.B.1; Garrafa, P.2; Dergovics, F.L.1; Mehnert, D.U.2; Barrela, K.M.2; Monezi, T.A.2; Barbosa, M.R.3; Garcia, S.C.3; Bonanno, V.M. dos S.3; Kisielius, J.J.1

1. IAL - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 - Cerqueira César, São Paulo - SP, 01246-000

2. ICB/USP - Instituto de Ciências Biomédicas da Universidade de São Paulo, Edifício III USP - Administração




- Av. Prof. Lineu Prestes, 2415 - Butantã, São Paulo - SP, 05508-900

3. CETESB - Companhia Ambiental do Estado de São Paulo, Av. Prof. Frederico Hermann Jr.,345, Av. Prof. Frederico Hermann Júnior, 345 - Alto de Pinheiros São Paulo - SP, 05459-010

Using a metagenomic approach, our group recently discovered two new viruses, which we named circo-like virus Brazil (CLV-BR) strains hs1 and hs2, in a sample of human feces collected in 2003, as published in Virus Research in 2013. The viruses contain a circular DNA genome approximately 2500 nucleotides long encoding a replication initiator protein (Rep) and were characterized by molecular biology and electron microscopy techniques, as well as bioinformatics analysis. In the phylogenetic tree, the viruses did not cluster with members of the already established viral families but with some circo-like viruses identified in reclaimed water and rodent and bat feces. Objective. To investigate the presence of CLV-BR viruses in various types of samples. Material and methods. We tested 11 pools of cerebrospinal fluid samples (55 individual samples) sent to IAL for viral detection; 5 pools of wastewater samples and 5 pools of reclaimed water samples collected between January 2005 and November 2006 (177 samples of each type) and provided by ICB-USP; 2 samples of wastewater (São Lourenço, SP, and Taboão da Serra, SP) and 2 raw water samples (Guarapiranga dam, SP, and São Lourenço spring, SP) collected in December 2013 and provided by CETESB. All samples were analyzed by PCR targeting the Rep gene of both CLV-BRs. Results. Among the samples tested, the target sequence was found in one pool of wastewater samples from ICB-USP (pool-2). This result was confirmed by sequencing and BLAST analysis. Discussion. Metagenomic studies have contributed to the detection of large numbers of new viruses. After the discovery of a new virus, further studies are required to evaluate different kinds of samples where the virus might be detected, to identify its specific host and to determine whether the virus can be associated with disease. The finding of CLV-BR in a pool of wastewater samples collected in 2005 supports the hypothesis that (a) this virus continued to circulate in the state of São Paulo two years after first being detected and (b) it may also have come from human waste.

EV350 - DETECTION OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) AND GYROVIRUS TYPE 2 (AGV2) IN SWINE SLURRYCorrea, R.A.1; Teixeira, T.F.2; Cibulski, S.P.2; Santos, H.F.2; Soliman ,M.4; Staggemeier, R.4; Henzel, A.4; Rigotto, C.4; Spilki, F.R.4; Roehe, P.M.3

1. UFPEL - Universidade Federal de Pelotas, Capão do Leão - RS, 96160-000

2. FEPAGRO - Fundação Estadual de Pesquisa Agropecuária, R. Gonçalves Dias, 570, Porto Alegre - RS, 90130-060



Environmental contamination by swine manure may represent a significant risk to human health. It is therefore important to analyze and detect potential viral contaminants in liquid waste of pigs. In the present study, pig slurry (PS) were tested for the presence of the following viruses: chicken anemia virus (CAV), avian gyrovirus type 2 (AGV2), porcine cirovirus type 2 (PCV2), porcine bocavirus type 1 (PBoV1), Torque teno sus virus 1 and 2 (TTSuV1, TTSuV2). The DNA was extracted with a commercial nucleic acid extraction. Different, previously described quantitative polymerase chain reactions (qPCR) were employed to examine 213 samples of slurry, of which 28 (13,1%) were found to contain PCV2 genome fragments. Nineteen (8,9%) contained AGV2 genome fragments. Only 6 samples (2,8%) were contaminated with both PCV2 and AGV2. CAV, PBoV1, TTSuV1 and TTSuV2 genomes were not detected in the samples examined. These results indicate that AGV2 and PCV2 can be detected in swine manure. Further experiments will be conducted in order to examine whether such samples might also contain infectious virus.

EV413 - HUMAN AND MURINE NOROVIRUS RECOVERY FROM TOMATOES SAMPLESCorrea, A. de A.1; Melgaço, F.G.2; Correa, A.3; Luz, I.2; Ganime, A.C.2; Miagostovich, M.P.2





2. Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Fiocruz

3. Laboratory of Virology, Fluminense Federal University

Norovirus (NoV) is the most important agent of food borne outbreaks of acute gastroenteritis and their identification in foods is difficult due to the food matrix complexity. This study evaluated the efficiency of two viral concentration methods, organic flocculation (skimmed milk) and polyethylene glycol (PEG), for Human NoV GII.4 (NoV GII.4) and Murine Norovirus (MNV-1) recovery from three tomatoes classifications samples (cherry, grape and common tomatoes). For organic flocculation, Glycine 0.05M/Tris-HCl 0.1M buffer was used as elution buffer; for PEG precipitation; Glycine 0.05M/Tris-HCl 0.1M with 3% beef extract buffer, according to the International Organization for Standardization (ISO/TS 15216-1:2013), was applied. All the samples were tested in the whole form; grape tomato samples were also tested sliced. The final concentrate obtained was treated or not treated with 1,3% cationic detergent cetyltrimetylammonium bromide (CTAB) for organic flocculation, and with equal volume of chloroform/butanol (1:1 vol/vol), for PEG precipitation, in order to remove inhibitory substances from the virus extract . The viral RNA was extracted by QIAamp viral RNA mini kit® (Qiagen) and the cDNA produced, using random primers. The viral quantification was performed by real time PCR using primers and probes previously described. For the flocculation method, cherry tomato samples showed low recovery efficiency for NoV GII.4 (5.0%) and MNV-1 (0.6%). Whole/sliced grape and common tomatoes samples presented recovery efficiencies for NoV GII.4 (16.9%, 47.7%, and 33.4%, respectively) and for MNV-1 (2.26%, 4.61%, and 4.05%, respectively). The samples treated with CTAB, showed better recovery efficiency than the untreated samples. When the PEG method was applied, cherry tomato samples showed a 0.06% and 2.1% recovery for NoV GII.4 and MNV, respectively. For the grape tomatoes whole/sliced and common tomatoes, the respective recovery efficiencies were 6.8%, 51.6% and 2.5% for NoV GII.4 and 11.2%, 13.8% and 8.8% for MNV. The results obtained suggest that the organic flocculation can be the method of choice for recovery those viruses from tomatoes samples. Since there is no standardized

methodology for detecting NoV in a broad range of food samples, the results observed in this work provides a new perspective for the study of salad vegetables as a vehicle for viral transmission in outbreaks of gastroenteritis associated to NoV. FINANCIAL SUPPORT: MCTI/CNPQ/ANVISA NO. 23/2012 .

EV530 - SHELLFISH DEPURATION TANKS FOR INACTIVATION OF RECOMBINANT HUMAN ADENOVIRUS AND MURINE NOROVIRUS IN ARTIFICIALLY SEEDED SEAWATERGarcia, L.A.T.; Nascimento, M.A.; Barardi, C.R.M.


Shellfish depuration is a process that aims to eliminate pathogens from mollusks tissues. The seawater disinfection during the depuration process is important and ultraviolet (UV) light treatment is the most used method worldwide. Viral models are usually employed as surrogates of fastidious viruses in viability studies. The aim of this study was to determine methods based on GFP-fluorescence able to detect recombinant adenovirus seeded in seawater and apply these assays to assess the inactivation of rAdV-GFP and murine norovirus in seawater in recirculation shellfish depuration tanks with and without UV light treatment. Kinetics of rAdV GFP-expression was previously measured by UV-spectrophotometer. Flow cytometry (FC) and fluorescence microscopy (FM) were used to determine virus titre and detection limit of each method. Traditional method of plaque assay was done in order to compare its efficiency with FC and FM. Disinfection of seeded seawater was evaluated with and without UV treatment. The time post-infection established for the detection of infected GFP-fluorescent cells rAdV was 24h. FC showed lower sensitivity, when compared with FM, which was similar to plaque assay. Seawater disinfection results showed that rAdV-GFP declined 99.99% after 24h and 48h with and without UV treatment respectively. MNV was completely inactivated after 24h in both treatments. As conclusion, the depuration tanks were effective to inactivate rAdV-GFP and MNV and the UV disinfection treatment accelerated the process. FINANCIAL SUPPORT: CAPES

HUMAN VIROLOGY -HV


Human Virology: HV116

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Human Virology: HV

HV6 - PREVALENCE OF HPV 18 DNA IN CERVICAL SAMPLES OBTAINED FROM WOMEN WITH ALTERED CITOLOGYGomes, A.C.C.; Vago, A.R.; Lopes, L.V.A.; Ebani, E.C.; Andrade, L.O.


Aims: Numerous epidemiologic and laboratory studies have confirmed a strong causal association between a persistent HPV infection and the development of cervical pre-malignant and malignant lesions. Several studies also demonstrated HPV DNA in more than 95% of cervical neoplasia (CIN) and cancer (CC). High-risk species, namely types 16 and, to a lesser extent, types 18, 45, 56, 31, 33, 35, 51, 52, 58, are types that are most frequently found in pre-malignant and malignant lesions, while low-risk types (namely types 6, 11, and rarely 42, 43 and 44) are most commonly associated with benign or condylomatous lesions are rarely identified within these lesions. This study was aimed to investigate the prevalence of HPV18-oncogenic type among a women-group from the Brazilian population which presented cervical cell abnormalities. Methods: By using a Hemi-nested PCR protocol and primers directed to HPV E6-E7 early-transcribed genes, we examined the prevalence of HPV18-DNA in 190 LBC (Liquid-based Cytology) cervical samples obtained from patients from Belo Horizonte, Minas Gerais state, which were diagnosed by Cytopathology analysis as ASCUS (67), CIN1(116) and CIN2/CIN3(07). Results: Almost ninety percent of samples were positive to HPV-DNA (169/190) by using a Nested- PCR protocol and primer-sets directed to the HPV L1 conserved gene sequence. Among the all analyzed samples, the general prevalence of HPV18-DNA was of 16% (31/190), with 15% (10/67) of positivity in ASCUS, 17% (20/116) in CIN1 cases and in 14% (1/07) of CIN2/CIN3 samples. Conclusion: Our results pointed out a relatively high incidence of HPV 18 type infection among the analyzed women population, who were mainly diagnosed as exhibiting low-grade cervical lesions. These patients would require a strict clinical accomplishment and/or treatment, in order to prevent a subsequent cervical lesion progression.

HV4 - LOW - RISK HPVS PREVALENCE IN CERVICAL SAMPLES OBTAINED FROM WOMEN WITH ABNORMALITIES IN THE UTERINE CERVIX BY USING LBC METHODOLOGYCalastri, L.P.; Vago, A.R.; Lopes, L.V. de A.; Fonseca, L.P.; Toppa, N.H.


Aims: Mounting evidence from both laboratory and epidemiological studies, indicate that sexually transmitted HPV infections may be the leading cause of cervical cancer (CC) world-wide. The presence of these HPV types is most often first detected by an abnormal Pap smear, however, a normal Pap smear does not prove the absence of HPV infections. According to the HPV phylogenetic tree, the so-called low-risk (LR) HPVs, namely types 6, 11, and rarely 42, 43 and 44, are most commonly associated with benign or condylomatous lesions on the anogenital tract. This study was aimed to investigate the prevalence of the LR-HPVs 6 and 11 types in a women-group from the Brazilian population which presented cervical low-grade squamous intraepithelial lesions, especially ASCUS and CIN1. Methods: By using a conventional PCR protocol and primers directed to HPV E6-E7 genes, we examined the prevalence of HPVs 6/11 in DNA samples isolated from 190 LBC (Liquid-based Cytology) cervical samples, obtained from patients from Belo Horizonte, Minas Gerais state, which were diagnosed as ASCUS (67), CIN1(116) and CIN2/CIN3(07). Results: Among the analyzed samples, the general prevalence of HPVs 6/11A was of 9% (18/190), with 7% (05/67) of positivity in ASCUS and 11% (13/116) in CIN1 cases. These LR HPVs were not found in any of the HSIL samples. Conclusion: Our results pointed out a relatively high incidence of LR-HPVs 6/11 among the analyzed women population who presented low-grade cervical intraepithelial neoplasia. By considering that 32% of women with CIN1 will exhibit lesion persistence and that, 10% of them will progress to CIN3, these data emphasize the importance of vaccination (especially the quadrivalent one, against HPVs 6, 11, 16 and 18 types) in preventing the establishment of pre-invasive cervical lesions.




HV10 - PREVALENCE OF HPV 45 DNA IN CERVICAL SAMPLES OBTAINED FROM WOMEN WITH CITOLOGY ABNORMALITIESVago, A.R.; de Freitas, G.V.; Lopes, L.V. de A.; Andrade, L.O.; Silva, M.X.


Cervical cancer is one of the most common causes of cancer-related death in women and is associated with persistent infections with a high-risk (HR) subset of human papillomavirus (HPV). According to the HPV phylogenetic tree, the HR HPVs, namely types 16 and, to a lesser extent, types 18, 45, 56, 31, 33, 35, 51, 52, 58, are primarily associated with CIN. Very recently, the preliminary results about immunogenicity and safety of a novel 9-valent HPV L1 Virus-like Particle (VLP) vaccine (Merck) were presented. This vaccine is directed against the L1VLPs from types 6/11, 16, 18, 31, 33, 35, 45, 52 and 58. However, there is a scarcity of data concerning the HPV 45 prevalence among the worldwide women, including the Brazilian ones. Aims: The purpose of this work was to investigate the HR-HPV 45 prevalence among women from Belo Horizonte city, MG state, Brazil. Methods: By using a conventional PCR protocol and primers directed to HPV E6-E7 genes, we examined the HPV45-DNA prevalence in 190 LBC (Liquid-based Cytology) cervical samples. These cases included 67 ASCUS, 116 CIN1 and 7 CIN2/CIN3. Results: HPV45 showed to be the second most prevalent type in each cervical lesion group (ASCUS, CIN1 and CIN2/3), when compared with other HR-HPV tested. Among the all analyzed samples, the general prevalence of HPV45-DNA was of 16% (31/190), with 16% (11/67) of positivity in ASCUS and 17% (20/116) in CIN1 cases. This HR-type was absent among the high-grade lesions. Conclusion: Our results pointed out a relatively high incidence of HPV 45 type infection among the analyzed women, who were mainly diagnosed as exhibiting low-grade cervical lesions. By considering that most of women submitted to the cytology cervical screening are diagnosed as CIN1, our data emphasize the relevance of the HPV45 type inclusion in newly designed vaccines to effectively prevent this HR-type dissemination among the sexually active women.

HV29 - ANIMAL MODEL OF ARTHRITIS MAYARO VIRUS-INDUCEDSantos, F.M.; Oliveira, M.D.; Dias, R.S.; Monteiro, J.M. de C.; de Paula, S.O.

UFV - Universidade Federal de Viçosa, Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa - MG, 36570-000

The Mayaro (MAYV) virus belongs to the genus Alphavirus and the family Togaviridae, and is considered an arbovirus of epidemiological importance for the countries of South America, once that is responsible for causing acute febrile illness, often followed by polyarthritis. In Brazil the MAYV is endemic in the Amazon region, but the risk of emergency in other regions of the country as well as in other continents has been demonstrated by the identification of travelers and animals infected outside the endemic areas. Thus, due to the potential of become a public health problem and the impact that the alphaviral disease has, not only in the human life quality, but also on the economy, the objective of this work is to develop an animal model of disease MAYV-induced for understanding the mechanisms by which arthritis is caused and the determination of the primary sites of infection. We utilized Balb/c mice as experimental models, which were divided into three groups. Being an infected in the right rear footpad and the other subcutaneously in the thorax below the right forelimb, to assess whether there is influence of the route, and the third was the negative control, which was inoculated only PBS. The animals were evaluated for weight loss and clinical signs at 24-hour intervals for a period of 21 days. In addition, the animals were subjected to physical tests that assess muscle strength. Observed that the group infected in the right forelimb lost weight and showed various clinical signs such as ruffled fur, abnormal march, curved body. The animals infected in the rear footpad did not lose weight and also did not show any of these clinical signs. However by strength test performed, we found that both infected groups showed significantly lower mean time compared to control. These results show that infected groups had loss of strength caused by arthralgia, and also that the route of infection is an important factor in determining the clinical signs to be developed by the animals.




HV35 - EVALUATION OF ANOGENITAL HUMAN PAPILLOMAVIRUS INFECTION IN ASYMPTOMATIC MEN FROM RIO DE JANEIRO, BRAZILMenezes, W. da R.1; Afonso, L.A.1; Dobao, E.2; Gouvea, T.D.3; Carestiato, F.N.1; Cavalcanti, S.M.B.1

1. Lab Diagn Virologico, UFF2. Setor De Dematologia Da Santa Casa De

Misericordia3. Setor de DST da UFF

Currently, the genital tract infection by human papillomavirus (HPV) is one of the most prevalent sexually transmitted diseases in the world. However, there are still gaps in the knowledge regarding the etiology of penile cancer, and the natural history of HPV infection in men is not yet fully elucidated. This study aimed to determine the prevalence of HPV infection in penile swab samples, derived from a clinically asymptomatic male population. For this purpose, 261 samples were collected between January 2011 and July 2013 in different institutions in the city of Rio de Janeiro, including a hospital, a dermatology clinic, a university and a metallurgical company. We also analyzed the epidemiological variables of these subjects through the application of a socio-demographic questionnaire to aid in the investigation of possible risk factors. The viral identification was made through the generic and type-specific Polymerase Chain Reaction, and Restriction Fragment Length Polymorphism (when needed) techniques. An overall prevalence of HPV infection was observed in 16.47 % (43 individuals). The most prevalent HPV type was HPV 6 (34.88%), followed by HPV 16 (23.25%), HPV 11 (16.27%), HPV 45 (9.30%) and HPV 58 (2,32 %). Hence, infection was associated with low-risk oncogenic types in 53.66% of the studied individuals, and high-risk oncogenic types were detected in 46.34 % of them. The age of the subjects studied ranged between 18 and 65 years with a mean age of 26.30 years. Among the epidemiological variables, statistical significant results were found for the group of men who have sex with men, for the group who have kept anal intercourse during sexual relationship, and for the group that using condom regularly (p<0.1). We described a lower prevalence of HPV infection among asymptomatic male population than those reported in some recently published studies. We believe that the results may contribute to a clearer view about the circulation of HPV in the general male population, as well

as to the identification of risk factors associated with the epidemiology of HPV infection in our state.

HV42 - SULFATED POLYSACCHARIDE OF ADENANTHERAPAVONINAINHIBITS HERPES SIMPLEX AND POLIOVIRUSIN HEP-2 CELLSGodoi, A.M.2; Lopes, N.2; Rechenchoski, D.Z.2; Faccin-Galhardi, L.C.2; Nozawa, C.2; Ricardo, N.M.P.S.1; Linhares, R.E.C.2



The herpes simplex 1 (HSV-1), member of Herpesviridae family, is an enveloped double-stranded DNA virus that affects various age group, causing lesions in the oro-facial region. The drug of choice for the disease treatment is the acyclovir(ACV), but its continued use can select resistant strains. Poliovirus (PV), member of Picornaviridae family, is a non-enveloped positive single-stranded RNA virus, known as the causative agent of poliomyelitis, a disease that affects the central nervous system, causing flaccid paralysis. There is no effective chemotherapy for its treatment. Several studies report the use of natural products with antiviral activity, including sulfated polysaccharides. This work evaluated the antiviral effect of a sulfated polysaccharide of Adenanthera pavonina (SPLSAp), in HEp-2 cells, against HSV-1 and PV-1. The 50% cytotoxiticy of the polysaccharide (CC50) was carried out by MTT method and the antiviral activity by plaque reduction assay (PRA), using different protocols of treatment. The SPLSAp CC50 was determined as 500µg/ml.A 50% inhibitory concentration (IC50)of 15µg/ml with a selectivity index (SI) of 33.3 were found for HSV-1, and for PV-1, an IC50 of 1.18 µg/ml and SI of 423.73. The inhibition of HSV-1 was demonstrated by the time-of-addition and time-of-removal tests, with the highest percentage of viral inhibition (%IV) of 100% and 99.3%, respectively, at the concentration 200 µg/ml. A maximum inhibition of viral protein synthesis was also demonstrated by the immunofluorescence(IF) assay at the highest concentration(200µg/ml). The HSV-1DNA synthesis was totally inhibited by SPLSAp at 25µg/ml as demonstrated by PCR.The combination index (CI)of




SPLSAp with ACV showed an antagonistic effect to the reference drug. The maximum %IV for PV-1 was found at the time 0 hour of infection and the SPLSAp also showed virucidal effect of 74.1% at the highest concentration tested (100 µg/ml). By IF assay the SPLSAp inhibited 100% of PV-1 at 100 µg/ml, in a dose-dependent response. These results suggested that the maximum inhibitory effect of SPLSAp in the replication of HSV-1 occurred after the step of viral penetration and for PV-1 in the initial stages.Besides, the SPLSAp low toxicity and high selectivity make the compound a potential candidate for further studies in the control of these infections.

HV44 - SEROPREVALENCE OF DENGUE IGM AND IGG ANTIBODIES AGAINST DENGUE VIRUS AMONG HEALTHY INDIVIDUALS IN JUIZ DE FORA, MINAS, BRAZILSiqueira, T.R.1; Mendonça, L.A.1; Veiga, R.2; Fernandes, G.C. Coelho, L.F.L.3; Drumond, B.P.1


2. LAWALL - Laboratório de Análises Clínicas em Juíz de Fora, Av. dos Andradas, 341 - Centro, Juiz de Fora - MG, 36036-000


Dengue virus (DENV) infection is the most important arboviral disease in the world. Dengue viruses are classified into four antigenically and genetically distinct serotypes designated DENV-1 to DENV-4. DENV infection may result in a range of disease,ranging from acute, febrile illness to severe, life-threatening hemorrhagic disease. Each serotype can cause human disease and mortality. Infection with one DENV serotype is believed to induce protective immunity against homotypic infection. However, immunity to one DENV serotype may be related to more severe disease in heterotypic infection through a process believed to be associated with antibody-dependent enhancement (ADE) of infection. Juiz de Fora (JF) is a medium size city and considered the regional capital and the main reference for health care of macro region Zona da Mata, in Minas Gerais, Brazil. JF has been facing dengue epidemics since 2010, with reports of

severe cases and deaths. Previous studies of our group demonstrated de co-circulation of DENV-1 and DENV-2 and data from City Hall also indicated the introduction of DENV-4 in JF. Given the hyperendemicity of DENV and the lack of data regarding seroprevalence of dengue in healthy population in JF, the aim of this work was to perform a serological study to check the presence of anti-DENV antibodies in healthy persons living in JF, Minas Gerais. Whole blood samples were collected from 340 individuals from September to October/2013 and from February to May/2014. A rapid immunochromatography test (Dengue Duo Cassete PanBio kit) to detect IgM and IgG antibodies to DENV in human serum samples was used. From 340 samples, 16.17% (55 samples) were positive: 19 samples were IgM positive and IgG negative, 12 samples were IgM negative and IgG positive and 24 samples were IgM positive and IgG positive. This data indicated a high seroprevalence in individuals living in Juiz de Fora, besides the occurrence of primary and possible secondary infections in individuals in this city. Further experiments will be performed in order to check the presence of neutralizing antibodies and the presence of antibodies that may facilitate DENV infection in a heterotypic infection through ADE like mechanism. Information about the DENV circulation as well population immune status are important factors for dengue control.

HV49 - PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION AND PHYLOGENETIC ANALYSIS OF HPV-16 E6 VARIANTS AMONG INFECTED WOMEN FROM NORTHERN BRAZILSilvestre, R.V.D.1; Lopes, B.P.T.2; Sousa Jr, E.C.1; Passetti, F.2; Ferreira, C.G.M.2; Mello, W.A.1


2. INCA - Instituto Nacional do Câncer, Praça Cruz Vermelha, 23, Centro, Rio de Janeiro - RJ, 20230-130

The main cause of cervical cancer in the world is high risk human papillomavirus infection (mainly represented by HPV-16 and HPV-18), that are associated to the development of malignant transformation of the epithelium. HPV prevalence exhibits a wide geographical variability and HPV-16 variants have been related to an increased risk of developing cervical intraepithelial lesion. The aim of this study was to describe DNA-




HPV prevalence and HPV-16 variants among a women population from Northern Brazil. Methods: One hundred and forty three women, during routine cervical cancer screening, at Juruti Project, fulfilled an epidemiological inquiry and were screened through a molecular HPV test. HPV-16 variants were determined by sequencing the HPV-16 E6 open reading frame. Results: Forty two samples were considered HPV positive (29.4%). None of those had abnormal cytology results. HPV prevalence varied between different age groups (Z(U) = 14.62; p = <0.0001) and high-risk HPVs were more frequent among younger ages. The most prevalent type was HPV-16 (14%) and it variants were classified, predominantly, as European (87.5%). Conclusions: HPV prevalence in our population was higher than described by others and the most prevalent HPV types were high-risk HPVs. The European HPV-16 variant was the most prevalent among HPV-16 positive samples. Our study reinforces the fact that women with normal cytology and a positive molecular test for high-risk HPVs should be submitted to continuous follow up, in order to verify persistence of infection, promoting an early diagnosis of cervical cancer and/or its precursors.

HV53 - FIRST COMPLETE GENOME SEQUENCE OF SAINT LOUIS ENCEPHALITIS VIRUS (SLEV) ISOLATED FROM A HUMAN HOST IN BRAZILVedovello, D.1,2; Drumond, B.P.3; Rafael, E.4; Ullmann, L.S.2; Favaro, E.A.1; Terzian, A.C.B.1; Figueiredo, L.T.M.5; Teixeira, M.M.4; Araújo Jr, J.P.2; Rahal, P.6; Nogueira, M.L.1

1. FAMERP - Faculdade de Medicina de São José do Rio Preto,Av. Brigadeiro Faria Lima, 5416 - Vila São Pedro, São José do Rio Preto - SP, 15090-000





6. UNAERP - Universidade de Ribeirão Preto, Av. Costábile Romano, 2201, Ribeirania. Ribeirão Preto - SP, 14096-000

St. Louis encephalitis virus (SLEV), a member of the Flaviviridae family, Flavivirus genus, is a causative agent of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the first outbreak of SLEV in Brazil was identified only in 2007, concomitant to a Dengue virus (DENV), serotype 3 outbreak. This finding, along with other reports, indicates that SLEV circulation in Brazil is largely unknown, and there may be epidemiological implications of the co-circulation of SLEV, DENV and other flaviviruses in Brazil. Here, we describe the first complete genome sequence of a SLEV strain isolated from a human patient in Brazil, strain BeH 355964. Phylogenetic analysis was performed to determine BeH 355964 genotype using the full-length genome and the envelope (E) gene sequences, separately. Both analyses classified BeH 355964 as within genotype V. Although the number of single gene sequences available is greater (such as the E gene), but the complete genome phylogenetic shell offers best supports and provides further information about the virus.

HV57 - PROTEOME CHANGES IN A549 CELLS INFECTED WITH HADV-40Guissoni, A.C.; Parente, A.F.; Badr, K.; Souza, K.; Soares, C.; Cardoso, D.


The viral infection may cause a lot of modifications to the host cells. Determination of these changes can be observed from cell-virus relationship which is not important only in the direction of viral biology, but also at the cellular, which support to elucidate of various phenomena, including the discovery of new targets for antiviral therapy. In the context of viral infection, which considered one of the fundamental possible aspects that lead to protein changes in functional levels for many cells such as, cell signaling pathways, protein degradation, apoptosis, and cytoskeletal rearrangement. These proteins are extremely important since it responsible for many of biological functions of the cell. Thus, the viruses are evolved to reverse cellular proteins for their benefit, which includes maintenance, replication and proper release. For the study of these protein changes that induced by viruses, proteomics analysis, which has been widely used to study these changes, was our




methodology tool that used in this study. In this context, we infected A549 cells (human lung carcinoma) with HAdV-40 to observe the cellular proteins differentially which expressed after viral infection (treated) versus to uninfected cells (control). After this, the proteins were extracted by osmotic lysis and quantified by the Bradford method in Nanodrop, followed by digestion. The extracted peptides were separated on the LC system in two dimensions, nanoUPLAC (Waters) coupled to a mass spectrometer Q-TOF (Syanpt, Waters). The ionization of the peptides was performed on nano-eletronspray source in positive mode (nanoESI+) and examined by MS, aiming to identification and quantification of proteins. 321 differentially expressed proteins were observed, 202 from the treated and 119 from control. The differentially expressed proteins of the treated were classified into the following functional categories: regulation of cellular immunity, mechanisms of signal transduction, cellular metabolism, protein degradation, cell organization, cell cycle, and DNA processing. These data suggest that, the infection of A549 cells with HAdV-40 caused changes in the cellular machinery and prioritizing pathways that promote efficient viral progeny.

HV58 - MOLECULAR AND SEROLOGICAL INVESTIGATION OF DENGUE INFECTION, IN JUIZ DE FORA, MINAS GERAISBotelho, J.1; Mendonça, L.A.1; Siqueira, T.R.1; Sacchetto, L.P.1; Rezende, I.M.1; Fernandes, G.C.1; Kroon, E.G.2; Drumond, B.P.1



Dengue is the most important arboviral disease caused by Dengue virus (DENV), family Flaviridae, genus Flavivirus. DENV comprises four genetically and antigenically distinct serotypes, termed DENV-1 through DENV-4. Infection by any DENV serotype can cause a wide range of clinical symptoms that vary from classic dengue to life-threatening syndromes characterized by hemorrhage and capillary leakage, dengue hemorrhagic fever and dengue shock syndrome. Brazil has the highest incidence of dengue cases in the Americas and Minas

Gerais state often presents high indices of dengue. The city of Juiz de Fora, MG, has been facing dengue epidemics since 2009/2010, with the record of severe cases and deaths. This work aimed to investigate the circulation of dengue virus (DENV) in clinical samples from patients with clinical symptoms of dengue, in 2012 and 2013, using molecular and serological approaches. Samples of serum from patients were used for total RNA extraction, and the total RNA was used for detection of DENV by RT-PCR. Clinical samples from patients who had fever for up to six days were also tested for Dengue virus NS1 protein. Furthermore, analysis of patient records was performed in which we could check the day the fever began, the hematological data (hematocrit, total leukocyte count, and platelet count) and serologic response (IgM and/or IgG). A total of 166 serum samples were analyzed for molecular detection, being six DENV positive (one serum sample was DENV-1 positive and five serum samples were DENV-2 positive). Sixty clinical samples were tested for NS1 and 11 (18.33%) were positive. From the analysis of patient records 39 (29.32%) were IgM positive, nine (6.77%) were IgG positive and 15 (11.28%) were IgM and IgG positive. In addition, from 109 patients hematological data were available. Among these, 18 (16.51%) patients had less than the reference value, 32 (29.35%) developed leukocyte hematocrit below 3.500/mm3 and 25 (22.94%) had platelet counts below the reference value. The results indicated the co-circulation of serotypes 1 and 2 in Juiz de Fora and the combination of molecular and serological tests allowed the identification of 50 patients in the acute phase of the disease and 22 in the convalescent phase. The knowledge of viral types circulating in the municipality and the diagnosis of dengue in patients using different techniques constitute valuable information to control dengue. Financial support: FAPEMIG, CNPq, CAPES, UFJF, PROPESQ/UFJF.




HV59 - MOLECULAR CHARACTERIZATION OF HEPATITIS C VIRUS IN END-STAGE RENAL DISEASE PATIENTS UNDER HEMODIALYSISde Carvalho, I.M.V.G.1; Alves, R.2; Bernardino, J. de S.T.2; Feldner, A.C. de C.A.2; Ferraz, M.L.C.G.2; Silva, A.E.B.2; Filho, J.R.C.2


2. Departamento de Gastroenterologia/UNIFESP - Universidade Federal de São Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-0

Several new direct-acting antiviral agents are in development or already approved for the treatment of chronic hepatitis C virus (HCV) infection. The effectiveness of many of these drugs is still dependent on whether resistance-associated variants pre-exist before therapy. Certain clinical conditions can change immunological characteristics of the host and modify genetic features of viral populations. The aim of this study was to perform a molecular characterization of HCV isolates from end-stage renal disease (ESRD) patients under hemodialysis (HD). Nested PCR and Sanger sequencing were used to obtain genetic information on the NS5B region from a cohort of 73 treatment-naïve subjects with chronic HCV infection was selected (31 ESRD-HD and 42 with normal renal function). Genomic sequences from the Los Alamos databank were used for comparative analysis. Bioinformatics methodologies such as phylogenetic reconstructions and informational entropy were used to analyze datasets separately by geographical location, HCV genotype and renal function status. ESRD-HD patients presented HCV genotypes 1a (n=14), 1b (n=12), 2a (n=1), 2b (n=2) and 3a (n=2). Control subjects were infected with genotypes 1a (n=9), 1b (n=21), 2b (n=4) and 3a (n=8). Each genotype exhibited typical branching patterns, already described. Separation of HCV subtype 1a into two distinct clades was observed in most Brazilian sequences, in contrast to subtype 1b isolates. The entropy analysis of genomic sequences from the ESRD-HD group reveled two amino acid peak positions which were absent in most control sequences. These amino acid positions are closely related to a particular epitope for cytotoxic T lymphocytes and T helper cells recognition (CCDLDPQARVAI; positions 2663–2673 in the H77 reference sequence). The identification of specific mutant epitopes within the NS5B HCV protein

in patients on hemodialysis can be related to host immunological features and geographical patterns.

HV64 - DEVELOPMENT OF REAL TIME PCR PLATFORM FOR VIRAL MENINGOENCEPHALITIS DIAGNOSIS AND MOLECULAR EPIDEMIOLOGY OF MENINGOENCEPHALITIS AT CHILDREN HOSPITAL IN MINAS GERAIS STATE, BRAZILOliveira, D.1; Candiani, T.2; Almeida, G.1; Luiz, A.P.F.1; Alvarenga, P.1; Castro, F.1; Abrahão, J.1; Coimbra, R.1; Kroon, E.1


2. FHEMIG - Fundação Hospitalar do Estado de Minas Gerais, Alameda Vereador Álvaro Celso, 100, Santa Efigênia, Belo Horizonte - MG, 30150-260

Meningitis is a worldwide disease, which main etiologic agents are viruses, bacteria and fungus. Viruses are the main causes of central nervous system (CNS) infection around the world. In Brazil, there are 11,500 cases / year putative reported cases of viral meningitis. However, for most cases there is no identification of the etiological agent. Human Herpesvirus 1 (HHV-1), Human Herpesvirus 2 (HHV-2), Human Herpesvirus 3 (HHV-3), viruses from the Enterovirus genus (ENTV) and viruses from the Flavivirus genus(FLAV) are the main etiological agents of viral meningitis. The objective of this work was to develop a real-time PCR platform for HHV 1, HHV 2, HHV 3, ENTV and FLAV diagnosis in cerebrospinal fluid (CSF) of patients with clinical suspect of viral meningitis and apply that platform on CFSs collected of hospitalized children from Hospital InfantilJoão Paulo II (HIJPII). Primers were designed (or based on literature), targeting conserved regions in the genome of these virus (HHV 1, HHV2 and HHV 3)or genus (ENTV and FLAV). The viral isolates HHV 1 EK, HHV 2 (ATCC VR 590), HHV 3 HC05, Enterovirus C (Sabin) and Yellow fever virus(17D strain) were used during the initial primer tests. Then 57 samples of CSFs(10ul -140 ul) from patients with meningoencephalitis of probably viral etiology were collected from 2010 to 2013 at HIJPII for tested in our platform.The reactions used in this platform showed high efficiency114% for HHV-1 and HHV-2, 113% for HHV -3, 110% ENTV and 92% FLAV.When an analytical sensitivity test was performed, our data demonstrated




high sensitivity, with detection limit up to 1 PFU/µl for HHV 1, ENTV and FLAV. When the samples were tested our platform was able to identify the viral etiologic agent in 71.9% of cases (41 cases). Among these positive samples 29(50.87%) were positive for ENTV, 6 for HHV-3, 5 for FLAV (4 DENV-2 and 1 co-infection DENV-2/DENV-3) (8.7%) and 1 positive for HHV-1/2 (1.7%). ENTV was most prevalent agent what is consistent with others works in the literature. The detection of DENVs in 8.7% of cases is an important result because these are emerging neurotropic agents. Finally this platform could be useful for the rapid diagnosis of viral meningoencephalitis. The identification of viral agent of neurologic infection is important for therapy, severe cases surveillance and viral emerging agents surveillance.

HV65 - DENGUE VIRUS 3 GENOTYPE I INFECTION WITH NEUROLOGICAL MANIFESTATIONS, BRAZILOliveira, D.1; Machado, G.3; Almeida, G.1; Abrahão, J.1; Campos, M.2; Kroon, E.1


2. FIOCRUZ MINAS - Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002

3. Hospital Risoleta Tolentino Neves, Rua das Gabirobas, 1 - Vila Clóris, Belo Horizonte - MG, 31744-012

Dengue virus(DENV) is the most important mosquito-borne disease of humans. Worldwide approximately 2,5 billion of people live in areas with risk of dengue transmission and estimated 50 million dengue infections occur annually. In Brazil more than 200.000 cases were reported in the first 45 days of 2013 (105,5 cases/100.000 habitants).The distinct DENV (1,2,3 and 4) might be usually associated to a systemic and dynamic disease. Among the plethora of clinical symptoms caused by DENV, neurological manifestations has been undernotified or neglected. In this work we describe a case report of a patient presenting neurological manifestations likely associated to DENV-3.This case occurred in Belo Horizonte, Minas Gerais State, Brazil. In October 2012, our lab was contacted by a local Hospital to perform the diagnosis of a 21 old patient that presented dengue symptoms and also neurological symptoms. The patient stayed in hospital for 27 days presented wide range of

neurological manifestation as confusion and behavior alterations. With the aim of characterizing the etiological agent, cerebrospinal fluid (CSF) samples from the patient were sent to our laboratory. The samples were submitted to RNA extraction (Viral RNA QIAamp Extraction Kit, Qiagen, USA) followed by reverse transcription using random primers (MMLV, Promega, USA). The cDNA was used as the template in a real time PCR designed to amplify the NS5 region of virus of genus Flavivirus. Specific amplification in the real time PCR with a melting temperature of 79,5ᴼ C was observed for the sample (CSF). To identify the member of genus Flavivirus that was the etiological agent of such infection, a specific PCR for Dengue virus was performed confirming DENV as the etiological agent.The optimal alignment of the C-prM region using ClustalW and MEGA version 4 showed high identity among the nucleotide sequences of the sample and other DENV-3sequences deposited in GenBank. Phylogenetic trees of the C-prM region were constructed and showed that the DNA fragment sequence clustered with other sequences of DENV-3genotype I from Brazil, Asia and Central . Neurological manifestations of DENV are possibly underreported in Brazil, this report serves as an alert to the importance of DENV diagnosis in CNS infections. The number of cases and the impact in public health maybe more important than suspected because the viral pathogen is not identified in most cases of CNS viral infection in Brazil.

HV66 - GENOTYPING AND PHYLOGENY OF DENGUE VIRUS 1 AND DENGUE VIRUS 4 IN DIVINÓPOLIS/MG, BRAZIL, 2013Dutra, K.R.1; Corrêa, V.M.1; Lopes, D.O.1; Drumond, B.P.2; Nogueira, L.M.3; Ferreira, J.M.S.1; Santos, L.L.1

1. Laboratório de Biologia Molecular da Universidade Federal de São João Del-Rei (Campus Centro Oeste Dona Lindu)

2. Laboratório de Virologia da Universidade Federal de Juiz de Fora

3. Laboratório de Pesquisa em Virologia, Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do Rio Preto (FAMERP)

Dengue is a disease transmitted to humans through the bite of infected female mosquitoes of the genus Aedes. The Dengue virus (DENV) is an enveloped virus belonging




the family Flaviviridae with four distinct serotypes: DENV1, DENV2, DENV3, DENV4. Currently, these four serotypes are circulating in almost all states of Brazil and an increase in genetic variability between each serotype has been described. In Minas Gerais, the number of cases has been grown, therefore the genotyping will provide information on dengue virus distribution in human populations over time and place. Using phylogeny studies is possible to identify mutations and recombination events accumulated over the years. The most common gene used in studies of molecular epidemiology of DENV is E gene, due to the importance of its product in the binding and entry of the virus into the cell, especially in the humoral response of the human host. Mutations in this gene may be related to the increased virulence of viral strains. In this study, 100 samples of whole blood from patients with suspected dengue, from Divinópolis/MG , in the year 2013 were analyzed. Viral RNA was extracted from the serum with QIAamp ® Viral RNA Kit ( QIAGEN ®, USA) and viral detection was performed by reverse transcription (RT - PCR). The serotyping was performed by sequencing the viral fragment of 511 pairs obtained by RT - PCR. The sequences obtained were compared to reference sequences deposited in GenBank using the BLASTN software. Of the 100 samples analyzed, 26 were positive for DENV. 12 samples were sequenced, ten were positive for DENV 1 and two positive for DENV 4. One second RT - PCR was performed with specific primers for each serotype to amplify the envelope protein gene. Phylogenetic analyzes are being performed by MEGA 6.0 software. These results explain the epidemic occurred in the city, since DENV1 do not circulate in the municipality for almost 10 years, and there was no report of circulation of DENV4 in Divinópolis history before 2013. The results of this work will improve the evolution analyze and flow of DENV in Brazil and its relations with DENV isolated from other continents, besides the determination of circulating genotypes in Divinópolis. This study can be applied to surveillance programs of health services and preventive interventions. This study will also provide the screening of molecular markers, associating them with the pathogenicity and immunogenicity of DENV. Financial support: FAPEMIG

HV75 - DETECTION OF NOROVIRUS RECOMBINANT STRAINS ISOLATED FROM ACUTE GASTROENTERITIS CASES IN BRAZILFumian, T.M.; Alves, R.; Assis, M.; Simões, M.; Andrade, J.; Leite, J.P.G.; Miagostovich, M.P.

FIOCRUZ MINAS - Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715 - Barro Preto, Belo Horizonte - MG, 30190-002

Norovirus (NoV), a major cause of acute gastroenteritis (AGE) outbreaks worldwide, are constantly evolving. This ability is reflected in the speed and efficiency with which these viruses spread and remain in human population. The present study aimed to investigate the presence of recombination events – one of the main driving forces that shapes NoV evolution – among different genotypes in Brazil. Fecal samples were tested using primers targeting region B (ORF1) and region D (ORF2) of NoV genome by using specific primers. Samples yielding positive results with both methodologies were subjected to investigate the possibility of a recombination event, and ORF1/2 junction region was amplified with primers Mon 431/432 and G2SKR. Amplicons obtained were purified and sequenced using the Big Dye Terminator Reaction Kit® and an ABI Prism 3130xl DNA sequencer. Assignment of the strain to specific NoV genotypes was made according to the Noronet (http://www.rivm.nl/mpf/norovirus/typingtool) and the phylogenetic analysis was performed using MEGA version 5.05. In total, we analyzed 37 samples, and found recombination events in 19 (51%) of these. We detected eight different combinations of NoV recombinant genotypes, being: GII.Pe/GII.4 (n=12); GII.P7/GII.6 (n=7); GII.P7/GII.14 (n=1); GII.Pe/GII.17 (n=2); GII.P13/GII.17 (n=1); GII.P21/GII.3 (n=1); GII.Pg/GII.12 (n=5); GII.P16/GII.3 (n=2). These results were confirmed by phylogenetic and Simplot analysis with references strains, and identified the breakpoint located near the ORF1/2 overlap. Our study revealed a high circulation of NoV recombinant strains in Brazil, revealing that this type of event is not uncommon among divergent genotypes. In addition, we highlight the importance of this mechanism for successfully NoV maintenance and spread in human population. To our knowledge, this is the first description of a large study concerning NoV recombination from cases of AGE in Brazil.




HV76 - MOLECULAR INVESTIGATION OF DENGUE VIRUS INFECTION IN GOIANIA, GOIAS, BRAZIL, 2012–2013Oliveira, A.C.R.; Guimarães, V.N.; Cunha, M. dos P.; Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Almeida, T.N.V.; Fiaccadori, F.S.

IPTSP/UFG - Instituto de Patologia Tropical e Saúde Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor Universitário, Goiânia - Goiás, 74605050

Dengue is an important public health problem worldwide and it is spread for tropical and subtropical countries. Dengue virus (DENV) is an arbovirus classified in four serologically related viruses designated as DENV-1-4. Since the introduction of DENV-1 in Brazil, the epidemiological scenario is characterized by the co-circulation of four DENV, resulting in successive epidemics with alternating prevalence among serotypes. The pathogenesis of this infection has been associated with the occurrence of a cross-serotype immune response in secondary infections. Thus, the identification of serotypes circulating as well as the characterization of the genomic variants have been considered important perspectives on disease control, since the introduction of new variants can be a risk factor for severe dengue. In this context, the aim of this study was perform a molecular investigation of DENV infection in Goiania, Goias, during the epidemic period 2012- 2013, and the identification of serotypes/genotypes of the virus. 278 samples were collected from patients with symptoms of dengue fever in basic health centers, which were submitted to RNA extraction followed by a hemi-nested RT-PCR for the C-prM genome region to detect the viral RNA and to identify the serotype. The molecular characterization was performed for all RNA positive samples by nucleotide sequencing of the junction region E/NS1 and phylogenetic analysis. It was observed a positivity rate of 20.5% (57/278). From the 57 DENV samples, 18 (31.6%) were DENV-1 and 39 (68.4%) DENV-4, which is in agreement with findings from Brazil and Goias that demonstrated this co-circulation with a DENV-4 predominance in epidemiological surveillance of 2013. Phylogenetic analysis classified the DENV-1 samples as genotype V, with two different clades, which may reflect the occurrence of genetic variants with differences in evolutionary history. For DENV-4 samples, classified as genotype II, a phylogenetic proximity between the

isolates in this study and other isolates from Brazil and Latin America was observed. Therefore, molecular monitoring is important to establish a complete data base for surveillance studies in hyperendemic population, collaborating with investigations of the virus dynamics and the prospective pattern of future epidemics.

HV79 - HANTAVIRUSES VERSUS DENGUE DISEASE: RETROSPECTIVE DIAGNOSIS OF A HANTAVIRUSES CASE CLINICALLY MISTAKEN WITH DENGUE DISEASEda Costa, V.G.; Silva, A.C.M.; Floriano, V.G.; Moreli, M.L.


Viral diseases result in nonspecific clinical picture. In this context, we can include diseases caused by dengue virus (Family Flaviviridae) and hantavirus (Family Bunyaviridae). The dengue causes a flu-like clinical picture, or a more severe disease, and it is constantly associated with epidemics in urban areas. Conversely, the hantaviruses is primarily a rural disease, it is transmitted to man by inhalation of aerosols waste from infected wild rodents. The cases of hantaviruses occur sporadically, and, as well as dengue fever, there is seasonal distribution. In view of this, our objective was to report a case of hantavirus disease that went unnoticed in municipality of Jataí-Goiás. Thus, the present study was approved by the ethics committee of Federal University of Goiás (n°348/2010). Blood samples of 202 patients suspected dengue were screened, retrospectively, which were collected between the years of 2012 and 2013. Thus, the samples were stored in the serum bank of the medical center municipal health from Jataí. For the processing of the samples was used an in house ELISA using a recombinant N protein of Araraquara virus. Thus, was found a case with positive anti-hantavirus IgM antibody. The patient was a 44-year-old Brazilian male with fever and myalgia presented. Sample collection occurred on March 18, 2012. For this case the optical density was 0.92, which was obtained in triplicate, while the cut-off value was 0.12. The detection of anti-dengue IgM antibodies by ELISA was negative. Regarding to laboratory data obtained, during hospital admission, was found abnormal, only mild thrombocytopenia (115x103/mm3), but two days after was observed elevated hematocrit (64%), thrombocytopenia (89x103/mm3) and leukocytosis, however without left




shift (segmented neutrophils=11218/mm3). Finally, after supportive symptomatic treatment the patient progressed to the stage of convalescence. The record of this case shows the difficulty in the laboratory diagnosis of hantavirus, since that laboratory tests require a qualified diagnostic referenced system, as the central public laboratories (LACEN). However, this may delay diagnosis or discourage that samples are sent to diagnostic centers, mainly because of hantaviruses to be a severe acute disease. In conclusion, this case of hantavirus disease, retrospectively diagnosed, serves as a warning and indicates that the number of cases of disease can be been significantly underestimated in the country.

HV81 - INFLUENZA VIRUS AND RESPIRATORY SYNCYTIAL VIRUS INFECTIONS AMONG EXACERBATED ASTHMATIC CHILDREN FROM GOIANIA-GOIASCastro, Í. de A.1; Costa, L.D.C.2; Oliveira, A.C.R.1; de Souza, K.M.C.1; Cardoso, D. das D. de P.1; Souza, M.B. de L.D.1; Fiaccadori, F.S.1

1. IPTSP/UFG - Instituto de Patologia Tropical e Saúde Pública/ Universidade Federal de Goias, Rua 235 - s/n, Setor Universitário, Goiânia - Goiás, 74605050

2. FM/UFG - Faculdade de Medicina da Universidade Federal de Goias, Faculdade de Medicina, 235 c/ 1a. s/n, S. Universitário,Goiânia - Goiás, 74605-020

Acute respiratory infections (ARIs) are among the most significant causes of morbidity and mortality in early childhood worldwide. Many aetiological agents are related with ARIs, although more than 80% of these infections are caused by viruses, mainly by Influenza Virus (FLU) and Respiratory Syncytial Virus (RSV). The symptoms range from dry cough, wheeze, runny nose and fever to severe episodes of pneumonia. Asthma is a chronic condition that affects a large number of children and its emergence has been associated with an interaction of genetic predisposition and environmental factors. Furthermore, episodes of asthma exacerbation, clinical condition with rising prevalence over the past few years, may be related to respiratory viral infections. Considering ARIs as a key factor of asthma exacerbations, there are few studies in Brazil reaching the pediatric population. In this context, this study aimed to investigate the occurrence of FLU and RSV infections

in asthmatic and non-asthmatic pediatric population from Goiania-Goias, as well as to evaluate the possible role of these infections in asthma exacerbations. The study enrolled four to fourteen-old children from four healthcare centers, divided into three groups according to the clinical symptoms and the physician’s evaluation: stable asthma, exacerbated asthma and just ARI. Between August/2012 through August/2013, 225 nasal samples were collected (aspirates – 193, nasal swabs – 32). For the molecular screening, a Multiplex Nested-PCR protocol was performed, using specific set of primers targeting the nucleoprotein gene for FLU (FLUA, B and C) and the F gene for RSV (RSVA and B). It was observed a global detection rate of 5.77% (13/255), being FLUA and RSVA the most prevalent, with five (5/13 – 38.46%) and four (4/13 – 30.76%) positive samples respectively. Most of the positive samples belonged to the group of exacerbated asthmatic children (7/92 – 7.6%) and no coinfection cases were detected. The results indicated the circulation of viral pathogens among asthmatic children, especially among asthma exacerbation cases, although the comparison between the population groups was not statistically significant. Hence, further analysis with broader assays including other frequent respiratory viruses will improve the knowledge of viral ARIs in this specific population, and help the establishment of new treatment guidelines.

HV91 - VIRUS SURVEILLANCE IN PATIENTS WITH CLINICAL DIAGNOSIS OF DENGUE FROM A MEDIUM-SIZED CITY OF RONDONIA STATE, BRAZILPereira, W.V.E.G.; Gusmão, A.F.; Guioti, F.; Ferreira, A.C.; Mondini, A.

FCFAR/UNESP - Faculdade de Ciências Farmacêuticas da Universidade Estadual de São Paulo, Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902

The infection by any of the four serotypes of the dengue virus (DENV 1-4) can be asymptomatic or produce a disease that ranges from a mild febrile illness to life threatening hemorrhagic manifestations. In Brazil, the majority of dengue disease diagnostics are obtained by serology and clinical and epidemiological criteria. Virus isolation, identification of DENV by polymerase chain reaction or the detection of the nonstructural protein 1 are usually performed in reference laboratories for a limited number of cases. No further tests to detect other




arthropod borne viruses (arboviruses) are performed in febrile patients. In our study, we are testing samples with clinical diagnostic of dengue from a medium sized city from the west part of Brazil for DENV 1-4 and other flaviviruses, along with orthobunyaviruses and alphaviruses. We collected blood samples from febrile patients that had clinical diagnostic of dengue from August 2013 to January 2014 in Ji-Paraná (Rondonia), which is allegedly an area with the circulation of several arboviruses. Viral RNA was extracted and a Multiplex-Nested-RT-PCR with genus-specific primers to detect members of the Flavivirus, Alphavirus and Orthobunyavirus genera and species-specific primers to identify DENV 1-4, yellow fever virus, mayaro virus, oropouche virus and seven other viruses was performed. We were able to collect 103 blood samples for the study. So far, we have tested 12 samples and we could not detect DENV or any other arboviruses. However, 89% of the samples still remain untested. It is undeniably important to provide differential diagnostic for febrile illnesses, not only for a better management of the patients but also to understand the epidemiology of the viruses that may be circulating in a certain location and the control measures that should be taken to control their spread.

HV93 - CO-CIRCULATION OF TWO DIFFERENT LINEAGES OF DENGUE VIRUS TYPE 1 IN WEST CENTRAL REGION OF BRAZILCunha, M. dos P.; Guimarães, V.N.; Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Castro, Í. de A.; de Souza, K.M.C.; Fiaccadori, F.S.


Dengue virus (DENV) is the most important arboviral pathogen in the world and has been spread throughout the tropical and subtropical regions. It is a positive single strand RNA virus belonging to genus Flavivirus, family Flaviviridae, classified in four antigenically distinct serotypes DENV-1-4. In Brazil, DENV-1 was first detected in the years 80, and after a low or silent co-circulation with other serotypes, DENV-1 re-emerged in 2008 becoming predominant in later years. Nevertheless, the population structure of the viruses transmitted in this region is not well understood. In this context, the phylogeny analysis of DENV-1 strains from Goiás was performed in order to

identify the phylogeny pattern of the virus circulating in the epidemic period of 2013, using the complete sequence of envelope gene (E gene). DENV-1 positive samples were subsequently subjected to RNA extraction and RT-PCR amplification using DENV-1 specific primers by the “primer walking” method for the sequencing of the entire envelope gene (1485bp). All nucleotide sequences of Goiás were aligned with other sequences available at the GenBank, representing all DENV-1 genotypes, using the ClustalW algorithm and edited using Jalview software. The evolution model that best fits the dataset was inferred with jModelTest program according the Akaike information criterion (AIC) and a Neighbor-Joining method available in the software MEGA6 was used in order to analyze the phylogenetic relationship. The analyzes demonstrated that all isolates of DENV-1 Goiás belong to genotype V. The set of isolates form two clades within the genotype V, revealing the co-circulation of two distinct lineages. The first clade is composed of sequences isolated in Rio de Janeiro and Espírito Santo - 2010, while the second is formed by isolates from Americas countries like Venezuela - 2006 and Puerto Rico - 2010, and together other Brazilian states such as Roraima - 2009, Pernambuco - 2010 and Rio de Janeiro - 2011. Furthermore, these results indicate that despite an endemic viral serotype/genotype in a particular geographic region over a prolonged period, this does not mean that only one variant are associated with this condition, revealing the role of co-circulation and replacement lineages as responsible by hyperendemicity dengue in urban centers isolates. This is the first report of co-circulation of two lineages of DENV-1 detected in West Central region of Brazil.

HV99 - MONITORING OF CYTOMEGALOVIRUS INFECTION IN RENAL TRANSPLANT RECIPIENTSCosta, A.P.F.1; Souza, L.M.S.1; Joventino, K.M. de S.1; Santos, W.K. da S.1; Pereira, M.G.1; Quintana, V.H.A.2; Araújo, J.M.G.1; Freitas, J.C. de O.C.1; Farias, K.J.S.1; Machado, P.R.L.1

1. UFRN - Universidade Federal do Rio Grande do Norte, Campus Universitário Lagoa Nova, Natal - RN, 59078-970





The cytomegalovirus (CMV) is responsible for high morbid-mortality in solid organs transplanted patients and it is associated with the increase in the predisposition to acute and chronic rejection to allograft. The objective of this study was to carry out the monitoring of an infection by CMV through the polymerase chain reaction (PCR) in mononuclear peripheral blood cells (PBMC) samples and serum from patients who underwent renal transplant in the Onofre Lopes University Hospital (HUOL), Natal, Rio Grande do Norte. In the prospective study 45 patients who underwent the renal transplant at HUOL between February 2013 and January 2014 were included. Peripheral blood was collected on the 2nd, 4th, 8th, 12th and 24th weeks after the transplant to obtain the PBMC and serum. The DNA extraction was carried out according to the QIAamp® DNA kit protocol and the molecular detection of CMV according to the nested-PCR. It was observed in the pre-transplant that 93.3% (42/45) presented IgG anti-CMV antibodies, being that 89% of the receptors were IgG+/IgM-, 4.4% IgG+/IgM+ and 6.6% IgG-/IgM-. In relation to the CMV molecular detection in serum and PBMC samples, 62.2% (28/45) presented positivity in PBMC, and 24.4% (11/45) positive result both in the PBMC and in the serum, in at least five of the monitoring collections. Detection of CMV DNA in serum correlates with retarded graft function (27.3%), fever (45.5%) and gastrointestinal disease (54.5%). In relation to the monitoring of the infection by CMV, it observed that 2.7% of the patients included in the first collection presented positive detection. On the 4th week this positivity increased to 10.5%, on the 8th week, to 16.6%, on the 12th week, there was a reduction to 11.4% and on the 24th week, none of the patients presented positivity in the serum. In PMBC, the positivity found was of 16.2% on the 2nd week, with an increase to 26.3% on the 4th week, 36.1% on the 8th week and 45.7% on the 12th week, with a reduction in the detection on the 24th week to 36.3%. Finally, the results demonstrated an increased seroprevalence of IgG anti-CMV in the group studied, in relation to the molecular detection, an increased positivity was found in PBMC or serum, with the association between the detection of the viraemia in serum and the clinical manifestations associated with the infection. Financial support: FAPERN.

HV102 - LACK OF ASSOCIATION OF TNF-ALPHA AND IL-6 GENES METHYLATION WITH CLINICAL OUTCOME IN DENGUE INFECTED PATIENTSMorais, S.M. de S.1; Gomes, A.V.B.T.1; Filho, S.L. de M.1; Ferreira, J.M.S.2; Malaquias, L.C.C.1; Coelho, L.F.L.1



Dengue virus (DV) is an enveloped virus, positive single-stranded RNA, transmitted to humans through the bite of infected female Aedes aegypti mosquito. There are two main clinical manifestations caused by DV infection named Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF). DNA methylation is the best characterized epigenetic modification in DNA exerting great importance in silencing and gene regulation. In eukaryotes, it is found predominantly in the carbon 5 position of cytosine followed by a guanine in the CpG dinucleotide. This modification is biologically relevant when it occurs in regions rich in CpG dinucleotides known as CpG islands, there are common in promoter regions of certain genes. This work aims to analyze the methylation status in the promoter region of TNF-α and IL-6 in symptomatic and asymptomatic dengue patients.These two cytokines were involved in DHF pathogenesis and the promoter methylation was investigated using methylation specific PCR (MS-PCR). The DNA was extracted from 23 symptomatic and 18 asymptomatic dengue patients and submitted to sodium bisulfite treatment. The converted DNA was used to amplify the methylated and unmethylated regions using two sets of different primers for each gene in MS-PCR reaction. Promoter methylation of the TNF-α gene was detected in 65,22% of symptomatic patients and in 66.67% of asymptomatic patients and, for the IL-6 gene, in 30.43 and 61.11% of symptomatic and asymptomatic dengue patients, respectively. Promoter methylation of TNF-α and IL-6 was not statistically different in symptomatic compared with asymptomatic cases (p values of 0.8267 for TNF- α and 0.6536 for IL-6). Our findings indicate no association between methylation status of TNF- α and IL-6 genes and clinical outcome in dengue infection.




HV104 - CMV GLYCOPROTEIN B GENOTYPES OF CHILDREN WITH CONGENITAL INFECTION HOSPITALIZED IN THE INTENSIVE CARE UNITCardoso, E.S. de C.; Gadelha, S.R.; Marin, L.J.

UESC - Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900

The cytomegalovirus (CMV) is the most common agent of congenital infection in the world. It is not clear why some newborns with congenital CMV infection are symptomatic or not. One of the major glycoprotein of the virus is the glycoprotein B (gB). This protein is polymorphic and highly immunogenic. Most of the wild viral strains are grouped in four major gB genotypes. Recognizing the vital role of this glycoprotein in virus-host interaction, different genotypes can be associated with virulence and different clinical outcomes. In fact, it has been suggested that these genotypes could have specific tissue tropism or some mechanism to facilitate viral replication. In this study, it has been performed a neonatal screening for detection of symptomatic congenital CMV infection. It has been included all newborns under three week who were admitted to the Intensive Care Unit of the Hospital Manoel Novaes, in Itabuna, Bahia. Urine samples were collected in collectors bags, and subsequently, saliva samples also were collected with sterile swabs. Samples of saliva and urine collected were subjected to PCR without prior DNA extraction. It was used a positive and negative controls in all PCR reactions. Children with congenital infection will be assessed and monitored by a medical team until the second year of life. If necessary, they will be treated. The genotypes of CMV glycoprotein B will be determined by RFLP. For quantification of viral load, it will be carried out a real-time PCR. By the time, it was collected samples of 17 children. There was no positivity in any samples. This fact can be explained because the small number of children included in the study until this moment. Financial support: UESC

HV107 - VISUAL INSPECTION OF THE CERVIX: PRECISE, EFFECTIVE AND INEXPENSIVE METHOD USEFUL IN THE CANCER PREVENTION IN PUBLIC HEALTH UNITS FROM REGIONS WITH HIGH PREVALENCE OF HPVBarreto, D.M.V.S.; Almeida, S.M.V.S.; Mariano, A.P.M.; Gomes, L.G. da S.; Costa, G.B.; Marin, L.J.; Gadelha, S.R.

UESC - Universidade Estadual de Santa Cruz, Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho Ilhéus-Bahia, 45662-900

The human papillomavirus (HPV) is responsible for one of the most prevalent sexually transmitted diseases and is the major cause of death from cervical cancer worldwide. Development of simple and inexpensive approaches to screening early lesions of cervical cancer in low-resource regions is essential and should be investigated. The aim of this study was to evaluate the use of the technique of visual inspection of the cervix for screening of cervical lesions and consequent prevention of cervical cancer. The definition of a positive test result for the visual inspection was the presence of white lesions in the transformation zone near the squamous columnar junction (JEC), 1 minute after direct application of a 5% solution of acetic acid. This technique increases screening for cervical lesions without additional costs. For cervical cytology smears were prepared and stained by Pap smear and classified according to the Bethesda System. The virus was detected by nested PCR. It was used the primers MY09 and MY11, followed by primer GP5+ and GP6+. In his study, clinical and epidemiological data of 195 women from public health units in southern Bahia region were analyzed. The prevalence of HPV in the region was 47.7% by nested PCR and cytology detected 35.4% of intraepithelial lesions and atypical. The test visual inspection showed high specificity and high positive predictive value, 85% and 83%, respectively. The performance was calculated using the standard method of detecting the nested PCR. In fact, it was observed that the techniques (cytology, molecular detection and visual inspection) are complementary and it would be ideal that these methods could be performed together, in order to perform a better screening. However, in the basic health units and in areas with less resource, this is not feasible. The visual inspection method proved been effective in detecting lesions caused by HPV, an inexpensive tool and precise, demonstrating that this approach can be




included in a routine gynecological visit. In addition, this method can be applied in large-scale screening and may be an alternative to cytology exam (in our study, visual inspection showed better performance when compared to cervical cytology). Moreover, its application provides a simple and safely early detection of subclinical infections and premalignant lesions, reducing cases of invasive cancer and metastasis. Financial suport: PR

HV110 - MOLECULAR DETECTION OF OROPOUCHE VIRUS IN MATO GROSSO, BRAZILCardoso, B.F.1; Serra, O.P.1; Heinen, L.B. da S.; Zuchi, N.; dos Santos, M.A.M.2; Slhessarenko, R.D.1

1. UFMT - Universidade Federal de Mato Grosso, R. Guadalajara, 1 - Jardim das Américas Cuiabá - MT 78060-624

2. MT-Laboratório

Oropouche fever is the second most frequent arbovirus in notifications in Brazil, reported mainly in the Amazon region. Oropouche virus (OROV) is an Orthobunyavirus found in neighboring states of Mato Grosso (MT). In Central and South America genotypes I, II and III of OROV have been reported. This study aimed to investigate the circulation of orthobunyaviruses from Simbu serogroup in MT. To achieve that, viral RNA extracted from the serum of 529 patients with acute febrile illness obtained between 2011-2012 in 20 cities of MT and total RNA from 145/319 pools of Culex quinquefasciatus and 57/102 of Culex spp. mosquitoes captured in 2013 from 200 censitary sectors in the urban area of Cuiabá, MT were reverse transcribed to cDNA using a primer designed to the S segment of Orthobunyavirus genus. Nested RT-PCR for Simbu serogroup (300 pb) was followed by nucleotide sequencing and phylogenetic analysis. 4/529 (0.75%) human samples from Cuiabá, Várzea Grande and Nova Mutum and 3/202 (1.48%) mosquitoes pools, two of C. quinquefasciatus and one of Culex spp. negative for other 11 flaviviruses and five alphaviruses were positive for the serogroup Simbu. The nucleotide sequences showed homology with strains from Belém-PA and Manaus-AM belonging to genotype I of OROV. The preliminary phylogenetic analysis revealed that the samples belong to subgenotype IA, similar to strains from Pará obtained from humans, sloths and Ochlerotatus serratus and C. quinquefasciatus mosquitoes and to subgenotype IB, similar to human isolates from Pará, Maranhão and

Acre. C. quinquefasciatus, the culicidae found in higher abundance in Cuiabá, is considered a secondary vector for OROV transmission. Culicoides paraensis, considered the main vector for OROV, was not captured during the study. Serology to OROV was already identified in humans from cities of Pará affected by Cuiabá-Santarém highway and in primates in the Pantanal of Mato Grosso do Sul recently, corroborating the molecular identification of OROV in Mato Grosso. *Financial support: CAPES/CNPq.

HV112 - SEROPREVALENCE OF DENGUE AMONG THE ASSYMPTOMATIC INDIVIDUALS IN ALFENAS, MINAS, BRAZILPrado, A.A.O.; Gomes, A.V.B.T.; Rodrigues, N.F.; de Lima, M.M.; Malaquias, L.C.C.; Coelho, L.F.L.

UNIFAL - Universidade Federal de Alfenas, R. Gabriel Monteiro da Silva, 714 - Centro, Alfenas - MG, 37130-000

Dengue is an arboviral disease transmitted to humans through the bite of infected female mosquitoes of the genus Aedes. Among the most common clinical manifestations caused by Dengue virus (DV) infection are the Dengue Fever and Dengue Hemorrhagic Fever. The disease is now endemic in 112 countries in tropical and subtropical regions and Brazil is one of the most affected regions in the world accounting for approximately 60% of notifications in the Americas. The lack of studies covering systematically the population exposed to DV (non-infected, symptomatic and asymptomatic) hinders the knowledge of predisposing factors which may contribute to the development or maintenance of the disease in a specific population. This study aims to verify the seroprevalence of IgM/IgG anti-DV in the population of Alfenas, southern Minas Gerais. Three hundred serum samples were collected to verify the presence of IgM/IgG anti-DV in using Panbio Dengue Duo Cassette (Alere Australia) . The results showed that from 300 samples, 239 (79.67%) were negative; 37 (12.33%) were positive and 24 (8%) were indeterminate (the presence of a weak band in the IgM or IgG result). Among the samples that were positive, 24 (64,86%) are IgM positive, 4 (10.81%) are IgG positive and 9 (24,32%) are positive for IgM and IgG. The Panbio Dengue Duo Cassette can detect the high levels of IgG characteristic of secondary infections and IgM levels associated with primary dengue with high sensitivity and specificity. So, among the positive samples 24 (64,86%) were derived from




primary infection and 13 (35,12%) were derived from secundary infection. This data showed a high prevalence of anti-dengue antibodies in the population of Alfenas, MG and also the circulation of the virus in the region.

HV116 - MOLECULAR CHARACTERIZATION OF A DENGUE VIRUS 1 ISOLATED IN ALFENAS, MINAS GERAIS, BRAZILFranco, I.R.1; Rocha, R.P.1; Fumagalli, M.J.1; da Silveira, N.J.F.1; Elias, T.C.1; Fagundes, L.G.1; Nogueira, M.L.2; Drummond, B.P.3; Malaquias, L.C.C.1; Coelho, L.F.L.1




Dengue is a major public health problem worldwide, especially in the tropical and subtropical areas with around 2.5 billion people living in areas at risk regions of the world. The disease is caused by a positive single strand RNA virus that belongs to genus Flavivirus, family Flaviviridae. There are four serologically related viruses designated as DENV-1, 2, 3 and 4. Infection with one of these serotypes causes a mild, self-limiting febrile illness called dengue fever. However, a reduced number of patients could develop the severe forms of disease called dengue hemorrhagic fever and dengue shock syndrome. In this study, we performed a molecular characterization of envelope gene from a DENV-1 strain isolated from a dengue hemorrhagic fever patient from Alfenas, South of Minas Gerais (BR/Alfenas/2012). The phylogeography analysis using nucleotide sequences from the DENV-1 E gene shows that these samples clustered within the genotype V, lineage L1 together with other Brazilian DENV-1 isolates and also with the Venezuelan isolates. Thereby, it was observed that the introduction of this lineage probably occurred from Rio de Janeiro state, Southeast Brazil and after, the virus has spread to Minas Gerais state where it had a local evolution evidenced by the distance between the BR/Alfenas/2012 and the other related isolates. Fifteen conservative and/or non-conservative aminoacids substitutions were observed in the deduced polyprotein sequence. Two aa substitutions

were unique to this isolate were not observed in the other isolates from the same genotype/lineage. Among these aa substitution one is conservative at position 222 (located in domain II of the E protein) and one is non-conservative at position 306 (located in domain III of the E protein). To check the virulence of the isolate, Swiss mice were infected intraperitonally with 1 x 104 pfu or inoculated with PBS. After 7 days of infection the liver of infected animals showed the presence of inflammatory infiltrate around the central veins and also focal points of edema, hemorrhage and necrosis. It is also observed the presence of steatosis when compared to uninfected animals, suggesting the presence of a viral replication in the liver of these animals. This is the first report of molecular characterization and analysis of a virulence potential of a DENV-1 strain isolated from South of Minas Gerais, Brazil.

HV117 - INVESTIGATION OF DENGUE VIRUS INFECTION IN GOIANIA, GOIAS, BY MOLECULAR METHODS AND SEROLOGICALOliveira, T.S.; Guimarães, V.N.; Cunha, M. dos P.; e Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Carneiro, R. dos S.; Fiaccadori, F.S.


Dengue is a major challenge to public health in Brazil and worldwide. Dengue virus (DENV) is an arbovirus of the family Flaviviridae, genus Flavivirus, classified into four antigenically distinct serotypes DENV-1-4. Infection with one of these serotypes may causes a disease whose spectrum ranges from clinically asymptomatic or a mild and self limited febrile illness called dengue fever to severe clinical forms. In Brazil, the co-circulation of four serotypes has led to successive epidemics and Goias state, usually present high number of dengue cases. In 2013 were related alarming statistics, with an increase of 401% in the number of reported cases and 25% in the number of deaths. The Epidemiological Surveillance System plays an important role in the continuous monitoring infections, nevertheless, it has been shown the relevance of laboratory diagnosis in the confirmation of new cases. To increase the detection of DENV in early infections, the NS1 research was introduced in Public Health Laboratories. However, the time of infection has




a decisive influence on the results regarding the method of choice. Therefore, the present study evaluated the occurrence of DENV infection in Goiania, Goias in the epidemic period of 2012-2013 among the population with clinical suspected of dengue infection (up to seven days) using a combination of serological and molecular methods. Serum samples were collected from 278 symptomatic patients who were attended in basic health centers between October/2012 and May/2013. The samples were submitted to the research of serological markers NS1, IgM and IgG using a commercial kit (DuoTest-Bioeasy), as well as viral RNA detection by RT-PCR for the C-prM region. The positivity for infection was 43.9% (122/278), with rates of 30.5%, 13.6% and 20.5% for NS1, IgM and RNA markers, respectively. In 24.5% of DENV infection cases, NS1 was the only marker detected, demonstrating its advantage mainly favoring early diagnosis with higher rate of positivity between 4 and 5 days. The IgM was detected singly in 16.4% with statistical significance for the sixth and seventh days. For the RNA, the highest rate was observed between 1 and 3 days. The combination of these three markers, notably constitute a powerful tool in the diagnosis DENV. Monitoring of dengue epidemics by laboratory confirmation, with an active and effective notification system, it is relevant to establish a complete data base to be used for surveillance studies.

HV120 - MOLECULAR INVESTIGATION OF NATURAL INFECTION BY FLAVIVIRUSES AND ALPHAVIRUSES IN ADULT MOSQUITOES CAPTURED IN CUIABÁ, MATO GROSSO, BRASILSerra, O.P.1; Cardoso, B.F.1; dos Santos, F.A.L.1; Heinen, L.B. da S.1; Zuchi, N.1; Ribeiro, A.L.M.2; Rodrigues, J.S.V.1; Myiazaki, R.D.1; Slhessarenko, R.D.1


2. HUJM - Hospital Universitário Júlio Müller, Av. Fernando Corrêa da Costa, nº 2367, Bairro Boa Esperança, Cuiabá - MT, 78060-900

Arboviruses belonging to Flavivirus and Alphavirus genus are considered an important public health issue in Brazil, mainly in tropical areas.The aim of the study was to identify the frequency of hematophagous mosquitoes naturally infected by flaviviruses and

alphaviruses in Cuiabá, MT.Culicidae specimens were captured in 3 locations from 200 censitary sectors from Cuiabá in 2013, identified with dichotomy key, allocated in pools (1-10 mosquitoes) according to sex, species, data and collection point.Minced pools were subjected to total RNA extraction, duplex-RT-PCR followed by multiplex-semi-nested-RT-PCR to five alphaviruses and 11 flaviviruses.Positive samples for Saint Louis encephalitis (SLEV), dengue 1 (DENV-1) and mayaro (MAYV) viruses were amplified by single RT-PCR and subjected to nucleotide sequencing.A region of the SLEV envelope gene was amplified for phylogenetic analysis.Positive pools for MAYV were confirmed by RT-qPCR.The minimum infectious rate (MIR) was calculated for positive species.Between 11.090 mosquitoes, 4.556 females classified in 13 species constituted 593 pools, 1/137(MIR=1.04) of Aedes aegypti positive for DENV-1 and 1/67 (MIR=2,6) Culex sp. for SLEV. For MAYV, 10/67(MIR=26,2) Culex sp., 7/162(MIR=3.7) Cx. quinquefasciatus, 4/137(MIR=4.1) Ae. aegypti and 5/54(MIR=24) Cx. comp. pipiens were confirmed by RT-qPCR. For DENV-4, 55/137(MIR=57,1) Ae. aegytpi, 34/54(MIR=162.7) Cx. comp. pipiens, 68/162(MIR=36) Cx. quinquefasciatus, 46/67(MIR=120,4) Culex spp., 1/6(MIR=142.8) Limatus sp., 2/4(MIR=500) Psorophora spp., 3/7(MIR=375) Ps. varipes albigenu, 1/1(MIR=1000) were positive.In previous studies, SLEV, MAYV and the four serotypes of DENV were identified in patients from the urban area of Cuiabá during a large outbreak of dengue.The SLEV genotype V-A, previoulsy reported in humans from Cuiabá, was identified in a Culex sp. female.Culex and Aedes species presented log of 3.52-4.08 copies/µL of MAYV.Experimentaly, these species have been demonstrated as competent vectors for the virus, but their participation in the epidemiological cycle of MAYV transmission is unclear.DENV-4, responsible for the majority of the human cases in MT during 2012-2013, was identified in several species, possibly due to hematophagy in humans.Cuiabá presents a variety of culicidae species, associated to environmental conditions favorable to the occurrence of arboviral outbreaks, emphasizing the importance of entomological and virological surveillance. FINANCIAL SUPPORT: CAPES, FAPEMAT, UFMT




HV121 - IMPROVEMENT OF NOROVIRUS GII/4 SUBTYPING PROTOCOLOliveira, L.M.1; Fumian, T.M.2; Miagostovich, M.P.2; Andrade, J.S.R.2; Colombo, A.C.F.1; Paixão, S.1; Nagata, T.1



Norovirus (NoV) are a major cause of acute gastroenteritis (AGE), responsible for almost 50% of AGE outbreaks worldwide, affecting all ages. Despite the high genetic diversity, the genogroup II, genotype 4 NoV (GII.4), are the main agent of these outbreaks. Every two years a new GII.4 variant arises by the capsid protein (VP1) amino acids changes as a new pandemic strain, resulting in a high antigenic variability of this NoV group. It had been common to determine the subtype (or strain) by sequencing the part of VP1 gene, however, recent study of NoV taxonomy suggested the use the whole VP1 gene sequences for GII genotyping and GII.4 subtyping analysis. In this study new primer set were designed by multiple alignment of NoV GII.4 sequences for complete VP1 gene recovery by RT-PCR (GII-4 Subtyping 5019 For: 5´-GGC AAG AGC CAA TGT TCA G-3´ and GII-4 Subtyping 6821 Rev: 5´-TGT TAT TTT CAA AAT CAA YYT TTT GGT T-3´). Six NoV positive stool samples previously confirmed by RT-qPCR were used for RT-PCR with the primer set described above and five samples amplified successfully. The amplicons were gel-purified and sequenced using the same primer set. All five NoV samples were classified as NoV GII.4, Sydney 2012 variant, using multiple alignment and phylogenetic tree constructed by MEGA6 with representative sequences of each GII.4 subtypes. The possible presence of recombination was investigated using RDP4, and our results showed that no recombination was found among the samples analyzed. The use of new primer set was successful to determine the subtype of NoV GII.4. FINANCIAL SUPORT: CAPES

HV123 - DIFFERENCES IN THE INCIDENCE OF HPV TYPES AT A NORTHERN POPULATION IN BRAZIL, BASED IN THE WORLDWIDE PROFILESilva, A.K.1; Silvestre, R.V.D.1; da Paixão, C.G.S.1; Ferreira, L.S.S.1; Lima, S.F.2; Junior, L,B.D.2; Mello, W.A.1


2. Laboratório Paulo C. Azevedo, Avenida Comandante Brás de Aguiar, 99 - Batista Campos, Belém - PA, 66035-000

The Human papillomavirus (HPV) is a sexually transmitted infectious agent, commonly found worldwide. It causes asymptomatic infections in mucous membranes that oft are eliminated naturally about two years, but can cause important clinically diseases depending of the viral type, with persistence or progression of infection, such as high grade lesions and cancer. Among the diseases caused by HPV, cervical cancer represents one of biggest challenge to public health. Taking into account assessments conducted by the National Cancer Institute (INCA), in 2014, it was estimated that cancer of the cervix already ranks third overall in Brazil, with the highest rates found in North region as 23.57/100000 women. Specifically in the metropolitan area of Belém/Pará. The gross rate cases is about 35/100000 women, becoming the second cancer in incidence in Pará capital. For prophylactic use, two vaccines have been developed trying to cover the major circulating types, with the intention of decrease the incidence of genital HPV-related disease. Brazil was adopted quadrivalent vaccine that combat subtypes 6, 11, 16 and 18 of which the first two are related to 90% of genital warts and the other two are causally implicated in 70% of cervical cancers. The aim of this study is to identify which HPV types are circulating in a routine gynecology treatment in Belém city, Pará. Was used 84 samples collected from women aged 20-50, submitted to the Pap test, previously detected as HPV positive by Hybrid Capture second generation. These samples were tested by PCR with biotinylated specific primers for a region of L1 in viral genome. Then, they were subjected to the test “Linear Array HPV” that is able to identify 37 different HPV types from hybridization of the amplified products with oligonucleotide probes and detection by colorimetric determination. The study identified 10 different types of high risk HPV with prevalence of 59




(18.7%), 31 (16.7%), 16 (12.5%) and 58 (10.4%). We also identified 15 different types of low risk HPV, with prevalence of HPV 55 variants (14.58%) and 81 (10.4%). Finally, we conclude that the occurrence of HPV in our population shows a trend different from the worldwide prevalence types included in quadrivalent vaccine. With these founds we suggest the strengthening of networks tests of patients by cytology and, if possible associated to a molecular HPV test, to reduce the incidence of cervical cancer in our quite different epidemiological scenario. FINANCIAL SUPPORT: MS / SVS / IEC

HV124 - PRELIMINARY WORK WITH CHIKUNGUNYA VIRUS IN A BIOSAFETY LEVEL 3 (BSL-3) LABORATORYde Figueiredo, M.L.G.1; Amarilla, A.2; Romeiro, F.3; Badra, S.J.3; Aquino, V.H.2; Figueiredo, L.T.M.3

1. Evandro Chagas Institute, Brazilian Ministry of Health

2. School of Pharmaceutical Sciences of The University of São Paulo

3. Virology Research Center, School of Medicine of Ribeirao Preto of The University Of Sao Paulo

Chikungunya (CHIKV) is an Alphavirus (Togaviridae) that has reemerged in tropical and subtropical regions of Asia and Africa. The main vectors of this virus are Aedes aegypti and Aedes albopictus. Last year, CHIKV has been introduced in the Americas, in the Caribbean. However, an autoctonous case was found in North America. Therefore, probably, this virus will reach Brazil and will produce large outbreaks of acute febrile illness with arthritis. Thus, it is important to develop diagnostic methods for this virus in our country. We describe here a preliminary work with the prototype of CHIKV (S27-African strain), isolated in 1953. In a biosafety level 3 laboratory, the virus has been rehydrated from a 30 years old ampoule (ATCC, V548-001-522), inoculated into C6/36 (Aedes albopictus) cells and also intracerebrally in baby mice. Five days after infection, despite not showing cithopatic effect, the infection of CHIKV in C6/36 cells was confirmed based on immunofluorescent test and real-time RT-PCR. Forty eight to 72 hours after infection, 70% of the infected mice developed encephalitis and after 5 days all animals were dead. Cell culture fluids of the infected cells and brains of mice with encephalitis were collected and stored at -70 oC as virus seeds. These seeds will be used in further experiments that will include the

production of an antiserum and the development of a real time RT-PCR for diagnosis of infections by CHIKV in humans and mosquitoes. FINANCIAL SUPPORT: CNPQ- 150815/2014-0

HV128 - ADENOVIRUS INFECTION IN ALLOGENEIC STEM CELL RECIPIENTS: EVALUATION OF VIRAL EXCRETION AND VIREMIASantos, H.C.P.1; Fiaccadori, F.S.1; Cardoso, D. das D. de P.1; Arantes, A. de M.2; Silva, L.P.2; Almeida, T.N.V.1; Souza, M.1


2. Unidade de Transplante de Medula Óssea do Hospital Araújo Jorge/ ACCGO

The human adenoviruses (HAdV) infect people of all ages worldwide, causing a wide range of clinical syndromes, depending on the viral type. In immunocompromised hosts, mainly those undergoing allogeneic stem cell transplant (ASCT), persistent and severe infection are common, resulting in a bad prognosis. HAdV diagnosis is not included in routine laboratory exams of ASCT patients in public hospitals in Brazil. Therefore, studies involving HAdV infection in this group of patients are still scarce. Thus, the objective of this study was to monitor ASCT recipients (admitted to Hospital Araújo Jorge in Goiânia, Goiás) for HAdV occurrence and also correlate viral positivity with clinical symptoms and patients’ prognosis. For this, one fecal and one total blood sample were obtained from each patient prior to the ASCT and, after that, samples were obtained weekly. Patients were monitored for a minimum of one month and a maximum of 19 months. Up until now, 98 serum samples and 62 fecal samples, obtained from 19 ASCT recipients, have been tested for HAdV. Serum and fecal samples were screened by Nested-PCR, using primers targeting a partial region of the hexon gene. Fecal samples were further screened by a commercial enzyme immunoassay (EIA) (RIDASCREEN Adenovirus, R-Biopharm). All samples were negative by the EIA. However, ten stool samples from four patients were positive for HAdV by Nested-PCR, and ten serum samples from seven patients, were also positive. In total, ten patients (53%) had at least one positive sample (serum or fecal) for HAdV. Five HAdV-positive patients presented diarrhea, and five patients




presented graft versus host disease. In one patient, HAdV-positivity in fecal samples preceded viral positivity in the serum. So far, three positive samples, from three distinct patients, were sequenced. Two of those samples were characterized as HAdV F serotype 40 and one as HAdV C type 57. This data highlights the importance of the inclusion of HAdV testing in the routine laboratory exams of ASCT patients. Preliminary results also suggest that fecal screening for HAdV could be potentially used to predict HAdV viremia, and thus contribute to an early clinical intervention and prevention of disseminated infection. FINANCIAL SUPPORT: FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE GOIÁS (FAPEG); PROGRAMA DE PÓS GRADUAÇÃO EM BIOLOGIA DA RELAÇÃO PARASITO-HOSPEDEIRO (PPGBRPH)

HV129 - DEVELOPMENT OF A REAL TIME-PCR BASED IN SYBR GREEN FOR RAPID DETECTION OF CHIKUNGUNYA VIRUSRomeiro, M.F.; de Souza, W.M.; Figueiredo, M.L.G.; Tolardo, A.L.; Figueiredo, L.T.M.


Chikungunya virus (CHIKV- S27-African strain) is a mosquito-borne Alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia and Africa. The main vectors of this virus are Aedes aegypti and Aedes albopictus. Last year, CHIKV has been introduced in the Americas, in the Caribbean. However, an autoctonous case was found in North America. Therefore, probably, this virus will reach Brazil and will produce large outbreaks of acute febrile illness with arthritis. We show here a real-time RT-PCR based on SYBR green I, using primers that amplify part of the gene of NS1 of CHIKV. This new real-time RT-PCR was validated using a CHIKV in Vero cells and in suckling baby mouse brain, both passaged in a biosafety level 3 laboratory. This RT-PCR, used as a routine, could become a useful tool for rapid diagnosis of infections by CHIKV in Brazilian patients and mosquitoes, allowing manage its disease, and monitor the spreading of CHIKV through Brazil.

HV131 - EVALUATION OF SERA OF MICE IMMUNIZED WITH A CHIMERIC ANTIGEN CONTAINING SEGMENTS OF E1 AND E2 ENVELOPE PROTEINS OF HEPATITIS C VIRUSOliveira, M.M.1; Freitas, G.R.O.2; Froelich, L.2; Yokosawa, J.1

1. Laboratório de Virologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia (UFU), Uberlândia, MG.

2. Programa de Pós-Graduação em Imunologia e Parasitologia Aplicadas, ICBM, UFU, Uberlândia, MG.

Hepatitis C is a disease that affects the liver and is a major cause of chronic liver disease worldwide, progressing frequently to cirrhosis and liver cancer. Approximately 3% of the world population is infected chronically with hepatitis C virus (HCV). The transmission occurs through contact with contaminated blood and until now there is no effective vaccine. The difficulty in the development of a vaccine is due to the HCV genetic diversity, and the existence of only one system for virus replication in cell culture, based on a single genotype (2a). Conserved segments of HCV E1 and E2 glycoproteins that had been reported to elicit neutralizing antibodies were selected to construct the chimeric antigen GST-E1E2, which was expressed in bacterial system and used to immunize mice. Sera obtained from these mice showed to be reactive, by ELISA, against synthetic peptides corresponding to HCV segments present in the GST-E1E2. To confirm this reactivity, in this study, we transfected CHO-K1 cells with the plasmid EFJFH c-NS2, which encoded for the HCV proteins core, E1, E2, p7, and NS2. Zeocin-resistant clones were selected and expression of the HCV core protein was confirmed by indirect immunofluorescence assay (IFA) by using an anti-core monoclonal antibody. Out of five sera that showed to be reactive by ELISA, four also showed reactivity by IFA against pEFJFH c-NS2-transfected cells. In conclusion, these results indicated the capacity of GST-E1E2 to elicit specific humoral response in mice. FINANCIAL SUPPORT: CNPQ, FAPEMIG, PROPP-UFU, CAPES, UFU.




HV139 - CIRCULATION OF DIFFERENT SEROTYPES OF DENGUE VIRUS IN SAO JOSE DO RIO PRETO, SAO PAULOColombo, T.E.1,2; Vedovello, D.1; Biselli, J.1; Drumond, B.3; Cabrera, E.1; Nogueira, M.L.1




The four serotypes of dengue virus (DENV 1–4) (family Flaviviridae, genus Flavivirus) are antigenically and genetically distinct. The emergence of epidemic dengue in the Americas, as well as worldwide, has been characterized by a rise to hyperendemicity. In the present work we looked the DENV transmission in Sao Jose do Rio Preto from 2011 to 2014. Were used serum samples of suspected and confirmed DENV patients provided by the Public Health Authority to profile DENV circulation. The viral surveillance was performed with multiplex RT-PCR using Flavivirus generic primers based on non-structural protein (NS5), followed by nested assays with species-specific primers. The DENV serotypes were as follows: 65,41% (522/798) DENV-4, 22.56% (180/798) DENV-1, 11,28% (90/798) DENV-2 and 0.75% (6/798) coinfection, showing a complex pattern of serotypes circulation. Statistical analysis of the mean ages (years) of DENV-1 (38,12 ± 18,82), DENV-2 (38,06 ± 18,78), DENV-4 (36,57 ± 16,69) and coinfection (29,66 ± 18,94) cases showed that there was no significant difference (P = 0,4470, F = 0,8876) between means. The correlation between of the gender of DENV patients according to infecting serotype also showed that there was no significant difference, DENV-1 (P = 0,4868), DENV-2 (P = 0,5634) and DENV-4 (P = 0,7781). Up to now, 9 DENV-1, 15 DENV-2, 33 DENV-4 have been subjected to sequencing of the envelope gene, and 8 DENV-1, 7 DENV-2, 7 DENV-4 had their genome completely sequenced. The phylogenetic analyses of serotypes 1, 2 and 4 show that the samples identified in this study grouped with genotypes that circulating in Brazil (genotypes V, Asian American and American, for types 1, 2 and 4 respectively). Looking inside the genotypes two distinct

clades formed in DENV-1 phylogenetic reconstructions, indicating two possible lineages. For DENV-2 and DENV-4, one clade grouped all SJRP samples, indicating a single possible lineage. This data shows that the phylodinamics of dengue circulation can be much more complex than expected even in a small city, with circulation not only of different serotypes but also different strains as we already showed before for DENV-3. These data reinforce the importance and need for surveillance programs to detect and trace the evolution of these viruses. FINANCIAL SUPPORT: PRONEX; INCT-DENGUE; FAPESP; CAPES

HV141 - GASTROENTERIC VIRUS SHEDDING BY ASSYMPTOMATIC DAY-CARE CHILDREN FROM GOIÂNIA, GOIÁSCorrêa, T. dos S.; Souza, M.; Santos, H.C.P.; Fiaccadori, F.S.; Souza, K.M.; Abdelhaleem, K.R.; Almeida, T.N.V.; Cardoso, D. das D. de P.


Gastroenteric viruses, such as rotavirus A (RVA), human adenovirus (HAdV), human calicivirus (norovirus and sapovirus), human astrovirus (HAstV), are important acute gastroenteritis agents. These agents affect mainly children, elderly, and immunocompromised individuals. Outbreaks of AGE are common in semi-closed environments, such as hospitals, schools and day-care centers; however, asymptomatic viral excretion has been reported by children and immunocompromised individuals. The main objective of this study was to screen fecal samples (previously tested and negative for norovirus and sapovirus genogroups I and II by RT-PCR) obtained from children less than five years of age, for RVA, HAdV and HAstV detection. Sample collection took place in a day-care center in Goiânia, Goiás, from October 2009 to September 2011. For RVA and HAdV screening, all samples were tested by a commercial enzyme immunoassay (EIA) (RIDASCREEN Rotavirus/Adenovirus – R-Biopharm); samples were further tested by polyacrylamide gel electrophoresis (PAGE) for RVA-genome detection. For HAstVs screening, an RT-PCR reaction was performed, using primers targeting the ORF2 region. All positive samples were submitted to molecular assays for characterization. From all 42




children, 10 (23.81%), between two and three years old, were positive for one of the viral agents. From those, two (4.7%) were positive for RVA, by both methodologies, and characterized by RT-PCR (targeting the VP7 and VP4 genes) as G2/Pnon-typable. Three (7.14%) were positive for HAdV and characterized, by PCR/NestedPCR followed by genomic sequencing of a partial region of the hexon, as species F (serotypes 40/41). Five (11.9%) were positive for HAstV, and characterized as serotype 1. Data reveal the occurrence of asymptomatic viral excretion by the children in the day-care environment constituting a potential risk for viral dissemination and the occurrence of outbreaks. FINANCIAL SUPPORT: UNIVERSIDADE FEDERAL DE GOIÁS

HV142 - IDENTIFICATION AND BIOLOGICAL CHARACTERIZATION OF DENGUE SEROTYPE 1 ISOLATES (DENV-1) DETECTED IN SÃO JOSÉ DO RIO PRETO, SÃO PAULOPinheiro, T.M.1; Biselli, J.M.1,2; Colombo, T.E.1,2; Drumond, B.P.3; Ullmann, L.S.2; Araújo Jr, J.P.2; Batista, I.C.A.4; Silva, C.E.C.4; Vedovello, D.1,2; Nogueira, M.L.1





Dengue is an infectious viral disease transmitted by the bite of infected Aedes mosquitoes (Aedes aegypti and Aedes abopictus). It is considered a serious public health issue in tropical and subtropical regions where the vector is best adapted. Dengue virus (Flaviviridae family, Flavivirus genus) has four antigenically distinct serotypes (DENV 1-4) subdivided into genotypes and lineages with different degrees of virulence. Therefore, the aim of this study was to perform an investigation to correlate the molecular and biological characteristics of samples of DENV-1 identified in São José do Rio Preto. A viral surveillance was performed with Multiplex RT-PCR using Flavivirus generic primers based on non-structural protein (NS5), followed by Nested assays

with species-specific primers in serum from samples from dengue suspected patients. We amplified 544 samples for DENV in SJRP from 2008 to 2012 and 373 (68.56%) were positive for DENV-1. 20 DENV-1 have been subjected to sequencing of the envelope gene and/or full genome sequencing and were used for phylogenetic reconstruction. Our analyses showed that the all samples belong to Genotype V and formed two well defined groups divided into two lineages identified as L1 and L6. Comparing the two lineages, the following amino acid (aa) substitutions are observed: L338S (leucine to serine, aa position 338), K394R (lysine to arginine, aa position 394), L428V (leucine to valine, aa position 428) and I436V (isoleucine to valine, aa position 436). The I457R (isoleucine to arginine, aa position 457) was recognized only one sample (BR/SJRP/287/2011). Additionally, epitope analysis of samples showed that the two lineages are recognized by different receptors of T and B lymphocytes indicating that exhibit differences in immune response. Our preliminary data show the existence of differences between the samples, evidencing the importance of understanding the biological DENV characteristics in vitro and in vivo correlating to the likely epidemiological consequences.

HV148 - CHARACTERIZATION OF AN ATYPICAL DENV-2 STRAIN ISOLATED IN BRAZILSalvador, F.S.1; Amorim, J.H.2; Ferreira, L.C. de S.2; Romano, C.M.1

1. IMTSP - Instituto de Medicina Tropical de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, Jardim América, São Paulo - SP, 05403-000


Dengue virus is a flavivirus, and is responsible for the main arboviruses that infect man worldwide. From a patient presenting dengue without warning signs in Brazil it was isolated a strain of Dengue Virus (named JHA) with capacity of neurovirulence in mice using 100 PFU Amplification and Sanger sequencing of a small NS1 fragment of this sample characterized it as DENV 2. To study this virus in more detail, we performed the complete sequencing of viral genome using NGS technology in Ion Torrent platform. Viral RNA was




reverse transcribed and three fragments of 4kb with overlap were amplified using the SuperScript III high-fidelity one-step reverse transcription-PCR (RT-PCR) kit (Invitrogen). Reads were assembled and analyzed in CLC genomic Workbench. Consensus complete genome was aligned to 208 globally sampled DENV genomes and genotyping was performed by maximum likelihood reconstructions. A second dataset comprising only envelope region of DENV2 was built to estimate genetic distance among sequences using PAUP. Recombinant analysis was done in RDP software. We also visually inspected JHA genome by comparing it to well-known neurovirulent DENV2 strains. Phylogenetic analysis of complete genomes showed that JHA strain belongs to American genotype of DENV2, but it is more divergent than expected from viruses of the same genotype. While the intra- and inter-genotype average genetic distance was around 2.5% and 10% respectively, JHA was almost 6% divergent from American genotype viruses. No evidence of recombination was found that could explain such divergence. Although this strain was quite divergent from general American viruses, JHA was almost identical to a single strain isolated in Trinidad Tobago in 1953 (Trin53). To identify specific sites that would be related to neurovirulence, the genome was then compared to others (neurovirulent and non-neurovirulent American). Several non-synonymous changes were observed throughout the genome in comparison to American viruses consensus, but two sites found in envelope gene were suggestive of neurovirulence ability (E126 and E390) since they differ from other American but are similar to neurovirulent viruses. Others already reported the importance of these sites for neurovirulence in mouse model. Ongoing neurovirulence reversion experiments using cell culture and mice should better clarify the particular characteristics of JHA genome neurovirulence.

HV149 - EVALUATION OF THE EXPRESSION LEVELS OF GENES INVOLVED WITH INNATE IMMUNITY IN HUMAN CELLS INFECTED BY APEU VIRUSFerreira, J.G.G.1; Villani, F.N.A.2; Oliveira, J.G.1; Ferreira P.C.P.2; Calzavara Silva, C.E.1



Bunyaviridae family viruses are considered important emerging and reemerging infectious agents, capable of causing different clinical features in humans, including fever, encephalitis and hemorrhage. Little is known about the interaction of some of these viruses with the human immune system, justifying the need for studies on the influence of Orthobunyavirus and innate immunity. This study aimed to quantify the expression of a set of 5 genes related to human innate immunity (TLR3, TLR7, TLR9, IFNg and IFNb) after infection by the Apeu virus (APEUV). Peripheral blood mononuclear cells (PBMC) collected from 5 healthy individuals were exposed to APEUV in two distinct conditions: PBMC infected with m.o.i.=1 and m.o.i.=3; and uninfected controls (mock infected PBMC; uninfected PBMC and fresh PBMC (T0 which were not submitted to any process of incubation). After infection and 4 hours of incubation, total mRNA was extracted from cells and used to obtain a cDNA pool, for qPCR assays based on TaqMan system. The group T0 was used as “normalizer group”. Expression of TLR9 mRNA was increased in the infected group (m.o.i. 1 and m.o.i. 3). IFNb messenger expression was even higher in these groups (15-fold higher than in T0 group). There was no significant changes in the expression of TLR3, TLR7 and IFNg genes in all tested groups, as well as any significant changes in expression of all tested genes on mock and uninfected groups. These results indicate that TLR9 and IFNb genes are related to changes in the human immune response in the early stages of APEUV infection and further studies with these and other genes are being conducted aiming to elucidate the molecular processes involved in the infection of human cells by APEUV.

HV153 - IDENTIFICATION OF G AND P GENOTYPES OF GROUP A ROTAVIRUS AMONG CHILDREN IN RIO BRANCO, ACRE, BRAZILNeves, M.A.O.1; Menezes, E. de F.C.2; Silva, M.C. de M.2; Silva, L.D.2; Loureiro, E.C.B.2; Gabbay, Y.B.2; Rodrigues, I.C.2; Silva, R.U.2; Soares, L. da S.2; Mascarenhas, J.D.P.2

1. UEPA - Universidade do Estado do Pará, Rua do Una, nº 156, Belém - Pará, 66.050-540





In developing countries, group A rotavirus (RVA) cause death in more than 197,000 children each year and is recognized as a major viral agent of acute gastroenteritis in infants and young children. Its structure is composed by 12 proteins, two structural proteins are located at outer capsid of the virion; the VP7 (Glycoprotein) and the VP4 (Protease-sensitive) are widely studied once that induce immunological response and are involved in viral neutralization. To date, 27 G and 37 P genotypes have been reported by sequence analysis. This study aimed to identify G and P genotypes of RVA in fecal samples of children from Rio Branco, Acre. Material and Methods: For RVA identification, 488 stool samples recovered from children with and without diarrhea were subjected to enzyme-linked immunosorbent assay (RIDASCREEN). The viral RNA of positive samples was extracted and subjected to reverse-transcription polymerase chain reaction (RT-PCR). The primers used were Beg9/End9 and 4con3/4con2 for the G and P type, respectively. For RVA genotyping the product of the first round of amplification (RT-PCR) was subjected to a second round of amplification (seminested PCR) with the same primer used in RT-PCR in combination with specific primers for G (G1, G2, G3, G4, G9, and G12) and P-types (P[4], P[6], P[8], and P[9]). Results: RVA positivity was observed in 9.6% (47/488) of the samples. The predominant G types detected in 2012 in Rio Branco were: G2 40.4% (19/47), G3 23.4% (11/47), G12 21.3% (10/47) and G1 4.2% (2/47), and with regard to the P types: P[4] 40.4% (19/47), P[8] 29.8% (14/47) and P[6] 21.3% (10/47). Five samples were not typed for VP7 gene and four for VP4. Genotype G2P[4] was the most prevalent combination accounting for 40.4% (19/47), mostly in January with 58% (11/19) of the cases. Conclusion: This study allowed the identification of RVA genotypes circulating in the post-vaccine period in Rio Branco, the capital of the Acre State, Brazil, and showed similar results if compared with other studies conducted in northern Brazil where a significant predominance of G2P[4] has been observed after the introduction of the Rotarix vaccine. Furthermore, the epidemiological basis of the cyclic phenomenon of rotavirus genotypes is not fully elucidated, and this represent challenges with regard to the effectiveness of rotavirus vaccines against the variability of genotypes detected. FINANCIAL SUPPORT: INSTITUTO EVANDRO CHAGAS

HV157 - RELATIONSHIP OF HPV GENOTYPES FROM CERVICAL AND ANAL SITES AMONG HIV SEROPOSITIVE WOMENVolpini, L.P.B.; Boldrini, N.A.T.; Miranda, A.E.; Freitas, L.B.; Spano, L.C.

UFES - Universidade Federal do Espírito Santo, Av. Fernando Ferrari, 514, Goiabeiras, Vitória - ES, 29075-910

The human papillomaviruses (HPV) have 198 described genotypes and approximately 40 have tropism for the anogenital region. Infection with oncogenic or high-risk HPV types (HR-HPV) in the anus or cervix sites can lead to invasive squamous cell carcinoma and coinfection with HIV can increase the risk of cancer in both sites. Furthermore, the HPV cross-infection between cervical and anus is common, both acting as a reservoir to another. The aim of this study is to describe HPV types found in cervical and anal specimens from HIV-positive women attending the Reference Center for STD/AIDS, Vitória-ES, from September 2013 to June 2014. Cervical and anal samples were obtained with cytobrush from 73 HIV-seropositive women, aged between 18 and 65 years. Viral DNA was extracted using the QIAGEN QIAamp® DNA Mini Kit. HPV was screened with the sets of PGMY09/11 primers and genotyped by Reverse Line Blot (RLB) with probes for 32 genotypes. The results showed HPV detected in 74% (54/73) of women in cervical and/or anal sites, being 45.2% (33/73) and 65.8% (48/73) at each site, respectively. In 50% (27/54) of the positive cases, HPV was present at both sites. Twenty six different genotypes were found and the most frequently detected in the anal and cervical sites were, respectively, HPV-16> 6> 51 and 53 and HPV-45> 31> 16, 52, 53 and 69. Infection with the same genotype at both sites was present in 48.1% (13/27), and 69.2% (9/13) of them were HR-HPV. Mixed infection occurred in 57.4% (31/54). These preliminary data show an important and partial correlation between HPV genotypes detected in both sites and suggest a common source of infection or even, spread between these anatomical sites. Detection and typing of HPV in HIV-positive women are important for monitoring of anal and cervical lesions. The persistence of HPV infection and the presence of lesions will be analyzed, which will generate information that will clarify the natural history of HPV infection and persistence in this vulnerable group of women. FINANCIAL SUPPORT: UFES, FAPES




HV158 - STANDARDIZATION OF THE REAL TIME POLYMERASE CHAIN REACTION IN DETECTION AND MONITORING OF EARLY BETAHERPESVIRUS REACTIVATION IN HEMATOPOIETIC STEM CELLS TRANSPLANT PATIENTSBonon, S.H.A.; Oliveira, R.S.; Dellariva, T.C.; Lima, R.G.; Costa, S.C.B.

University of Campinas/Faculty of Medical Sciences/Laboratory of Virus

Molecular methods for the detection and quantification of infectious agents are being deployed in routine, to minimize the consequences and avoid possible clinical manifestations of herpesviruses, providing early treatment with appropriate antiviral, especially CMV. Real Time Polymerase Chain Reaction (qPCR) in plasma is one of the options to be used in monitoring of patients. Also, it is necessary to develop such techniques “in house” to minimize costs with commercial kits. The objective of this study is to standardize an qPCR “in house” using Taqman technology for the detection and quantification of betaherpesvirus DNA (CMV, HHV-6 and HHV-7) in HSCT patients. An nested PCR and CMV antigenemia was also performed and the comparison between these techniques was evaluated. Plasma samples from 57 patients were prospectively obtained weekly from the day of transplant until day +100 after the transplant. Nested PCR for betaherpesvirus and CMV antigenemia were used in all samples. The qPCR technique is being developed and standardized. The results will be compared to healthy group matched for sex and age and with cardiac transplant patients group. Fifty seven patients were analised by Nested-PCR and CMV antigenemia. Active CMV infection was detected by N-PCR in 37/57 (64.9%) with a median of 34 days (range 2-91 days) and CMV antigenemia in 24/57 (42.1%), median 42 days (range 0-91 days) after transplantation. Five patients (13.5%) with active CMV infection died in less than 100 days after transplantation (median 50 days, range 46-100 days) and two of them (40%) had CMV disease. Fourteen with active CMV infection (24.6%) had acute GVHD and overlap. Five patients (8.8%) had CMV disease in the gastrointestinal tract (GIT), biopsy proven. Active HHV-6 in plasma was diagnosed in 12/57 patients (21%) for a median of 24 days after transplantation (range 0-84 days). A patient with HHV-6 positive (8.3%) died by CMV disease in less than 100 days after transplantation.

Four patients with HHV-6 (33.3%) had acute GVHD and overlap. HHV-7 occurred in 31/57 (54.3%) in a median of 15 days after transplantation (range 0-98 days). In 3/31 patients with HHV-7 (9.7%) died in less than 100 days after transplantation and their main cause of death was acute GVHD and bacterial infection. Eight patients with HHV-7 (25.8%) had acute GVHD and overlap. Active betaherpesvirus infection monitoring using sensitive techniques are very important to use preemptive therapy. Supported by FAPESP.

HV162 - IDENTIFICATION AND MONITORING OF INFLUENZA VIRUS A AND B, IN POPULATION MACEIÓAntão, K.L.; de Sá, J.P.O.; de Lima, M.C.; Pinheiro, T.M.L.; Pinheiro, M. da S.; de Barros, L.H.R.; Maria, F.H. de O.S.

UNCISAL - Universidade Estadual de Ciências da Saúde de Alagoas, Rua Doutor Jorge de Lima, 113, Trapiche da Barra, Maceió - AL, 57010-300

Viral agents, such as influenza viruses A and B, parainfluenza 1, 2 and 3, adenovirus and respiratory syncytial virus (RSV) are responsible for outbreaks of acute respiratory infection (ARI), causing a high rate of morbidity and mortality, especially in children and the elderly. This work aimed to study these viruses as etiologic agents of ARIs in patients of all ages. Also aimed to assess the association between seasonality and respiratory virus. The samples of nasopharyngeal secretion (NPS) were used for patients under 5 years of age and the combined swab (nasal and oral) for patients above 5 years of age. These samples were collected at the Center Sentinel Health Unit Dr. Ib Gatto Hawk, from lower and / or upper respiratory tract by up to three days. ARI and / or in the period November 2012 to November 2013 a total of 12 months of observation. The analyzes of 488 samples of male (203) and female (285) sexes were performed by IIF in virology sector LACEN-AL. With the indirect immunofluorescence using a panel of monoclonal antibodies (MAbs). Of the 488 samples, 42.4% (207) showed IFA positive for influenza A and B virus, parainfluenza types 1, 2 and 3, adenovirus and RSV. It is observed that 25.8% (126) of the cases were positive for influenza A; 10.0% (49) for adenovirus; 4.1% (20) for RSV; 3.5% (17) for parainfluenza 3; 2.5% (12) for parainfluenza virus 2; 1.8% (9) for parainfluenza 1; and 0.6% (3) influenza B. The monthly distribution of




positive cases shows a distinct epidemiological profile for each virus. This fact makes clear the need to maintain continuous surveillance virology, in order to mitigate the impact of these viruses during periods of peak activity. Suporte Financeiro: LACEN/AL - Laboratório Central de Saúde Pública de Alagoas UNCISAL - Universidade de ciências da saúde de alagoas PROBIC/UNCISAL - Programa Institucional de Bolsas de Iniciação Científica

HV163 - PARADIGM SHIFT IN DIAGNOSIS OF PATIENTS ATTEND FOR GYNECOLOGIC ROUTINE EXAM IN BELÉM METROPOLITAN REGION, PARÁSilva, A.K.1; Paixão, C.G.S da1; Silva, A.K.1; Ferreira, L.S.S.1; Lima, S,F.2; Junior, L.B.D.2; Mello, W.A.1; Silvestre, R.V.D.1


2. Laboratório Paulo C. Azevedo, Avenida Comandante Brás de Aguiar, 99 - Batista Campos, Belém - PA, 66035-000

In order to improve the efficiency of diagnosis of precursor lesions of cervical cancer, new technological advances are being implemented. In screening programs for cervical cancer is utilized for decades the Pap test, which is the primary method for diagnosis, however, depends on several factors, getting too subject to results bias. Currently new microscopic and molecular tools have united to complement these results with greater accuracy. In this study, we investigated the relationship between cytological and molecular diagnostics for Human Papillomavirus (HPV), about high and low oncogenic HPV risk in patients aged 20-50 years attend for routine gynecologic exams in Belém / PA. For results comparison, the Pap test and Hybrid Capture second generation (CH2) molecular method are used. For diagnosis, 184 samples were tested. In the CH2 tests, a total of 19.5% (36/184) were positive for HPV being 12% (22/184) positive for high risk HPV; 3.8% (7/184) tested positive for low risk HPV and 3.2% (6/184) positive samples are mixed infections caused by types of high and low risk or more than one viral type. In the analysis of cytological examinations of 184 samples, 176 tests are considered without cellular atypia (usual default), being 17% (30/176) of these specimens revealed positive for HPV in CH2 (56.6% HR / LR 20% / 23.4% mixed infections); two samples

were cytologically classified as atypical, squamous or glandular cells of undetermined significance (ASCUS / ASGUS), whereas 50% (1/2) for high-risk HPV compared to CH2; five presented intraepithelial lesions low-grade (including HPV suggestive alterations and cervical intraepithelial neoplasia grade I), 80% (4/5) of these was positive for high-risk, and a single sample classified as invasive squamous cell carcinoma revealed a high-risk oncogenic in CH2. According to the findings, we note that a large percentage (17%) of HPV detection in molecular tests that are not reported in cytological tests being classified as one of several biases that may occur by the subjectivity of the Pap smear, thus showing necessary to implement a molecular tool to complement routine gynecologic examinations, thus increasing the capacity to predict changes arising from HPV infection. FINANCIAL SUPPORT: MS / SVS / IEC

HV165 - DIVERSITY OF HADV IN HUMAN STOOLSVicentini, F.1; Gomes, Y.M.1; Rodrigues, M.C.1; Mello, I.O.1; Leite, J.P.G.2; Miagostovich, M.P.2; Spano, L.C.1

1. UFES - Universidade Federal do Espírito Santo, Av. Fernando Ferrari, 514, Goiabeiras, Vitória - ES, 29075-910


Adenoviruses (HAdV) have long been associated with diarrhea, but to establish a causal relationship with different types has been difficult due for the detection in feces of other types not enteric. In order to characterize the types of HAdV excreted in the feces of black children that living in Quilombola communities in Espírito Santo state, the hexon gene was amplified by PCR (nt 21-322) and sequenced. Between August 2007 and May 2010, 212 fecal samples were obtained from 133 (62.7%) children with (symptomatic) and 79 (37.3%) without (asymptomatic) diarrhea. The overall frequency of positivity was 27.8% (59/212), with 30.1% (40/133) and 24.1% (19/79) among the symptomatic and asymptomatic children, respectively. Fifty samples were sequenced. Thirty three samples of the symptomatic cases belonged to the following species: 3 (9.1%) A, 6 (18.2%) B, 18 (54.5%) C, 2 (6.1%) D, 4 (12.1%) F; E and G were not found. Among the C species, types 1, 2, 5 and 6 were found. The HAdV-C (n=27) was found in the feces with a frequency of four times that of the enteric HAdV-F




(n=7). The HAdV-A and HAdV-F species accounted for approximately 20% of the total of the HAdV types and infected only children up to 5 years of age and 70% of these samples were present in the stools of symptomatic children. The HAdV-F41 was excreted equally between symptomatic and asymptomatic. This study showed a very large diversity of HAdV in human feces.

HV169 - ASEPTIC MENINGITIS IN NEONATE BY COXSACKIEVIRUS B5 GENOGROUP B IN BRAZILCarmona, R. de C.C.1; Vieira, H.R.1; Machado, B.C.1; Sousa, C.A.1; Lo, D.S.2; Ibidi, S.M.2; Gilio, A.E.2; Alves, M.R. de M.1; Carmona, R. de C.C.1


2. HU/USP - Hospital Universitário da Universidade de São Paulo, Av. Prof. Lineu Prestes, 2565 - Cidade Universitária, São Paulo - SP, 05508-000

Enteroviruses (EVs) comprise a large genus in the Picornaviridae family, with more 100 serotypes delineated into four species (A–D) based mostly on their phylogenetic relationships. Coxsackievirus B5 (CV-B5) belongs to the species Enterovirus B, considered one of the most prevalent serotypes in humans, often associated with sporadic cases of neurological diseases and epidemics of meningitis aseptic. EVs neonatal infections are associated with a broad spectrum of signs and symptoms, ranging from febrile illness not specific to a multisystem disease potentially fatal. The aim of this study was to report the first detection of aseptic meningitis in neonate by CV-B5 in Brazil, and to understand of the genetic relationships with strains detected in others countries by phylogenetic analysis of the protein VP1 gene. The cerebrospinal fluid (CSF) from neonate with suspect of meningitis aseptic was collected and subjected to cell culture isolation. Subsequently, enterovirus-positive was identified as serotype CV-B5 (IAL-E1186) by indirect immunofluorescence assay (IFA), and carried out RT-PCR and sequencing the VP1 region. Genetic analysis of IAL-E1186 strain revealed that the VP1 displayed a great relatedness to other selected human CV-B5 strains already defined to genogroup B. The VP1 sequence of IAL-E1186 strain displayed the highest nucleotide sequence identity to STU27/DEU/09 strain (96.5%), which was detected in Germany in 2009. Also, the highest nucleotide sequence

identity was observed with strains detected from Denmark (96.3%), and France (96.1%). The occurrence of aseptic meningitis caused by EVs in newborns is rarely detected, probably due to the difficulty in collecting clinical samples for conducting viral research. In this study, we document the rare occurrence of a CV-B5 in neonate. This result draws attention to the importance of an etiologic diagnosis in early disease. Furthermore, it demonstrates the importance of routine laboratory diagnosis of EVs contributing to the management of neurological disease, the broader genomic knowledge of EV, and epidemiological study of the circulation of CV-B5 in the pediatric population. Financial support: Fapesp 2012/50234-5. FINANCIAL SUPPORT: FAPESP 2012/50234-5.

HV173 - DETECTION OF RARE REASSORTANT G5P[6] ROTAVIRUS STRAIN IN CHILD WITH DIARRHEA, BRAZILCarmona, R. de C.C.; Cilli, A.; Luchs, A.; Morillo, S.G.; Gregório, D. de S.; Carmona, R. de C.C.; Timenetsky, M. do C.S.T.


Group A rotavirus (RVA) genotype G5, which are common in pigs but also detected in horses and cattle, were first identified in humans in Brazil in the 1990´s. Following its first detection, this genotype has also been reported in children with severe diarrhea worldwide. Since 90´s, G5 RVA strains has been rarely isolated from humans and commonly found in combination with P[8] genotype. The aim of this study was to analyse the rare G-P combination G5P[6], identified in a stool sample collected from a 12-years-old boy in May 2013, state of Goiás, Western Brazil. RVA genotype G5P[6] was detected using a commercial immunoenzymatic assay, PAGE, genotyped by RT-PCR and sequencing of the VP4 and VP7 genes. The comparison of the VP7 sequence of IAL3029 strain showed 94-95% of nucleotide (nt) identity compared to human Brazilian G5 strains of 90´s decade, including prototype IAL28 (Lineage I) (bootstrap value 99%). Comparing with others human and animal VP7 sequences from Europe, America, Asia, and Africa, the similarity varies from 78-88%nt. The VP4 sequence of IAL3029 clustered within Lineage I showing the highest nucleotide identity (95%nt) compared to Brazilian




porcine strains (PGRV33 and 898/07) and ranged from 82 to 92%nt comparing to animal RVA P[6] strains from other countries. The identification of a rare reassortant human G5P[6] genotype supports the hypothesis of a dynamic interaction between human and animal RVA, indicating that there is a constant flux of genetic materials among co-circulating human and animal RVA. Recently, uncommon RVA strains with similarity to animal strain have been detected in Brazil and selective vaccine pressure could increase the circulation of unusual strains. The continued RVA surveillance in Brazil is vital to monitoring the possible reemergence of strains, such as genotype G5, after the introduction of the vaccine against RVA. In addition, simultaneous surveillance of animal RVA infections is necessary for understanding the evolution of RVA. FINANCIAL SUPPORT: INSTITUTO ADOLFO LUTZ CTC/53-2005.

HV188 - DETECTION OF ARBOVIRUS IN MOSQUITO POPULATION OF RIO DE JANEIRO CITY: MONITORING IN PREPARATION FOR 2016 OLYMPICSFerreira, D.F.; Santos, L.L.R.; da Silva, J.L.1; dos Reis, A.S.1; Dias, C.M.G.2; Ribeiro, M.S.3; Meire, G.L.S.4; Chanasit, J.S.5; Campos, R.M.4

1. Gerencia de Pesquisa em Antropozoonoses - GPA/LACEN/SVS/SESRJ.

2. Gerencia De Doenças Transmitidas Por Vetores E Zoonoses - GDTVZ/DTI/CVE/SVS/SESRJ

3. Subsecretaria de Vigilância em Saúde - SVS/SESRJ

4. Laboratório de Interação Vírus Célula, Departamento de Virologia, Instituto Microbiologia Prof. Paulo de Góes. Centro de Ciência e Saúde, Universidade Federal do Rio de Janeiro.

5. Instituto Bernhard Nocht para Medicina Tropical – Colaborador da OMS para Arboviroses e Febres Hemorrágicas

The last two decades have been marked by the large number of human diseases caused by arboviruses. An arboviral depends on the replication in two hosts: vertebrate and arthropod vector. In this scenario, climatic changes, the increasing area of deforestation, the increasing speed in transportation of hosts and vectors and the disorganized urbanization are contributing factors for the emergence and re-emergence of arboviral diseases in various regions of the world. One example was the recently introduction (December of 2013) of the

virus Chickungunya in America. Since the first autoctone transmission detected, around 61,864 suspected cases were reported by Pan American Health Organization until the epidemiological week 20 of 2014. Meanwhile, in the same region Dengue epidemics still happen and are most related to the introduction of new serotypes or the ones not in circulation recently. In this context, the city of Rio de Janeiro, which is hosting major events, and increasing foreigners visiting, is vulnerable to import arboviruses. The aim of this study is to monitor mosquitoes infected by arboviruses in the region where it is being built Olympic Village, residence of athletes during the Olympic and Paralympic Games in 2016. Adult mosquitoes are collected in traps BG Sentinel ® and Aspirator. Insects are separated by gender, species, sex and analyzed by RT-PCR for detection of Dengue virus, Chikungunya and other arboviruses. From October 2013 to January 2014, 2,056 mosquitoes were collected 32 Aedes sp. (7 males and 25 females) and 2.023 Culex sp. (538 males and 1,486 females). The area investigated presented a prevalence of mosquitoes of the genus Culex, however, it was possible to detect within the sampling Aedes sp a positive female for Dengue virus serotype 4. The production of information concerning rates of infection vectors is a pioneering activity in health services in the country and may contribute to the assertiveness of the models used to predict outbreaks and epidemics scenarios and decision making in relation to vector control actions. FINANCIAL SUPPORT: FAPERJ; BNI; INBEB

HV196 - FREQUENCY OF SEROLOGICAL MARKER FOR HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV-I/II) IN INMATE POPULATION OF A CITY OF PERNAMBUCOMorais, V.M.S.1; Lopes, T.R.R.1; Cahu, G.G. de O.M.1; Silva, D.M.2; Rabelo, D.C.C.2; Lucena, W.A.T.2; Lima, P.C. de S.2; Albuquerque, A.C.C.2; Coelho, M.R.C.D.2


2. ASCES - Associação Caruaruense de Ensino Superior e Técnico, Av. Portugal, 584, Bairro Universitário- Caruaru - PE, 55016-400

Prisons are known to be favorable environments for the transmission of sexual and blood diseases. The inmate population is considered high risk for infections such as




human immunodeficiency virus, hepatitis B, hepatitis C and sexually transmitted diseases. Factors such as marginalization, illicit drug use and low socioeconomic level contribute towards dissemination of infectious and contagious diseases in prisons. However, the inmate population has needed to investigate other virus, such as human T-cell lymphotropic virus (HTLV-I/II). The HTLV-1 can be transmitted by horizontal, vertical and parenteral route, staying asymptomatic in approximately 97% of patients. In Brazil, data based on the prevalence of blood donors, it was estimated that 2.5 million people are infected with HTLV-I in 2002. On the other hand, a study realized in Minas Gerais found a frequency of 1, 6% for HTLV, however, there are few studies of the prevalence of HTLV in the inmate population of a city of Pernambuco. The aim the study was to estimate the frequency of positive anti-HTLV I/II markers in male population of a prison in the state of Pernambuco, in the period May to July 2011. The datas from each individual were collected through an interview, after 5 mL peripheral blood was collected in without anticoagulant tube. The samples were centrifuged at 1500 rpm for 10 minutes to separate serum, after, the samples were forwarded to the Department of Virology of Laboratory Immunopathology Keizo Asami (LIKA) of Universidade Federal de Pernambuco (UFPE) to perform the anti-HCV by the commercial kit (Murex HTLV I+II, DiaSorin). The serology was realized in 1085 samples and the frequency of the anti-HTLV I/II was 0.72% (8/1085). The population study has less 30 years old, 75% (6/8) were married, 37.5% (3/8) had tattoos and have used cocaine. None inmates used injection drugs or had sex with another man, 50% (4/8) did not use condom, 25% (2/8) had a sexually transmitted disease and 12.5% (1/8) had received blood transfusions. The frequency of anti-HTLV (I/II) was lower in this population than in Minas Gerais State, this may be due to the low frequency of risk factors, such as injection drugs and use cocaine. However, data about HTLV among Brazilian prisoners is still scarce, and it draws attention to the need for epidemiological studies and policies to prevent transmission of infectious and contagious diseases during incarceration. FINANCIAL SUPPORT: CNPq.

HV206 - HUMAN GROUP C ROTAVIRUS IN HOSPITALIZED CHILDREN FOR GASTROENTERITIS IN NORTHERN REGION, BRAZIL: EPIDEMIOLOGICAL, CLINICAL AND PHYLOGENETIC ANALYSISLobo, P. dos S.2; Guerra, S. de F. dos S.1; Siqueira, J.A.M.1; Soares, L. da S.1; Gabbay, Y.B.1; Linhares, A. da C.1; Mascarenhas, J.D.P.1


2. Centro Nacional de Primatas

Acute gastroenteritis (AG) is a major cause of morbidity and mortality and rotavirus is one of the causative agents of AG and it is responsible for about 38.3% of the hospitalizations, resulting in 197,000 deaths of children under five years annually, mainly in developing countries. Rotavirus belongs to the Reoviridae family, genus Rotavirus, have genome composed by 11 segments of double-stranded RNA (dsRNA) and are classified into eight groups/different species (A to H). Most of the cases belongs to the group/species A (RVA), however rotavirus group/species C (RVC) has assumed importance in gastroenteritis and is usually relate to self-limiting childhood diarrhea, with possible transmission from pigs. This study aims to detect the RVC in children less than three years of age hospitalized with AG in Belém city, Pará state, Brazil. From May 2008 to April 2011, an intensive surveillance for AG was performed in a pediatric hospital of Belém. A total of 279 fecal samples with negative results to RVA and norovirus had been tested for RVC. The nucleic acid was extracted from a 10% suspension using silica methodology. The cDNA was obtained by reverse transcription and after amplified by polymerase chain reaction (PCR) using primer pairs specific for the VP7 gene (G8S/G8A); for VP6 (C1/C4); for VP4 (T434/T435); and NSP4 (+)/(-) specific for NSP4 gene. Positive control (prototype Cowden) and negative control (water RNAse free) were used in all the tests. Automatic sequencing and subsequent phylogenetic analysis were performed. Data were analyzed using statistical chi-square partition, Fisher’s exact test and linear regression with p values ≤ 0.05. The positive rate for RVC was 2.1% (6/279) by PAGE and RT-PCR. The sequencing classified these positive samples as I2 (VP6), G4 (VP7), P[2] (VP4) and E2 (NSP4) genes. The frequency of RVC was higher in the age group between 24 to 36 months (5.7%) with p= 0.0493. Male




were more affected by RVC with a frequency of 83.3%. Signs and/or symptoms most frequent in hospitalized children with RVC were fever (80%), vomiting (83.3%) and dehydration (100%). In summary, in this study it was possible the characterization and phylogenetic analysis of RVC VP6, VP7, VP4 and NSP4 genes, providing important data regarding the presence of these agents in hospitalized children with AG in the metropolitan region of Belém, Pará.

HV211 - DETECTION OF ANTIBODIES FOR YELLOW FEVER VIRUS AND OTHER ARBOVIRUS IN NON-HUMAN PRIMATE AT THREE URBAN PARKS IN GOIÂNIA-GOBadr, K.R.A.1; Gibrail, M.M.1; Vasconcelos, P.F.C.2; Fiaccadori, F.S.1; Guissoni, A.C.P.1; Cardoso, D.D.P.C.1



Arboviruses are transmitted by hematophagous arthropod vectors. They are maintained in nature through cycles involving vectors, hosts and their reservoirs, such as vertebrates, and are thus present in the wild environment. In Goiânia, an outbreak of yellow fever in 2007/2008, with records of epidemics, infections and death in the population of non-human primates (NHP’s) urban parks in the township occurred. In this context, the present study aimed to carry one sero-epidemiological research on NHP’s the city of Goiania. The study Were involved all species present in NHP Screening Center for Wild Animals of the Brazilian Institute of Environment and Renewable Natural Resources - Regional Goiânia (CETAS-IBAMA), Thus, blood samples were collected by femoral venipuncture, or brachial vein from 24 animals. After processing and storage, the samples were sent to LACEN-GO for forwarding to the Instituto Evandro Chagas - IEC. The samples were subjected to reactions of the Hemagglutination Inhibition (HI) using antigens from 19 different arboviruses: Eastern Equine Encephalitis; Western equine encephalitis; Mayaro; Mucambo; Guaroa; Maguari; Tacaiuma; Yellow Fever; islanders; Icoaraci; Utinga; Sao Luis; Cacipacore; Bussuquara; Rocio; Belem; Caraparu; Oropuche; Catu and Dengue virus (DEN-1-4). NHP’s species investigated, only the species C. libidinosus

Caraya A. and C. penicillata were positive by HI. From the total samples, five (20.8%) were positive for one or more of the following viruses: Bussuquara (n = 3), Mayaro (n = 1), Cacipacore (n = 2), Oropouche (n = 3) and Dengue (DEN-2) (n = 2). Noteworthy is the fact that one of the animals was positive for specific antibody Oropouche Virus (VORO), featuring an animal not checked out the urban region and origin of Goiânia, which may suggest the existence of this movement as arboviruses epizootic promoted by a competent vector for the transmission of the virus. This is the first investigation of arboviruses in NHP in the region, and the record of positivity for antibodies against VORO in Goiás state becomes relevant in the context of public health. FINANCIAL SUPPORT: CNPQ

HV214 - A HIGH-THROUGHPUT DNA MICROARRAY PLATFORM FOR THE DIAGNOSIS OF VIRUSES TRANSMITTED BY ARTHROPODS AND RODENTSTrabuco, A.C.1; Khan, M.J.1; Alfonso, H.L.1; Badra, S.J.2; Figueiredo, L.T.M.2; Quintana, V.H.A.1

1. FCFRP/USP - Faculdade de Ciências Farmacêuticas de Ribeirão Preto/ Universidade de São Paulo, Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903


Brazil is a tropical country where several viruses transmitted by arthropods (arbovirus) and rodents (robovirus) are circulating. Considering the public health significance of these viruses, there is a great interest for the development of rapid diagnostic methods. The DNA microarray platform has emerged recently as a high-throughput method for pathogen detection. The objective of this study was the development of a DNA microarray platform (RoboArbovirusChip) for the detection of roboviruses and arboviruses. Based on the sequences of 466 viruses (roboviruses and arboviruses) deposited in the GenBank, 4715 probes with 60 nucleotides were designed. Approximately 10 probes per virus were designed. The microarray slide was prepared by Agilent (USA) in an eight array per slide format. Each array contained the 4715 probes with at least three replicates, plus positive and negative controls. The DetectiV software (http://detectiv.sourceforge.net/) was used to analyze the fluorescence




data. To evaluate the RoboArbovirusChip, 15 viruses was tested: 15 laboratory viruses from 4 families were detected: Flaviviridae – Dengue virus type-1 (DENV-1), Dengue virus type-2 (DENV-2), Dengue virus type-3 (DENV-3), Dengue virus type-4 (DENV-1), Saint Louis encephalitis virus (SLEV), Rocio virus (ROCV), Yellow fever virus (YFV), Bussuquara virus (BSQV), Iguape virus (IGUV) and Ilheus virus (ILHV) – Alphaviridae – Mayaro virus (MAYV) – Rabdoviridae – Piry virus (PIRV) – and Bunyaviridae – Oropouche virus (OROV), Rio Mamoré virus (RIOMV) and Guaroa virus (GROV). The RoboArbovirusChip was also able to detect DENV-1, DENV-2, DENV-3 and DENV-4 from human serum samples. No cross-hybridization was observed with RNA obtained fro C6/36 cells, mouse brain or dengue negative patients. These results strongly suggest the potential of this platform for the used as a rapid diagnostic method for high-throughput monitoring of roboviruses and arboviruses.FINANCIAL SUPPORT: FAPESP.

HV217 - COMPARISON OF SYMPTOMS OF INCLUSION AND OTHER CLINICAL DENGUE DRIVE BETWEEN PRIMARY AND TERTIARY HEALTH UNITPontes, C.M.L.1; Monta, M.J.1; Neto, L.L.S.1; Igreja, R.R.1; Filho, F.A.X.M.1; Souza, N.V.2; Parente, A.M.B.3; Colares, J.K.B.4; Lima, D.M.1

1. UNIFOR - Universidade de Fortaleza, Avenida Washington Soares, 1321 - Edson Queiroz, Fortaleza - CE, 60811-905

2. Departamento de Patologia e Medicina Legal/ UFC - Universidade Federal do Ceará, Rua Monsenhor Furtado, s/n, Rodolfo Teófilo, Fortaleza - CE, 60441-750

3. MSC/UNIFOR - Mestrado em Saúde Coletiva da Universidade de Fortaleza, Av. Washington Soares, 1321, Edson Queiroz, Fortaleza - CE, 60811-905

4. HSJ - Hospital São José de Doenças Infecciosas, Rua Nestor Barbosa, 315, Parquelândia, Fortaleza - Ce, 60455-610

Dengue is an acute infectious disease currently considered the most important arboviral disease in the world affecting in particular the tropical countries.Thus, the aim of this study was to identify the clinical manifestations, based on the new WHO classification, adopted in Brazil since January 2014, through the comparison of signs and symptoms presented by patients.Methodology:Cross-sectional study, conducted through interviews of patients with suspected dengue.

The information was collected in the interview in order to complete a questionnaire prepared by researchers, based on the symptoms of individuals with suspected dengue. Results: From January 2012 to January 2013, 96 patients were recruited, of which, 47 were treated in a tertiary unit and 49 in two primary health centers in Fortaleza, Brazil.The mean duration of disease of the patients registered in tertiary healthcare was approximately seven days, while primary units, was three days.The basic units, the main symptoms of inclusion had the following frequencies in descending order: headache: 81.63%(40), retroorbital pain: 76.65%(37), myalgia: 72.80%(36), vomiting: 42.85%(21) nausea: 38.77%(19), arthralgia: 32,04%(16) and rash: 15.38%(8). And the tertiary unit, the main symptoms include headache were 80.85%(38), myalgia 76.59%(36), arthralgia 63.82%(30), retroorbital pain 59.57%(28), nausea: 57.44%(27), vomiting: 55.31%(26), rash 44.68(21) and petechiae 37.5%(18). With respect, the other clinical manifestations that may be associated with dengue patients recruited in the basic units had the following percentage: 30.61% diarrhea(15), cough 26.53%(13), odynophagia 20.40%(10), rhinorrhea 16.32%(8), meningism 8.16%(4), 10.20% sputum (5) and pruritus 4.08%(2).While, in tertiary units, 51% patients had cough (24), pruritus 35.41%(17), diarrhea 35.41%(17), odynophagia 29.16%(14), meningism 25%(12), 20.83% sputum (8) and rhinorrhea 12.50%(6).Conclusion:The principal criterion for cases of suspected dengue recommended by the Ministry of Health (2013) is fever, and with respect to this signal, it was observed that patients seek hospital when they are with a longer disease than patients recruited the basic health unit. Regarding the inclusion criteria, no differences between units, demonstrating the importance of follow the new classification for dengue cases in which symptoms have been added, such as vomiting and nausea that had representative prevalence. FINANCIAL SUPPORT: MCTI; CNPQ; MEC; SISU




HV220 - IDENTIFICATION OF HUMAN BOCAVIRUS IN STOOL SAMPLES FROM CHILDREN WITH ACUTE GASTROENTERITISSampaio, M.L. da S.1; Brandão, C.J. de F.2; Sardi, S.I.1; Campos, G.S.1; Dorea, A.1; Tigre, D.M.1; Paula, F.L.1; Andrade, A. de S.1; Costa, L.F. de M.1

1. UFBA - Universidade Federal da Bahia, Rua Augusto Viana , s/n, Palácio da Reitoria, Canela , Salvador - BA, 40110-909

2. Hospital Aliança, Avenida Juracy Magalhães Junior, 2096 - Rio Vermelho, Salvador - BA, 41920-900

Gastroenteritis is an important cause of mortality and morbidity among children worldwide, especially in children. This disease can be caused by bacteria, fungi, protozoa and virus infections. Among the viral infections include: Norovirus (NoV), Human Rotavirus (HRV), Adenovirus (AdV) and, more recently identified, the Human Bocavirus (HBoV). The HBoV has a single stranded DNA, non-enveloped, belongs to the Parvoviridae family, genus Bocavirus and encodes four proteins: two structure proteins (VP1 and VP2), one nonstructural protein (NS1) and one unknown function nucleoprotein (NP1). The objective of this study was to detect HBoV in stool samples of children under 5 years old with clinical manifestation of gastroenteritis. Initially, stool samples were tested for the presence of NoV, AdV and HRV by ELISA using the kits of RIDASCREEN® 3rd. Generation Norovirus (R-Biopharm, Germany), RIDASCREEN® Adenovirus (R-Biopharm, Germany) and RIDASCREEN® Rotavirus (R-Biopharm, Germany), respectively. Subsequently the identification of HBoV was made by extraction of viral DNA in the stool samples with the QIAamp DNA kit (QIAGEN, Germany) and submitted to Nested-PCR using the Pan bocavirus primer that amplifies a fragment of approximately 576 bp region of the gene encoding the polyprotein, VP1-VP2. The results of these tests demonstrated that from 105 samples analyzed, 41.9% (44-105) were positive for HBoV, 19.04% (20-105) for NoV, 3.8% (4-105) for AdV and 3,8% (4-105) for HRV . Among the positive samples for HBoV, it was detected co-infection in 27.3% (12-44) stool samples, of which 91.6% (11-12) were co-infection with NoV and 8.4% (1-12) with AdV; in contrast, the HBoV infection was detected exclusively in 72.7% (32-44)patients. The results of this study show that HBoV has high incidence of infection compared to the other viral

agents on study, moreover, it was found as a co-infection etiologic agent. This work contributes to the surveillance of gastroenteritis affecting children, showing the presence of HBoV as a possible viral etiologic agent. FINANCIAL SUPPORT: FAPESB ( Fundação de Amparo à Pesquisa do Estado da Bahia)

HV223 - RESPIRATORY INFECTIONS CAUSED BY PARAINFLUENZA VIRUS IN HOSPITALIZED CHILDRENOcadaque, C.J.; Pereira, S.A.R.; Oliveira, F.M.S.; Florencio, C.M.G.D.; da Silva, F.E.R.; Alves, A. de A.; Moura, F.E.A.

UFC - Universidade Federal do Ceará, Avenida da Universidade, 2853 - Benfica, Fortaleza - CE, 60020-181

The hospital respiratory infections (HRI) pediatric are responsible for high rates of infant morbidity and mortality. Due to non-implementation of routine laboratory methods for detecting virus little is known about the role of these agents in the etiology of HRI. International studies have highlighted several virus associated with these infections, including parainfluenza virus (HPIV) type 3. Objective of the study is to verify the detection rate of types 1, 2, 3 and 4 of HPIV in hospital respiratory infections in hospitalized children Hospital Infantil Albert Sabin, located in Fortaleza-Ce. Nasopharyngeal aspirate samples collected were used in the study from January to December 2013, hospitalized and started a children of respiratory infection for at least 72 hours after your hospital stay. The samples were analyzed by the technique of polymerase chain reaction preceded by reverse transcription (RT) for detecting the HPIV 1, 2, 3 and 4 plus chain respiratory syncytial virus (RSV), metapneumovirus, Influenza A and B (FluA and FluB) and adenovirus (without RT). A total of 120 cases of HRI were included in the study and, of these, 31 (32.2%) were positive for HPIV, and 5 HPIV-1 (5/31), no HPIV-2 (0/31), 14 HPIV-3 (14/31) and 12 HPIV-4 (12/31). The co-detections of HPIV with other virus were found in 10 cases, 4 with adenovirus; 3 with RSV, 2 with influenza A and 1 to CoVH-OC43. Patients that were detected HPIV had been hospitalized for neurological diseases (40%), lung (13.33%), heart (8.33%) and immunosuppression (8.33%). Children up to 24 months were the most HRI presented by HPIV. Infections were predominantly diagnosed as upper respiratory tract (82.67%), and pneumonia (10.33%) and bronchiolitis (7%) were




predominant in infections of the lower airways. Three children with positive samples for HPIV (two with HPIV-3 and 1 to VPI-4) had complications from pneumonia and died. The occurrence of IPV-3 was predominant in the driest months of the year as already described. The VPI-4 showed greater movement when compared to the HPIV-1 and 2. This study shows that the HPIV can join HRI commonly the case in our city. FINANCIAL SUPPORT: FUNCAP-PPSUS 2009

HV231 - SCREENING OF MULTIPLE BIOLOGICAL ACTIVITIES OF MARINE INVERTEBRATES AND ALGAE FROM SOUTH AMERICAN COASTBoff, L.; Kratz, J.M.; Almeida, M.T.R.; Costa, D.T.M.; Gomes, M.; Sarda, F.N.; Steindel, M.; Reginatto, F.H.; Schenkel, E.P.; Simões, C.M.O.


Natural products remain an important source of active substances, even though the development of high-throughput screens based on molecular targets proved valuable for drug discovery. The shift away from large combinatorial libraries towards more diversity-oriented small compounds collections, containing natural products features, highlight their potential as drug leads. Marine natural products in special represent a spring of unique structures with diverse biological properties. In this study, we evaluated multiple in vitro biological activities of 39 marine samples collected off South American coast, containing crude extracts and fractions from marine invertebrates (sponges, corals and ascidians) and algae. Cytotoxic potential was evaluated on A549 cells (lung tumor) by sulforhodamine B assay. Antiviral activity (herpes simplex virus type 1, KOS strain) was investigated by plaque reduction assay. Antifungal and antibacterial activities were investigated by disk diffusion method against several pathogens. Antiprotozoal activity (Leishmania amazonensis amastigotes) was assessed by a colorimetric assay employing parasites expressing beta-galactosidase. Results were expressed as mean percentage of inhibition or 50% inhibitory concentrations (IC50) of three independent experiments. Results showed a very diverse profile of activities. Regarding the antiviral activity, the hydroalcoholic fraction from the sponge Raspailia sp.

presented the most promising antiherpes activity, with an IC50 value of 39.20 µg/mL and no toxicity (>50 µg/mL) against the cells used in the antiviral assay. This preliminary parallel screening directed the selection of the most promising species. Phytochemical studies are now being carried out to support the bioguided fractionation of these samples, in order to isolate the natural products responsible for the biological activities identified, and further mechanistic in vitro/in vivo experiments. FINANCIAL SUPPORT: CNPq and CAPES.

HV233 - MOLECULAR EPIDEMIOLOGY OF HUMAN ADENOVIRUS IN ACUTE CHILDHOOD RESPIRATORY INFECTIONS IN FORTALEZA-CEARÁ, AN EQUATORIAL AREA OF BRAZIL, BETWEEN 2001-2011Ocadaque, C.J.1; Pereira, S.A.R.1; Florêncio, C.M.G.D.1; Harsi, C.M.2; Marinheiro, J.C.2; Moura, F.E.A.1



Human adenovirus (HAdV) is commonly viral agent associated with infections of the respiratory tract, both upper and lower, mainly in children under the age of five. HAdV are classified into seven species (A-G) and divided into 54 types. HAdV are important respiratory pathogens, found in between 2 and 27% of acute respiratory infection (ARI) cases. Few studies have analyzed the diversity of species and types of HAdV associated with ARI in Brazil. The purpose of this study was to determine the circulation patterns of the different HAdV species and respective types associated with ARI in children in the city of Fortaleza, northeastern Brazil. Nasopharyngeal aspirates were collected from children with ARI symptoms and were submitted to the indirect immunofluorescence assay (IFA) for initial detection of HAdVs. Then the HAdV strains were identified to the level of species and type through PCR, nested-PCR and sequencing of the hypervariable regions of the hexon gene (HVR1 to HVR6). HAdV were detected 290 (3.41%) of the 8,517 samples, 60 (20.69%) of those found in coinfection with one, two or three viruses. A total of 190 (65,52%) HAdV were molecularly characterized: 162 HAdV belonging to species B (85.72%), 21 to HAdV- C (11.11%) and 6 to HAdV-3 (3, 17%), getting




a strain without identification. The species circulated throughout the period the predominance of species B in almost all years analyzed. Seven different types were identified circulating in Fortaleza during the analysis period. The predominant types were 3 (67.73%) and 7 (17.80%). HAdV-3 was found in all the years and the HAdV-7 was not observed in years 2006, 2010 and 2011. Only in 2011, all kinds of species identified C circulated, and the only representative of the species and the HAdV- 4, began to be identified in our population only from 2007. The detection rate of HAdV in this study is similar to the findings of other studies using IFA to detect HAdV. The predominance of HAdV-B, type 3, observed in this study, is similar to that reported in recent studies in Colombia and South Korea. In turn, in southeastern Brazil there are reports of the predominant circulation of HAdV-C and HAdV-B, with type 7 being the major type. Prevalence of HAdV-C over HAdV-B has been reported in many other Latin American countries. In conclusion, we identified seven different types of HAdVs in the pediatric population of Fortaleza, associated with different respiratory complaints, in a period of 11 years. FINANCIAL SUPPORT: CNPQ

HV234 - DO YOU KNOW BOVINE VACCINIA? PERCEPTIONS AND KNOWLEDGE OF ORTHOPOXVIRUS INFECTIONS IN RURAL BRAZILCalixto, R.S.1; Costa, G.B.1; Calixto, R.S.1; Augusto, L.T.S.1; Bonjardim, C.A.1; Ferreira, P.C.P.1; Abrahão, J.S.1; Moreno, E.C.2; Kroon, E.G.1; Trindade, G. de S.1

1. IMA - Instituto Mineiro de Agropecuária, R. Piauí, 639, Poços de Caldas - MG, 37701-024


3. Departamento de Microbiologia/ICB/UFMG - Laboratório de Vírus localiza-se no Departamento de Microbiologia, no Bloco F4, sala 258, do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, MG, 31270-901

In Brazil, Vaccinia virus (VACV), which is included in Orthopoxvirus (OPV) genera, is responsible for a disease called Bovine Vaccinia (BV), characterized by vesiculopustular lesions in dairy cattle and horses, as well as in humans. Infections can occur after direct contact with animals naturally infected. Furthermore,

wild and peridomestic rodents can be involved in viral transmission. The aim of this study was to investigate the knowledge of a rural population which lives in an endemic rural area to BV occurrence, evaluating perceptions about virus spread in environment, as well as measures to prevent infections. It was conducted an epidemiological survey and 240 individuals were interviewed about BV outbreaks in region and exposure factors. The population comprises 127 men (52.9%) and 113 women (47.1%), aged from 5 to 90 years. A total of 124 individuals (51.7%) know or heard about BV. Of these individuals, 100 (80.7%) have contact with dairy cattle and horses, 46 (19.2%) have contact with wild environment, 67 (54%) practice milking. All of these variables were statistically significant, p < 0.001. People who never had heard outbreaks were asked about main form they know BV, over the radio (2.5%), or TV (2.9%), if a veterinarian or health professionals explain to them (2.9%), over the internet (0.4%) or in their neighborhood (7.9%). After smallpox eradication, the vaccination that confers immunity against OPV was discontinued and several outbreaks have been described worldwide. In Brazil, BV occurs in several regions of the country, but little is known about its epidemiology. At the present moment, outbreaks are associated to rural environments affecting dairy cattle and humans. Therefore, individuals located in these areas are vulnerable. Knowledge regarding BV infection and/or transmission is helpful to avoid virus spread in cattle herds and neighborhood, preventing outbreaks or even its contention. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG, MAPA, PPG-MICROBIOLOGIA UFMG.

HV236 - IN VITRO ANTIHERPES EFFECTS OF A STANDARDIZED EXTRACT OBTAINED FROM CECROPIA GLAZIOVII SNETHLSilva, I.T.; Santos, T.C.; Silva, I.T.; Battisti, M.A.; Ortamnn, C.F.; Reginatto, F.H.; de Campos, A.M.; Simões, C.M.O.


Herpes Simplex Viruses cause mild to severe labial and genital infections and represent a good enveloped virus model for antiviral in vitro research. Natural products have been evaluated due to the efficacy as an alternative




for chemical antiviral agents. Pharmacological studies using Cecropia glaziovii (embaúba) have described antiviral activity, and quantification of the chemical constituents showed C-glycosylflavonoids as the major phenolic compounds. Thus, the increased need for predicting the optimal extractive conditions has become a growing challenge in the search for phytopharmaceuticals with high level of uniformity, reproducibility, stability, and biological activity. The aim of this study was to optimize the efficiency of the preparation of a standardized extract obtained from the leaves of embaúba as well as to evaluate the cytotoxicity and the antiherpes activity. Standardized extract (SE) was prepared by the turboextraction technique. The Response Surface Methodology (RSM) design was used to evaluate the effects of the variables: (A) solvent type (water, ethanol 20% and 40%), (B) stirring speed (9500 and 13500 rpm) and (C) extraction time (5, 10 and 15 min). The SE was evaluated in terms of its phytochemical markers, isoorientin (ISOO) and isovitexin (ISOV), by HPLC. The cytotoxicity of the SE was investigated on VERO cells using a trypan blue exclusion assay, and antiviral activity was tested against HSV-1 (KOS strain) by viral plaque number reduction assay. Results showed that the Factor A had significantly influence (p<0.0001) on the content markers, where there was an increasing of the markers extraction using ethanol 20% (similar results were obtained for 40%) (ISOO=8.14 mg/g and ISOV=4.12 mg/g). Factor B exhibited a significant effect on the ISOV content (p<0.0184) and Factor C had no influence. The experimental design allowed to predict the ideal extraction conditions using ethanol 20%, 9500 rpm and 5 min. (desirability = 1). Cytotoxicity and antiviral activity results were expressed as 50% cytotoxic concentrations (CC50) and 50% viral replication inhibitory concentrations (IC50), respectively, in order to calculate the selectivity index (SI=CC50/IC50). SE inhibited HSV-1 replication showing a SI value of 50.13. These results indicate that SE might be a promising candidate for the development of new pharmaceuticals for the treatment of herpetic infections. FINANCIAL SUPPORT: CAPES/CNPQ

HV237 - MOLECULAR AND SEROLOGICAL DETECTION OF CYTOMEGALOVIRUS INFECTION IN CHILDREN ATTENDING DAY CARE CENTERSantos, W.K. da S.; Costa, M.A.; Joventino, K.M. de S.; Miranda, F.J.A.; Costa, M.C.M.; Araujo, L.; Costa, A.P.F.; Souza, L.M.S.; Souza, L.B.F.C.; Rocha, N. de S.P.D.; Farias, K.J.S.; Machado, P.R.L.

UFRN - Universidade Federal do Rio Grande do Norte, Campus Universitário Lagoa Nova, Natal - RN, 59078-970

Cytomegalovirus (CMV) infection is the most prevalent in the world with variable incidence according to the population studied. The aim of this study was to evaluate the prevalence of CMV in children attending day care center of Natal city, Rio Grande do Norte, Brazil. The prospective study included 106 children with 1-4 years of age, 50% males and 50% females, besides twelve daycare educators, with median of thirty-one years old. Anti-CMV IgG and IgM antibodies were determined using ELISA and CMV DNA was detected by nested PCR using viral DNA extracted from peripheral blood cells. Viral DNA was extracted from serum with AxyPrep Body fluid viral DNA/RNA miniprep kit from positive samples to molecular detection inside peripheral blood cells. Questionnaires recollected epidemiologic-clinical data. Of the studied sample, 83% children were confirmed positive anti-CMV IgG, and 17%, negative. As what concerns the detection of anti-CMV IgM antibodies, 34.9% were positive, and 64% negative. The twelve daycare educators presented anti-CMV IgG antibodies. No difference emerged from the comparison of positive and negative child groups in the research of anti-CMV IgG antibodies as what respects sex, age, income, mother school degree, and water and nutrition status. At the molecular detection, 6.6% samples viral DNA obtained from peripheral blood cells were positives and no sample of serum was positive. This study found a high prevalence of infection by CMV in children attending day care center, and viral detection in peripheral blood cells in children with asymptomatic infection. FINANCIAL SUPPORT: Ministério da Educação (MEC).




HV238 - PREVALENCE OF EPSTEIN-BARR VIRUS INFECTION IN GASTRIC ADENOCARCINOMA IN PARA STATECosta, I.B.1; Paixão, L.C.F.1; Barros, I.C.1; Santos, L.F.P.1;Polaro, A.A.1; Monteiro, T.A.F.1; Burbano, R.M.R.2


2. UFPA - Universidade Federal do Pará, Rua Augusto Corea, nº 1, Belem Do Pará - Para, 68440-000

Epstein-Barr virus (EBV), formally named Human hespesvirus 4 (HHV-4), primarily infects B lymphocytes and epithelial cells and is present in over 90% of the adult population. The virus is associated with about 10% of gastric adenocarcinomas, ranging according to geographic region. Estimates by the National Cancer Institute indicate 20,390 new cases of gastric cancer in Brazil in 2014, ranking the sixth most common cancer in the Brazilian population. In the North Region, is the second most frequent cancer in men and the third in women. Gastric adenocarcinomas can arise in different regions of the stomach and are classified into two main histological types: intestinal and diffuse. This study aimed to show the EBV prevalence in gastric adenocarcinomas, compares two detection techniques and associate the results with epidemiological characteristics. Was used 307 samples of gastric adenocarcinomas. Slides from paraffin embedded tissue were prepared for in situ hybridization technique (ISH), which was performed with one commercial kit according to the manufacturer’s instructions. The DNA extracted from the tumors was used in quantitative PCR (qPCR) for detection of EBV, using the kit “EBV Q-PCR Alert”. Of the 307 samples, 252 had results in ISH, being two (1%) positive samples, 230 (91%) show lymphoid infiltrate label and 20 (8%) without labeling. For qPCR, 228 results were obtained: 133 (58%) were positive and 95 (42%) undetectable. The results by the techniques of ISH and qPCR did not achieve an agreement (p <0.0001) use McNemar test. The association between the results of ISH or qPCR and variables such as sex, histological type and primary site were not statistically significant (p> 0.05). The ISH is considered gold standard for studies of association of EBV and cancer, and in this study it was considered for determining prevalence. It is much more specific for the presence of the virus in tumor cells. The prevalence

of EBV infection in gastric adenocarcinoma was 1%. There wasn’t concordance in results of qPCR and ISH techniques. There isn’t association of EBV detection tests with sex, histological type and primary site. In our study, we found a high frequency of lymphoid label, but it was not possible to infer if these infected cells have some influence on the development of gastric cancer. Then, it is suggested to make further studies to understand the role of these cells and if it can produce effects in tumor tissues. FINANCIAL SUPPORT: MS/SVS/IEC.

HV239 - DENGUE TRANSMISSION IN A MEDIUM-SIZED CITY FROM BRAZIL, 2008 – 2012: DESCRIPTIVE EPIDEMIOLOGYMondini, A.1; Ferreira, A.C.1; Neto, F.C.2

1. FCFAR/UNESP - Faculdade de Ciências Farmacêuticas da Universidade Estadual de São Paulo, Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902

2. FSP/USP - Faculdade de Saúde Pública da Universidade de São Paulo, Av. Dr. Arnaldo, 715 - Consolação, São Paulo - SP, 01255-000

Dengue has been a public health problem in tropical regions for decades but autochthonous transmission has now been reported in countries like USA, France and Croatia. In 2013, 2.35 million dengue cases were reported in the Americas and Brazil alone was responsible for more than 50%. The country has been presenting outbreaks for more than three decades and important lessons can be learned. Thus, a detailed analysis of dengue transmission in Araraquara, a medium-sized city at the central portion of São Paulo, which is a critical area for dengue transmission, may clarify transmission patterns and entail effective control measures. Dengue has been circulating in the city since 1990s at low incidences. However, the number of cases has increased in recent years and we will be describing the scenario of five years of transmission herein. Official data on dengue reports from 2008 to 2012 were recovered from the Information System on Diseases of Compulsory Declaration. Data from 5,253 reported cases were analyzed. The majority occurred at the first five months of the year - January to May -which is final portion of the rainy season. Severe dengue or dengue with complications was a rare event in the city. The incidence in Caucasians (78%) was higher than in other ethnic groups, a pattern that has been described worldwide. Another important observation




is that dengue transmission has become endemic in the city, with cases being officially reported in all months of the year, with the exception of 2009, which was atypical in the whole state of São Paulo. This is part of an ongoing project that is also focused on classical and spatial epidemiology involving a survey of risk factors such as socioeconomic and climatic variables. Corresponding author: [email protected] FINANCIAL SUPPORT: FAPESP 2013/02338-9

HV241 - MOLECULAR SURVEILLANCE OF DENV IN MOSQUITOES FROM A MEDIUM-SIZED CITY OF THE CENTRAL PORTION OF SÃO PAULO STATE, BRAZILGusmao, A.F.1; Guioti, F.1; Mota, T.Q.1; Bergo, E.S.2; Ferreira, A.C.1; Mondini, A.1

1. FCFAR/UNESP - Faculdade de Ciências Farmacêuticas da Universidade Estadual de São Paulo, Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902

2. Superintendência de Controle de Endemias – SUCEN/SR-06

Infections by the four dengue serotypes (DENV 1-4) have been reported in more than 100 countries and it is considered one of the most important arthropod borne viruses in terms of public health. Aedes aegypti is the main vector of DENV in Brazil due to its adaptation to the urban environment. Araraquara, at the central portion of São Paulo State/Brazil, has a high quality of life (HDI 0, 830), which is comparable to developing countries. However, no studies on DENV circulation were available for the city. The aim of this study is to evaluate the presence of DENV 1-4 in samples of mosquitoes collected in Araraquara from February 2014 to January 2015. We have installed 150 ovitraps in a preestablished grid of census tracts that covered the entire municipality. The ovitraps were collected five days after installation and the eggs in cardboard paddles were hatched. Mosquitoes were identified and pooled according to collection site, species and gender. A Multiplex-Nested-RT-PCR was used to detect DENV in our mosquitoes. So far, we have collected 4041 eggs that produced 2712 adults and 336 pools. We have assessed 20 pools (10 pools of each gender). We could not detect the presence of DENV in any of the pools. It is important to notice that DENV is actively circulating in the city and not even 10% of the pools were analyzed. We are still collecting eggs and we will be collecting samples from febrile patients with

clinical diagnosis of dengue. These are preliminary data from a study that is being conducted in Araraquara to detect DENV in mosquitoes and in humans.

HV245 - INTERLEUKIN-6 PROMOTER POLYMORPHISM IN RENAL TRANSPLANT PATIENTS INFECTED BY CYTOMEGALOVIRUSSantos, W.K. da S.1; Minnicelli, C.1; Souza, L.M.S.1; Joventino, K.M. de S.1; Costa, A.P.F.1; Freitas, J.C. de O.C.1; Pereira, M.G.1; Quintana, V.H.A.2; Farias, K.J.S.1; Lemos, T.M.A.M.1; Hassan, R.3; Machado, P.R.L.1



3. INCA - Instituto Nacional do Câncer, Praça Cruz Vermelha, 23, Centro, Rio de Janeiro - RJ, 20230-130

The cytomegalovirus (CMV) infection is an important cause of morbid-mortality in solid organ transplant patients. CMV reactivation from latency occurs after immunosuppression, thus immune surveillance is required to maintain constant persistent infection under control. The single nucleotide polymorphism (SNP) located at position -174 in the promoter region of interleukin (IL)-6 gene is associated with differential expression of this cytokine. The aim of this study was to investigate the role of IL-6 -174C/G with CMV infection in patients undergoing renal transplantation at the University Hospital Onofre Lopes (HUOL), Natal, RN, Brazil. From August 2012 to January 2014 samples were collected from the peripheral blood of organ recipients after transplantation. DNA was extracted from peripheral blood mononuclear cells (PBMC) and serum. Genotyping of IL-6 -174C/G was performed using a validated allele specific PCR in real time using SYBR⎕ Green. Clinical information were obtained from medical records of patients. A total of 52 patients, 27 (51.9%) male, were included in the study with median age= 45y (22-69). Of them, 1 (1.9%) and 48 (92.3%) were CMV IgM and IgG +, with similar frequencies for donors. Three patients (10.7%) presented rejection to the graft. Viral DNA was detected in PBMC of 20/52 (38.5%) and in serum of 10/52 (19.2%) patients. A possible association was observed between detection of CMV in recipients PBMC and the serological status of donors with all the 20 CMV + being




related IgG + donors (p= 0.068; OR= 1.2; 95% CI= 1.04 – 1.49). The frequencies of genotypes CC, CG and GG were 30 (57.7%), 32 (61.5%) and 0 (0%), respectively, Hardy-Weinberg equilibrium (HWE) (⎕²= 7.5, p= 0.006, df= 1). The CC genotype was present 7 (70%) of the 10 patients with CMV infection in serum and in 23 (54.8%) from the 42 patients negative for the virus in serum, although this difference was not statistically significant (p= 0.48, Fisher test). In this cohort, the SNP-174C/G genotypes were not associated to serological or molecular status of CMV or clinical symptoms of reactivation. A possible reason for the HWE deviation found in our study is in the small number of patients and the low frequency of the G allele already described worldwide. A possible association was observed between serological status of donors and viral reactivation in recipients of renal transplant which describes a specific panorama of our population that requires further investigation.

HV249 - AAV-HPV COINFECTION AND THE EVOLUTION OF THE INTRAEPITHELIAL CERVICAL LESIONSFreitas, L.B.; Volpini, L.P.B.; Boldrini, N.A.T.; Miranda, A.E.; Spano, L.C.

UFES - Universidade Federal do Espírito Santo, Av. Fernando Ferrari, 514, Goiabeiras, Vitória - ES, 29075-910

The cervical cancer is one of the most incident types of cancer in women worldwide, being human papillomavirus (HPV) recognized as the etiologic agent. Some cofactors related to viruses and hosts may influence the progression of malignant lesions. The adeno-associated virus (AAV) is a parvovirus that depends on the helper virus for productive infection to occur, and HPV was the helper virus reported more frequently in the genital tract. A bidirectional interaction between these viruses was demonstrated, which AAV could repress the gene expression of HPV, subsequently may reflect on a protective role for this parvovirus in the development of HPV-induced cervical carcinoma. Therefore, this study aimed to investigate the role of the coinfection AAV-HPV in the progression of intraepithelial lesions of the cervix in women attended at the University Hospital of Espírito Santo. To identify the persistence / clearance of the viral infection and the progression / regression of the lesion, two samples of each woman were collected with one year of interval.

Cytopathological examinations were performed of all samples and the treatment was made as recommended. DNA was extracted using QIAamp ® DNA Mini Kit commercial kit, following manufacturer’s instructions. AAV DNA was investigated by PCR and nPCR and HPV by PCR and hybrid capture. AAV and HPV genotyping was performed by RFLP and RLB, respectively. About 50% of cases referred to the colposcopy clinic had normal cytology, followed by low-grade lesions and fewer, high-grade lesions. Out of the normal group, 78% remained with normal cytology, while 22% progressed to cervical lesion; 74% of the low-grade group regressed to normal cytology, and 78.6% of the high-grade lesion group regressed to low-grade or normal cytology. Prevalence of AAV2 was 30% at both times of collection and HPV was positive for 56% of the samples, at first visit, and in the second moment, 36.5% were positive. The PCR test showed good correlation with hybrid capture for HPV detection. Using hybrid capture and RLB, HR-HPV were the most frequent, with HPV16, 58, 51, 52 and 53 the genotypes mostly identified in the two moments of the study. Logistic regression analysis demonstrated the presence of AAV was inversely related to the progression of HPV-induced cervical lesions. FINANCIAL SUPPORT: CAPES, FAPES

HV250 - VIRUS SURVEILLANCE IN PATIENTS WITH CLINICAL DIAGNOSIS OF DENGUE FROM A MEDIUM-SIZED CITY OF RONDONIA STATE, BRAZILPereira, W.V.E.G.; Fagotti, A.; Guioti, F.; Ferreira, A.C.; Mondini, A.

FCFAR/UNESP - Faculdade de Ciências Farmacêuticas da Universidade Estadual de São Paulo, Rodovia Araraquara - Jaú Km 1, Araraquara - SP, 14801-902

The infection by any of the four serotypes of the dengue virus (DENV 1-4) can be asymptomatic or produce a disease that ranges from a mild febrile illness to life threatening hemorrhagic manifestations. In Brazil, the majority of dengue disease diagnostics are obtained by serology and clinical and epidemiological criteria. Virus isolation, identification of DENV by polymerase chain reaction or the detection of the nonstructural protein 1 are usually performed in reference laboratories for a limited number of cases. No further tests to detect other arthropod borne viruses (arboviruses) are performed in febrile patients. In our study, we are testing samples




with clinical diagnostic of dengue from a medium sized city from the west part of Brazil for DENV 1-4 and other flaviviruses, along with orthobunyaviruses and alphaviruses. We collected blood samples from febrile patients that had clinical diagnostic of dengue from August 2013 to January 2014 in Ji-Paraná (Rondonia), which is allegedly an area with the circulation of several arboviruses. Viral RNA was extracted and a Multiplex-Nested-RT-PCR with genus-specific primers to detect members of the Flavivirus, Alphavirus and Orthobunyavirus genera and species-specific primers to identify DENV 1-4, yellow fever virus, mayaro virus, oropouche virus and seven other viruses was performed. We were able to collect 103 blood samples for the study. So far, we have tested 12 samples and we could not detect DENV or any other arboviruses. However, 89% of the samples still remain untested. It is undeniably important to provide differential diagnostic for febrile illnesses, not only for a better management of the patients but also to understand the epidemiology of the viruses that may be circulating in a certain location and the control measures that should be taken to control their spread.

HV253 - PREVALENCE AND DIVERSITY OF BETA-PAPILLOMAVIRUS IN ANOGENITAL AND ORAL SAMPLES OF MEN IN THE HIM STUDYNunes, E.M.1; Ferreira, S.1; Campbell, C.M.P.2; Gheit, T.3; Tommasino, M.3; Baggio, M.L.1; Galan, L.4; Silva, R.J.C.; Ponce, E.L.5; Giuliano, A.R.; Villa, L.L.6; Sichero, L.1

1. Center of Translational Oncology, Cancer Institute of São Paulo

2. Department of Cancer Epidemiology, H. Lee Moffitt Cancer Center and Research Institute

3. International Agency For Research On Cancer (IARC)

4. Ludwig Institute for Cancer Research, São Paulo Branch

5. National Institute of Public Health6. Center of Translational Oncology, Cancer

Institute of São Paulo; Department of Radiology and Basic Oncology, School of Medicine of The University of São Paulo and HPV Institute, School of Medicine, Santa Casa de São Paulo

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. However little is known about the natural history of

HPV infection in men. The HPV Infection in Men (HIM) Study is a prospective study of the natural history of HPV infection in men from Brazil, Mexico, and the USA. At baseline, 22% of the genital specimens HPV-positive by Roche Linear Array were considered HPV unclassified because the viral type could not be identified. Among these unclassified samples, a high prevalence of Beta-HPV types was further observed using a PCR-sequencing protocol, and multiple Beta-HPV types were found in most specimens by a bead-based multiplex genotyping methodology. Our aim was to identify the prevalence and distribution of Beta-HPV types detected in samples concurrently collected from the genitals, anal canal, and oral cavity of the HIM Study participants. Samples were analyzed using a bead-based multiplex genotyping methodology capable of simultaneously identifying 43 distinct Beta-HPV types. We analyzed 198 genital, anal, and oral samples concurrently collected among 66 participants (22 men per country). We observed that the overall prevalence and detection of multiples infections by different Beta-HPV types was higher in the genital region from the 3 study sites. Multiple infections were less common in the USA. Mexican men presented the highest prevalence of the same HPV type across all three anatomic sites analyzed. Concomitant detection of the same HPV types in the anal canal and oral cavity was rare. Beta-HPVs were highly prevalent in the male genital region. The detection of the same Beta-HPV simultaneously in the 3 anatomical regions analyzed is suggestive of digital transmission or auto-inoculation. Further research is needed to determine how these Beta-HPVs are transmitted. FINANCIAL SUPPORT: Ludwig Institute for Cancer Research; FAPESP [Grants numbers 13/01440-4 to LS, 13/ 20470-1 to EMN and 08/57889-1 to LLV]; CNPq [Grant number 573799/2008-3 to LLV]. The infrastructure of the HIM Study cohort was supported through a grant from the NCI-NIH to ARG (CA R01CA098803).




HV263 - LACK OF TRANSMITTED MAJOR HIV-1 INTEGRASE RESISTANCE MUTATIONS AMONG HIV-1 INFECTED CHILDREN AND ADOLESCENTS FROM RIO DE JANEIROAzevedo, S.S.D.; Fernandez, J.C.; Morgado, M.G.

FIOCRUZ - Fundação Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-900

Raltegravir, an integrase inhibitor (INI), is one of the salvage therapy options offered by the Brazilian Ministry of Health for HIV-1 infected children from six years old and adolescents failing first and second line highly active antiretroviral therapy (HAART). As this drug has been in use in Brazil since 2009, in this study, we evaluated the potential occurrence of INI transmitted resistance mutations and the HIV-1 subtype diversity among 72 HIV-1 seropositive children and adolescents from Rio de Janeiro, Brazil. The viral RNA was obtained from plasma samples of 24 drug-naïve and 48 failing HAART children and adolescents from Rio de Janeiro. Integrase region was amplified by nested polymerase chain reaction and automatically sequenced. Subtype was determined by phylogenetic analyses and the profile of resistance mutations was performed using the HIV Drug Resistance database. Most samples were classified in integrase region as subtype B (73.5%), followed by F1 (10%), C (5.5%) and A1 (2.7%). Five samples were classified as URF_BF (7%) and one as URF_BU (1.3%). No differences in HIV-1 subtype frequencies were observed between drug-naïve and failing HAART groups, except for subtype F1 that was not identified in the drug-naïve group. Major INI resistance mutations were not observed in the two groups, but accessory mutations were detected, with higher frequency in the drug-naïve patients (33%). The most prevalent mutation was V151I, present in 20.8% in drug-naïve group and 8.3% in failing HAART group. This mutation was only detected in samples classified as HIV-1 subtype B. Mutation R263K was present in 8% and 4% among drug-naïve and failing HAART groups, respectively. The mutation L74V (4%) was only detected drug-naïve group, while mutations E157Q (12.5%) and G163K (2%) were only detected in failing HAART group. The absence of major resistance mutations in both groups emphasizes the importance of INI in the salvage therapy and these results highlight the relevance of the continuous surveillance of integrase genetic diversity and transmitted mutations in the country.

HV264 - SPATIOTEMPORAL DYNAMICS OF DISSEMINATION OF NON-PANDEMIC HIV-1 SUBTYPE B CLADES IN THE CARIBBEAN REGIONGonzalo, B.; Cabello, M.; Mendoza, Y.Y.


The Human immunodeficiency virus type-1 (HIV-1) epidemic in the Caribbean region is mostly driven by subtype B; but information about the pattern of viral spread in this geographic region is scarce and different studies point to quite divergent models of viral dissemination. In this study, we reconstructed the spatiotemporal and population dynamics of the HIV-1 subtype B epidemic in the Caribbean. A total of 1,806 HIV-1 subtype B pol sequences collected from 17 different Caribbean islands between 1996 and 2011 were analyzed together with sequences from the United States (n = 525) and France (n = 340) included as control. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in the Caribbean are driven by dissemination of the pandemic clade (BPANDEMIC) responsible for most subtype B infections across the world, and older non-pandemic lineages (BCAR) characteristics of the Caribbean region. The non-pandemic BCAR strains account for > 40% of HIV-1 infections in most Caribbean islands; with exception of Cuba and Puerto Rico. Bayesian phylogeographic analyses indicate that BCAR strains probably arose in the island of Hispaniola (Haiti/Dominican Republic) around the middle 1960s and were later disseminated to Trinidad and Tobago and to Jamaica between the late 1960s and the early 1970s. In the following years, the BCAR strains were also disseminated from Hispaniola and Trinidad and Tobago to other Lesser Antilles islands at multiple times. The BCAR clades circulating in Hispaniola, Jamaica and Trinidad and Tobago appear to have experienced an initial phase of exponential growth, with mean estimated growth rates of 0.35-0.45 year-1, followed by a more recent stabilization since the middle 1990s. These results demonstrate that non-pandemic subtype B lineages have been widely disseminated through the Caribbean since the late 1960s and account for an important fraction of current HIV-1 infections in the region.




HV265 - EVALUATION OF A POSSIBLE SENTINEL SURVEILLANCE SYSTEM FOR DENGUE TRANSMISSION IN A NEIGHBORHOOD FROM SÃO JOSÉ DO RIO PRETO (SP): EPIDEMIOLOGICAL AND MOLECULAR APPROACHESParra, M.C.P.1; Favaro, E.A.1; Dibo, M.R.1; Neto, F.C.2; Eiras, A.E.4; Kroon, E.G.4; Mondini, A.3; Nogueira, M.L.1





Dengue (DENV 1-4) is the most important vector-borne virus worldwide and Aedes aegypti mosquitoes are responsible for the urban transmission of dengue. São José do Rio Preto (SJRP), a medium-sized city located at the northwestern region of São Paulo State, has been presenting high incidences of the disease for more than 20 years and 2013 was not different. Geographical Information System (GIS) and spatial analysis tools may aid understanding how dispersion of mosquitoes can impact dengue incidences. Spatial data concerted with traps for capturing adult mosquitoes may enable the analysis of fundamental variables in dengue transmission. In our study, we propose the use of adult traps associated with molecular diagnosis as a sentinel surveillance system for the early detection of dengue cases in a region of SJRP. MosquititoTM and BG-SentinelTM traps were installed weekly from epidemiological week 15 of 2012 until epidemiological week 22 of 2013. The mosquitoes were tested with Hemi-Nested-Multiplex-RT-PCR for the presence of DENV. TerraView and ArcGIS softwares were used to perform spatial analysis and to produce thematic maps. We were able to analyze 893 mosquito pools and 2.8% were positive for DENV-4. Thematic maps demonstrate clusters of vector infestation. However, they indicate that traps associated with molecular surveillance in mosquitoes were not suitable for the early detection of DENV circulation once human cases in the study area were detected previously in health facilities. Thus, the

use of mosquito traps associated with RT-PCR does not work as a sentinel event for early detection of DENV human infections but it can be used as an important instrument for the entomological and viral surveillance. FINANCIAL SUPPORT: FAPESP, PRONEX, INCT-DENGUE, CAPES, CNPQ. EVOLVING INSTITUITIONS: FAMERP, UNESP, UFMG E USP

HV266 - EXPERIMENTAL MODEL OF INFECTION BY OROPOUCHE VIRUS IN MICEMamani, P.R.; Souza, W.M.; Badra, S.J.; Figueiredo, L.T.M.

Virology Research Center, School of Medicine of The University of São Paulo in Ribeirão Preto, Brazil.

Oropouche virus (OROV) of the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, and is transmitted to mammals and birds by Aedes serratus, Culex quinquefasciatus and the Culicoides paraenses is the primary vector in urban cycle. The OROV is the etiological agent of Oropouche fever in humans, which is clinically characterized as an acute febrile disease, that is endemic in north of Brazil and all Amazon region The OROV is the second arbovirus most important, where estimated more than 500,000 cases has been reported in Brazil only supplanted by dengue virus. We show here preliminary results of an experimental model of infection by OROV in young adult Swiss albino mice (Mus musculus) by intraperitoneal inoculation. The OROV strain BeAn 1999 was grown in C6/36 (Aedes albopictus) cells by 4 days, it was confirmed by immunofluorescent assay. The stock was centrifugation at 5,000xg, filtered through a 0.22 μm membrane and titrated by detection of cytopathic effect (CPE) in serial 10-fold dilutions inoculated in quadruplicates of Vero cell cultures, and obtained a final titer of OROV of 1.5 x 107 plaque forming units (PFU) in Vero cells per ml. A number of 24 female Swiss albino mice, with 3 weeks old, were inoculated intraperitoneally with 3 x 106 PFUs of OROV in 200 ul of the virus stock and observed for 14 days. The develop of clinical symptoms were observable after 6 days post inoculation, the animals started to show signals of encephalitis characterized by loss of equilibrium, circular march and seizures, and a mortality rate of 50%. This experimental model, is a necessity to understand pathogenesis of OROV, and




is very important for preclinical testing of vaccine or therapeutic intervention for OROV.

HV269 - SEROPREVALENCE AND RISK FACTORS OF HCV IN PEOPLE LIVING WITH HIVCahú, G.G.O.M.; Silva, D.M.; Lopes,T.R.R.; Morais, V.M.S.; Silva, J.L.A.; Coêlho, M.R.C.D.

UFPE - Universidade Federal de Pernambuco, Av. Prof. Morais Rego, 1235 - Cidade Universitária, Recife - PE, 50670-901

Coinfection by hepatitis C virus (HCV) in people living with the human immunodeficiency virus (HIV) is often observed due to the similar transmission route, particularly in relation to the parenteral route, illicit injecting drug use and blood transfusions. It is estimated that approximately 30% of all HIV-positive individuals are coinfected with HCV in the United States and Europe. In Brazil, prevalence depends on the geographic area, ranging from 4.1% to 54%. The prevalence of cirrhosis is three times higher in patients with HCV/HIV coinfection than in patients exclusively infected with HCV. The aim of the present study was to estimate the prevalence of HCV among HIV-positive individuals and evaluate the risk factors associated with the coinfection. This cross-sectional study evaluated individuals attended at the University Hospital of Pernambuco, Northeastern Brazil, between November 2013 and July 2014. The patients answered a standard questionnaire about socio-economic data, like the socio-economic status based on CritérioBrasil, which considers very poor people, classes D and E, whose income is under U$ 150.00 per month. The samples were tested for HCV antibodies byenzyme-linked immunosorbent assay (ELISA) using Murex anti-HCV kit (version 4.0; AbbottDiagnostics Division), according to the manufacturer’s instructions. A total of 500 individuals were screened, of whom 181 (36.2%) were female and 319 (63.8%) were male, with ages ranging from 18 to 98 years (mean = 43.1 years); 29.2% were white, 21.4% were black, and 49.4% representing other races. Regarding sexual orientation, 28.4% declared to be homosexual or bisexual. The overall prevalence of HCV antibodies was 2% (95% CI 1.02%–3.77%) in people living with HIV. This serological marker was observed in 8/10 (80%) males, the age group more frequent was from 40 to 49 years (4/10, 40%) and the socio-economic status in 8/10 (80%)

could be classified as classes B and C. Regarding sexual orientation, 40% (4/10) declared to be bisexual. No use of injected drugs was reported. Having had a tattoo and piercing were reported by 80% and 60%, respectively. The frequency HCV/HIV coinfection in this study was lower than in others Brazilian regions, this may be due to the low frequency of risk factors, such as injectable drugs. FINANCIAL SUPPORT: SCHOLARSHIP FROM CNPQ.

HV271 - IMPACT OF A MULTIPLEX MOLECULAR PANEL FOR DIAGNOSIS OF RESPIRATORY VIRAL INFECTIONSSena, A.; Oliveira, C.P.; Silva, A.L. de S.; Trindade, A.C.G.; Nogueira, G.B.; Laurindo, M.F.L.; Bueno, V.D.C.; Neto, M.M.; Moura, M.E.G.; Ricci, E.; Lazari, C. dos S.; Granato, C.

FLEURY - Fleury Medicina e Saúde

Viral infections are amongst the main causes of respiratory diseases. They are usually diagnosed using direct fluorescence assays (DFA). However, this method detects only a few number of pathogenic viruses and does not include emergent ones. Conversely, there are molecular panels currently available, which identify a large amount of viral agents, with higher sensitivity and faster performance. In order to evaluate the impact of simultaneous molecular detection of multiple respiratory viruses for etiologic diagnosis of respiratory infections, we analyzed 372 samples from respiratory tract processed in a private laboratory from 2011 October to 2013 July. A commercial kit has been used (CLART PneumoVir®, Genomica, Spain), based on a multiplex polymerase chain reaction (PCR) step, and the subsequent analysis of the amplified material on a low density microarray. This platform detects and identifies, simultaneously, adenovirus, bocavirus, coronavirus type 229, enterovirus, influenza A (pandemic and seasonal H1N1, H3N2) B and C, metapneumovirus A and B, parainfluenza 1, 2, 3, 4a and 4b, rhinovirus, respiratory syncytial virus (RSV) A and B. Of all samples, 56% were positive for at least one virus, a larger proportion compared to the 30% positivity of DFA found in previous studies performed in the same institution. Rhinovirus was the most frequent (34% of positive samples), followed by RSV (19%), metapneumovirus (9%) and influenza A pandemic H1N1. Thirteen co-infections had




been detected (6% of positive samples). Additionally, we identified 43 samples which had also been analyzed by DFA up to 14 days before, which detects adenovirus, influenza A and B, parainfluenza 1, 2 and 3, and RSV, with negative results. The molecular panel was able to detect at least one virus in 28 of them (65%), identifying not only viruses which were not detectable by DFA (15 samples) but also those which DFA should have been able to detect (16 samples). There were 4 co-infections amongst these samples (14% of positive samples). We concluded that molecular assays which detect multiple viruses simultaneously may have positive impact for diagnosing respiratory infections, once they detect a larger variety of viral agents and, in addition, they are more sensitive when compared to DFA, including cases of co-infection. Future studies with larger number of samples may show even more robust epidemiologic data.

HV273 - GEOGRAPHIC DISTRIBUTION OF HEPATITIS C VIRUS GENOTYPES IN BRAZIL, 2013-2014Reys, C.; Sacramento, P.R.; Moreira, C.M.; Lazari, C. dos S.; Granato, C.; Bueno, V.D.C.; Moura, M.E.G.

FLEURY - Fleury Medicina e Saúde

Hepatitis C virus (HCV) is endemic worldwide and infects more than 185 million people around the world. In Brazil, a prevalence of 1.82% has been reported. HCV is a positive-stranded RNA-enveloped virus which has a high genetic variability and is classified into seven major genotypes and over 100 subtypes that vary with regard to their geographic distribution and response to treatment. The aim of this study was characterize HCV genotypes circulating in some states of Brazil. For this project, 1218 sequential samples from HCV infected patients were analyzed, from January 2013 to June 2014. HCV-RNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and the 5’ UTR were sequenced using Big Dye® Direct Cycle Sequencing Kit (Applied Biosystems, USA). The samples were obtained in different cities from several Brazilian States, with the following distribution: 874 samples from Southeast region (São Paulo, Rio de Janeiro and Minas Gerais states); 259 samples from South region (Paraná and Rio Grande do Sul states); and 85 samples from Northeast region , (Bahia, Pernambuco and Maranhão states). Genotypes 1, 2, 3 and 4 were found among these samples, and the general frequencies were 52.93% for

genotype 1, 4.32% for genotype 2, 18.65% for genotype 3 and 0.73% for genotype 4. The distribution of HCV genotypes was different among Brazilian regions. In the Northeast states, subtype 1b was found in 35.79% of samples, followed by subtype 1a, with 21.05%. Subtype 3a was found in 16.84% of the samples and 2b in 1.05% in this region. Other subtypes as such 2a, 3b and 4 were not detected. In the South states, in 26.64% of samples subtype 3a was detected, followed by 1a in 21.62%, 1b in 16.22%, 1a/1b in 2.32%, 2b in 10.04%, 2a in 1.93%, and 4 in 0.39%. In the Southeast states, subtype 1b was present in 27.80% of samples, followed by 1a in 23.11%, 1a/1b in 5.38%, 3a in 16.36%, 2b in 1.49%, 2a in 0.92%, 4 in 0.92%, and 3b in 0.11%. Although genotype 1 has predominated in all regions, a larger proportion of genotype 3 was seen in South region. These results are similar to those found in a previous survey in Brazil (Campiotto et al, 2005), which analyzed 1688 samples from 1995 to 2000 and demonstrated a 64.9% frequency for genotype 1, 4.6% for genotype 2, 30.2% for genotype 3, 0.2% for genotype 4. In all regions, genotype 1 was the most frequent (51.7% to 74.1%), while genotype 3 was more common in the South (43.2%).

HV276 - VIRAL CONJUCTIVITIS CAUSED BY HUMAN ADENOVIRUS: COMPARISON AND STANDARDIZATION OF MOLECULAR DETECTION METHODS IN EYE SWAB SAMPLESBonon, S.H.A.; Baldi, C.; Costa, S.C.B.

University of Campinas/Faculty of Medical Sciences/Laboratory of Virus

Adenovirus (AdvH) is a major pathogen associated with eye infections worldwide. Therefore, a pattern for detecting AdvH in ocular swab specimens is desirable and may aid in therapeutic management to minimize the impact of these infections. Thus, we analyzed 175 samples collected from 79 patients who presented signs and symptoms of viral conjunctivitis to the Ophthalmology Department at the Clinics Hospital/UNICAMP. Three eye swab samples were collected from 48 patients: one on the day of the first visit (day 0), one on the second day of the visit (day +5), and one on the third day of the visit (day +10). This study used a randomized design which was created by ophthalmologists to study the efficacy of dexamethasone 0.1% / povidone-iodine 0.4%, administered on the days +5 and +10, in the treatment of




patients with acute viral conjunctivitis. The main goal of this work was the standardization the detection of AdvH infections in eye swab samples using a Polymerase Chain Reaction (PCR) with the primers H1 and H2 and a Nested Polymerase Chain Reaction (Nested PCR) with the primers externals AdTU7 e AdTU4 and internals AdNUS e AdNUA. This was done to determine the prevalence of infections caused by AdvH in patients with signs and symptoms of conjunctivitis viral and to compare the efficiency of both techniques. Was performed Nested PCR detection for HSV-1, HSV-2, VZV, CMV and EBV to verify the incidence of other herpesvirus in all samples on the day 0. AdvH detection by PCR was positive in 29/48 (60.4%) on the day 0 and on the days +5 and +10, 12/48 (25%) and 5/48 (10.4%), respectively. Nested PCR was positive in 37/48 (77%) day 0, and day +5 and +10, 18/48 (37.5%) and 10/48 (20.8%), respectively. The remaining 31 patients had only a single visit, on the day 0, because they skipped the others days of visits (days +5 and +10). This patients were taken together with the 48 patients on the day 0 to verify the prevalence of conjunctivitis viral, being 33/79 (41.7%) positive for AdvH, 4/79 (5%) positive for HSV-1 and 79/79 (100%) negative for the others herpesvirus tested (HSV-2,VZV, CMV and EBV). Of accord with the seasons, the major occurrence of conjunctivis viral was in the spring 34/79 (43%), being 23/34 (67,7%) positive for AdvH. Nested PCR was more effective that PCR for ocular detection of AdvH on the three days of the collected, with standard deviation of 1on the days +5 and +10, and 0 on the day 0. The results were statistically significant (p ≤ 0.0001).

HV279 - MAPPING THE INTEGRATION SITES E1-E2 OF HPV-16 AS A TOOL TO EVALUATE DIFFERENT STAGES OF CERVICAL DISEASE PROGRESSIONMello, F.C. do A.1; Carestiato, F.N.1; Mello, F.C. do A.1; Cordeiro, T.I.1; Silveira, F.A.2; Cavalcanti, S.M.B.1



Persistent infection with oncogenic genotypes of genital Human Papillomavirus (HPV) is associated with the development of cervical cancer, a significant cause of morbidity and mortality of women worldwide.

Precancerous cervical disease is classified by cytological (low [LSIL] or high grade [HSIL] squamous intraepithelial lesions) and histological stages (cervical intraepithelial neoplasia [CIN] grades 1 to 3). There are about a dozen HPV types associated with the development of cervical cancer, but a major role is attributed to the high-risk HPV types 16. Integration of the viral genome into the host DNA is related to lesion persistence and progression and is considered a late event in cervical carcinogenesis. Integration usually occurs within fragile areas of the viral E1 and E2 ORF, with the latter being the most frequently described. Elimination of E2 expression results in transcriptional over-regulation of the viral oncogenes E6 and E7, leading to increased expression of both viral oncoproteins that target tumor suppressor proteins, p53 and pRb respectively, among others, resulting in loss of cell cycle control, DNA alterations and telomerase activation. In order to evaluate the physical state (episomal or integrated) of HPV 16 genome, a PCR combining 8 primers pairs were used, covering the E1-E2 region, as described by Vernon et al (1997). Our preliminary results involved 45 samples from patients infected by HPV16, harboring cervical lesions in different stages of progression. Among them, 8 (18%) presented episomal HPV16, 17 (38%) mixed forms and 20 (44%) were integrated. The upper region E1a were the most frequently absent (disrupted) (19/42%), as well as for mixed states (18/40%); followed by the downstream E2c region, counting for 11 (24%) cases of complete disruption and 8 (18%) combined episomal+integrated forms. It is interesting to observe that literature points out a predominance of disruption in E2 region but our results suggested a highly prevalent E1 inactivation. The approach here described is a very specific methodology that can successfully map the HPV 16 genome fragile areas. Data are being analyzed in order to search for statistical correlation between integration and severity of the lesion but it has been observed that E1-E2 absences were suggestively frequent in HSIL and cancer. In a few cases, episomal forms were observed in cancer samples, suggesting additional biomarkers as responsible for carcinogenesis.




HV281 - EVALUATION OF REAL TIME QPCR ASSAY FOR ENTEROVIRAL MENINGITIS DIAGNOSIS IN PUBLIC HEALTH LABORATORY FROM SÃO PAULO STATEMachado, B.C.1; Vieira, H.R.1; Alves, M.R.M.1; da Silva, M.V.2; Carmona, R. de C.C.1


2. Instituto de Infectologia Emílio Ribas, Av. Dr. Arnaldo, 165 - Cerqueira Cesar, São Paulo - SP, 01246-900

Enterovirus (EV) genus is the most frequent etiological agent involved in viral meningitis cases worldwide. Implementation of a fast method to diagnose enteroviral meningitis became a very important requirement in public health services, because it quickly provides information to clinicians allowing better medical management. The aim of this work was to evaluate preliminary results of the real-time reverse transcription-PCR (RT-qPCR) assay implantation for detection of EV based on the conserved 5’untranslated region. A total of 616 cerebrospinal fluid (CSF) samples were selected from meningitis cases, sent to the Enteric Diseases Laboratory, Adolfo Lutz Institute for EV diagnostic between 1998 and 2013. These samples have been stored at – 70ºC. The RNAs were extracted directly from CSF by QIAamp® Viral RNA Mini Kit, and a TaqMan® single-tube fluorogenic 5’ nuclease RT-qPCR assay was applied. Evaluation was made by comparing the RT-qPCR results with it previously obtained by cell culture viral isolation method. EV RNA was detected in 94 of 616 (15.2%) samples using RT-qPCR assay and EV was isolated from 58 of 616 (9.4%) samples by viral culture. RT-qPCR had a sensitivity, specificity, positive predictive value and negative predictive value of 89.7%, 92.4%, 55.3% and 98.9%, respectively. Accuracy was 92.2% and incorrect classification 7.8%. Cell culture viral isolation and RT-qPCR showed a Kappa value of 0.78, demonstrating a good and substantial agreement. In our study, RT-qPCR assay was slight superior to viral culture for detecting EV from CSF samples. These results demonstrate that TaqMan RT-qPCR assay is a fast and sensitive assay for detection of EV infection. The test is easily performed, which makes it an excellent alternative to the laborious and time-consuming viral culture. EV RT-qPCR with CSF samples was highly sensitive and specific and has a potential to contribute substantially to the epidemiological surveillance of viral meningitis disease with incorporation into routine public health

laboratory in São Paulo State. FINANCIAL SUPPORT: Fapesp 2012/50234-5

HV282 - MOLECULAR DETECTION OF FLAVIVIRUSES IN TICKS (ACARI: IXODIDAE) FROM CAPYBARAS AND ENVIRONMENT IN AN URBAN PARK OF UBERLÂNDIA, MINAS GERAIS, BRAZILVieira, R. de S.; Pascoal, J. de O.; Pascoli, G.V.T.; Oliveira, T.F. de M.S.; Yokosawa, J.; Szabo, M.P.J.


The genus Flavivirus of Flaviviridae family comprises more than 70 virus species including many arthropod-borne viruses. Tick-borne encephalitis virus (TBEV), a pathogen that infects human central nervous system, has spread extensively in some regions of the world. Human cases of TBEV infections have increased in the past 30 years dramatically posing a danger to public health in several European countries. The main hosts for the virus are small rodents and humans are accidental hosts. In endemic areas, recreational or occupational exposure to rural or outdoor settings is a potential risk for infection from infected ticks. The Flaviviridae includes several other tick-borne viruses affecting humans that are closely related to TBEV. In Brazil the survey for virus in ticks is overall scarce. We herein report a survey for flavivirus in ticks, potentially responsible for zoonoses, in an urban park in Uberlândia city, state of Minas Gerais, Brazil. Ticks were collected from natu¬rally infested capybaras and from environment and stored at -70ºC. RNA was extracted from 60 ticks by using Trizol (Invitrogen) and polymerase chain reaction (PCR), in two rounds, using cDNA (RNA reverse transcribed into complementary DNA) was performed. The assay utilized degenerate primers targeting the flavivirus NS5 gene (encoding RNA-dependent RNA polymerase) and detects a range of flaviviruses. Two samples of Amblyomma dubitatum tick produced amplicons of approximately 242 bp and were submitted to sequencing. Nucleotide sequences were edited using Lasergene program (DNAstar) and compared with those available in the GenBank database using the BLAST algorithm. The sequences obtained were not similar to any known sequence. Other tick samples will be investigated for the presence of flavivirus. In addition, the isolation and purifica¬tion of the virus in




Vero cell culture will be attempted. Further research is important to determine potential emerging tick-borne zoonotic pathogens in Brazil. FINANCIAL SUPPORT: FAPEMIG AND CNPQ.

HV283 - DETECTION AND GENOTYPING OF SAPOVIRUS IN FECAL SAMPLES FROM CHILDREN HOSPITALIZED IN SÃO LUÍS, MARANHÃO, BRAZIL, FROM JUNE 1997 TO JUNE 1999Resque, H.R.1; Costa, L.C.P. das N.1; Portal, T.M.1; Siqueira, J.A.M.1; Linhares, A. da C.1; Luz, C.R.N.E. da2; Gabbay, Y.B.1; Resque, H.R.1


2. UFMA - Universidade Federal do Maranhão, Av. dos Portugueses, 1966 - Anjo da Guarda, São Luís - MA, 65080-805

Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide. Among the viruses that cause AGE the sapoviruses (SaVs), members of the Caliciviridae family, seems to be very important since they are associated with sporadic cases and outbreaks of AGE occurring in schools, day care centers and cruise ships. The most frequently observed symptoms on a SaV infection are diarrhea, vomiting and abdominal pain. The virus transmission occurs by the fecal-oral route, primarily through contact person-to-person, intake of contaminated water and/or food, and aerosolized particles generated during episodes of vomiting. The study aimed to detect and genotype SaVs in fecal samples from hospitalized children with and without gastroenteritis in São Luís, MA, during the period of June 1997 to June 1999. Nucleic acids were extracted from SaVs by the silica method and were subsequently tested by RT-PCR using the primers pair P289 and P290. Specimens were considered positive when showing amplicons of 331 bp. The positivity rate was 8.1% (11/136), with 15.2% (7/46) in the diarrheal specimens and 4.4% (4/90) in non-diarrheal ones (p <0.04). One sample was sequenced and classified as GII.1. Of the positive cases, 27.3% were associated with fever, vomiting and anorexia, and 18.2% with fever, anorexia and abdominal pain. The presence of asymptomatic cases reinforces the suggestion that even in the absence of clinical symptoms the virus continues to spread. SaVs detection in samples of children under two years corroborates published data

that this age group is the most affected, as well as the highest percentage of positive cases are in the group with clinical manifestation. The genotype found (GII.I) in this study was also detected in Belém, in samples of diarrheic children collected in different years (1998-2000 and 2003) and in Parauapebas in 2006, both in Pará state. Therefore, the results of this research are relevant as they demonstrate the circulation of SaVs in São Luís and reinforce that further studies should be developed, as there is still lack of information regarding the epidemiology of this virus. Financial Support: PIBIC/CNPq Keywords: sapovirus, gastroenteritis, children, São Luís

HV284 - CIRCULATION OF NOROVIRUS PANDEMIC VARIANT GII.4 SYDNEY 2012 AND NEW ORLEANS 2009 IN CHILDREN FROM MANAUS, AMAZONAS, BRAZILGabbay, Y.B.; Hernandez, J. das M.; da Silva, L.D.; Rodrigues, E.A.M.; Bandeira, R. da S.; Lucena, M.S.S. de; Soares, L. da S.; Mascarenhas, J.D’A.P.; Resque, H.R.; Gabbay, Y.B.

IEC - Instituto Evandro Chagas, Rodovia BR-316 km 7 s/n, Levilândia, Ananindeua - Pará, 67030-000

Norovirus (NoV) is a enteric pathogen known to cause pandemics of acute gastroenteritis worldwide. In developing countries, its estimated cause 200,000 deaths in children under 5 years old. In the EUA, some authors related that after the increased use of rotavirus vaccine, NoV become the primary cause acute gastroenteritis. Due NoV suffer a dynamic process of mutation and recombination, providing a great genetic diversity and this is the basis for its adaptability. These events have led to the emergence of variant strains, mainly of linhage GII.4, which has caused epidemics globally. In 2012, fecal specimens were collected from children until five years old from the city of Manaus, Amazonas, Northern Brazil. The samples were tested for NoV initially by EIE and the positive samples by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers with target the polymerase gene. The positive samples identified as GII.4 NoV were tested by PCR and sequenced using the primers EVP2F and EVP2R. These primers were designed to amplify the antigenic region of the viral capsid (VP1 protein), subdomain P2, allowing identification of variants. Samples were sequenced in




ABI Prism 3130XL DNA Sequencer (Applied Biosystems, Foster City, USA), aligned and analyzed by the Bio Edit program (v.7.1.3.0) and compared with others registered in GenBank. A dendrogram was generated by the neighbor-joining method, with 2,000 replicates in the bootstrap analysis, using MEGA 6.0 software. From the 51 samples positive by PCR (polymerase region), 16 were characterized by the capsid region, demonstrating the predominance of the strain GII.4 Sydney 2012 (62.5%) in the amazonian population, but the GII.4 New Orleans 2009 was also detected in lower percentage (37.5%). Nucleotide analysis showed difference of 1% among the Sydney ones and 7% among the all positive samples. When compared with the other GII.4 variants, as Farmington Hills 2002 and the DenHaag 2006b prototypes, the nucleotide difference was 15%. The results demonstrate the circulating of the pandemic strain Sydney 2012 in Manaus and are relevant if considered the limited data about the variants of NoV in the North region and also in Brazil. The surveillance of NoV variants is also important and necessary because each two to three years, it is being observed a new variant that replace the antecessor. FINANCIAL SUPPORT: Evandro Chagas Institute, PIBIC/IEC/CNPq, PPGV/IEC/FAPESPA.

HV286 - DEVELOPMENT OF A REAL TIME-PCR BASED IN SYBR GREEN FOR RAPID DETECTION OF BRAZILIAN VESICULOVIRUSTolardo, A.L.1; Romeiro, M.F.1; Souza, W.M.1; Siqueira, C.E.H.2; Bronzoni, R.V. de M.3; Figueiredo, L.T.M.1


2. Laboratório Municipal de Análises Clínicas , SINOP, MT

3. Universidade Federal do Mato Grosso, SINOP, Mato Grosso, Brazil

Vesiculovirus is a genus in the family Rhabdoviridae. In the Americas, Indiana and New Jersey serotypes of vesiculovirus, are both endemic and responsible for vesicular stomatitis, a disease of cattle, horses and pigs, responsible for important economic losses. In Brazil, it have been reported other vesiculovirus, such as Piry, Carajas, Cocal, Alagoas and Maraba. Curiously, serologic surveys in human populations of different regions of

Brazil, have shown approximately 10% of seropositivity to Piry virus. However, the disease produced by Piry virus has not been reported, except for one accidental case in the laboratory. There is a poor knowledge on the human infection and the disease by vesiculovirus as well as a limitation on the diagnosis which is based in serologic tests and performed in few laboratories. Ten years ago, our group reported a conventional RT-PCR to vesiculovirus. As we show in this on going study, the conventional RT-PCR evolved into a SYBR green I-based real-time RT-PCR that amplifies 221 bp of the G gene of Brazilian vesiculovirus. Our preliminary results show that it was able to amplify genomes of 4 Brazilian Vesiculovirus (Piry, Carajas, Alagoas and Indiana) with a higher sensitivity than the conventional RT-PCR. The melting curve of the real time RT-PCR, 81.41 ± 0.50 °C, has shown that it is specific for Brazilian vesiculovirus and do not generate primer-dimers or non-specific products. The sera of 165 patients with acute febrile illness, from Sinop city, in Mato Grosso State, were collected during the dengue outbreak of 2011 - 2012 and tested by the real time RT-PCR and no positive samples were found. This real time RT-PCR is reproducible, specific, and could become a useful tool for the diagnosis of infections. It could also be used in a surveillance program to better evaluate the importance of Brazilian vesiculovirus in human disease. FINANCIAL SUPPORT: FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo Nº. 13/14929-1, Fellowship Nº. 13/02256-2, 12/02836-6, 12/24150-9.

HV288 - INFLUENZA A H1N1 PDM09: LABORATORY SURVEILLANCE OF SUSPICIOUS DEATHS OF SEVERE ACUTE RESPIRATORY SYNDROME IN THE STATE OF SAO PAULO, BRAZIL - 2013Cirqueira, C. dos S.; Martines, R.B.; Iglezias, S.D.A. Kanamura, C.T. Ramos, A. da C.; Aguiar, A.


In April 2009 a new influenza A virus called H1N1pdm09 virus was identified in humans resulting in the first influenza pandemic of the 21st century with 5,700 deaths. After this time the pandemic activity is declining, however, the transmission of the virus remains. The disease presents clinical and transmission characteristics similar to those of seasonal influenza




and keeps as an etiological agent of flu cases in Brazil. So its vigilance with seasonal period and the risk of co-movement with other influenza viruses, as well as the risk of the emergence of new viruses become important laboratory surveillance along with cases of suspected deaths from severe acute respiratory syndrome (SARS). The objective of the study is demonstrate the prevalence of etiological diagnosis of influenza A H1N1 pdm09 in São Paulo in 2013 in tissue samples fixed in formalin and embedded in paraffin (FFEP) of suspected from deaths of SARS. Routine hematoxylin-eosin stains were performed for histopathological evaluation. The immunohistochemical assay for Influenza A H1N1 pdm09 was performed on 3-mm sections of FFPE lung and airways tissues using a monoclonal anti-influenza A H1N1pdm09 antibody, appropriate positive and negative controls were run in parallel. The results were evaluated and the data interpreted toghether with clinical, epidemiological and laboratory information available. Immunohistochemical staining for H1N1pdm09 was seen in 21 of 198 suspected deaths to SARS, with 28.5% of those elucidated only by IHC. 75.4% were male and 24.6% female, no pregnant woman; Ages ranged from 15 to 70 years. Concomitant bacterial infectious agent was observed in 28.57% of cases. Other viral etiologic agents in 19 cases of Influenza A not subtyped, 1 case positive for both influenza H1N1 pdm09 and not subtyped influenza A, 2 deaths by influenza B and 2 deaths from hantavirus were also identified. Although we observed a decrease in the incidence of cases of H1N1 influenza pdm09, even in 2013 detected the transmission of the virus in the state of Sao Paulo and its association with mortality. The vaccination and laboratory surveillance must be maintained and the sustained transmission of the disease may enable genetic recombination and virulence alteration.

HV296 - INVESTIGATION OF DENGUE VIRUS INFECTION IN GOIANIA, GOIAS, BY MOLECULAR AND SEROLOGICAL METHODSde Oliveira, T.S.; Guimarães, V.N.; Cunha, M. dos P.; Souza, M.B. de L.D.; Cardoso, D. das D. de P.; Carneiro, R. dos S. Fiaccadori, F.S.


Dengue is a major challenge to public health in Brazil and worldwide. Dengue virus (DENV) is an arbovirus of the family Flaviviridae, genus Flavivirus, classified into four antigenically distinct serotypes DENV-1-4. Infection with one of these serotypes may causes a disease whose spectrum ranges from clinically asymptomatic or a mild and self limited febrile illness called dengue fever to severe clinical forms. In Brazil, the co-circulation of four serotypes has led to successive epidemics and Goias state, usually present high number of dengue cases. In 2013 were related alarming statistics, with an increase of 401% in the number of reported cases and 25% in the number of deaths. The Epidemiological Surveillance System plays an important role in the continuous monitoring infections, nevertheless, it has been shown the relevance of laboratory diagnosis in the confirmation of new cases. To increase the detection of DENV in early infections, the NS1 research was introduced in Public Health Laboratories. However, the time of infection has a decisive influence on the results regarding the method of choice. Therefore, the present study evaluated the occurrence of DENV infection in Goiania, Goias in the epidemic period of 2012-2013 among the population with clinical suspected of dengue infection (up to seven days) using a combination of serological and molecular methods. Serum samples were collected from 278 symptomatic patients who were attended in basic health centers between October/2012 and May/2013. The samples were submitted to the research of serological markers NS1, IgM and IgG using a commercial kit (DuoTest-Bioeasy), as well as viral RNA detection by RT-PCR for the C-prM region. The positivity for infection was 43.9% (122/278), with rates of 30.5%, 13.6% and 20.5% for NS1, IgM and RNA markers, respectively. In 24.5% of DENV infection cases, NS1 was the only marker detected, demonstrating its advantage mainly favoring early diagnosis with higher rate of positivity between 4 and 5 days. The IgM was detected singly in 16.4% with statistical significance for the sixth and seventh days. For the RNA, the highest rate was observed between 1 and 3 days. The combination of these three markers, notably constitute a powerful tool in the diagnosis DENV. Monitoring of dengue epidemics by laboratory confirmation, with an active and effective notification system, it is relevant to establish a complete data base to




be used for surveillance studies. FINANCIAL SUPPORT: CNPQ; FAPEG

HV298 - PREVALENCE OF HPV-16 VARIANTS IN REPRODUCTIVE AGED WOMEN ATTENDED AT PRIMARY HEALTH CARE UNITS IN BOTUCATU, SP, BRAZILCandeias, J.M.G.1; Kurissio, J.K.1; Silveira, A.C.1; Ferreira, S.2; Bolpetti, A.3; Sichero, L.2; Silva, M.G.3; Villa, L.L.4,5; Candeias, J.M.G.1

1. INSTITUTO DE BIOCIêNCIAS/UNESP - Instituto de Biociências da Universidade Estadual Paulista, Instituto de Biociências, Distrito de Rubião Junior S/N, Botucatu - SP, 18618-970

2. ICESP - Instituto do Câncer do Estado de São Paulo, Av. Dr. Arnaldo, 251 - Sumaré, São Paulo - SP, 01255-000

3. FMB/UNESP - Faculdade de Medicina de Botucatu da Universidade Estadual de São Paulo, Av. Prof. Montenegro Bairro: Distrito de Rubião Junior, s/n, Botucatu - SP, 18618970

4. FM/USP - Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, 01246903

5. FCMSCSP - Faculdade de Ciências Médicas da Santa Casa São Paulo,Rua Doutor Cesário Motta Júnior, 61 - Vila Buarque, São Paulo - SP, 01221-020

It is known that cervical cancer is the second leading cause of death in women worldwide usually due to persistent infection with high-risk Human Papillomavirus (HPV). Among those, HPV-16 molecular variants have been shown to be differentially involved in viral persistence and development of cervical lesions. The aim of this study was to determine the prevalence of HPV type and HPV-16 molecular variants in a cohort consisting of 1601 women aged 18-50 years attended at Primary Health Care Units in Botucatu, Brazil. Ecto and endocervical samples were taken during physical exam using a non-lubricated speculum. HPV was detected by polymerase chain reaction (PCR) and HPV genotyping was conducted by Linear Array HPV Genotyping Test (Roche Molecular Systems Inc.). HPV-16 intratypic variation in single or mixed infections was evaluated by sequencing a fragment of the viral long control region (LCR). The overall frequency of HPV DNA detection was 33.0% and HPV-16 (17.0%) was the most frequent HPV genotype followed by HPV-58 (8.3%), HPV-52 (8.2%)

and HPV-31 (7.2%). Among samples for which it was feasible to analyze intratype variability, we identified four different molecular variants belonging to four branches of geographical and phylogenetic relatedness. European variants (87%) were the most prevalent and diverse group, followed by the Asian-American (6.4%), African (4.8%) and North-American (1.6%). Within the European branch, 75.0% of the samples were prototype and the remaining 25.0% were B-12. Although we conducted cytology examination in all samples, we did not observe correlation to any lesion grade and molecular variants detected. However, since this is an ongoing study where multiple samples are being collected from the same women, we hope to obtain more information regarding this specific question.

HV301 - DETECTION OF JC POLYOMAVIRUS (JCV) AND HUMAN CITOMEGALOVIRUS (HCMV) IN GLIOMASSilva, B.F.G.; Stangherlin, L.M.; Castro,L.F.F.; Silva, M.C.C.


The John Cunningham virus (JCV) infects a large proportion of the population worldwide. The virus usually remains latent on the kidney cells but can reactivate under immunosuppressive conditions and migrate to the brain, resulting in progressive multifocal leukoencephalopathy, a fatal demyelinating disease of the central nervous system. JCV can transform cells in culture and is oncogenic in laboratory animals. In addition, viral DNA sequences have been detected in several kinds of human malignancies, including brain tumors of glial origin, medulloblastomas and colon carcinoma, suggesting a possible relationship between JCV and cancer, Intringly, a study has demonstrated that the Human Cytomegalovirus (HCMV), a Beta-herpesvirus possibly implicated with brain cancer malignancy, is capable of induce JCV replication in vitro, in cells not permissive for this virus. In the present study we aimed to verify the presence of JCV and HCMV viral DNA sequences in glial brain tumors. Paraffin-embedded and fresh tumor samples were tested for the presence of HCMV and JCV by real time PCR and nested PCR. From 19 samples analyzed HCMV DNA was detected in all samples (100%) and JCV in 13 samples (68%). Despite a low number of specimens tested, the results




suggest a positive association between HCMV and JCV in glioblastomas. We are currently testing more samples for the presence of viral DNA and RNA in tumor tissues. Funding: UFABC

HV302 - VALIDATION OF TWO SINCICIAL RESPIRATORY VIRUS RAPID TESTSda Silva, E.R.M.; Lazari, C.S.; Granato, C.F.H.

Grupo FLEURY

Acute respiratory infections caused by respiratory syncytial virus (RSV) are important health burdens that affect infants worldwide. RSV infections are a common cause of pneumonia and bronchiolitis in children up to 5 years. Currently, RSV infection is diagnosed using direct fluorescence assays (DFA), which require specific laboratorial facilities and trained staff. Conversely, immunochromatographic test (ICT) for antigen detection may be useful as a point-of-care method of simple execution, which may provide rapid and reliable results. In order to validate this method in our population, we have evaluated two different ICT commercial kits – BinaxNow® RSV Card (Alere, USA) and RSV Test Bioeasy® (Bioeasy, Brasil) – and compared to results of a DFA panel which detects RSV, Influenzavirus A (IVA) and B (IVB), Parainfluenzavirus 1, 2, 3 (PIV1-3) and Adenovirus (AD). Sixty-six respiratory tract samples were tested. Compared to DFA, BinaxNow® has shown 90.4% sensitivity (19/21 RSV-positive samples in DFA). Twenty RSV-negative samples were tested using BinaxNow®, 10 of which were negative and the other 10 were positive to other viruses in the DFA panel. The internal control of the ICT did not work in three of them. The result was negative in 13, corresponding to an overall specificity of 76.4%. Four false-positive results were detected, suggesting cross-reactivity, in positive samples to IVB, PIV3 and AD, according to DFA. Regarding Bioeasy®, 84.2% sensitivity was demonstrated (16/19 RSV-positive samples in DFA). All of 30 RSV-negative samples tested with Bioeasy® remained negative, showing 100% specificity, without any cross-reactivity in 10 of them which were positive to other viruses in DFA. We did not have to discard any test of this brand due to inconclusive results. We concluded that RSV Test Bioeasy® has better performance, with acceptable sensitivity and superior specificity; therefore, it is appropriate for point-of-care use in children healthcare settings.

HV304 - SEROPREVALENCE OF HEPATITIS E VIRUS AMONG SCHISTOSOMIASIS PATIENTS IN RECIFE, PE, BRAZILSena, A.1; Passos, A.M.1; Reinaldo, M.R.1; Ferraz, M.L.1; Lopes, E.2; Granato, C.1

1. UNIFESP - Universidade Federal de São Paulo, R. Sena Madureira, 1500 - Vila Mariana, São Paulo - SP, 04021-001


Hepatitis E virus (HEV) infection is a worldwide disease usually presented as an acute self-limiting hepatitis, but in immunocompromised patients it may cause chronic infection with cirrhosis. Brazil has been classified as moderate endemic to HEV, with seroprevalence from 1% to 5% in the general population and 10% in renal transplant recipients. Schistosomiasis may play a role in virus infection by altering the immune system. However, the seroprevalence of HEV in this population group is unknown. The aim of this study was to evaluate the prevalence of past or present HEV infection in schistosomiasis patients in Recife, PE. One hundred nineteen schistosomiasis patients were enrolled in this study. Clinical and laboratory data were available for eighty individuals. The mean age of patients was 50 years old (median 51, range from 14 to 78 years old) and 52 (65%) were female. Serum samples were tested for the presence of anti-HEV IgG antibodies by enzyme immunoassay (Mikrogen, Germany) and for the presence of HEV RNA using real time reverse transcriptase-polymerase chain reaction with primers targeting the HEV ORF2 and ORF3. A third set of primers and probe targeting the human RNAse P gene was used as endogenous internal amplification control to certify specimen quality and extraction. Anti-HEV IgG was positive in 7 (6%) of the 119 serum samples tested. None of the samples showed the presence of HEV RNA. Seropositive patients aged from 35 to 70 years old (mean 56). The mean AST and ALT in patients without IgG antibodies were 31 U/L (range from 11 to 132 U/L) and 33 U/L (range from 6 to 132 U/L). Patients with IgG antibodies did not show significant increase in of ALT or AST levels. The mean platelet level was significantly lower in patients with IgG antibodies (83,667) when compared to patients without IgG (138,535) (p =




0.03). The same was observed for the mean leucocyte levels, 3,533 in patients with IgG and 4,993 in patients without IgG (p = 0.02). Alkaline phosphatase, gamma-glutamyl transpeptidase, albumin and protein levels were also decreased in patients with IgG antibodies, although not significantly. This study demonstrates that the seroprevalence of HEV may be higher in patients with schistosomiasis than in some imunocompetent populations in Brazil. Also, schistosomiasis patients with more impaired immune system and more severe disease seem to be more at risk of HEV infection. Hepatitis E should be further investigated among schistosomiasis patients. FINANCIAL SUPPORT: FAPESP 2012/22925-3 E 2013/03701-0

HV307 - MOLECULAR INVESTIGATION OF NATURAL INFECTION BY FLAVIVIRUSES AND ALPHAVIRUSES IN ADULT MOSQUITOES CAPTURED IN CUIABÁ, MATO GROSSO, BRASILSerra, O.P.1; Cardoso, B.F.1; dos Santos, F.A.L.1; Heinen, L.B. da S.1; Pacheco, T. dos1; Zuchi, N.1; Ribeiro, A.L.M.2; Rodrigues, J.S.V.1; Miyazaki, R.D.1; Slhessarenko, R.D.1


2. HUJM - Hospital Universitário Júlio Müller, Av. Fernando Corrêa da Costa, nº 2367, Bairro Boa Esperança, Cuiabá - MT, 78060-900

Arboviruses belonging to Flavivirus and Alphavirus genus are considered an important public health issue in Brazil, mainly in tropical areas.The aim of the study was to identify the frequency of hematophagous mosquitoes naturally infected by flaviviruses and alphaviruses in Cuiabá, MT. Culicidae specimens were captured in 3 locations from 200 censitary sectors from Cuiabá in 2013, identified with dichotomy key, allocated in pools(1-10 mosquitoes) according to sex, species, data and collection point.Minced pools were subjected to total RNA extraction, duplex-RT-PCR followed by multiplex-semi-nested-RT-PCR to 5 alphaviruses and 11 flaviviruses.Positive samples for Saint Louis encephalitis(SLEV), dengue 1(DENV-1) and mayaro(MAYV) viruses were amplified by single RT-PCR and subjected to nucleotide sequencing. A region of the SLEV envelope gene was amplified for phylogenetic analysis.Positive pools for MAYV were confirmed by

RT-qPCR.Minimum infectious rate(MIR) was calculated for positive species.Between 11.090 mosquitoes, 4.556 females classified in 13 species constituted 593 pools, 1/137(MIR=1.04) of Aedes aegypti positive for DENV-1 and 1/67(MIR=2,6) Culex sp. for SLEV.For MAYV,10/67(MIR=26,2) Culex sp.,7/162(MIR=3.7) Cx. quinquefasciatus,4/137(MIR=4.1) Ae. aegypti and 5/54(MIR=24) Cx. comp. pipiens were confirmed by RT-qPCR and 1 pool was confirmed by isolation.For DENV-4, 55/137(MIR=57,1) Ae. aegytpi,34/54(MIR=162.7) Cx.comp. pipiens,68/162(MIR=36) Cx. quinquefasciatus, 46/67(MIR=120,4) Culex spp.,1/6(MIR=142.8) Limatus sp.,2/4(MIR=500) Psorophora spp.,3/7(MIR=375) Ps. varipes albigenu,1/1(MIR=1000) were positive.In previous studies,SLEV,MAYV and the 4 serotypes of DENV were identified in patients from the urban area of Cuiabá during a large outbreak of dengue.The SLEV genotype V-A, previoulsy reported in humans from Cuiabá, was identified in a Culex sp. Female.Culex and Aedes species presented log of 3.52-4.08 copies/µL of MAYV.Experimentaly, these species have been demonstrated as competent vectors for the virus, but their participation in the epidemiological cycle of MAYV transmission is unclear.DENV-4, responsible for the majority of the human cases in MT during 2012-2013, was identified in several species, possibly due to hematophagy in humans.Cuiabá presents a variety of culicidae species, associated to environmental conditions favorable to the occurrence of arboviral outbreaks,emphasizing the importance of entomological and virological surveillance. FINANCIAL SUPPORT: CAPES, FAPEMAT E UFMT

HV309 - OCCURRENCE OF PICOBINAVIRUS GENOGROUP I IN WORKERS OF PIG FARMS FECES IN THE WESTERN REGION OF PARANAGallego, J.1; Bittencourt, L.H.F.B.2; Kunz, A.F.1; Macedo, R.1; Lustosa, J.M.1; Evers, F.1; Navarro, I.T.2; Takiuchi, E.1

1. UFPR - Universidade Federal do Paraná, Rua XV de Novembro, 1299 - Centro, Curitiba - PR, 80060-000


Picobirnavirus (PBVs) are small, non-enveloped, bisegmented double-stranded RNA genomic viruses of




vertebrate hosts. Despite first detections of PBV have occurred in children’s feces and pigs with diarrhea, their role as etiologic agent in this pathogenesis are still unclear. In humans, PBVs are classified as emerging opportunistic agents primarily affecting immunocompromised. Human PBV strains are currently classified into two distinct genogroups, denominated genogroup I and genogroup II. Comparative studies of PBV genome sequences detected in humans and pigs has been suggested the possibility of inter-species transmission, highlighting the zoonotic potential associated with this virus. The intent of this study was to detect PBV in workers feces and contacts of pig farms in western Paraná. One hundred sixty five feces samples were collected from workers between 2012 and 2013, in the Western Region of Paraná. The PBV diagnosis was carried out in fecal samples by silver-stained polyacrylamide gel electrophoresis (SS-PAGE). The positive samples were amplified by RT-PCR using the primer pair PicoB25/PicoB43 to genogroup I gene that encodes the RNA-dependent-RNA-polymerase-protein. Of the 165 samples processed 7.3% (12/165) were positive for PBV from the PAGE technique. Of these, 9 samples were amplified in RT-PCR, generating the product of 201pb. The samples not amplified in RT-PCR may belong to the PBV genogroup II. Considering the close interaction between humans and pigs in swine production chain of the western region of Paraná and the possibility of cross-species infection, the need for further investigation must be reinforced to elucidate the epidemiology and pathogenesis of PBV in humans and animals.

HV314 - INVESTIGATION OF FACTORS THAT INFLUENCE THE SPONTANEOUS RESOLUTION OF HEPATITIS C VIRUS INFECTIONProvazzi, P.J.S.1; Miura, V.C.1; Carvalho, L.R.1; Rosa, P.C.R.1; Nogueira, M.L.2; Grotto, R.M.T.3; Silva, G.F.3; Rahal, P.1

1. São Paulo State University - UNESP, Department of Biology

2. São José do Rio Preto Medical School, Laboratory of Virology

3. São Paulo State University - UNESP, Department of Internal Medicine

The hepatitis C virus (HCV) is associated with chronic and active hepatitis, cirrhosis, and hepatocellular

carcinoma. Approximately 20% of HCV infections are spontaneously resolved, and the mechanism of HCV clearance is associated with polymorphisms in immune response genes. HCV can prevent the innate immune response of the host through the action of the NS3/4A protease. A catalytic interaction between amino acid Cys-508 of the MAVS protein and S139 of the NS3/4A protease can impair the interferon cascade, and mutations at Cys-508 can confer the ability to resolve HCV infection in some individuals. The aim of this study was to search for substitutions in the coding sequence from MAVS and investigate demographic and risk factors associated with HCV infection in an attempt to correlate the spontaneous resolution of hepatitis C with the presence of mutations at position 508. The region of the human MAVS gene containing residue 508 was amplified and sequenced. Demographic and risk factors were collected from chronic and spontaneously resolved patients, and the data were subjected to hypothesis testing with a 95% confidence interval, revealing a statistically significant association between female gender and venereal disease with patients who achieved spontaneous hepatitis C resolution. Because all patients carried a cysteine at position 508, it was not possible to establish a relationship between hepatitis C clearance and the presence of mutations at position 508 of the MAVS protein in the patient groups selected for this analysis. FINANCIAL SUPPORT: FAPESP, CNPQ E CAPES.

HV324 - SEROSURVEY OF HANTAVIRUS INFECTION IN SINOP, MTda Silva, D.J.F.; Vieira, C.J. da S.P.; Siqueira, C.E.H.; Barreto, E.S.; Moreli, M.L.; Bronzoni, R.V. de M.


2. UFG - Universidade Federal de Goiás, Av. Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970

Hantaviruses are rodent borne pathogens included in the genus Hantavirus (Bunyaviridae family), and transmitted to humans through aerosols of infected wild rodents excreta. Brazil has reported in the last two decades nearly 1,800 cases of hantavirus cardiopulmonary syndrome (HCPS). Mato Grosso state has reported 15.2% of these cases within the same period, with a case-fatality rate of ~40%. Sinop municipality is located




in the middle north of Mato Grosso state, in a region covered by Amazon rainforest. Since its foundation, in 1974, the landscape has undergone intense deforestation due to population growth, lumber commerce, and agroindustry development. It has been suggested that such changes favors the emergence of hantaviruses in the human population. Sinop is located in a region considered as endemic for the disease, though there are no epidemiological studies performed in the city. From January 2011 to February 2012, we developed an arboviruses surveillance study, and sera from 198 patients with acute febrile illness were collected. These sera were also assessed for anti-hantavirus antibodies by ELISA using Araraquara virus recombinant N protein (ARAV rN) as antigen. Thirty-nine (19.7%) clinical samples were IgG positive, with titers from 100 to 3,200, but none IgM positive. Mean age of seropositive patients (29 female, 10 male) was 32.9 years (95% CI: 29.9-35.9). Serological surveys of Hantavirus, using the same methodology, were performed in all Brazilian regions, showing 0.6% to 13% seropositive individuals in general populations. Since the first record of HCPS in Brazil, 14 cases were notified in Sinop, however they were all considered imported cases. Our findings demonstrate serological evidence of Hantavirus infection in humans from Sinop municipality, where these viruses can be causing minor or subclinical infections. Deforestation due to population growth and extensive agricultural activity in Sinop could be associated with this high seroprevalence. Furthermore, public health policies targeting to raise population awareness about the disease are necessary.

HV326 - HIGH HIV-1 DIVERSITY AND MODERATE PREVALENCE OF TRANSMITTED DRUG RESISTANCE MUTATIONS AMONG ANTIRETROVIRAL-NAIVE HIV-INFECTED PREGNANT WOMEN IN RIO DE JANEIRO, BRAZILDelatorre, E.1; Pilotto, J.H.2; Morgado, M.G.1

1. Laboratório de Aids e Imunologia Molecular, Instituto Oswaldo Cruz

2. Hospital Geral de Nova Iguaçu, Ministério da Saúde

The increase in the access of antiretroviral (ARV) drugs in Brazil has strong impact in the reduction of morbidity and mortality of HIV-1 positive individuals. However, it

may also favors the emergence of HIV-1 drug-resistant variants, due to low genetic barrier of some drugs as well as poor adhesion to treatment regimens, among others. These variants may limit the therapeutic options available for people who were newly infected with them. The aim of this study was to evaluate the viral diversity and prevalence of transmitted drug resistance mutations (TDRM) among ARV-naive HIV-1-infected pregnant women followed at Hospital Geral de Nova Iguaçu (HGNI) in Rio de Janeiro – Brazil, between 2012 and 2013, and to compare the results with a similar previous study including pregnant women recruited from 2005-2008. The viral RNA was extracted and the HIV-1 protease and reverse transcriptase (PR/RT) regions of the pol gene were sequenced from 41 plasma samples using an in house genotyping method. HIV-1 subtypes were determined by phylogenetic analyses and TDRM were detected using the Calibrated Population Resistance Tool-CPR v.6.0. The HIV-1 subtype B was identified in 58.5% of the sequences, representing a decrease in the prevalence found in the previous period (81%). The frequencies of the subtypes F1 (12.2%) and C (2.4%) remained similar, while there was a 3-times increase in the recombinant forms frequency (from 8.0% to 26.8%) since the previous study. The overall prevalence of any HIV-1 TDRM was 12.2%, in accordance to the previous estimates (10.7%). TDRM for nucleoside reverse transcriptase inhibitors remained similar (from 5.6% to 4.9%), while protease inhibitors TDRM increased from 3.0% to 7.3%. No TDRM for nonnucleoside reverse transcriptase inhibitors were identified in the analyzed samples collected between 2012 to 2013, contrasting with the 2%-prevalence previously found. Multidrug-resistant strains were not identified in both periods. These results indicate an increase in the HIV-1 diversity and a probable change in the TDRM profile in Rio de Janeiro between the two periods. The moderate prevalence of HIV-1 TDRM in this population could affect the virological outcome of the standard first-line ARV regimens to prevent HIV vertical transmission, reinforcing the importance of continuous monitoring of the HIV-1 genetic diversity and TDRM in Brazil.




HV327 - ESTIMATING THE ORIGIN OF THE HIV-1 CRF02_AG LINEAGES CIRCULATING IN BRAZILDelatorre, E.1; de Castro, C.A.V.2; Pilotto, J.H.3; Bello, G.1; Morgado, M.G.1

1. Laboratório de Aids E Imunologia Molecular, Instituto Oswaldo Cruz

2. Laboratório de Virologia, Departamento de Patologia Clínica, Instituto Fernandes Figueira

3. Hospital Geral de Nova Iguaçu, Ministério da Saúde

The HIV-1 CRF02_AG is one of the most prevalent circulating recombinant forms (CRF) in the world and is responsible for at least 8% of the HIV-1 infections worldwide. This CRF is distributed mainly in West Africa and, to a lesser extent, in the Middle East and North Africa. Due to the migrations from these endemic regions, CRF02_AG has been reported recently in countries where this recombinant is not native, including Brazil. In a previous study, including six CRF02_AG samples identified in Rio de Janeiro from 2004-2011, we found at least four introductions of this clade in Brazil, probably from Western African countries. As more CRF02_AG samples have been identified since then, in this study, we aimed to review the geographic origin and to date the introduction of some lineages circulating in Brazil using a larger dataset. A total of 20 Brazilian (18 from Rio de Janeiro and two from São Paulo) and 1,505 African HIV-1 CRF02_AG pol sequences were analyzed using Maximum Likelihood (ML) and Bayesian phylogeographic methods. The ML analysis showed that the 20 CRF02_AG Brazilian sequences were distributed in five different lineages. The Bayesian phylogeographic analysis of the Brazilian sequences and their most closely related African sequences (n = 212) placed the origin of all Brazilian lineages in West Africa, probably Ghana (lineages BR-I, BR-II and BR-III), Senegal (BR-IV) and Nigeria (BR-V). Lineages BR-I and BR-II comprise >1 sequence, all from the Rio de Janeiro state, and their dates of origin were estimated at 1985 (95% highest posterior density: 1979 – 1992). These results support the existence of at least five independent introductions of the CRF02_AG lineage from West Africa into Brazil and further indicate that at least two of these lineages have been disseminated in the Rio de Janeiro state for about 30 years.

HV329 - GENOTYPING OF HPV IN SAMPLES OF HIGH GRADE CERVICAL INTRAEPITHELIAL NEOPLASIADias, M.C.1; Calmon, M. de F.1; Provazzi, P.J.S.1; Camilo, H.P.1; Badial, R.M.1; Kobayashi, M.T.2; Quintana, S.M.2; Yamamoto, A.Y.3; Rahal, P.1

1. Laboratório de Estudos Genômicos, Instituto de Biociências, Letras e Ciências Exatas – UNESP

2. Departamento de Ginecologia e Obstetrícia, FMRP-USP

3. Laboratório de Virologia Clínica, HCFMRP-USP

The human papillomaviruses (HPV) infect the mucosal and squamous epithelium, remaining asymptomatic or causing disease. Persistent infection with HPV is responsible for almost all cervical cancers, as well as other anogenital carcinomas, therefore, it has a large expression in public health. During the development of cervical carcinoma, multiple premalignant stages can be distinguished, including cervical intraepithelial neoplasia of low (CIN I) and high grade (CIN II and III). In this way, the present study aimed the genotyping of the human papillomavirus present in 95 samples of high grade cervical intraepithelial neoplasia (CIN II and III) and correlates the HPV types detected in the samples with the clinical and pathological data of the lesions. Genotyping was made by PCR with PGMy09/11 and GP5+/6+ primers, followed by sequencing according to Sanger method. In the sequence analysis, the electropherograms were observed in the BioEdit software, and the sequence quality was assessed by Phred/Phap/Consed. The HPV types were identified using the BLAST tool in the NCBI website. To calculate the correlation between genotypes versus clinical and pathological data from samples, the Minitab software was used. The findings showed that lesions classified as CIN III were predominant and that 86 samples were positive for HPV and nine samples were negative. The HPV16 was the most common type and proved to be correlated with CIN III, whereas HPV33 proved to be correlated with CIN II. Among the evaluated samples, 17 showed co-infection with HIV, but a positive correlation was not found between HIV and CIN II or III. On the other hand, CIN II lesions were positively correlated with patients aged between 18 to 28 years. Some patients had follow-up samples and the same genotype of HPV as well as different genotypes in different samples from




the same patient was observed. In this way, the analysis allowed to observe the prevalence of HPV genotypes and their relationship with high-grade lesions, together with clinical and pathological data. These data are important for the formulation of preventive measures, such as vaccines against the HPV types most commonly found in cervical cancer, thus helping prevent the development of the disease. FINANCIAL SUPPORT: FAPESP, CNPQ, CAPES.

HV337 - MOLECULAR DETECTION IN HPV-16 AND HPV-18 NATURALLY INFECTED CELL LINES, SIHA AND HELA RESPECTIVE ULTRASTRUCTURAL CELL MORPHOLOGYSimoes, R.S.Q.S.1; Fernandes, A.T.G.2; Carvalho, M.O.O.1; Silva, M.A.N.1; Silva, L.M.1; Braga, R.M.1; Roma, E.H.2; Almeida, M.G.B.2; Barth, O.M.1

1. Laboratory of Viral Morphology and Morphogenesis, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Helio e Peggy Pereira

2. Aboratory of Immunology and Immunogenetic, Evandro Chagas National Institute of Infectious Diseases, FIOCRUZ

Human papillomavirus (HPV) is the primary factor of HPV-types 16 and 18 cervical cancer causing an average of 70% of cases worldwide. Prophylactic quadrivalente HPV vaccine against HPV-types 6 and 11 (non-oncogenic) and HPV 16 and 18 (oncogenic types) was developed. Few studies have assessed the transmission electron microscopy in different cells lines. The present study investigated molecular detection by PCR using consensus primers in standard samples of SiHa and HeLa cell lines and respective ultrastructural cell morphology. In order to optimize the current cell lines, a screening process of 3x106 cells was counted and DNA extracted using DNA blood mini kit (Qiagen). For molecular detection, DNA of SiHa and HeLa cells naturally infected with HPV 16 and 18, respectively, were used as positives controls and the amplification misture without DNA as a negative control. MY09/11 consensus primers that amplify a 450 bp DNA sequence specific for HPV L1 ORF were used to positive standard samples. For ultrastructural analysis, the SiHa and HeLa cells were embedded in epoxy resin, fixed in 1% glutaraldehyde and post-fixed in 1% osmium tetroxide. Later steps followed by washes in cacodylate buffer 0.2 M in sodium sucrose 0.7% and distilled water.

The dehydration step was performed using 70% acetone in 1% uranyl acetate followed by 90% acetone and 100% acetone. Cells were included in epoxy resin and kept at 60C to complete polymerization. Ultra-thin sections, 50nm thick, were performed to electron microscope observation. Semi-thin sections, 0.5 µm thick, were performed to optical photonic microscopy. The ultra-thin sections were stained with uranyl acetate and lead citrate, the semi-thin sections with methylene blue. By optical photonic microscopy we observed in both cell lines similar features such as presence of very large nuclei with highlighted nucleoli, reduced cytoplasm, cell membranes presenting filopodia, few vacuoles and interconnected cells by desmosomes. Very electrodense cells were detected by electron microscopy presenting well developed mitochondria and rough endoplasmic reticulum, many vesicles and ribosomes in HeLa and SiHa cell lines. Furthermore, HPV-DNA was amplified by PCR method using different amount of template (1-5µL). These preliminary results suggested a strong cellular activation in these cervical keratinocytes. More studies are needed to evaluate the morphological alterations induced by HPV in those cell lines. FINANCIAL SUPPORT: CAPES/PROGRAMA DE PÓS-GRADUAçãO EM MEDICINA TROPICAL/BRASIL SEM MISÉRIA, IOC AND INI-FIOCRUZ

HV339 - STANDARDIZATION OF A PCR PROTOCOL IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS PREVIOUSLY GENOTYPED USING HPV TYPE 3.5 LCD-ARRAYSimoes, R.S.Q.S.1; Carvalho, M.O.O.1; Fernandes, A.T.G.2; Rocha, N.2; Russomano, F.B.3; Barth, O.M.1; Roma, E.H.2; Almeida, M.G.B.2

1. Laboratory of Viral Morphology and Morphogenesis, Instituto Oswaldo Cruz, Fiocruz, Pavilhão Helio E Peggy Pereira

2. Laboratory of Immunology and Immunogenetic, Evandro Chagas National Institute of Infectious Diseases, FIOCRUZ

3. National Institute of Women, Children and Adolescent Healthy Fernandes Figueira, FIOCRUZ

Human papillomavirus (HPV) is critical in the pathogenis of cervical cancer, the second most common cancer among women in Brazil. The mucosal types are classified as high-risk and low-risk depending upon the associated disease intensity. This study was designed to evaluate a




standard PCR protocol in positive samples previously genotyped. DNA was extracted from eight samples of cervical biopsies using DNA tissue mini kit (Qiagen). Samples with squamous intraepithelial lesions of cervix low grade (LSIL) and high grade (HSIL) were previously genotyped using HPV type 3.5 LCD-Array kit (Chipron). Multiple HPV types per sample were detected from specific capture probes according to the manufacturer’s protocol. A PCR protocol using consensus primers MY09/MY11 that amplify a 450-bp fragment of the L1 open reading frame of genital HPV was proposed. Amplification was developed in a 25µL reaction mixture (10X PCR buffer, 2,5mM dNTPs, 50mM MgCl2, 10µM each primer, 5 unit of Taq polymerase, and 5µL of sample). After activation step at 94ºC for 5 min, 35 cycles of amplification were run. Each cycle included a desnaturizing step at 94ºc for 1 min, an annealing step at 55ºC for 30 s, and a chain elongation step at 72ºC for 1 min using Gene Amp PCR (Applied Biosystems). A final elongation at 720C for 10 min was done. DNA isolated from HPV-16 positive SiHa and HPV-18 positive HeLa cells were used as positives controls. The integrity of the DNA isolated from cervical specimens was confirmed using a β-globin gene set of primers (GH20/PC04) that amplify a 268-bp, and all samples showed positive detection. HPV was detected in 83.3% (5/6) of positive samples and negative in 100% of negative samples. The discordant sample was positive for β-globin gene and specific HPV-DNA may be at low level and could not be detected. Several modifications were performed in the standardized protocol using the common set of primers (MY09/MY11) for HPV detection, such as the modification of the extension and annealing time. Adjustment in concentrations of reagents in the PCR reaction mix was also applied. Therefore, the results showed that the protocol used was able to detect HPV in accordance of previous commercial PCR protocol. Clinical identification of HPV types requires a gold standard protocol able to detected HPV even in the absence of cytological alterations. Molecular epidemiological studies are need to identify HPV types in general population before and after the HPV-vaccination. FINANCIAL SUPPORT: CAPES, INI AND PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA TROPICAL/BRASIL SEM MISÉRIA

HV341 - GENETIC DIVERSITY OF INFLUENZA VIRUS STRAINS CIRCULATING IN THE AMAZON REGION, BRAZILSantos, M.C.1; Barbagelata, L.S.1,2; Ferreira, J. de A.1; Júnior, E.C.S.1; de Pinho, R.A.P.1; Gomes, É.R.1; Filizzola, E.M.A.1; Gonçalves, M. dos S.1; Brabo, M.V.V.1; Rodrigues, S.V.1,2; Mello, W.A.1; Medeiros, R.1,2



Flu or influenza is a highly contagious viral disease that affects the respiratory tract and the etiologic agent that causes it is the Influenza viruses, which belong to the Orthomyxoviridae family. The high genetic diversity of these viruses provides antigenic changes, allowing them to escape from host defenses. Such mutations occur mainly on the glycoproteins surface, hemagglutinin (H) and neuraminidase (N). In this context, the constant monitoring of mutations, associated to the variability of the Influenza viruses plays an important role in obtaining data to subsidize interventions for prevention and control of the infections by this pathogen. In order to characterize Influenza strains circulating in the Amazon region in the year 2013, as well as analyze the occurrence of genetic rearrangement among circulating strains and verify the incidence of HA and NA genes mutations associated to escape the host immune response, pathogenicity and antiviral resistance, a total of 2198 samples of patients with signs or symptoms suggestive of flu, attended by health units in the States of Acre, Amapá, Amazonas, Pará and Roraima was analyzed, from January to December 2013. The samples were analyzed by extraction and detection of viral nucleic acid by Polymerase Chain Reaction preceded by real-time reverse transcription (qRT-PCR). Positive samples were subjected to conventional RT-PCR for gene amplification of HA and NA and then sequenced. Influenza virus was detected in 401 samples, being 229 A (H1N1) pdm09, 51 A (H3N2), and 121 Influenza B. The HA gene analysis showed the genetic similarity of the Influenza A virus strains circulating in our region with the flu vaccine strains. Most strains of Influenza B virus belongs to Victoria lineage, however




the contemplated one in the vaccine was Yamagata. It was also demonstrated the presence of amino acid substitutions in the HA gene, related to increased virulence in Influenza A (H1N1) pdm09, A (H3N2) and B. Relating to NA analysis, in the A (H1N1) pdm09 strains it was found the I106V replacement, which is associated with decreased sensitivity to antiviral drugs. However, no important substitution in A (H3N2) samples was verified. Our study highlights the variability of Influenza virus strains circulating in our region, as seen in strains analyzed worldwide. Amino acid replacements that were presented, which was related to pathogenicity and antiviral resistance, reinforce the need for monitoring these viruses. FINANCIAL SUPPORT: SVS/MS,CNPQ,PPSUS,FAPESPA

HV348 - OPTIMIZATION OF A TAQMAN MOLECULAR PLATFORM FOR HEPATITIS B VIRUS (HBV) DETECTION IN BLOOD DONORSSantos, D.F.1; Junior, M.C.R.1; Rodrigues, E.S.1; Slavov, S.N.1; Salustiano, S.G.1; Azevedo, R.1; Martinelli, A. de L.C.2; Covas, D.T.1; Kashima, S.1

1. Regional Blood Center of Ribeirão Preto, Faculty of Medicine of Ribeirão Preto, University of São Paulo

2. Division of Gastroenterology, Department of Clinical Medicine, Faculty of Medicine of Ribeirão Preto, University of São Paulo

Although Nucleic Acid Technologies (NAT) have been implemented in the routine practice for the detection of different viruses threatening blood transfusion, such as HCV and HIV in Brazil, there is no NAT technique for HBV in this platform. Our aim was to optimize a real-time TaqMan® PCR test for detection of the major HBV genotypes (A-F). Materials and Methods. Primer and probe sequences for the conserved region among all HBV genotypes (gene S) were obtained from previous studies. Their specificity was confirmed by sequencing analysis. DNA from plasma was extracted using two commercial kits (A and B). Several concentrations of primers (250 and 500nM) and probe (100–400nM) were evaluated in order to obtain a better efficiency of amplification. The reaction was controlled using HBV DNA standards from Worldwide HBV DNA Performance Panel PHD301 (BBI Diagnostics) containing the genotypes A, B, C, D and E. Genotype F was obtained from plasma of a patient with chronic hepatitis B. As a negative control,

plasma DNA obtained from healthy individuals was applied. In order to define the limit of HBV detection, eight replicates of a standard curve (1,000 to 31.25 UI/ml) were diluted from standard plasma (3rd WHO International Standard for HBV, NIBSC code 10/264). Results. The ability of the primers to detect all known HBV genotypes was confirmed by their search in a global HBV alignment. Two commercial extraction kits (A and B) were compared. We verified that kit B provided more efficient extraction due to the higher quantity of DNA and the earlier cycle threshold (Ct) observed during real-time PCR amplification of the same samples. We also compared the most suitable samples for HBV-DNA extraction: plasma, ultracentrifuged plasma and buffy coat. Ultracentrifuged plasma was chosen because it allowed HBV detection, with lower Ct, in all samples of HBV patients. For the detection of HBV DNA, we defined a concentration of 500nM for primer and 250nM for probe. A commercial reaction buffer tested was also able to reduce the Ct. Moreover, genotypes A, B, C, D, E, and F were detected by real-time PCR. The sensitivity of the reaction was 225.84 UI/ml of DNA, using the Probit analysis. Conclusion. The optimization of the real-time PCR demonstrated that it detects all known genotypes. However, a suitable validation must be still performed in order to improve its sensitivity.

HV354 - VIRAL METAGENOME OF THE HUMAN INTESTINAL TRACT – PRELIMINARY RESULTSSilva, P.A.; Nogueira, R.; de Lima, L.M.P.; Nagata, T.

UCB - Universidade Católica de Brasília, W5 Norte, Brasília - DF, 70790-160

Diarrheal diseases are major public problem worldwide, especially in developing countries. The knowledge of these etiological agents is important for the treatment and epidemiological studies. In order to identify viral population and new viral pathogens including the non-cultivable, the intestinal metagenome aims the sequencing of the entire genetic material present in the fecal sample. The study proposal is a metagenomic investigation of the human intestinal tract viruses from 12 Brazilian individuals to analyze the viral population and identify new potential viruses that cause diarrheal diseases. Methodology: A previous experiment was done with one sample from a healthy individual. The fecal sample (~ 500 mg) was suspended in PBS and centrifuged




at 9700g for 10 minutes, the supernatant was collected and filtered in 0.45μm and 0,2μm, and ultracentrifuged at 30.000 rpm for 2.5 hours and the genome form the pellet was extracted using PureLink ® Viral RNA/DNA Mini Kit (Invitrogen). The reverse transcriptase and amplification using the illustra GenomiPhi V2 DNA amplification kit (GE Healthcare) was performed, and the library constructed with the TruSeq DNA Sample Preparation kit V2 (Ilummina). For the sequencing was used HiSeq 2000 paired end (Ilummina), To alignment the sequences was used Bowtie aligner. Results: The analysis of sequencing in comparison with the viral data base resulted in 25.032.073 reads. 8,548 reads were aligned with viral sequences. The alignment there was a greater number of alignments with baculoviruses and bacteriophages, without the presence of enteric viral pathogens. Conclusion: There was no detection of any virus pathogens and this fact can be explained by the rarity of sequences of eukaryotic viruses in samples of viromes gut of healthy individuals and the detection of baculovirus is the result of contamination during ultracentrifugation. The study with the other samples is in progress. Perform viral metagenome of samples from the intestinal tract is an innovative initiative, considering that it is a country that has high biodiversity, feeding habits and natural life and may bring results completely new to the scientific community.

HV355 - MOLECULAR IDENTIFICATION OF HUMAN RHINOVIRUS AND CLINICAL MANIFESTATIONS IN A CHILD POPULATION IN THE STATE OF RONDONIAAlves, F.A.G. dos S.1,2,3; dos Santos, A. de O.1,2,3; Baffini, V.R.1,2,3; Souza, L.F.B.1,2,3; Tarboda, R.L.M.1,2; Salcedo, J.M.V.1,2; Vieira, D.S.1,2,3

1. UNIR - Universidade Federal de Rondônia, Av. Presidente Dutra, 2965, Centro, Porto Velho - RO, 76801-974

2. CEPEM - Centro de Estudo e Pesquisas da Mulher, R. Barão de Lucena, 71 - Botafogo, Rio de Janeiro - RJ, 22260-020


Human Rhinovirus (HRV) is among the leading causes of infections in the upper respiratory tract. The HRV is an RNA virus, which is classified in three species: A, B, and C. Generally, the infection is involved with more serious

infectious conditions in children, such as the lower respiratory tract disease and asthma exacerbation. Therefore, this study aimed to identify the prevalence of HRV in samples collected from children with symptoms of respiratory infection. Methods: From February to December 2013, 195 nasal secretion samples were collected from 0-6 years old children who have presented symptoms of respiratory infection at the Hospital Infantil Cosme e Damiao (HICD) considered as a reference in the state of Rondonia, Brazil. The biological samples were submitted to molecular techniques for identification of viruses by real-time PCR using the SYBR Green system. Results: Molecular detection rate of Human Rhinovirus was 86% (168/195). The more frequent symptoms related to patients HRV positive were cough with 90.4%, followed by rhinorrhea (76.7%), pulmonary secretions (71.4%) and nasal obstruction (70.2%). Children who are from 0 to 1 years old were more susceptible to infection by HRV. The seasonal distribution during the months from February to December 2013 has showed peaks in the warmer months, specially from June to September. Conclusion: Data has demonstrated that the detection of HRV was significant, observed by the high prevalence found. These data are really relevant once there is no published works in the region. It is important to know the causative agent of infection that is of great value to aid in prognosis and therapeutic management of these children. Therefore, the development of prospective studies to define the epidemiology of this virus and the severity of the disease in children is needed. Financed by: FIOCRUZ-RO, FAPERO, CNPQ.

HV356 - INVESTIGATION OF HEPATITIS E VIRUS IN CASES OF ACUTE FLACCID PARALYSIS OF UNKNOWN ETIOLOGYMorgado, L.N.1; Vitral, C.L.1; da Silva, É.E.2; Pinto, M.A.2



Hepatitis E is transmitted by the fecal-oral viral infection and is considered as the main cause of acute liver infection in several areas of Asia, Africa, and the Middle East. Brazil is considered as an area of low endemicity for hepatitis E virus (HEV). The possibility of circulation of HEV in our country has been demonstrated in numerous




seroepidemiological studies, which observed prevalence of infection rates ranging generally 2-8%. It was also reported an acute case of the disease by researchers at the Instituto Oswaldo Cruz (Fiocruz). Complementing these findings, the possibility of zoonotic infection with HEV was also confirmed by the detection of specific antibodies and HEV genome sequences in different species of animals, especially pigs, in which the infection showed almost universal. Hepatitis E usually presents itself indiscriminately of other viral hepatitis and progresses to healing spontaneously after a few weeks of disease onset. However, some cases may progress to more severe forms, including the appearance of neurological syndromes, with a predominance of peripheral nerve disorders such as Guillain-Barré syndrome (GBS) and brachial neuritis. Each year, the IOC Enterovirus Laboratory, which represents a National Reference Laboratory of the Ministry of Health of Brazil, receives around 500 fecal samples from individuals with acute flaccid paralysis (AFP) or Guillain-Barré syndrome (SGB), with a large proportion (> 80%) of these cases remains undetermined etiology. The objective of this project is to investigate the HEV research by the viral genome in fecal samples from cases of acute flaccid paralysis that were negative for the presence of enteroviruses as well as those in which enteroviruses were detected. Fecal samples from healthy individuals without clinical signs and symptoms of neurological syndromes were used as a control group.

HV359 - CIRCULATION OF INFLUENZA A AND B VIRUS IN THE NORTH AND NORTHEAST REGIONS OF BRAZIL, FROM JANUARY 2013 TO JUNE 2014Gomes, E.R.1; Barbagelata, L.S.1; Ferreira, J. de A.1; Santos, M.C.1; de Souza, E.M.A.1; Gonçalves, M. dos S.1; Medeiros, R.2; de Melo, W.A.1



Influenza is a viral disease that generates major concerns in health authorities, due to its high transmissibility and impact on morbidity and mortality rates, which increase substantially during seasonal epidemics in

addition to the risk of pandemics. In Brazil Influenza virus infection has been described as an infection outbreak usually starting in April in the northern equatorial region and gradually spreading to the other regions of the country. In this context, this study aims to determine the pattern of circulation of influenza A viruses (H3N2 and H1N1pdm09) and influenza B in population segments located in the North and Northeast of Brazil. From June 2013 to June 2014, the Respiratory Virus Laboratory of Instituto Evandro Chagas, belonging to National Influenza Surveillance Network, and accredited as Reference Laboratory by World Health Organization (WHO) received 5,432 clinical specimens (nasopharyngeal aspirate or swab combined) of patients presenting signs or symptoms of influenza, for investigation / confirmation of viral etiology. These samples were from states in the North region (AC, AM, AP, BP, RR) and Northeast region (CE, MA, PB, PE, RN). Diagnosis was received by detection of viral genome involving the extraction of viral RNA (RNAv), using the PureLink TM Viral RNA / DNA Mini Kit (Invitrogen) and the amplification of RNAv by Polymerase Chain Reaction preceded by Reverse Transcription (qRT-PCR) in real Time, using SuperScript IIITM One-step RT-PCR with Platinum ® Taq - (Invitrogen Life Technologies). Of the total patients analyzed, 877 (16.1%) were positive for influenza viruses, 394 (45%) were positive for Influenza A (H1N1pdm09), 241 (27.5%) for influenza B, 218 (26%) for Influenza A (H3N2), three (0.34%) patients with coinfection (one H1N1pdm09 / B, two H3N2 / B) and 22 patients presented inconclusive influenza A typing. In this study, all types of human influenza viruses circulated more intensively in the first half of 2013 (65% of all infections) compared to the first half of 2014 (35% of all infections), contrary to what was expected, since we just experience the World Cup in our country and a large number of people (locals and foreigners) circulated in the territory under study. The peak prevalence occurred in April and May both in 2013 and in 2014 with relevance in May 2013, when Influenza A infection (H1N1pdm09) represented 16% (n = 142) of cases, and the Influenza B extended its peak by the end of June 2013. FINANCIAL SUPPORT: IEC/SVS/MS




HV361 - GENETIC DIVERSITY AND TRANSMITTED DRUG RESISTANCE OF HIV-1 IN MOZAMBIQUEVubil, A.2; Jani, I.2; Bhatt, N.2; Gudo, E. S.2; Mabunda, N.2; Ismael, N.2; Bila, D.2; Morgado, M.1; Fernandez, J.C.C.1

1. IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - RJ, 21040-360

2. INS-MISAU

Sub-Saharan Africa accounts with 69% of global HIV/ADS infections. The HIV-1 epidemic in African countries is characterized by high degree of genetic diversity. Subtype C followed by subtype A1 are the most prevalent and occurring respectively, in South and Central Africa. In addition, transmitted drug resistance mutations are increasing in many countries as a result of the universal access to antiretroviral therapy (TARV) and lack of adherence to treatment. The aim of this study is to improve knowledge about HIV-1 genetic diversity and transmitted drug resistance (TDR) of the viruses circulating in different regions of Mozambique. METHODS: Blood samples were collected from 200 HIV-1 positive blood donors in three cities in northern Mozambique, between November 2009 and June 2010. Genotyping of HIV-1 resistance was performed using Trugene HIV-1 Genotyping assay. Phylogenetic inferences using maximum likelihood were applied on protease and reverse transcriptase segments derived from the genotyping of HIV-1 resistance. RESULTS: A total of 95 HIV-1 genotyping sequences were analyzed, 79% was classified as subtype C. Followed by subtype A1 (10.5%), recombinant forms CA1, DA1, DU and CU (5.3%), 3.2% subtype D and 2.1% subtype G. Transmitted drug resistance mutations associated to the non-nucleoside reverse transcriptase inhibitors (NNRTI) were detected in 4.2% of the samples and one individual show protease inhibitor (PI) resistance mutation associated to typranavir. CONCLUSIONS: This study shows that subtype C remains the most prevalent in northern of the country and supply evidences of low levels of TDR in Mozambique. Phylogenetic analysis has shown striking genetic similarity between South African isolates and autochthonous subtype C strains identified in Mozambique, suggesting a continuous introduction and circulation of closely related strains in the country. However, new subtypes such as D, G and recombinants

have been integrated in the local epidemiology. This might probably due to the high number of emigrants mainly from Central Africa and neighboring countries (Tanzania and Malawi) where HIV diversity is known to be high. Considering the HIV-1 diversity and the rapid expansion of TARV in Mozambique, continuous molecular epidemiology surveillance is important to monitor HIV-1 infections, that might impact in the dynamics of the AIDS epidemic in Mozambique.

HV372 - EX VIVO RHINOVIRUS REPLICATION IN HUMAN TONSILLAR EXPLANTSJunior, R.B.M.; Criado, M.; Gagliardi, T.; Escremim, F.; Silva, M.; Carenzi, L.; Tamashiro, E.; Valera, F.; Lima, W.A.; Arruda, E.


Acute respiratory infections (ARI) caused by human rhinovirus (HRV) have great impact in public health, but little is known about HRV persistence/latency. In a previous study we detected HRV by TaqMan Real Time PCR (qPCR) in 38% of children with chronic tonsillar hypertrophy without ARI symptoms, who underwent tonsillectomy at the Otorhinolaryngology Clinic, University of São Paulo Hospital, in Ribeirão Preto. High frequency of HRV detection in tonsils in the absence of ARI symptoms suggests that these agents may persist in human tonsils. The present study is based on two experimental approaches: immunohistochemistry (IHC) of tonsillar tissues naturally infected with HRV and ex vivo inoculation of tonsillar explants with stocks of HRV-16 (major receptor group) and HRV-1A (minor receptor group). Roughly 50% of all IHC tested with antibody for picornavirus VP1 capsid protein, indicating the presence of virus capsid protein in lymphoid tissue, within and outside of the lymphoid follicles of tonsils and adenoids, and also in epithelial cells in adenoids. This prompted us to try a model of ex vivo infection of adenoid explants. Tissue fragments of ~3 mm3 were placed in 6-well tissue culture plates, and cultured over 7 days in RPMI-1640 with 10% BSA. Explants were considered viable when epithelial outgrowth developed around the tissue fragment. A 5µL droplet (HRV-16 at 107and HRV-1A at 106,25 TCID50/mL) of virus stock was carefully inoculated on top of the tissue. After 24 hours, tissue was




washed and re-incubated at 37ºC in a 5% CO2 atmosphere for 3 more days. After that, fixation, dehydration and paraffin embedding were done for later IHQ with anti-VP1. In addition, a different experimental approach was taken, with ex vivo infection of dissociated lympho-epithelial cells from adenoids and palatine tonsils with rhinovirus HRV-16 and HRV-1A. HRV capsid protein VP1 was detected by indirect immunofluorescence on day 5 post infection. Results indicate that lymphoid cells from tonsillar tissues are susceptible and permissive to infection and may function as possible reservoirs of HRV. Assays to quantitate continuous progeny production in lymphocytes are underway. FINANCIAL SUPPORT: FAPESP and CNPq.

HV375 - COMPARISON OF THE NSP4 DEDUCED AMINO ACID SEQUENCES FROM GROUP A HUMAN ROTAVIRUS G2P[4]E2 STRAINS CIRCULATING IN SÃO PAULO, BRAZILBertol, J.W.1; Fregolente, M.C.D.2; Caruzo, T.A.R.1; Gatti, M.S.V.1


2. UNICID - Universidade Cidade de São Paulo, Rua Cesário Galeno, 448/475, Tatuapé - São Paulo - SP, 03071-000

Rotaviruses (RV) are the main agents of gastroenteritis in humans and animals. Their non-structural protein NSP4, encoded by segment 10, is a viral enterotoxin and is involved in the viral morphogenesis and pathogenesis. The gene encoding NSP4 has been characterized into 15 genotypes (E1-E15) and this diversity of observed amino acid sequences could alter human RV-A (HuRV-A) strains virulence. It is unclear whether NSP4 should be included in RV vaccination strategies; however, NSP4 does appear to play a role in immunity and protection. The aim of this study was to determine the deduced amino acids sequences of NSP4 from G2P[4] HuRV-A. From 113 positive stool samples for HuRV-A by enzyme immunoassay or polyacrylamide gel electrophoresis, 40 samples were characterized as G2P[4] using semi-nested multiplex RT-PCR (samples from 1994, 2005 and 2006) or using semi-nested RT-PCR (samples from 2009 and 2010). The NSP4 gene of 13 HuRV-A strains from 1994, 17 from 2006 to 2007 and 10 from 2009 to

2010 were amplified by RT-PCR. The 731-bp segments were sequenced and classified as E2 genotype by RotaC software. The amino acid sequences and the alignment of them were obtained by BioEdit software. The results showed the prevalence of variations at antigenic sites II and III, VP4-binding domain, enterotoxin domain and at ECM-protein binding domain. Some nucleotide polymorphisms led to amino acid changes in all strains collected in a given year. Only samples collected in 2006 encoded different amino acid at position 87 (E→D). The same kind of change was observed at positions 136 (V→M) and 139 (T→I) for strains from 1994. Positions 103 (V→I) and 131 (Y→H) had amino acid changes in 2007 and 2009, respectively, and these were maintained for the later years. Finally, in position 135 the samples from 1994 has the same amino acid as RV reference strain DS-1 (M), and the samples from other years have the same amino acid as RV patterns TB-Chen and B1711 (I). Since DS-1 and TB-Chen strains were collected in 1976 and 1996, respectively, this mutation probably occurred between 1994 and 1996. Most of the variation in NSP4 amino acids occurs in C-terminal region, including the VP4 binding domain. This fact reinforce the idea that somehow NSP4 is involved in morphogenesis and pathogenesis activities and more studies are necessary to understand how these variation may interfere in structural conformation on interaction sites between NSP4, VP4 and VP6 of RV.Financial support: FAPESP

HV376 - EVALUATION OF DETECTION OF VIRAL RESPIRATORY INFECTION CAUSED BY RESPIRATORY SYNCYTIAL VIRUS (RSV)Pedrosa, C.F.; Campos, A.D.; Carraro, E.

UNICENTRO - Universidade Estadual Centro Oeste, R. Padre Honorino João Muraro, 875 - Santa Cruz, Guarapuava - PR, 85015-430

Human respiratory syncytial virus (RSV) is an important viral agent that causes serious diseases of the respiratory tract in the paediatric population worldwide especially in children under 5 years of age. Cause symptoms ranging from common influenza frames to cases of severe respiratory disease, such as bronchiolitis with recurrent wheezing and pneumonia. Methods: Collection of respiratory samples occurred in the city of Guarapuava PR, in winter the years 2013 and 2014. Inclusion of individuals was about the acute respiratory




disease and nasopharyngeal secretion collection, with people of all ages. To detect RSV applied RT-PCR test in front of RSV controls strain, known to be positive, obtained from the laboratory of Virology of the UNIFESP, who had the genetic material extracted “QIAamp RNA mini kit (Qiagen). Used amplification primers forward: RSVAB-GTCTTACAGCCGTGATTAGG (5’- 3’) and reverse: RSBAB-GGGCTTTCTTTGGTTACTTC (5’- 3’), amplifying 838 base pair fragment. Reverse transcription reaction (RT-PCR) with RNA extracted from the sample, dNTPs, Buffer, molecular grade H2O, forward and reverse primers, reverse transcriptase enzyme “Moloney Murine Leukemia Virus (MMLV-RT) and RNase inhibitor, incubated 42°C for 1 hour and 72°C for 15 minutes in Multigene clusters TC 9600 thermal cycler G. The reaction of amplification polymerase chain reaction (PCR) cDNA had addition Taq DNA polymerase, dNTPs, -MgCl2, Buffer, primers, final volume of 25µl reaction completed with molecular grade water. Amplification reaction in thermal cycler, with 95°C for 5 minutes, 40 cycles to 94°C per 30 seconds, 54°C for 45 seconds, and 72°C for 1 minute. Last step 72°C for 7 minutes. A result confirmed by electrophoresis in agarose gel amplified material to 2% and stained with ethidium bromide for visualization in ultraviolet light. Results and Discussion: The reaction was optimized for each primer in relation to the gain in the detection capability of the dilutions of strains controls. The hybridization temperature of the primers with best performance was 54ºC. RT-PCR for RSV was held in 50 nasopharyngeal samples collected, and there was no detection of RSV in none of the samples analyzed. Conclusion: The standardization of PCR for the detection of RSV control strain showed maximum capacity strains detection controls, however there was no detection among the 50 samples collected for the completion of the study. Financial support: Capes.

HV384 - SEROEPIDEMIOLOGY OF EPSTEIN-BARR VIRUS INFECTION IN PATIENTS WITH SUSPECTED INFECTIOUS MONONUCLEOSIS REFERRED TO EVANDRO CHAGAS INSTITUTE, ANANINDEUA, PARÁ, BRAZILBarros, I.C.; Polaro, A.A.; Raissa, L.; Monteiro, T.A.F.; Costa, I.B.


Infectious Mononucleosis (IM) is characterized by a triad of high fever, pharyngitis and tonsillitis and cervical lymphadenopathy. The clinical symptoms are mild/moderate in most cases. The transmission is oral-oral route through contact with the saliva of an infected host, being nasopharyngeal infection a primary event. It affects both sexes, all ages, especially children under 3 years, with significant numbers also in the range between 15 to 25 years and is uncommon after age 30. Epstein-Barr virus (EBV) is the etiologic agent of IM. The present study aimed to describe the seroepidemiology of EBV infection in patients suspected of IM. The study included 1,666 serum samples from patients with suspected IM (SIM) from January to December 2013, sent to the EBV Laboratory, at Evandro Chagas Institute, State of Pará, Brazil. The serological diagnosis was carried out by enzyme immunoassays (EIA) for the semi-quantitative determination of IgM anti-VCA, following the by manufacturer’s instructions. EIA shoed that 23.40% of the samples were positive for IgM antibodies, 70.70% were negative and 5.9% were indeterminate. Among the positives (n = 390), 48.46% were from male patients. Overall, 47.17% of positives were in the age range of 0-5 years, 27.43% between 11-30 years, 21.02% in the group above 30 years of age, and 4.38% lacked information on age. Regarding clinical findings, positive patients had higher prevalence of fever (55.12%) followed by headache (21.02%), lymphadenopathy (13.84%), cough (11.79 %) and myalgia (11.28%). The present study showed the low positivity for EBV IgM class antibodies anti-VCA, important marker used as a diagnosis of IM. The high prevalence of infection in children and young adults suggests that the infection occurs early in life, which is in accordance with other populations of developing countries, and lower in patients older than 30 years. There was no sex difference.As to clinical findings, the profile found is suggestive of IM, such as fever, headache, lymphadenopathy, cough and myalgia. The occurrence of childhood infection tends to be asymptomatic or clinically not specific, with an infection occurrence in young adults that leads to the development of classical IM in most cases. FINANCIAL SUPPORT: MS/SVS/IEC




HV386 - INVESTIGATION OF THE EPIDEMIOLOGY OF HEPATITIS A IN THE MUNICIPALITY OF CAMPOS DOS GOYTACAZES-RJ BEFORE THE INTRODUCTION OF THE VACCINE IN THIS CITYKury, C.M.H.1; Vitral, C.L.1; Cruz, O.G.2; Pinto, M.A.2; de Oliveira, J.M.2; Merlone, M.P.1; Kury, C.M.H.4; Freixo, H. de O.3; Santos, T.V.3; Lima, G.A.3; Araujo, B.R.3; Menezes, I. de Q.3



3. FMC - Faculdade de Medicina de Campos, Av. Alberto Torres, 111 - Centro, Campos dos Goytacazes - RJ, 28035-580

4. Secretaria Municipal De Saude

Brazil is currently considered a country of intermediate endemicity for hepatitis A virus (HAV).The prevalence of HAV infection has decreased in several Brazilian regions, a fact that has led to an increase in the number of susceptible individuals at risk for HAV infection.The introduction of vaccination against HAV in this epidemiological profile would be of great importance. Campos dos Goytacazes is the first and unique city in Brasil that implemented the vaccine against HAV, which started in march 2011 to all children under the age of two years old. This study aims to evaluate the epidemiology of hepatitis A in this municipality so that the impact of vaccination can be further evaluated.From august, 2011 to july 2012, individuals under 19 years old were randomly selected in schools from all of 14 districts of the municipality. Calculation of sample size was based on an estimated prevalence of HAV in the order of 40%, an accuracy rate of 5% and a level of confidence greater than 99%.After informed consent, capillary blood samples on filter paper (DBS) were obtained for anti-HAV detection.Each participant or legal guardian underwent an interview using a standardized questionnaire. The EpiData ® version 3.1 software and the program “R Archive Network ®” were used for univariate and multivariate data analysis Analysis of 585 children and 377 adolescents children showed an overall prevalence of anti-HAV of 20.6% and 94.1% of children under 5 years were seronegative.Univariate analysis showed that the statistically significant correlation between the risk factors associated with seropositivity were:age, black or

mulatto ethnic, contact with flood water, use bottled water in relation to water spout, number of people living in the house larger than 5, educational level of the mother and father and bath in the river, pond and marsh.There was no statistical difference in gender, type of sewage device, wages of family grouping and type of district property of the individual, among others. Multivariate analysis demonstrated a statistically significant correlation for age, non-white ethnic, maternal educational level and number of household members.The results of this study support the decision of the municipality of Campos to vaccinate children against hepatitis A before the start of school activity and corroborate data from other Brazilian seroprevalence studies showing that a large proportion of children under five years of age are susceptible to HAV infection

HV387 - HIGH FREQUENCY OF ASSOCIATION OF HUMAN RHINOVIRUS AND BOCAVIRUS ASSOCIATED WITH BACTERIA IN PATIENTS WITH CHRONIC RHINOSINUSITISPrates, M.1,2; Souza, J.1; Paula, F.E.1; de Jesus, B.L.S.; Júnior, R.B.; Saturno, T.H.; Gagliardi, T.B.1; Leite, M.2; Valera, F.C.P.2; Tamashiro, E.2; Arruda, E.2; Anselmo-Lima, W.T.2

Departments of Cell Biology1 and Otorhinolaryngology and Head and Neck Surgery2, University of Sao Paulo School of Medicine, Ribeirao Preto, SP, Brazil, 14049-900

Chronic rhino-sinusitis (CRS) is an inflammatory disease of paranasal sinuses mucosa with great public health impact. Previous studies have shown the presence of viruses and bacteria in paranasal sinuses of CRS patients, but the simultaneous detection of viruses and bacteria has not been investigated. In the present study secretions and tissue samples from CRS patients undergoing surgery at the University of Sao Paulo Hospital in Ribeirao Preto, Brazil, were tested simultaneously for viruses and common bacteria. A total of 481 samples were tested from 103 CRS patients (including 31 patients with nasal polyps) and 34 patients without CRS who underwent rhinoplasty, as a control group. Real-time PCR was used to test for a panel of common respiratory viruses and bacteria. Of 137 patients 37 (27%) were negative for all tested bacteria, including 15 (10.9%) who were also negative for any virus. Twenty-two patients were positive only for viruses, with higher




frequencies of human metapneumovirus (HMPV) (24.3%) and human parainfluenza virus 3 (HPIV3) (16.2%). One hundred patients had at least one sample positive for bacteria, and most of them had only CRS (n=25) or CRS with nasal polyp (n=61). The viruses most frequently detected in association with bacteria were human rhinovirus (HRV) (36%) and human bocavirus (HBoV) (23%). Staphylococcus aureus was detected in 27 patients, and 11 of them (40.7%) were negative for viruses; Streptococcus pneumonia was detected in 30 patients, and 9 of them (30%) were negative for viruses; Haemophilus influenza was detected in 51 patients, and 16 of them (31.4%) were negative for viruses; Moraxella catarrhalis was detected in 16 patients (37.5%) and none of them had viruses detected. Finally, 58 patients were positive for Pseudomonas aeruginosa, including 18 (31%) only positive for this bacteria. The present study has shown a high frequency of bacteria in patients with CRS associated with respiratory viruses, mostly HRV and HBoV.

HV388 - DETECTION OF RESPIRATORY SYNCYTIAL VIRUS FREQUENCY IN DISTINCT GROUPS OF ASYMPTOMATIC PATIENTSMoreira, L.P.; Watanabe, A.S.A.; Camargo, C.N.; Granato, C.; Bellei, N.


The human respiratory syncytial virus (HRSV) has a great impact as a causative agent of acute respiratory infection in young children, immunocompromised patients and the elderly. Individuals with asymptomatic infection could play an important role in the virus transmission chain. In this study a Real-time PCR technique was used to evaluate the frequency of HRSV in 534 samples from distinct groups of patients who attended in Sao Paulo hospital during the period of 2009 to 2012 with high risk of infection acquisition. Of all the samples analyzed, 94 were from health care workers (HCW), 100 from patients with immunosuppression due to HIV infection (HIV), 171 from symptomatic children (SC) and 169 from their asymptomatic caregivers (AC). In the HCW group, the median age was 33.5 years, and a HRSV frequency of 1% was found. In the HIV group, the median age was 46 years and the frequency found was 4%, with no statistically significant difference between

CD4 values for patients with positive and negative samples. Regarding the group of SC, the median age was 3.8 years, and the result showed a positivity of 25.7% for HRSV. In the AC group the presence of the virus was detected in 7.7% of samples. Comparing the AC and SC groups, we observed that 22.7% of the caregivers of children infected with HRSV were also infected with the virus. The odds ratio showed that having a child infected with HRSV at home increases the chance of infection for the caregiver in 12 times [OR 12.1 (95% IC 3.1 – 46.6)]. The HCW that have children infected with HRSV at home seems to play a significant role on the HRSV transmission chain. The presence of children infected with HRSV at home can represent an important factor for the virus acquisition, even for subjects without symptoms. These findings show the need to maintain the medical staff and hospital visitors informed about the prophylactic measures against the virus, mainly in the HRSV season in order to prevent the onset of outbreaks coming from the community.

HV389 - EVALUATION OF THE ROLE AND IMPACT OF ACUTE RESPIRATORY VIRAL INFECTIONS IN THE CITY OF GUARAPUAVA PARANÁCampos, D.A.; Pedrosa, F.C.; Carraro, E.


Acute respiratory infections are the most common disease in all individuals. Influenza A and B have been reported as the etiology of more than 50 of the acute respiratory infections around the world. The characteristic of influenza virus in frequent and unpredictable variants suffer, puts him in a prominent position among emerging diseases. The laboratory confirmation of influenza is important for infection control measures and to optimize treatment. Objectives: To evaluate the role and impact of acute respiratory infection caused by respiratory viruses, identifying and characterizing the respiratory viruses that infect the people of the city of Guarapuava Paraná frequenters of the Village Health Center Hospital Laboratory Carli and Santa Teresa with symptoms of respiratory infection, and so assess the clinical and epidemiological data Association of the disease with the virus identified. Methods: We conducted a study between 2013 and 2014 in 50 patients with symptoms of




respiratory infection, associated with seasonal climatic variables such as temperature, precipitation and relative humidity. We use samples of nasal secretion and these were tested by reverse transcriptase polymerase chain reaction (RT-PCR), which was chosen as the method for viral identification. Results: All 50 samples collected were analyzed and of these, two were positive for the respiratory virus Influenza A. Respiratory infections were characterized by the presence of mild symptoms of upper respiratory tract, the most common of which were runny nose and cough. In these two years of study, most cases of infection occurred in autumn and winter with more infections in the months of May to July, mas os vírus respiratórios foram detectados ao longo de todo o período do estudo. Conclusions: Our results show that respiratory infections have certain impact on population, Since this region is rather cold, with mild temperatures extreme being a factor susceptible to respiratory infections, This suggests that more attention should be paid to viral pathogens, increasing healthcare during the flu season. FINANCIAL SUPPORT: Capes

HV390 - PHYLOGENY OF HEPATITIS B VIRUS (HBV) CIRCULATION IN RORAIMA STATEGranja, F.1; Júnior, W.P.L.1; Barros, J. de A.1; de Sousa, D.D.1; de Souza, V.C.2; Acosta, P.O.A.1; Naveca, F.G.2

1. UFRR - Universidade Federal de Roraima, Avenida Capitão Ene Garcez, 2413 - Aeroporto, Boa Vista - RR, 69310-000

2. ILMD/ FIOCRUZ

The HBV belongs to Hepadnaviridae family, Orthohepadnavirus genus; it has a circular DNA partially double strand with approximately 3.2 kilobases that encode four viral genes. The virus has a large genetic diversity that divides it into 10 genotypes named from A to J and further divided into numerous subgenotypes. This project objective was trace the phylogenetic origin of the HBV’s strains that circulate in Roraima State. The extracted samples were submitted to nested-PCR to amplify a segment from the S gene. The sequenced fragments were edited and submitted to BLAST to identity confirmation and genotype obtaining. We created a database containing sequences from many localities with emphasis in adjacent places to Roraima. The database and this study’s sequences were submitted to Jmodeltest software to choose the best evolutionary model and the

phylogenetic tree was build using the MEGA software.Among the samples, 65,67% were from Boa Vista , 5,97% from Mucajaí, 4,48% from São João da Baliza and 23,88% from the other counties of Roraima. Thirteen samples were positive to fragment amplification, this samples also shown positives results in the sequencing. The evolutionary model used to build the tree was the Maximum Likehood based on the Tamura-Nei (G+I) with 1000 of bootstrep. Two phylogenetic trees were made based in the found genotypes (genotypes A and D), both rooted in the genotype C, the oldest genotype. Our data presented various similarities in relation to the samples; we found two samples similar to genotypes A1 and A2 from Caribe and one next to American strain. This can be explained by the geographic proximity of these countries with Roraima State. Inside to Brazil, we found samples similar to Brazilian south, southeast and Amazonas state. This diversity of the same genotype in Roraima can be related to 49% of its population being made by immigrants, we can point this last data as the main factor to this high diversity in the same genotype. Based on the analysis of the genotype D tree, we obtained only one sample that shown similarity of 100% with a Colombian strain, and forming a clade with sequences from Colombia, Costa Rica and Amazonas. We can suggest that, considering the geographic proximity between Roraima and Colombia, the studded strain has as origin in the neighboring country. The circulating HBV’s strains of Roraima demonstrated many origins pointing the state as an important area to study the viral dispersion.

HV391 - FREQUENCY OF SEROLOGICAL MARKER FOR HEPATITIS C IN INMATE POPULATION OF A CITY OF PERNAMBUCOCahu, G.G. de O.M.1; da Silva, D.M.1; de Morais, V.M.S.1; da Silva, D.M.2; Rabelo, D.C.C.2; de Lucena, W.A.T.2; de Lima, P.C. de S.2; de Albuquerque, A.C.C.2; Coêlho, M.R.C.D.1


2. ASCES

The inmate population is characterized by social exclusion, drug use and terrible conditions of confinement resulting in a high prevalence of infectious




and contagious diseases. The hepatitis C virus (HCV) is a causative agents of these diseases and the parenteral route is their primary route of transmission, moreover the virus is to cause more common of liver transplantation. The inmate population is considered at high risk for HCV infection, because prison conditions, marginalization, drug abuse and low socioeconomic status contribute to the spread of HCV in prisoners. In Brazil, studies conducted in different countries showed frequencies ranging from 6.3% to 41%, however, there are not reports of the frequency of HCV infection in the inmate populations in the State of Pernambuco. Objective: The aim the study was to estimate the frequency of positive anti-HCV markers in male population of a prison of a city of Pernambuco, in the period May to July 2011. Material and Methods: The datas from each individual were collected through an interview, after 5 mL peripheral blood was collected in without anticoagulant tube. The samples were centrifuged at 1500 rpm for 10 minutes to separate serum, after, the samples were forwarded to the Department of Virology of Laboratory Immunopathology Keizo Asami (LIKA) of Universidade Federal de Pernambuco (UFPE) to perform the anti-HCV by the commercial kit (MUREX anti-HCV 4.0). Results: The serology was realized in 1085 samples and the frequency of the anti-HCV was 1.66% (18/1085). The mean age of these was 36.11 years (±12), 83.3% (15/18) were married, 55.56% (10/18) had tattoos, 16.67% (3/18) used injection drugs, 44.44% (8/18) have used cocaine, 11.1% (2/18) had sex with another man, 55.56% (10/18) did not use condoms during intercourse, 27.78% (5/18) had a sexually transmitted disease and 16.67% (3/18) had received blood transfusions. Conclusion: The frequency of HCV infection was lower in this population than in previous studies, this may be due to the low frequency of risk factors, such as injectables drugs. However, data on HCV among Brazilian prisoners is still scarce, and it draws attention to the need for epidemiological studies and policies to prevent transmission of infectious and contagious diseases during incarceration.FINANCIAL SUPPORT: SCHOLARSHIP FROM CNPQ

HV409 - DETECTION OF RESPIRATORY VIRUSES AND BACTERIA IN SECRETIONS FROM THE MIDDLE EAR AND THE RESPIRATORY TRACT FROM PATIENTS WITH CHRONIC SECRETORY OTITIS MEDIA AND TONSILLAR HYPERTROPHYSaturno, T.H.1; Buzzato, G.P.1; Gagliardi, T.2; Modena, J.L.P.2; Carenzi, L.R.1; Prates, M.1; Tamashiro, E.1; Valera, F.1; Lima, W.T.A.1; Arruda, E.2

1. Departamento de Oftalmologia, Otorrinolaringologia e Cirurgia de Cabeça e Pescoço, Faculdade de Medicina de Ribeirão Preto

2. Departamento de Biologia Celular e Molecular, Faculdade de Medicina de Ribeirão Preto

Chronic tonsillar hypertrophy (CTH) is a chronic inflammation of palatine tonsils and adenoids that is frequent in children worldwide, characterized by the presence of recurrent infections and tonsillar hypertrophy. CTH may be associated with nasal obstruction, recurrent sinusitis, snoring, auditory tube dysfunction, otitis media, obstructive sleep apnea and altered facial growth. Some CTH patients develop secretory otitis media (SOM), with accumulation of effusion in the middle ear that frequently requires surgery. SOM pathogenesis is not entirely clear, but chronic tubal obstruction by hypertrophic adenoid and the presence of viral infections may be contributing factors. The high frequency of respiratory viruses and bacterial biofilm recently recognized in hypertrophic adenoids prompted the present study. Samples from palatine tonsils, adenoids, nasopharyngeal aspirates and middle ear secretion from 37 patients with CHT and SOM were tested by real-time PCR (qPCR) for: human rhinovirus (HRV), human adenovirus (HAdV), human bocavirus (HBoV), human enterovirus (HEV), human metapneumovirus (HMPV), human respiratory syncytial virus (HRSV), human coronaviruses (HCoV), human influenza virus (Flu), human parainfluenza virus (HPIV), and for the bacteria Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Moraxella catarrhalis. The most frequently detected virus in middle ear secretion was enterovirus (22%), but the other respiratory viruses were also detected. Of note, HRV species C was detected in 3 patients (8%). S. pneumoniae was the most frequently detected bacterium, in 35% of patients. Curiously, in only 11 (29%) patients there was agreement between




viruses and bacteria detected in adenoid and middle ear secretion, and in only 5 (13%) there was agreement between middle ear and nasopharyngeal secretions. The data strongly indicate that SOM is not directly caused by any of the viruses or bacteria tested, but their presence is likely due to stasis of secretions and biofilm consequent to the mechanical obstruction of the Eustachian tube by hypertrophic adenoids. Financial support: FAPESP, CAPES and CNPq.

HV416 - GENOTYPIC CHARACTERIZATION OF ROTAVIRUS STRAINS IN CHILDREN HOSPITALISED FOR ACUTE DIARRHEA IN BELÉM, PARÁ, BRAZIL IN POST-VACCINE INTRODUCTION PERIODFecury, P.C.M.S.1,2; de Oliveira, A. do S.L.1; Soares, L. da S.1; Bezerra, D.A.M.1; Justino, M.C.A.1; Linhares, A. da C.1; Mascarenhas, J.D’A.P.1; Guerra, S. de F. dos S.1


2. FAMAZ - Faculdade Metropolitana da Amazônia, Avenida Visconde de Souza Franco, nº 72, bairro Reduto (Doca), Belém - PA, 78600-000

Gastroenteritis is the second leading cause of death, being rotavirus (RV) is the most common enteropathogen, accounting for an estimated 453,000 deaths in children <5 years in 2008. The RV is devoid of envelope with genome composed of 11 segments of double-stranded RNA (dsRNA) that codes for 12 proteins. The VP4 and VP7 proteins make-up the outer layer shell of the RV particle and define 37 P and 27 G genotypes, respectively. The public health impact of RV disease, its genotypic diversity and the need for assessing vaccine effectiveness are among current priorities. In addition, the monitoring of circulating strains rotavirus vaccine introduction is strongly recommended, since new or emerging strains may pose a threat to vaccination strategies. Objective (s): Genotypic characterization of previously untyped rotavirus strains obtained from children hospitalized for diarrhea after countrywide introduction of rotavirus vaccine. Material and Methods: Stool specimens were obtained during a case-control study to assess vaccine effectiveness in Belém, Pará, Brazil, from May 2008 to May 2011. A total of 122 previously G and/or P untyped, were selected. Of these we could examine 76 in this study, 44 of which only were not G-typed, 16 only were not P-typed and 16 were not typed for G and P. The viral

dsRNA of samples were extracted from fecal suspensions and submitted to RT-PCR and seminested PCR, using primers to G and P genotypes not usually detected. PCR products were subsequently purified and sequenced. Results: For G, 56 samples were possible typed by subjected to either seminested-PCR or sequencing reaction we identified: G1 types (5.4%, n=3), G2 (3.6%, n=2), G3 (1.8%, n=1) and G12 (89.3%, n=50). P genotype was characterized in 27 samples by sequencing reaction, with the following specificities: P[4] (29.6%, n=8), P[6] (11.1%, n=3), P[8] (44.5%, n=12) and P[9] (14.8%, n=4). The dual characterization was determined for 67 samples (88.2%), among which the unusual combinations G12P[6] (68.7%, n=46), G12P[9] (3%, n=2) and G3P[9] (1.5%, n=1) were identified. Conclusion: The identification of unusual genotypes among samples not previously typed underscores the importance of continuous monitoring and characterization of circulating RV strains, particularly in the current scenario of post-vaccine introduction in Brazil. Possible circulation of new or unusual genotypes may represent a challenge to current vaccination strategies. FINANCIAL SUPPORT: FAPESPA, IEC

HV420 - LOSS OF DISEASE CONTROL AFTER SUPERINFECTION OF A LONG TERM NON PROGRESSOR HIV-1 POSITIVE INDIVIDUAL FROM RIO DE JANEIRO, BRAZILCaetano, D.G.1; Côrtes, F.H.1; Vorsatz, C.2; Grinsztejn, B.2; Veloso, V.G.2; Bello, G.1; Morgado, M.G.1

1. Laboratório de Aids e Imunologia Molecular IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - RJ, 21040-360

2. Instituto de Pesquisa Clínica Evandro Chagas - Fiocruz, Av. Brasil, 4365, Rio de Janeiro, RJ, 21040-360

Long Term Non Progressor (LTNP) patients are a rare segment of the HIV-1+ population that maintain high levels of T-CD4+ cells for more than 8 years after infection in the absence of antiretroviral therapy. Some studies identified infected LTNPs with more than one viral lineage with divergent effects in pathogenesis, characterized by maintenance or loss of progression control. Here we report the case of a LTNP diagnosed in year 2000 and that has been shown control of the T-CD4+ cell levels (median of 1093 cells/mm3; IQR




960-1215) and viral load (median of 216 copies/ml; IQR 150-345) along 12 years of follow-up. In mid-2012, the patient started to show an increase in viral load, reaching about 10.000 copies/ml in early 2014. The genetic diversity of the viral quasispecies was studied longitudinally in 3 moments (2009, 2011 and 2014) by the amplification of the C2-C4 envelope region (env), using DNA extracted from cryopreserved PBMC samples and limiting dilution, to assess single sequences from the viral population. Phylogenetic analysis showed that, in 2009, 100% of the viral population (n=28 sequences) formed a single monophyletic group that we denominated cluster A. However, the quasispecies obtained in 2011 were distributed in 2 monophyletic groups with genetic distance of 16,8%. The previously identified cluster A grouped 83% of the sequences obtained (n=24 sequences) and a new cluster B with 17% of remaining sequences (n=5 identical sequences) was identified. The analysis of samples from early 2014 confirmed the persistence of the 2 viral populations, but with a change in their relative prevalence: the cluster A contained 9% of obtained sequences (n= 3 sequences) while cluster B had 91% of the remaining ones (n=30 sequences). Together, the data obtained allow us to conclude that loss of progression control in this LTNP coincides with the detection of a new viral population, which could be explained by a reinfection event between 2009 and 2011. The switch of the proportions of cluster A to B indicates a progressive substitution of the original virus by the new variant, which could be associated with a better fitness of variant B compared to variant A. These results alert for the negative consequences of reinfection to disease pathogenesis, even in patients with a long history of control of immunity, viremia and disease progression, reinforcing the need of continued use of adequate measures to prevent new infections. FINANCIAL SUPPORT: CNPQ/FAPERJ

HV421 - EXPRESSION IN INSECT CELLS OF PROTEIN PRM AND E FROM DENGUE-4 VIRUS FOR USE IN VACCINE AND DIAGNOSTICSAbrão, E.P.; Moraes, F.M.; Esposito, D.L.A.; Fonseca, B.A.L.


Dengue is a febrile condition caused by an arbovirus of the genus Flavivirus, which is transmitted by Aedes aegypti mosquitoes and occurs predominately in tropical countries. All four identified serotypes (DENV 1-4) show similar antigenic characteristics. The virus genome is a positive-sense RNA with 10,644 nucleotides that is translated into a polyprotein, which is subsequently cleaved into three structural – Capsid (C), pre-Membrane (prM), and Envelope (E) – and seven non-structural proteins. Two of the structural proteins, prM and E, are the primary antigens and therefore represent clinically important targets for vaccine production. There is no commercial vaccine available against dengue virus, especially because antibodies against one specific serotype can enhance the entry of heterologous serotypes into the cells and can lead to increased viremia. The Schneider 2 (S2) cell line, derived from Drosophila melanogaster embryos, is often used as a machinery to produce recombinant proteins, due to the ability to incorporate heterologous DNA into their genome. In addition, a metal-induced promoter assures high protein expression without compromising the regular cell cycle. In this study, we examined the expression of the recombinant complex prM-sE protein of dengue serotype 4, that can be used to improve diagnose and further vaccine studies. After induction with copper sulfate, the protein was found mostly in the supernatant and it was purified with a pre-packed nickel affinity column (that allows separation of HIS tagged proteins) and a gel filtration Superdex 200 column (size exclusion separation). To verify the expression and purity of this protein, Western Blot (using anti-his antibody) and SDS-Page gels were performed. The results showed a high yield protein expression and purity. In addition to this study, we identified samples of patients infected with dengue virus, using a commercial ELISA assay (IgM and IgG) and conventional PCR. In further studies, the positive samples will be analyzed against the prM-sE expressed in S2 cells and the results will be compared with the commercial available assay. Financial Support: FAPESP and CNPq.




HV425 - GENETIC CHARACTERIZATION OF SEROTYPE 1 DENGUE VIRUS ISOLATED IN RORAIMA, BRAZIL, FROM 2008 TO 2010Sousa, D.D.1; Granja, F.1; Silva, G.A.V.2; Naveca, F.G.2; Acosta, P.O.A.1

1. UFRR - Universidade Federal de Roraima, Avenida Capitão Ene Garcez, 2413 - Aeroporto, Boa Vista - RR, 69310-000

2. Centro de Pesquisas Leonidas e Maria Deane - FIOCRUZ - AM

Roraima is highlighted as a dengue virus hyperendemic state, with the four serotypes circulating and with a high incidence of this disease. Futhermore, its geographic localization has an important part on the entry of new genotypes/serotypes of dengue in Brazil.DENV-1, after a brief incursion in the state in 1981, was reintroduced in 2000 and it has been one of most isolated serotypes until 2011. Nevertheless, there is little data that shows the occurrence of genetic variability or any evolution in genetic composition of this serotype on infections occurred in different years. The objective of this project was to perform a molecular characterization of the E gene from DENV-1 isolated on infections occurred between 2008 and 2010 in the state. The samples were inoculated in C6/36 lineage cells from Aedes albopictus, and identified by indirect immunofluorescence and RT-Hemi-Nested-PCR techniques. For the obtainment of an amplicom from the envelope region, it was made an RT-PCR test with specific primers, generating a product with 1724 bp. The amplicons were sequenced and the consensus sequences were obtained using the program Geneious v.5.5.4. For the molecular analysis, the sequences were compared to reference sequences from the four serotypes of dengue virus, and to five genotypes of DENV-1 from different parts of the world available on GenBank. The phylogenetic reconstruction was made by the Maximum-likelihood method. All of the isolates were grouped in genotype V of DENV-1. The sequences used in this study were grouped on a phylogenetic tree forming a subclade with strains of DENV-1 from neighbor countries and states and Caribbean region, indicating Roraima as possible entry route of new strains of dengue in Brazil. The analyzed Brazilian sequences formed three distinct lineages. The isolates of this study were grouped in lineage III, different from the strain that circulated in the state in 2001, which belonged to lineage II. It was

found four amino acid substitutions, two of them (E428 e E436) which did not change the conformation of the envelope (Leucine x Valine; Isoleucine x Valine), and the other two substitutions (E227 e E338) with high probability of changing the envelope (Proline x Serine; Leucine x Serine). These results may indicate an adaptive evolution of DENV-1 or the entry of a new lineage in the state, emphasizing the importance of molecular monitoring strains of DENV circulating in the region. Financial support: CNPq - UFRR

HV433 - ANALYSIS HCV AND HIV VIRAL LOADS, AND THE CD4+ AND CD8+ T LYMPHOCYTES COUNTING HIV/HCV CO-INFECTED AND HIV MONO-INFECTED PATIENTS BEFORE AND AFTER THE USE OF HIGHLY ACTIVE ANTIRETROVIRAL THERAPY (HAART)Klein, T.M.; Abrão, E.; Miquelitto, F.; Fonseca, B.A.L.


Because HIV and HCV share the same route of transmission, approximately 30% of the world’s population infected with HIV is co-infected with HCV. With the onset of the HAART, there was a significant increase in the surveillance of AIDS patients, and nowadays the C Hepatitis has becoming one of the most important causes of death among these patients. The pathogenesis of hepatitis C occurs through of the host immunological response to viral infection that is mediated mainly for CD4+ and CD8+ T lymphocytes. However, the HIV infection causes a decrease in the CD4+ T lymphocytes counting and consequently a deregulation of the immunological response. Some studies have demonstrating that the HIV/HCV co-infection accelerate the AIDS progression, accelerate too the liver damage, the HCV viral load is higher when compared with HCV mono-infected patients, and co-infected patients using HAART have a compromised recovery of CD4+ T lymphocytes. That’s why, this research aimed to analyze the viral loads of HIV and HCV in 13 patients co-infected with HIV/HCV before and after the use of HAART. The HIV viral load and the CD4+ and CD8+ T lymphocyte counting were analyzed also in the same way. We compared all the results with the results obtained of 11 HIV mono-infected patients. Two serum samples of each patient (one before the use of antiretrovirals and another after the beginning of HAART) were used. The HIV viral load and the CD4+ and




CD8+ T lymphocytes counting were took of the patient medical records. There was no statistical difference between the HCV viral load before and after HAART in both HIV mono-infected and HIV/HCV co-infected groups. The HIV viral load was the same for both groups before HAART, and after HAART, as expected these viral loads decreased in both groups becoming undetectable for the most of the patients. It was observed a higher CD4+ T lymphocyte counting in the mono-infected group before and after the treatment. Both groups increased their CD4+ T lymphocytes counting after HAART, but the recovery of these cells in the co-infected group was lower. The CD8+ T lymphocytes counting was the same for the both groups before and after HAART. Our results suggest that the HIV therapy doesn’t have any effect on the HCV viral load. HAART is being effective in to inhibit HIV replication. A major CD4+ T lymphocytes recovery in mono-infected patients and the absence of alteration in the CD8+ T lymphocytes counting corroborate with the literature data.

HV434 - DETERMINATION OF HAPLOTYPES OF THE ENVELOPE OF HIV-1 IN INDIVIDUALS INFECTED WITH SUB-SUBTYPE F1 BY NEXT-GENERATION SEQUENCINGde Almeida, D.V.; Junqueira, R.M.; Guimarães, M.L.

IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - RJ, 21040-360

The high degree of HIV-1 genetic diversity and the structural complexity of its envelope glycoproteins were obstacles to the designing of an effective vaccine candidate, requiring the improvement of reagents able to induce a broad immune response. The HIV-1 diversity estimation is currently restricted to single-nucleotide variants; to hence knowledge on quasispecies populations comprehensively required novel methods that allow complete reconstruction of the individual viral haplotypes, which now is available by Next-Generation Sequencing (NGS). HIV-1 subtype B is currently the most spread in the aids pandemic, as well as the subtype C is the most prevalent, thus most of the generated information concern this subtypes. In Brazil, sub-subtype F1 and BF1 recombinants have a significant relevance. Thus, the aim of this study was to develop a workflow capable to characterize and define quasispecies population of

sub-subtype F1 by shotgun sequencing strategy in a NGS plataform. For this purpose, we analyzed 10 clinical isolates of HIV-1 sub-subtype F1 collected in the State of Rio de Janeiro, Brazil in 2012. Each of these DNA samples were subjected of an env gene nested PCR and sequencing generating a fragment of 2,500 bp by ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems), which was used as reference. The same PCR env protocol was applied, the products were purified by magnetic beads (MagSi-NGSPREP) and further fragmented (NEBNext® dsDNA Fragmentase). These products were quantified by fluorescence-based measurements (Qubit, Invitrogen) and assessed for quality using Agilent Technologies Bioanalyzer before library preparation with adapters for NGS in GS Junior Roche 454 Life Sciences. We initially eliminated PCR amplification bias by using CD-HIT-454 software, evaluated quality at NGS QC Toolkit Software, subsequently used the QuRe Software for capture minority variants, based on coverage and diversity to attempt to reconstruct all the individual sequences of the viral quasispecies. In this study, over 60 Mb of sequence data and > 150 haplotypes were generated. The number of haplotypes varied with a median of 19 (IQR=8-50) per sample with a median of 60% (20%-80%) of frequency for majority population of quasispecies. The definition of workflow and of parameters used in tools for checking, filtering and mapping haplotypes of NGS data will facilitate the analysis for better understanding quasispecies diversity and evolution, helping in a vaccine conception.

HV438 - IGG ANTIBODIES OF HIGH AVIDITY TO HANTAVIRUS IN HUMAN POPULATION FROM THE STATE OF ALAGOASBorges, A.A.; Santos, F.M.; Medeiros, N.P.T.; Santos Jr, J.A. dos; Melo, D.M. de; Borges, A.A.

LAPEVI/ICBS/ UFAL - Laboratorio de Pesquisas em Virologia e Imunologia/Instituto de Ciências Biológicas e da Saúde/Universidade Federal de Alagoas, Av. Lourival Melo Mota, Ciudad Universitaria - AL, 57072-900

Hantaviruses are zoonotic viruses associated with wild rodents, belonging to the family Bunyaviridae, that can cause human illnesses with a range of severities. Cases of human hantavirus infection are rarely reported in the Northeast region and the state of Alagoas is considered such silent area for hantavirus. However, our previous




results obtained with volunteers from the state of Alagoas (n=1324) showed the presence of specific IgG to hantavirus in 5,36% of them and suggested that some hantavirus could be circulating in this state. The IgG-avidity test is a very useful tool for differentiating between recent and past infections, besides to reinforce the specificity of the binding of the antibodies on the antigen in the ELISA test. Thus, our antibody avidity assay was used to test the positive serum samples for anti-hantavirus IgG from participants of the serological survey performed in Coruripe, Palmeiras dos Índios and OlhoD’água do Casado counties from Alagoas. Then, 79 positive sera samples collected during the survey were used to detect by enzyme immunoassay (ELISA) IgG antibodies against N protein of Araraquara hantavirus (rN ARAV) after a treatment with a caotropic agent such as urea. For this, in the ELISA test performed, after the stage of serum incubation with the antigen rN ARAV (tested in quadruplicate), two wells/sample were washed three times with 6M urea, while others two wells/sample were washed just with buffer solution. At the end of test, color development was quantified by reading the optical density (OD). The relative avidity index was calculated by dividing the OD of the urea-washed wells by that of the wells washed with buffer solution and expressed as a percent. Samples with an index ≥60% were considered high avidity and that with index between 30 and 60% were considered intermediate avidity. From the 79 samples tested, 77 (97,47%) was considered of high avidity with mean index of 93,18%. Only 2 samples (2,53%) were considered of intermediate avidity. In conclusion, with the use of IgG-avidity test we confirmed that IgG antibodies specific to hantavirus of high avidity are present in sera from individuals that had never lived or travelled out of state of Alagoas, demonstrating evidence for the presence of hantavirus in this region. Financial support: CNPq, FAPEAL, CAPES.

HV439 - SURVEILLANCE OF ACTIVE POLIOMAVIRUS INFECTION IN GLOMERULOPATY AND PEDIATRIC RENAL TRANSPLANTATION PATIENTSMenoni, S.; Bonon, S.; Costa, S.; Torres, S.

UNICAMP - Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970

In immunocompromised patients, reactivation of BKV in kidney leads to infection in the urogenital tract, especially in renal transplant patients. The BKV disease is quite variable, and this variability is determined by the quality and quantity of immune host factors. Despite having been described for many years, BKV nephropathy had not been clinically observed until the middle of last decade. However, in recent years there has been an increased incidence of cases of nephropathy. This increase is attributed to improved diagnostic techniques and mainly by the increasing capability of immunosuppression. Objectives: This work aims to use PCR and nested PCR (N-PCR) to detect polyomavirus DNA in samples of plasma and urine from renal pediatric transplantation patients and with glomerulopathy to assist the treatment of this infection. Patients and Methods: Were studied 81 biological samples, 66 patients with glomerulopaty and 15 renal transplantation, samples of plasma and urine of each patient was collected in different periods. DNA extraction was done using commercial kits (Axygen Scientific, Inc.),and subsequently was made a simple PCR to detect viral DNA using primers that identify the two types of polyomavirus (BK and JC). The PCR is considered the standard method to perform the detection and identification of viruses. Results: As a results, only 1/81 (1.2%) samples was positive for BK virus. Conclusion: viremia and disease can be caused by BKV. In the genital tract can produce failure renal and rejection to trasplanted organ. The rapid detection is very important to early treatment and the monitoring of these patients in relation to poliomavirus infections.

HV440 - MOLECULAR DETECTION OF DENGUE VIRUS IN SERA SAMPLES FROM PATIENTS WITH FEBRILE ACUTE ILLNESS IN MACEIÓ, STATE OF ALAGOAS, BRAZILBorges, A.A.1; Silva, J. de M.1; Lira, E.L.1; Muller, V.D.M.1; Melo, D.M. de1; Botelho, J.A.1; da Silva, A.A.S.1; Aquino, V.H.2; Borges, A.A.

1. LAPEVI/ICBS/ UFAL - Laboratorio de Pesquisas em Virologia e Imunologia/Instituto de Ciências Biológicas e da Saúde/Universidade Federal de Alagoas, Av. Lourival Melo Mota, Ciudad Universitaria - AL, 57072-900

2. FCFRP/USP - Faculdade de Ciências Farmacêuticas de Ribeirão Preto/Universidade de São Paulo, Avenida do Café, s/nº Ribeirão Preto - SP, 14040-903




Dengue virus (DENV) infection is the most important arthropod-borne viral disease in the world. DENV are positive single-stranded RNA viruses that belong to the family Flaviviridae, genus Flavivirus. There are four serologically distinct dengue virus serotypes (DENV-1, DENV-2, DENV-3, DENV-4). Dengue virus infection causes a spectrum of illness ranging from asymptomatic or mild febrile illness to classic dengue fever (DF) and to severe and fatal hemorrhagic disease. Specific laboratory tests performed in the diagnosis of dengue can be accomplished through the detection of the presence of antibodies, virus isolation, detection of the virus genome by reverse transcription followed by polymerase chain reaction (RT-PCR), NS1 antigen detection, and immunohistochemistry. In the State of Alagoas, the diagnosis is performed only by serology and virus isolation. Thus, the aim of this study was to perform the molecular detection of DENV in sera samples from individuals with febrile acute illness and clinical suspect of dengue admitted to hospitals from Maceió/AL, whose illness has comprised between 0-7 days following the onset of symptoms. Sera from 55 patients were collected and were tested both by one-step real time RT-PCR and by RT-PCR conventional, for detection of the 5’UTR region and for detection of parts of the C, prM and NS5 genes at the viral genome, respectively. Of the total samples, in 15 (27.27%) was detected DENV genome in at least one of the used tests. The viral serotype could be determined in 9 samples (60%), of which 8 (88.9%) presented DENV-4 and 1 presented (11.1%) DENV-3. The overall positivity was considered low for a region were dengue epidemics occur annually. This data suggests that other members from genera Flavivirus may be causing infections in Maceio, in spite of the patients are receiving a clinical diagnosis of dengue. However, more studies are necessary to confirm this supposition. For this purpose, detection tests for the genomes of other flaviviruses are in progress in these same samples. Financial support: Ministério da Saúde/CNPq/SESAU-AL/ FAPEAL, CAPES

HV442 - HIV-1 MOLECULAR EPIDEMIOLOGY AND PREVALENCE OF TRANSMITTED DRUG RESISTANCE MUTATIONS AMONG INDIVIDUALS FROM MATO GROSSO DO SUL STATE,BRAZILLeite, T.1; Tanaka, T.2; Zacalusni, S.2; Castro, A.R.3; Guimarães, M.1

1. Oswaldo Cruz Institute2. Laboratory of Clinical Immunology, Federal

University of Mato Grosso do Sul3. Laboratory of Clinical Immunology, Federal

University of Mato Grosso do Sul,Oswaldo Cruz Foundation – Mato Grosso Do Sul

Since the beginning of the AIDS epidemic in Brazil, molecular epidemiology studies have identified a high prevalence of HIV-1 subtype B followed by sub-subtype F1 and BF recombinants in almost the entire country. The exception is the South region where a co-circulation of subtype C, B and BC recombinants is verified. The prevalence of these subtypes varied according to the geographic region, genomic region and studied group involved in the study. In order to add a new piece in this puzzle we aimed to investigate the genetic diversity and the transmitted resistance mutations in individuals from Mato Grosso do Sul, Central Brazil. For this purpose 131 HIV-1 ART-naïve positive individuals were enrolled since November 2009 to July 2011. DNA were extracted, amplified in PR/RT region and sequenced. Sequences were edited in DNASTAR software and then aligned with reference sequences using Clustal W. Neighbor Joining phylogenetic inferences with Tamura-Nei substitution model and recombination analyses applying Bootscan were performed in Mega 6 and Simplot programs, respectively. In order to investigate transmitted drug mutations sequences were submitted to the Stanford HIV Database for Transmitted DRM (TDRM/CPR Tool) Code Version 6.0. Based on these analyses, 68.7% was classified as subtype B, 11.5% as F1, 6.9% BF1, 11.5% as C and 1.5% as D. All BF1 recombinant samples were classified as unique recombinant forms. This high percentage of non-B subtypes (31.3%) has been previously described in this geographic region. We also highlight a temporal trend of increasing of subtypes C and sub-subtype F1 even not statistically significant. Moderate transmitted drug resistance mutations were verified in 11.5% of our samples; being 7.8% of NRTI mutations, 3.9% of NNRTI mutations and 2.3% of PI. This found is in agreement with previous studies in ART-naïve Brazilian samples. Taken together the data herein generated add new pieces in the Brazilian molecular epidemiology of HIV-1 and emphasizes the importance of monitoring drug resistance patterns among ARV-naïve individuals in this region of Brazil.




HV449 - EPIDEMIOLOGICAL AND SPATIAL ANALYSIS OF DENGUE CASES OCCURRING IN THE MUNICIPALITY OF TRES LAGOAS- MS IN THE TRIENNIUM 2012-2014Machado, A.M.1; Gotardi, A.H.B.1; Silva, M.H.R.1; Machado, A.R.S.R.2; Oda, J.M.M.1; Mirandola, P.H.1; Guerra, O.G.1; Machado, A.M.1

1. UFMS - Universidade Federal de Mato Grosso do Sul, Cidade Universitaria - Universitaria, MS, 79090-900


Dengue is the most important arboviral disease found in Brazil and responsible for thousands of cases annually. Três Lagoas - MS in recent years, the dengue has become a serious public health problem. We aimed to analyze retrospectively and spasso temporally all the cases of Dengue in Três Lagoas, in the triennium 2012-2014 relating them to different socioeconomic and infrastructural areas. For this, the data of all dengue cases, occurred in the period were obtained from Epidemiological Surveillance and the indices of Aedes aegypti infestation (IAI) obtained by the Vector Control Department. The data were geocoded using SPRING 5.1.5 ® system. Simultaneously the temperature and rainfall data, for the period, were obtained and correlated to dengue cases. In 2012 we observed 1050 positive cases, with 404 men and 646 women and mean age 20-34 years (32.66%), with highest number of cases occurred between the months of January to April (84.1%). In 2013 was observed an increase in numbers of cases, with 3855 positive cases (1714 men and 2141 women) and mean age 20-34 years (30.6%) with the highest number of cases occurred between the months of March to May (85.5%). In the year 2014, until the present time, we observed a marked decrease in the number of cases with only 48 positive cases (20 men and 28 women) which, differently from the previous years, occurred mainly in the months of May and June (87.5%). Most cases of Dengue fever (75%) occurred in the western and southwestern region of the city, especially in neighborhoods considered of middle class, where are found countless vacant lots and numerous deposits of vector. The IAI (Dengue positive) in the city ranged from 1.58% to 2.73%, however in neighborhoods with higher incidence of dengue positive cases this rates ranged between 2.35% to 14.6%, which

is greater than the considered acceptable <0.99%. We observed that the rainfall and temperature rates were similar in the 3 years studied and is directly related to the increased of positives cases. Also observed that the IAI (Dengue positive) remained high throughout the triennium. Thereat we hypothesized that the decrease in the number of positive cases, in the year 2014, is related to circulation of the same serotype during all this period. Therefore, we conclude that the entry of a new serotype of dengue might lead to a major outbreak, because the city has favorable characteristics of the circulate and maintenance of the vector and the dengue.

HV458 - OCCURRENCE OF HUMAN RHINOVIRUS SPECIES AMONG DIFFERENT POPULATIONS DURING EIGHT YEARSWatanabe, A.S.A.; Moreira, L.; Silva, E.; Camargo, C.; Kamikawa, J.; Granato, C.; Bellei, N.


Human rhinoviruses (HRV) are the major etiologic agent of the common cold. HRV infections are common among persons of all ages and account for 25–50% of respiratory illnesses among individuals presenting influenza-like illness (ILI). Advances in molecular techniques improved the molecular epidemiology of HRV, resulting in description of new HRV C specie in 2006. The aim of the present study was to describe the molecular epidemiology of the HRV species among different populations during 8 years. Nine distinct populations were analyzed, including children, adults, elderly, hospitalized patients, and immunossupressed patients. Severity parameters (presence of ILI, wheezing, ICU hospitalization, and mortality) were evaluated. Two distinct diagnostic tests were used for HRV detection: conventional PCR and real time PCR. Amplicons were purified and sequenced with Sanger´s sequencing method. Multiple sequences were aligned using Clustal X (v2.0.11). The multiple-sequence alignment was subjected to phylogenetic analyses using TOPALi (v2.5) software. HRV was detected in 25.3% (304/1202) of studied samples. Species distribution was: 51.7% (134/259) HRV A, 14.3% (37/259) HRV B and 34.0% (88/259) HRV C. Among all studied populations the species distributions follow this pattern (Table), except for children, with higher frequency of HRV C, followed




by HRV A and HRV B, and renal transplant outpatients (HRVA>HRV B>HRV C). Severity parameters were statistically similar among HRV species. Frequency of codetection with other respiratory viruses using direct fluorescence assay and/or conventional PCR resulted in 42/1353 (3.10%) of HRV positive samples. Influenza virus, human adenovirus and respiratory syncytial virus were frequently found in codetection. The molecular distribution of HRV species were similar with the described by others authors, with higher frequency of HRV A, although our study excluded the previous reported association between HRV C and severe infection, including wheezing children.

HV467 - PREVALENCE OF HUMAN PAPILLOMAVIRUS (HPV) IN HEALTHY ORAL MUCOSA OF USERS AND NON-USERS OF DRUGS IN A STATE IN NORTHEASTERN BRAZILRibeiro, M.G.M.1; Marcolino, L.D.2; Miranda, E.A.3; Jain, S.A.1; Santos, I.G. de A.1; Trento, C.L.2; da Silva, M.G.2; Dolabella, S.S.1

1. LEPAT/UFS - Laboratório de Entomologia e Parasitologia Tropical da Universidade Federal de Sergipe, Av. Marechal Rondon, s/n Jardim Rosa Elze, São Cristóvão - SE, 49100-000


3. DOL/UFS

Infection with Human Papillomavirus (HPV) is the sexually transmitted viral disease most prevalent in world. The infections can range from asymptomatic establishment to induction of squamous cell carcinomas. The aim of this study was to evaluate and compare the prevalence of HPV and its genotypes in healthy oral mucosa of users and non-users of drugs in a state in Northeastern Brazil by PCR technique. METHODS: One hundred and six patients with healthy oral mucosa, between 11 and 79 years visiting the Dentistry Clinic of the Universidade Federal de Sergipe and alcohol and drug users frequenting the rehabilitation institutions located in the state of Sergipe were evaluated. The samples were collected by exfoliation of oral mucosa using sterile cytology brushes. For quality control of DNA extraction beta-globin PCR was used. DNA-HPV

was detected using degenerate primers MY09/MY11 and general primers GP5+/GP6 +. Viral genotyping was performed using multiplex PCR with specific primers to genotypes 6, 16 and 18. RESULTS: Smoking and alcohol consumption was reported by 85.13% (74/106) of patients and 56.6% (60/106) used other drugs. Positive results were found in 83.02% (88/106). An HPV prevalence rate of 90.24% was detected in young patients aged between 11 and 30 years. Among patients taking multiple drugs, 86.67% (52/60) were HPV positive and 23.08% (12/52) samples with multiple type infections were identified. Among “non users” HPV prevalence was 78.26% (36/46) and two HPV types were identified in 13.89% of samples. CONCLUSIONS: The prevalence of HPV found in this study differs markedly from studies conducted around the world, in which a lower HPV prevalence has been reported. Few data are available on the oral HPV infection frequency for the Brazilian population and multiple drug users, notably the crackers, exhibit risky sexual behavior that increases the exposure to sexually transmitted diseases. Additional studies about HPV prevalence between drug users are needed for better knowledge of their exposure to the virus and development of prevention strategies. FINANCIAL SUPPORT: Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC) e PROMOB CAPES/FAPITEC.

HV468 - LACK OF INTRA-HOST VIRAL EVOLUTION IN HIV CONTROLLERS: A 10-13 YEARS FOLLOW-UP STUDYCaetano, D.G.1; Côrtes, F.H.1; Vorsatz, C.2,3; Grinsztejn, B.2,3; Veloso, V.G.2,3; Bello, G.1; Morgado, M.G.1

1. Laboratório de Aids e Imunologia Molecular IOC/FIOCRUZ - Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro - RJ, 21040-360



HIV-1 elite controllers (EC) are a very rare fraction (<1%) of HIV-1+ infected patients that maintain undetectable levels of viral RNA in plasma (<50 copies/ml) for several years in the absence of antiretroviral therapy. Analysis of the HIV-1 genomes integrated in




host cells revealed that most EC patients shown HIV-1 quasispecies with low diversity and extremely low divergence rate, compatible with a true evolutionary latency. Those longitudinal studies, however, analyzed the viral evolution over relatively short time periods (≤5 years). Here we described the long-term evolution of the HIV-1 quasispecies in three EC patients, two of them with occasional viral load blips (ECB42, ECB46) and one with undetectable viral load along the follow-up (EC52). The genetic diversity of the viral quasispecies was studied longitudinally over an interval between 10 and 15 years (2000-2013) by the amplification of the C2-C4 envelope region (env), using DNA extracted from cryopreserved PBMC samples and limiting dilution. Phylogenetic analysis of viral quasispecies revealed the existence of some long branches that were associated to hypermutation probably due to the action of the cellular enzyme APOBEC3G. Hypermutated sequences represent 16% of sequences of patient ECB46, 9% of sequences of patient ECB42 and 0% of sequences of patient EC52. All hypermutated sequences were then removed from the subsequent genetic distance estimations. Analysis of viral quasispecies of patient ECB42 at years 2000/2001 (n = 15), 2004 (n = 11) and 2011 (n = 9) revealed a stable diversity between 1 and 2% and an increase in the viral divergence from 0.5% to 1.7%. Analysis of viral quasispecies of patient ECB46 at years 2000 (n = 18), 2004 (n = 20) and 2011 (n = 13) revealed a decreasing trend of both diversity (from 1.8% to 0.8%) and divergence (from 1.5% to 0.5%) over time. Analysis of viral quasispecies of patient EC52 at years 2000/2001 (n = 20), 2004/2005 (n = 19) and 2013 (n = 16), revealed, for this patient, an extremely low level of viral diversity (0.1%) and no increase in viral divergence over time. This study revealed that HIV-1 variants integrated into cellular genomes of EC patients displayed undetectable or extremely low turnover over long time intervals and that most variation seems to be due to hypermutation related to cellular restriction factors, as verified for those presenting occasional blips. These results also support a very low rate of decay of the HIV-1 reservoirs in EC. FINANCIAL SUPPORT: CNPQ/SAPERJ

HV469 - HIGH PREVALENCE OF HUMAN PAPILLOMAVIRUS (HPV) IN ORAL LESIONS IN THE STATE OF SERGIPE, BRAZILRibeiro, M.G.M.1; Marcolino, L.D.3; Miranda, E.A.2; Jain, S.A.1; Santos, I.G. de A.1; Piva, M.R.2; Trento, C.L.2; da Silva, M.G.3; Dolabella, S.S.1

1. LEPAT/UFS - Laboratório de Entomologia e Parasitologia Tropical da Universidade Federal de Sergipe, Av. Marechal Rondon, s/n Jardim Rosa Elze, São Cristóvão - SE, 49100-000

2. DOL/UFS - Departamento de Odontologia da Universidade Federal de Sergipe, Av. Marechal Rondon, s/n Jardim Rosa Elze, São Cristóvão - SE, 49100-000


Among the infectious agents, HPV infection in oral mucosa is often associated with the onset and/or development of malignancy. Detection rates of HPV in oral cavity reported in the literature varies markedly around the world and make the relationship between HPV and oral carcinogenesis still controversial. The aim of this study was to evaluate the prevalence of HPV infection and its genotypes in patients with oral lesions in the state of Sergipe, Brazil by PCR technique.METHODS: Analyses included 39 patients frequenting the Dental Clinic of the Universidade Federal de Sergipe. aged between 2 to 83 years, with clinically detectable lesions in oral mucosa The samples were collected by exfoliation of oral mucosa using sterile cytology brushes. For quality control of DNA extraction beta-globin PCR was used. DNA-HPV was detected using degenerate primers MY09/MY11 and general primers GP5 +/GP6 +. Viral genotyping was performed using multiplex PCR with specific primers for genotypes 6, 16 and 18. RESULTS: Our study evaluated pre-malignant (dysplasias), malignant (Oral Squamous Cell Carcinomas-OSCC), and benign lesions (hyperplasia, papillomas and other lesions), having found the virus in all groups. HPV prevalence was 76.92% (30/39). The most common virus type HPV-6 was present in 56.67%(17/30) positive samples, followed by HPV-18 in 26.67%(8/30) and HPV-16 in 6.67 %(2/30). Multiple infection was detected in 26.67% (8/30) of samples. In 40% (12/30) samples the virus type was not identified. The relative prevalence of HPV according




to samples histopathology were 66.67% in papilloma cases; 83.34% (5/6) in OSCC; 100% in hyperplasia cases (1) and 72.23% (13/18) in samples with other types of lesions. All samples with dysplasia were positive. Patients who were also associated with papilloma or hyperplasia showed a HPV prevalence of 50%. In patients with other associated lesions the rate was 100%.CONCLUSIONS: A high prevalence of HPV in oral lesions was found in our study. This differs from other studies conducted in Brazil where HPV rates observed ranging from 0% to approximately 30% in oral lesions. Because HPV was found in all lesions groups evaluated, additional studies concerning HPV distribution in oral lesions in this country are needed, to assess the role of human papillomavirus in the development of lesions in oral mucosa. FINANCIAL SUPPORT: Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe(FAPITEC) e PROMOB CAPES/FAPITEC.

HV472 - SPATIAL DISTRIBUTION OF POSITIVE CASES OF HANTAVIRUS IN THE STATE OF MATO GROSSO BETWEEN THE YEARS 2008-2012Godoy, H.P.1; Cruz, C.A.1; Santos, R.F.1; Siqueira, A.B.1; Pimenta, S.T.S.2; Buzinaro, M.G.1



Hantavirus disease is an emerging disease transmitted by the inhalation of excreta of wild rodents and mostly occurs in individuals who have contact with the countryside with these rodents. This is a serious disease, with a mortality rate above 50%, which can lead to respiratory failure and shock. Hantavirus belongs to the family Bunyaviridae and is enveloped viruses that measure approximately 120 nm, have single-stranded RNA and negative polarity. In Brazil, cases were reported in 14 states noting that most of these cases were detected in the south and the states of Minas Gerais, São Paulo, Mato Grosso and the Federal District. In Mato Grosso, the first cases occurred in 1999, according to the State Department of Health The state of Mato Grosso is composed of 141 municipalities, is the largest grain producer in Brazil, and the pursuit of productive land, resulted in the destruction the natural habitat of

many animal species, including the wild rodents, in the search for food and the conditions associated with the production system, possible the largest animal / human contact. The objective of this study was to analyze the spatial distribution of positive cases of hantavirus in the state of Mato Grosso between the years 2008-2012. Data were taken from DATASUL tabulated in Excel 2007, analyzed and identifying risk areas using the program MapInfo Professional 7.0 software. 20 municipalities in the state with 108 confirmed cases for hantavirus, with the largest number of infected in the municipalities of Sinop 14.81% (16), Tangara da Serra 13.89% (15), Campo Novo Parecis 11.11% were identified (12), Feliz Natal 8.33% (09) and Peixoto de Azevedo 8.33% (09). The infected were mainly males 70.37% (76) and residents of rural areas with 57.40% (62). The cases occurred in Mato Grosso focused on the Middle North, predominantly agricultural region and grain producer, who suffered change your cerrado biome to large tracts of monoculture plantations was observed that the high frequency region of the affected people were males and residents of rural areas, which can be explained by the fact that they are the predominant sex as rural workers, increase the risk of contact between humans and infected rodents.

HV475 - ADENOVIRUSES PREVALENCY IN CHILDREN WITH ACUTE GASTRENTERITIS BEFORE AND AFTER INTRODUCTION ROTAVIRUSES VACCINATION IN BRAZILMuller, E.C.A.; Oliveira, A. do S.; da Silva, L.S.; Guerra, S. de F.; Oliveira, D.B.; Mascarenhas, J.D’A.P.; Gabbay, Y.B.; Linhares, A. da C.


Gastroenteritis is one of the major causes of childhood illness worldwide. Adenoviruses may play a role in the etiology of gastroenteritis. Adenovirus contains a double-stranded DNA and is non-enveloped. It belongs to the Adenovidae family, genus Mastadenovirus. They are divided into six species (A-F) and 51 serotypes. Epidemiological studies have detected adenovirus in 2 to 22% of cases of acute diarrhea in pediatric hospitals and emergency departments. The main purpose of this study was to detect the presence and define the types of adenoviruses in stool samples from 1,160 children




under three years of age including hospitalized patients and outpatients during the rotavirus pre-vaccination period (March to September 2003) and post-vaccination period (May 2008 to April 2009). This was a surveillance carried out by Institute Evandro Chagas these children were hospitalized with gastroenteritis in Belém, Pará, Brazil. Our study placed emphasis on 40 and 41 adenovirus (AdE) serotypes (species F). We used EIA and immunochromatography for screening, and PCR and nucleotide sequencing for molecular identification and typing. The adenoviruses (Ad) were found in 7.24% (84/1160) of samples. AdE was present in 5% (58/1160) of samples, corresponding to 69% (58/84) of positive cases. Overall, 3.3% (25/760) were found during the pre-vaccination period and 8.2% (33/400) during the post-vaccination period study. Our data suggest an increase in the prevalence of AdE after introduction of the vaccine Rotarix®. As based on nucleotide sequencing, species F was characterized as the more prevalent in our region, accounting for 70% (21/30) of isolates, with type 41 accounting in 85.7% (18/21) of positive cases. There were no significant variations in the molecular epidemiology when comparing samples from the pre and post-vaccination period. Ad predominated in the age group of 18-24 months during the pre-vaccination period; no clustering of Ad into a specific age group could be found during the post-vaccination period. Furthermore, no significant differences could be seen in relation to gender and the temporal distribuition. Our results provide strong evidence of adenovirus belonging to species F being a cause of severe gastroenteritis among children under 3 years of age.

476 - INFLUENZA: DEVELOPMENT OF A GENETIC REVERSE SYSTEM BY YEAST-BASED HOMOLOGOUS RECOMBINATION AND ITS APPLICATION IN THE IMPROVED VACCINE PRODUCTION IN CULTURE CELLSilva Jr, J.V.J.1; Cruz, F. da S.P.1; Bertani, G.R.1; Machado, A. de M.V.2; Gil, L.H.V.G.1

1. CPQAM/FIOCRUZ - Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, Recife - PE, 50.740-465


Recombinant influenza vaccines are produced using genetic reverse system and grown in embryonated chicken eggs. The viruses are generated by cell co-transfection with cloned cDNA of hemagglutinin (HA) and neuraminidase segments from wild-type strain plus six segments from influenza A/PR/8/34 (PR8) strain. However, theses clones are obtained by use of specific restriction sites and in vitro ligation, which sometimes are difficult to perform. In advantage, the homologous recombination in yeast (HRY) cloning technique is an efficient and simple process, where DNA fragments containing homologous ends with the vector can be directly cloned using in vivo recombination. The currently influenza vaccine also is limited by capacity of the egg supply in pandemic situation, e.g. as occurred in 2009 (pnd2009). Additionally, the WHO recommends Vero cells as an alternative substrate for recombinant influenza vaccine production, however some viruses grow sub optimally in this cell line. Recently, were reported the enhanced growth of H1N1 in Vero cell by changing an amino acid residue in HA (117N>D). Thus, to overcome the difficulties of producing recombinant pandemic influenza vaccine, the objective this work was the development of the first reverse genetics system of influenza made by HRY and the insertion of the HA/117N>D mutation (by HRY) in a chimeric virus expressing HA from isolated pandemic (2009) capable of improved its replication in Vero cells. For this, the pJG-HW2000-2013 vector was constructed and the mutation effect was first evaluated in prototype influenza PR8 strain. The PR8 genome was subcloned by HRY from pJG-HW2000 to pJG-HW2000-2013. The pJG-HW2000-2013/PR8 was transfected in HEK 293T/MDCK co-culture and recombinant PR8 (IC-PR8) was generated. After, the HA/117N>D mutation was inserted by HRY into pJG-HW2000-2013/PR8 and mutant virus (IC-PR8/HA-117N>D) was generated. Plaque assay performed in Vero cell showed replication larger in IC-PR8/HA-117N>D than in IC-PR8. After, the HA segment of 2009 influenza isolate was cloned by HRY into pJG-HW2000-2013 and the clone was co-transfected with pJG-HW2000-2013/PR8 and chimeric virus IC-PR8/HApnd2009 was generated. The mutation HA/117N>D was inserted by HRY in IC-PR8/HApnd2009 and mutant virus (IC-PR8/HApnd2009-117N>D) was recovered. Comparison of the plaque assay and viral growth




kinetics between IC-PR8/HApnd2009-117N>D and IC-PR8/HApnd2009 in Vero cell is in progress. FINANCIAL SUPPORT: CAPES, CNPq, Fiocruz

HV478 - RARE PRESENCE OF LANGERHANS CELLS WITHIN INTRAEPITHELIAL CERVICAL LESIONS INDUCED BY HPV INFECTIONCarvalho, M.O. de O.1; Simões, R.S. de Q.2; Rodrigues, F.C. das C.1; Portari, E.A.3; Barth, O.M.S.2; Russomano, F.B.3; Fernandes, A.T.G.1; de Almeida, M. da G.B.1

1. INI - Instituto Nacional de Infectologia Evandro Chagas, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro - RJ, 21040-360

2. IOC - Instituto Oswaldo Cruz, Av. Brasil, 4365 - Manguinhos, Rio de Janeiro -RJ, 21040-900

3. IFF - Instituto Nacional de Saúde da Mulher, da Criança e do Adolescente Fernandes Figueira, Av. Rui Barbosa, 716 - Flamengo - Rio de Janeiro - RJ, 22250-020

Cervical cancer (CC) was the 4th cause of cancer death and is the second most frequent cancer in women worldwide. HPV is associated with cervical cancer and plays a crucial role in tumor formation. Over 170 types of HPV have been identified, each with preferential tropism to a particular location. Around 45 types of HPV infect the genital tract and may be divided in low and high oncogenic risk types. Of these, HPV-16 and -18 are the most frequently associated with anogenital cancer. Despite these infections being considered a risk factor, they are not sufficient to induce CC. However, other factors associated with HPV infection may favor the development of cervical squamous intraepithelial lesion (C_SIL), a cancer precursor lesion. The cervical cellular immune response is responsible for control of HPV lesion progression. Langerhans cells (LC), a professional antigen-presenting cell, play an important role in the immune response surveillance against viral infections. However, several studies are controverse about the role of LC in cervical HPV infection. The present study aims to detect LC presence in C_SIL. Patients, 19 to 50 years aged, were recruited from IFF-Fiocruz. Nine cervical with high C_SIL diagnosed biopsies were taken and processed for immunohistochemistry analysis using an anti-langerin antibody for LC detection. Positive stained cells were evaluated in the epithelium, basal layer of epithelium, stroma and perivascular fields. LC were detected in all samples, rarely inside the epithelium

(1.0±1.1), more frequently inside the stroma (2.3±2.3) and in perivascular (1.5±1.2) areas. The migration of LC into the epithelium presenting HPV lesions is important to induce a pro-inflammatory microenvironment by recruiting immune cells to eliminate the HPV-infected keratinocytes, initiating an effective acquired immune response. Moreover, studies on LC function in cervical lesions and cancer are necessary for understanding the mechanisms involved in HPV clearance, the immune evasion favoring the cancer progression and to further discovery of new drugs and chemotherapies.

HV484 - HERPESVIRUS AND PAPILLOMAVIRUS DNA DETECTION IN ADULTS AND ELDERLY PATIENTS WITH HEAD AND NECK CANCERBonon, S.H.A.1; Torres, S.V.S.1; Sbegue, A.2; Menoni, S.M.F.1; Costa, S.C.B.1


2. PUCCAMP - Pontifícia Universidade Católica de Campinas, Prédio H13 - Portão 2. Rod. Dom Pedro, km 136. Parque das Universidades, Campinas - SP, 13086-900

Viruses that have been identified as causative agents for a large proportion of these diseases have also been associated with various malignant states. Concomitantly, the number of cases of oral cancer (considered to occur usually around or after the fifth decade of life) reportedly has been increasing among young adults. The oncogenic potential of herpesvirus and papillomavirus and their possible role in the development of malignant conditions, in particular cancer of head and neck has been described. Material and Methods: The aim of this study is to evaluate the presence of DNA of human herpesvirus directly in biopsies removed surgically from twenty-six patients, aged 45-75 years. Fresh biopsies from 16 patients with head and neck cancer and 11 fresh biopsies from a healthy control group were studied. DNA extraction was executes and nested PCR technique was used for DNA detection in lesions to analyzed for the presence of DNA of Epstein-Barr virus (EBV), Human Cytomegalovirus (HCMV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Human Herpesvirus 8 (HHV-8) and Papillomavirus (HPV). Results: Biopsies from cancer patients showed 55,5 % positive samples




for virus in relation to 22,2 % control group. HPV occurred in 8/16 (50%) cancer biopsies and only patient of control group was positive. HHV-6 and HHV-7 was detected in 22% from cancer group and HHV-7 was the most prevalent with four individuals study group and one of the control group, followed by EBV with two of the study group and the control group and one patient in the study group showed co-infection of EBV and HHV-6 and eight patients in the study group had HPV against four in the control group. No CMV virus DNA was detected in both groups. Conclusion: HPV were the most prevalent virus found in biopsies of cancer followed by EBV and HHV-7, and there were statistically significant differences between groups. This finding suggests that the virus in these lesions can lead patients to a worse prognosis, as well as virus infections may be an etiologic agent of cancer. Financial Support: CAPES

HV492 - DETECTION OF HUMAN CYTOMEGALOVIRUS IN DIFFERENT TYPES OF GLIOMASCastro, L.F.F.1; Stangherlin, L.M.1; Kimura, L.M.2; Guerra, J.M.2; Shirata, N.K.2; Nonogaki, S.2; Medeiros, R.S.S.3; Silva, M.C.C.1

1. UFABC - Universidade Federal do ABC, Rua Catequese, 242 - Jardim, Santo André - SP, 09090-400


3. FM/USP - Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455 - Cerqueira César, São Paulo - SP, 01246903

Human Cytomegalovirus (HCMV), a member of the Herpesviridae family is a ubiquitous pathogen associated with several human malignancies in immunocompromised, transplanted recipients and newborns. In addition, evidence suggests that HCMV is associated with tumor malignancy based on sero-epidemiologial studies as well as on the presence of HCMV DNA, RNA and antigens in tumor tissues. In particular, we and others have detected the viral genome and viral proteins in glioblastoma multiforme (GBM), the highest-grade glioma (Grade IV) and most malignant form of astrocytoma. Studies searching for HCMV in different types of gliomas are still scarce. Therefore the aim of our work was to verify the HCMV presence and viral load in paraffin-embeded sections of glioma grades I-IV. Fifty-two tissue samples consisting of 27

astrocytomas (ACT) (Grades I-III), 10 glioblastomas (GBM) (Grade IV), and 15 oligodendromas (OLD) (Grades I-III) were tested for the presence of HCMV DNA, RNA and protein. Viral DNA was detected by quantitative real time PCR (q-PCR), using primers for the UL83 ORF, in 18/27 cases of ACT (66,6%), 06/10 cases of GBM (60%) and 10/15 cases of OLD (66,6%). The immediate early transcript (IE1) was detected by in situ hybridization (ISH), using a fluorescein-conjugated probe, in 13/27 cases of ACT (48,1%), 04/10 cases of GBM (40%) and 07/15 cases of OLD (46,6%). The IE1 viral protein was detected by immunohistochemistry (IHC) in 10/27 cases of ACT (37%), 4/10 cases of GBM (40%) and 8/15 cases of ODG (53,3%). These results suggest that qPCR is a more sensitive method of viral detection. In addition, the data indicate that the presence of HCMV does not differ significantly in different types of gliomas. Interestingly, however, quantification of viral DNA in the samples demonstrated that the viral load increases with the grading of tumors, up to grade III, and decreases in GBM (grade IV). Funding: FAPESP

HV497 - IDENTIFICATION OF RESPIRATORY VIRUS AND COINFECTIONS IN SAMPLES FROM CHILDREN ASSISTED BY THE CHILDREN HOSPITAL COSME DAMIAO – PORTO VELHO, RONDONIAAlves, F.A.G. dos S.1,2,3; Santos, J. das V.2,3; dos Santos, A.O.1,2,3; Baffini, V.R.; Pereira, A.V.C.2,3; Souza, L.F.B.1,2,3; Lima, N.C. da S.2,3; Matos, N.B.1,2,3; Salcedo, J.M.V.1,2,3; Vieira, D.S.1,2,3




Acute respiratory infections (ARIs) are an important cause of childhood morbidity and mortality worldwide, which present greatest impacts in developing countries. ARIs are caused by different etiologic agents like viruses and bacteria that are the major causatives of ARIs. Objective: To identify by immunofluorescence technique the major respiratory viruses and check for potential bacterial coinfections in samples from




children treated at the Hospital Cosme Damiao - Porto Velho, Rondônia. Methods: Sixty samples were collected by nasopharyngeal swab from children (0-6 years old) who had symptoms of acute respiratory infection from February to December 2013. These samples were tested by immunofluorescence technique for the identification of the following viruses: adenovirus, Human Respiratory Syncytial Virus (RSV), influenza A and B, parainfluenza 1, 2 and 3 (HPIV) using a commercial panel. Besides that, samples were tested in the Microbiology laboratory for the following bacteria: Escherichia coli, Delftia acidovorans, Rothia mucilaginosa, Streptococcus sp., Neisseria meningitidis, Staphylococcus aureus, Neisseria flava, Corynebacterium sp., Moraxella catarrhalis, Kocuria sp. and Streptococcus parasanguinis through the following tests: antibiogram, CAMP, catalase, biofilm, susceptibility to optochin, bile solubility, PCR and sequencing. Results: Until this moment thirty-four samples were analyzed by immunofluorescence, of these 88.2% (30/34) were found to be positive. Among the positive samples there was found the following viruses: 30% (9/30) Parainfluenza 1, 23.33% (7/30) Parainfluenza 2, 13.33% (4/30) Parainfluenza 3, 16.66% (5 / 30) Respiratory Syncytial Virus, 6.66% (2/30) Influenza A and 20.58% (7/30) Adenovirus. Multiple infections were observed in nine samples, two samples were positive for HPIV-1 + HPIV-3, one sample HPIV-1 + Adenovirus, one sample HPIV-2 + Influenza A, one sample HPIV-1 + RSV, one sample RSV + Adenovirus, one sample Adenovirus + Influenza A, one sample HPIV-1 + HPIV-2 + Adenovirus and one sample HPIV-2 + HPIV-3 + RSV infections. Five of the samples positive for respiratory viruses present coinfected with bacteria.Conclusion: The presence of viral and bacterial coinfection highlights the necessity for further research that can define the epidemiological profile in this region. It is important to know the causative agent of the infection since it serves as an aid in the prognosis and management of these children.

HV511 - DETECTION OF NON-INFLUENZA RESPIRATORY VIRUS IN HOSPITAL PATIENTS WITH SEVERE ACUTE RESPIRATORY INFECTION (SARI) IN BELEM, STATE OF PARA, BRAZILBarbagelata, L.1; Vergueiro, M.1; Santos, M.1; Ferreira, J.1; Gomes, E.1; Souza, E.1; Medeiros, R.2; Mello, W.1



Acute Respiratory Infection (ARI) are characterized by respiratory tract infections, sudden onset, and variable severity, one of the major public health problems worldwide. Viruses are the leading causes of ARI since it is estimated that 90% of those infections are related to those pathogens. In addition to Influenza A and B viruses, other viruses also play an important role in the induction of gravity in respiratory infections such as Human Rhinovirus (HRV), Adenovirus (AdV), Parainfluenza Virus (PIV), Human Coronavirus (HCoV) and Human Bocavirus (HBoV). The similarity of the symptoms make difficult the clinical diagnosis, becoming essential laboratory diagnosis. In this context, this study aims to detect non-influenza viruses in hospital patients with SARI in Belém, State of Pará, Brazil, determining which virus is the most frequently involved one; and to describe the seasonality of infection by those agents in Belém. A total of 271 samples were collected from patients with ARI, both sexes, and different ages, treated at hospitals in Belém, from January 2013 to January 2014, screened by Influenza Surveillance Network, but diagnosis for influenza virus was negative. The viral nucleic acid was extracted from the clinical specimen by a commercial kit. The detection of viral genome was performed by real-time polymerase chain reaction (qRT-PCR), using probes and specific oligonucleotides primers for those virus. Of the 271 samples tested, 95 (35%) were positive for one or more viruses investigated. And 56 (58.9%) were positive for rhinovirus; eight (8.4%) for adenovirus; seven (7.3%) for parinfluenza virus type 3; five (5.3%) for coronavirus HKU1; four (4.2%) for coronavirus OC43; three (3.1%) for bocavirus; two (2.1%) for coronavirus 229E; two (2.1%) for coronavirus NL63. Eight co-detections for rhinovirus were also found in seven of them. Regarding seasonality, circulation of those virus was predominant in the first half of the year, mainly May and June, which is usually associated with the period of highest rainfall in our region. The indicated data show the important role of non-influenza viruses for inducing severe cases of respiratory infection, highlighting the rhinovirus,




once was only indicated as an inducer of the common cold. Another important finding is the demonstration of the first evidence of HCoV HKU1, NL63 in the Amazon region. FINANCIAL SUPPORT: IEC/SVS/MS

HV513 - PREVALENCE ADENOVIRUS AMONG CHILDREN WITH ACUTE GASTROENTERITIS PRE AND POST ROTAVIRUS VACCINE INTRODUCTION IN BRAZILMuller, E.C.A.1; Oliveira, A. do S.1; da Silva, L.S.1; Guerra, S. de F.1; Mascarenhas, D.O.J.D’A.P.1; de Sousa, M.S.2; Linhares, A. da C.1


2. UFPA - Universidade Federal do Pará, Rua Augusto Corea, nº 1, Belem Do Pará - Para, 68440-000

Gastroenteritis is one of the major causes of childhood illness worldwide. Adenoviruses may play a role in the etiology of gastroenteritis. Adenovirus contains a double-stranded DNA and is non-enveloped. It belongs to the Adenovidae family, genus Mastadenovirus. They are divided into six species (A-F) and 51 serotypes. Epidemiological studies have detected adenovirus in 2 to 22% of cases of acute diarrhea in pediatric hospitals and emergency departments. The main purpose of this study was to detect the presence and define the types of adenoviruses in stool samples from 1,160 children under three years of age including hospitalized patients and outpatients during the rotavirus pre-vaccination period (March to September 2003) and post-vaccination period (May 2008 to April 2009). This was a surveillance carried out by Institute Evandro Chagas these children were hospitalized with gastroenteritis in Belém, Pará, Brazil. Our study placed emphasis on 40 and 41 adenovirus (AdV) serotypes (species F). We used EIA and immunochromatography for screening, and PCR and nucleotide sequencing for molecular identification and typing. The adenoviruses (Ad) were found in 7.24% (84/1160) of samples. AdV was present in 5% (58/1160) of samples, corresponding to 69% (58/84) of positive cases. Overall, 3.3% (25/760) were found during the pre-vaccination period and 8.2% (33/400) during the post-vaccination period study. Our data suggest an increase in the prevalence of AdV after introduction of the vaccine Rotarix®. As based on nucleotide sequencing, species F was characterized as the more prevalent in

our region, accounting for 70% (21/30) of isolates, with type 41 accounting in 85.7% (18/21) of positive cases. There were no significant variations in the molecular epidemiology when comparing samples from the pre and post-vaccination period. Ad predominated in the age group of 18-24 months during the pre-vaccination period; no clustering of Ad into a specific age group could be found during the post-vaccination period. Furthermore, no significant differences could be seen in relation to gender and the temporal distribuition. Our results provide strong evidence of adenovirus belonging to species F being a cause of severe gastroenteritis among children under 3 years of age.

HV515 - CO-CIRCULATION OF TWO DENV-1 LINEAGES IN SÃO JOSÉ DO RIO PRETO, SÃO PAULO, BRAZILSilva, G.C.D.1; Périco, J.M.B.1; Vedovello, D.1; Colombo, T.E.1; Pinheiro, T.M.1; Pires, L.M.1; Silva, G.C.D.1; Ullmann, L.S.1; Junior., J.P.A.2; Drumond, B.P.3; Nogueira, M.L.1




Dengue fever represents an important public health problem in Brazil. São José do Rio Preto (SJRP) has been presenting a hyperendemic circulation of dengue virus (DENV) since 2008, when three serotypes (I, II and III) started circulating in the city. The serotype 4 was introduced in SJRP in 2011, and nowadays the four serotypes already had been detected in the city. Dengue virus 1 (DENV-1) serotype re-emerged in São José do Rio Preto in 2008 (1,83% of cases), after over 10 years without its detection. In the subsequent years (2009-2014), DENV-1 was the predominant serotype in Dengue cases in the city, except for the year 2013, when DENV-4 was the main circulating serotype. The literature reports the circulation of multiple DENV-1 strains in some countries, including Brazil, and DENV-1 clade diversity was previously associated with an increase in the abundance of this serotype and enhanced mosquito transmission. Thus, the aim of this study was to identify




the predominant genotype strains (or clades) of DENV-1 that may have contributed to the large number of cases of DENV1 infection in SJRP. This study included serum samples sent to the Laboratório de Pesquisa em Virologia of FAMERP for Dengue diagnosis between 2008 and May 2014. In this period, of 1162 samples positive for DENV, 490 (42%) were positive for DENV1. Of these, we have sequenced the complete E gene directly from serum of 29 infected patients previously diagnosed with DENV-1 infection by capillary DNA sequencing using the BigDye v3.1 in ABI3130 automatic sequencer (Applied Biosystems) or by Illumina technology (Illumina). Consensus DENV-1 E gene sequences were obtained using Accelrys Gene 2.5 software (Accelrys) and alignment (MUSCLE) and phylogenetic analysis (Maximum Likelihood, TN93+G+I) were performed using Mega 6.0 software, using previously published E gene sequences from GenBank. The results showed that all DENV-1 belong to genotype V and are grouped in two separate clades, indicating the existence of two DENV-1 lineages in SJRP. We detected the co-circulation of these lineages at least for three years (2010-2012). Although the clinical and epidemiological significance of the co-existence of these two lineages described here is still not clear, these results highlight the genetic diversity of circulating DENV-1 in SJRP and the potential for DENV-1 lineages to persist in a hyperendemic city. FINANCIAL SUPPORT: INCT/CNPQ-DENGUE; FAPESP; CAPES; FAMERP/FUNFARME;SECRETARIA MUNICIPAL DE SAÚDE

HV522 - NOROVIRUS CIRCULATION IN COMMUNITY CHILDREN WITH HIGH ROTAVIRUS VACCINATION COVERAGECarraro, E.; Ambrosini, V.A.; Semaan, L.M.; Campos, D.A.; Pedrosa, F.C.


Diarrheal disease is a common cause of morbidity and mortality worldwide, estimates in the top 5 causes of deaths, with most occurring in young children in nonindustrialized countries. The group A rotavirus was considered the most important as a cause of gastroenteritis in children. In Brazil, in March 2006, the National Immunization Program included oral

human rotavirus vaccine with antigen specificity G1P8. After started the vaccination several studies showed the benefits in reducing the number of infections, hospitalizations and mortality caused by rotavirus. In contrast, the Norovirus has been emerging as the main etiological agent. Few studies in Brazil have investigated the circulation of Norovirus, and most of them are in closed places (hospitals, nursing homes, and day care centers), and few of them report the circulation in the community. The objective of this study was to investigate the infection caused by Rotavirus and Norovirus in children until to five years of old enrolled in the health system of with gastroenteritis in Guarapuava - PR. On hundred twenty stool samples were included our study between the months of March 2011 through February 2012. RT-PCR assays for VP7 and VP1 genes amplification were applied for Rotavirus and Norovirus detection, respectively. The children include had a mean of 2.8 and a median of 3 years, of old and the average of rotavirus vaccination rate was 83.85%. No rotavirus infection cases were detected and Norovirus was detected in 19 samples (15.8%) of stool investigated. The months with the largest Norovirus circulation were September and October 2011, suggesting the possibility of an outbreak during this period. This result suggests a reduction of rotavirus infection caused by rotavirus in children with high vaccination coverage rate and pointed that Norovirus is circulating in the community children as an agent of gastroenteritis, should now be considered in the differential diagnosis and in the institution of measures to control transmission.

HV523 - CHARACTERIZATION OF THE GENOTYPIC PROFILE OF HEPATITIS DELTA VIRUS: HDV GENOTYPE-1 ISOLATION IN THE REGION OF WESTERN AMAZON, BRAZILSouza, L.F.B.1,2; Vieira, D.S.2,3; dos Santos, A. de O.1,2; Pereira, A.V.C.2,3; Salcedo, J.M.V.2,3







Hepatitis Delta Virus (HDV) is a hepatotropic subvirus which is dependent on the hepatitis B virus. Viral genetic diversity is related to the geographical origin of the isolates, and yet eight genotypes were identified and classified from HDV-1 to HDV-8. The regions with high levels of endemicity are the center and north Africa, the Amazon Basin, Eastern European, Mediterranean, Middle East and parts of Asia. Methods: This study has included sera from 54 patients with HBV/HDV infection whose HDV-RNA and HBsAg were detectable. Clinical and epidemiological data were obtained from the Medical Records Outpatient filled by the specialist physician. HDV-RNA was extracted and converted to cDNA. The cDNA was amplified and genotyped by nested PCR-RFLP. Twelve HDV-3 and four HDV-1 samples were sequenced by nested PCR-RFLP. Processing, editing and production of the consensus sequences were performed using Phred, Phrap-Consed. The consensus sequences were aligned using the Clustal X2. A phylogenetic tree was generated by Maximum Likelihood method using MEGA5 based on HKY-substitution model. Results: Fifty-two samples were positive in Nested PCR-RFLP. A prevalence of 92.3% for HDV-3 and 7.6% for the HDV-1 has also been observed. In the analysis of the evolutionary lineage it was observed from 16 sequenced samples, twelve of them belonged to HDV-3 and four to HDV-1, supporting the nested PCR-RFLP results. The patients infected with HDV-1 had become symptomatic, two of whom had splenomegaly. They have showed values of 52 and 53 respectively for AST and ALT and three of them showed Chronic Liver Disease signs on Abdominal Ultrasonography and esophageal varices at endoscope as well. Only one patient underwent liver biopsy and has showed fibrosis=2 and activity-1 in Metavir system. Conclusion: Earlier studies in patients with hepatitis Delta in the State of Rondonia have shown higher prevalence of HDV-3 in Amerindian population, and in those studies no patient was indigenous. The cases series shows a relatively young age with relatively advanced Chronic Liver Disease, since 3 of 4 patients had already signs of portal hypertension with evidence of esophageal varices. We reinforce the thinking that the hepatitis Delta represents a public health problem in many countries worldwide. However, we cannot neglect the presence of HDV-1 that is generally diagnosed late

and determines advanced liver disease in young patients. Financial support: FIOCRUZ/CEPEM

HV524 - MOLECULAR DIAGNOSTIC OF HUMAN T-CELL LYMPHOTROPIC VIRUS IN WHOLE BLOOD SAMPLES OF PATIENTS WITH ANTIRETROVIRAL TREATMENT FOR HIV IN PERUHuatuco, E.M.M. ; Bustamante, F.C.2; dos Santos, T.A.4; Barbosa,M.L.3; Oliveira, D.B.3; Durigon, E.L.3; do Rosario, J.S.C. 4

1. UNMSM - Universidad Nacional Mayor de San Marcos, Calle Germán Amézaga N° 375 - Edificio Jorge Basadre, Ciudad Universitaria, Lima 1, Lima - Peru

2. INS - Instituto Nacional de Salud, Avenida calle 26 No. 51-20 - Zona 6 CAN. Bogotá, D.C.


4. FMH/USP - USP - Universidade de São Paulo, Av. Prof. Almeida Prado, nº1280 - Butantã, São Paulo - SP, 05508-070

Human retrovirus HTLV and HIV share similar routes of transmission but cause different diseases. In common share tropism for CD4 + lymphocytes, the immune system change .Increased co-infections in South America is evident. It is an emerging health problem in patients with antiretroviral treatment in Peru. The antiretroviral therapy has decreased mortality and morbidity in patients with HIV-1 prolonging its life, with consequences that have resulted in an increased number of inflammatory syndromes, these processes difficult the accurate diagnosis of HTLV-1. This work aims to report the progress in diagnosing HTLV-1 of 23 cases of patients living with HIV-1 and antiretroviral therapy HAART during 2012.-2013 at the National Institutes of Health. Serological studies were performed for the detection of anti-HTLV-1 antibodies in serum with ELISA (HTLV-I + Bioelisa II5.0-Biokit). For confirmation was used WESTERN BLOT additional test (Innogenetics INNO-LIA-HTLV-I/II). In the investigation of the viral genome and the proviral DNA purification from whole blood was used the kit protocol (invitrogem Purelink Genomic DNA), and the genomic material was stored at -20 ° C. Test for amplifying proviral DNA segment was used (Nested-PCR). The consensus primers were used for amplification of the virus HTLV-1in the first run SK43




and SK44, and for the second run were used tax1 and tax 2.Verificando agarose gel 3%. B-actin was used to confirm the presence of DNA. The analysis of restriction fragment length polymorphism (RFLP) were evaluated (Taq1 New England-Biolabs). Nucleases hydrolyze using specific sequences in DNA resulting from digestion, easily detectable by differences in their migration electrophoretic. We report are serologically positive for HTLV 43.5% (10/23) and 56.5% (13/23) indeterminate cases. In PCR 26.09% (6/23) of cases were positive for HTLV. The 73.91% (17/23) were negative for HTLV. In 100% (23/23) of samples evidenced positive B-actin. The protocol RFLP that were used allowed us to determine the 33.33% (2/6) of HTLV-1 positive befits a man of 63 years and a woman of 40years, we not found HTLV-2. Subtypes could not be determined entirely possibly due to HIV-1 infected CD4 + T cells induce signals that trigger the activation of cytokines, chemokines that can interfere diagnosis and even more may be related to the emergence of different phenotypes of HIV-1 that modulates the cellular microenvironment, difficult to differentiate subtypes. In the capital of Peru (Lima) was confirmed circulation of HTLV-1 in patients living with HIV and antiretroviral treatment, we assume that we should continue investigating, to contribute to the improvement of new diagnostic systems in public health.

IMMUNOBIOLOGICALS IN VIROLOGY - IV


Immunobiologicals in Virology: IV 201

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Immunobiologicals in Virology: IV

IV98 - EVALUATION OF IMMUNOGLOBULIN IGA AND IGG DETECTION AGAINST HPVCosta, A.P.F.1; Machado, P.R.L.1; Souza, L.B.F.C.1; Freitas, J.C. de O.C.1; Eleutério Jr, J.3; Giraldo, P.C.2; Gonçalves, A.K. da S.1




The interest in HPV seropositivity has increased considerably since HPV vaccines have become available worldwide. To assess the performance of ELISA in analyzing serum samples provided from women with and without genital DNA-HPV infection confirmed by PCR, for detection of specific antibodies of the isotypes IgG and IgA recognizing HPV-16, and 18 as well as virus-like particles. Fifty women sexually active female patients, between 18-35 years from the outpatient clinic at university hospital from August to December 2013, were enrolled In order to test them, positive controls were obtained from patients with HPV induced lesions and DNA-HPV positive confirmed by PCR. A specific assay was used to identify antibodies to HPV virus-like particles by ELISA. The samples were divided into HPV positive and negative, and an ELISA detecting IgA and IgG anti-HPV-VLP was carried out. The effectiveness of ELISA and the Kappa (k) index was obtained from the values entered in the ROC curves for IgG and IgA. IgG-VLP-HPV16 showed a good correlation between ELISA and PCR (k=0.75) and IgG-VLP-HPV18 showed a very good correlation between ELISA and PCR (k=0.84). While the IgA antibody correlation was also positive, although weaker: IgA-VLP-HPV16 was moderate (k=0.45) and IgA-VLP-HPV18 good (k=0.66). The efficacy of the assay concerning IgG was the following: sensitivity, specificity and accuracy were, respectively, 82.3%, 92% and 88% to IgG-VLP-HPV16, and 100%, 92% and 94% to IgG-VLP-HPV18. The assay concerning IgA was the following: sensitivity, specificity and accuracy were, respectively, 64.7%, 80% and 73.8% to IgA-VLP-HPV16, and 100%, 80% and 84.8% to IgA-VLP-HPV18. IgG and

IV34 - EXPRESSION OF HEPATITIS A VIRUS PROTEINS IN BACULOVIRUS EXPRESSION VECTOR SYSTEMda Silva Jr, H.C.; de Azevedo, M.L.B.; Galler, R.; Medeiros, M.A.


The hepatitis A virus (HAV) is the primary etiologic agent of acute viral hepatitis and is estimated to cause tens of millions of new infections each year worldwide. Currently, there are commercially available vaccines against HAV based on inactivated viruses. However, the high cost of production hinders the introduction of these vaccines into the routine of developing countries. In this context, the use of recombinant proteins of HAV may represent an alternative model to existing vaccines. Given this, the present study aimed to evaluate the expression of VP1 and P1-2A proteins of hepatitis A virus in the baculovirus expression vector system (BEVS). The VP1 and P1-2A genes were cloned into the polylinker of pFastBac™ Dual vector, under the control of polyhedrin promoter. The recombinant baculoviruses were subsequently generated and used to express the recombinant proteins in Spodoptera frugiperda 9 (Sf9) cells. In order to verify the expression and evaluate solubility of VP1 and P1-2A, the infected cells were harvest, disrupted and analyzed by Western blotting. The nickel affinity chromatography (IMAC) was used to purification. The recombinant proteins VP1 (rVP1) and P1-2A (rP1-2A) were expressed with molecular mass of about 35kDa and 110kDa, respectively. The rVP1 was detected in both the soluble fraction and in the insoluble fraction of the lysate, whereas rP1-2A was detected only in the insoluble fraction. The soluble fraction of rVP1 was purified successfully, but the recovery was relatively low, around 0.5 mg/L of infected culture. The difficulty of obtaining rP1-2A in soluble form precluded purification. These results indicate that solubility may represent a drawback for obtaining large amounts of structural proteins of HAV. Further investigations are needed to limit the formation of aggregates and optimize extraction conditions. FINANCIAL SUPPORT: CAPES, FIOCRUZ




IgA antibodies against HPV-16 and 18 can be detected in unvaccinated individuals by using the VLP that serve as the basis for bivalent HPV vaccine. The values for ELISA assays and the values found for IgG correlate good/very good with HPV16 /18 detected by PCR. FINANCIAL SUPPORT: FAPESP

IV198 - DEVELOPMENT, CHARACTERIZATION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST BRAZILIAN DENGUE VIRUS ISOLATESZanluca, C.1,2,3; Mazzarotto, G.A.C.A.1,2; Bordignon, J.1,2; dos Santos, C.N.D.1,2

1. FCC - Fundação Carlos Chagas, Av. Prof. Francisco Morato nº 1565 Jd. Guedala, São Paulo - SP, 05513-900



Dengue is the most prevalent human arboviral disease, and dengue virus (DENV) infection symptoms are not sufficiently specific to allow clinical differentiation from other acute febrile illnesses. So an early diagnosis is crucial to reducing morbidity and mortality from dengue hemorrhagic fever and dengue shock syndrome. Although Brazil is a hotspot for dengue, no serological diagnostic test has been produced using Brazilian DENV isolates. This study aims to improve the development of immunodiagnostic methods for DENV detection through the production and characterization of monoclonal antibodies (mAbs) against Brazilian DENV isolates. B lymphocytes producing antibodies against DENV were obtained from spleens of Balb/c mice immunized with DENV-1 BR-01/MR, DENV-2 BR/01-01 or DENV-3 BR 290-02. Spleen cells were fused with P3X63Ag8.653 myeloma cells by PEG (MW 3000-3700), and hybridomes secreting anti-DENV antibodies were screened by indirect immunofluorescence on DENV-infected C6/36 cells. Two freeze-thaw cycles and limiting dilutions were performed and 22 stables clones were obtained. mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. To investigate whether the mAbs could be used for diagnostic and epidemiological purposes, mAbs were assessed for specificity to the

four DENV serotypes and to other flaviviruses. Different reactivity patterns were identified: flavivirus cross-reactive, dengue-group specific, dengue subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus, West Nile virus and Saint Louis encephalitis virus. None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus. Furthermore, mAb D3 424/8G was successfully conjugated to horseradish peroxidase and used to develop a capture enzyme-linked immunosorbent assay for anti-DENV IgM detection in sera from patients with acute dengue. The results were consistent with results from the commercially available PanBio IgM capture assay kit. To our knowledge, these are the first mAbs against DENV isolates circulating in Brazil to be developed and characterized. These mAbs may be of special interest in the development of diagnostic assays, as well as for basic research, and have the potential to increase the specificity of dengue diagnosis in Brazil. Financial support: Fiocruz, CNPq, CAPES, Fundação Araucária

IV201 - PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES FOR DENGUE VIRUS-4Andrade, A. de S.1; Sardi, S.I.1; Costa, L.F.M.1; Sampaio, M.L. da S.1; Brandão, C.2; Campos, G.S.1

1. UFBA - Universidade Federal da Bahia, Rua Augusto Viana , s/n, Palácio da Reitoria, Canela , Salvador - BA, 40110-909

2. Hospital Aliança, Avenida Juracy Magalhães Junior, 2096 - Rio Vermelho, Salvador - BA, 41920-900

Dengue virus (DV) is an arbovirus belonging to family Flaviviridae, genus Flavivirus, with four serotypes named DENV-1, DENV-2, DENV-3 and DENV-4. In Brazil, the infection by DENV-4 reemerged in 2010 after 30 years, exposing the population at high risk for developing severe diseases because of the co-circulation of the four serotypes. The monoclonal antibodies (MAbs) are an important tools due to its potential application to viral diagnostics. The aim of this work was to produce and to characterize of MAbs against DENV-4. The methodology to produce MAbs was developed by Köhler and Milstein (1975). Briefly, experimental animals were hyperimmunized with DENV-4 viral antigen obtained to virus multiplication in C6/36 culture cells




and subsequent concentration and precipitation with polyethylene glycol 8000. ELISA test was performed to screen hybridoma supernatants for reactivity to DENV-4, and the characterization of MAbs was obtained by Indirect Immunofluorescence (IFA) and Western Blot techniques. A total of ten hybridomas were obtained producing MAbs named E4, C11, F12, H12, A12, A2, B4, C12, D2 and F10. The characterization of MAbs by IFA showed a high positive reaction to DENV-4 in infected cells, especially F10, and weak reaction for C11, C12 and F12. The characterization of MAb F10 was then performed by Western Blot showing recognition to viral protein of approximately 20 kD, compatible with the molecular weight of protein M (prM). In conclusion, this study shows the use of MAbs produced against DENV-4 as a potential diagnostic tools to detect viral antigen or viral proteins. FINANCIAL SUPPORT: FAPESB-Bahia-Brazil

IV235 - GENETIC CHARACTERIZATION OF THE SEED LOTS OF INFLUENZAVIRUS FOR VACCINE PRODUCTIONBotosso, V.F.; Comone, P.; Oliveira, R. das N.; Tenório, E.C.N.; Miyaki, C.

Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São Paulo - SP, 05503-900

Since 2012, the Butantan Institute is responsible for the production of part of the vaccine used in the Brazilian annual influenza vaccination campaign. It is a trivalent split and inactivated vaccine from a mixture of Influenzavirus A, subtypes H1N1pdm and H3N2, and Influenzavirus B, which have been grown in embryonated hen´s eggs. The differences in the protective efficacy of the vaccines may be due to the continuous antigenic variation in the epidemic strains. Because of this, the composition of the influenza vaccine is reviewed annually by WHO that publishes the recommendations on the strains to be used in each influenza season. A prerequisite for the production and supply of an ideal influenza vaccine is the selection and development of optimal candidate vaccine viruses, adapted to grow in eggs or cells, that gives high yields of the surface antigens (HA and NA). In order to guarantee the characteristics of the strains included in the vaccine production, each Working Seed Lot used was analyzed by nucleotide sequence of Haemagglutinin (HA) and Neuraminidase (NA) genes using the Sanger

Methodology and specific primers for each strain. These analyses showed a significant change in the Haemagglutinin gene of A/Victoria/361/2011 (H3N2)-like virus that was recommended to be used in the 2013 season. The deduced amino acid sequence of the HA protein has shown four mutations as compared with the wild virus sequence: R172Q, E206D, S235Y e K262N, starting from methionine. Two of these mutations (sites 172 and 235) were described in egg-adapted prototype but not in the cell-propagated prototype. In addition, it has been shown that the mutation in the site 172 was responsible for alteration in the antigenicity of the strain, leading a less effective protection against the circulating viruses. Although the circulating virus didn´t change in the forthcoming season it was necessary change the egg adapted H3N2 strain to another that was antigenically like the circulating virus and the cell-propagated prototype virus. This change may cause a delay in the production of the vaccine that could be critical for the manufactures because of the schedule production to provide the vaccines for the first day of the campaign cannot be changed. Sequencing shows an important tool in monitoring changes in circulating strains and also assisting in the production of an effective flu vaccine.FINANCIAL SUPPORT – FUNDAÇÃO BUTANTAN

IV437 - COMPARISON OF Q AND DEAE MEMBRANE AND MONOLITH SUPPORTS IN DNA REMOVAL FROM HEPATITIS B VACCINEGouvea, M.N.; Sciani, J.M.; Souza, C.; Rocha,B.A.; Santos, J.R.; Oliveira, D.C.A.; Botosso, V.F.; Tenório, E.C.N.

Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São Paulo - SP, 05503-900

Butantan Institute’s hepatitis B vaccine (VRHB) is composed by the virus recombinant surface S-antigen (HBsAg), expressed by the transformed yeast Hansenula polymorpha. The harvesting of cells is followed by washing and disruption of the cells to release the HBsAg, which is purified by several physico-chemical steps to eliminate host cell-derived proteins. One impurity targeted for clearance during downstream process is residual host DNA. The regulatory guidance for our product specifies that DNA content in the final product should be less than 100 pg/dose. DNA is mainly released in cell disruption step and removed along the following




purification steps, including adsorption of negative molecules in DEAE based resin, which is separated from the semi-purified product by rough filtration. This in batch procedure shall be replaced by a convective media chromatography (flowthrough mode), in compliance with regulatory issues. Sartorius and BIA Separations chromatographic supports (Sartobind Q and Sartobind D membrane filters; DEAE CIMmultusTM monolithic columns) were compared to the DEAE resin (DE52, Whatman) used in the vaccine production process. The starting material was the supernatant from a precipitation intermediate purification step (PS, >80% purity). The goal of this study was to select the media with the best ratio between DNA removal/HBsAg adsorption (a drawback due to its low pI). To investigate the influence of salt concentration in the binding step, PS (2CV) were applied to Sartobind filters or monolithic columns, in five NaCl concentrations from 0 to 600 mM, and bound molecules were eluted with 2 M NaCl. Whole flowthrough and elution peaks were analysed: HBsAg content was evaluated with passive hemagglutination tests and SDS-PAGE; DNA removal was tested with a threshold assay. PS samples were also prepared with same five NaCl concentrations and incubated with the DE52 resin, in conditions similar to the production process. Any salt concentration had no led to dramatic lower adsorption of HBsAg to the DE52 resin, while concentrations up to 300 mM NaCl resulted in gradative less retention of HBsAg and DNA in DEAE chromatographic supports (Sartorius or BIA Separations). Similar behavior was observed in Sartobind Q filtrations only for the DNA – the HBsAg protein was adsorbed with any salt concentration. With some adjusts, both platforms may be used to replace the current process. FINANCIAL SUPPORT – BUTANTAN FOUNDATION

IV503 - MONOCIONAL ANTIBODIES AGAINST ADENOVIRUS TYPE 2: PREPARATION AND PRELIMINARY CHARACTERIZATIONPaulini, I.J.1; Silva, J.S.2; Thomaz, L.2; Rocha, L.B.2; Harsi, C.M.2; Belley, N.2; Granato, C.F.H.2



Human adenovirus (HAdV) presents 52 serotypes which can cause respiratory, gastrointestinal, urogenital, and systemic infections both in children and adults. The aim of this study is to produce monoclonal antibodies (McAbs) against HAdV-2 hexon protein which will be applied in a imunochromatografic test (IC). Polyclonal rabbit antiserum used in IC tests was prepared against purified Ad2 hexons. Hybridoma cell lines were prepared by the fusion of mouse myeloma cells (Sp2/0-Ag14) with lymphocytes from female BALB/c mice immunized with Ad 2 by a standard technique . Hybridomas were screened for antibody production by the indirect ELISA . Selected hybridomas were cloned by limiting dilution. Four monoclones reacted specifically with purified Ad2 hexon and expanded in large scale. After purification, the Ig subtypes in were determined ELISA isotype assay. All the McAbs produced from the four strains were IgG1 subtypes. In this study we showed shows that McAbs was specific for adenovirus serotypes 2,3,5 and 41 by indirect ELISA . This suggested that those Mabs recognized a conformational epitope on the protein that might be conserved in all sorotypes. Mixture of the McAbs not resulted in high reaction. The rapid assay was performed and results were interpreted by the presence or absence of visually detectable colored lines . An immobilized polyclonal rabbit capture antibody on a membrane formed colored line at the specimen if the specimen contained adenovirus antigen. An internal control line C, containing antimouse antibody that captured the colored conjugate antibody,was also built into the device to serve as a procedural quality. Here, we can demonstrate that monoclonal antibodies reacted against several serotypes adenovirus stocks. If so, this rapid test may provide an advantage over cell culture in samples containing noncultivatible adenoviruses but further studies evaluating this test still are necessary.




IV525 - STABILITY EVALUATION OF DENGUE VIRUS ANTIGEN (DENV-2) PRODUCED IN SUCKLING MOUSE BRAIN FOR APPLICATION IN ELISA IGM TESTPeña, B.P.1; Huatuco, E.M.M.1; Zapana, E.M.1; Garcia, P.2

1. UNMSM - Universidad Nacional Mayor de San Marcos, Calle Germán Amézaga N° 375 - Edificio Jorge Basadre, Ciudad Universitaria, Lima 1, Lima - Peru

2. INS - Instituto Nacional de Salud, Avenida calle 26 No. 51-20 - Zona 6 CAN. Bogotá, D.C.

Enzyme immunoassays, such as ELISA, are important and analytical methods that are widely used for various purposes. The proteins have the correct activity when its conformation is appropriate, those found in its native state, are more stable .There are several factors that cause protein denaturation within which was the temperature. Therefore, it is very important that the proteins to be stored and used in an environment which stabilizes the native structure. Currently, there is a range of stabilizers sold by different marks which ensure the maintenance of protein stability. As these products are designed to ensure the conservation of proteins in their native structure. In the present study, the stabilizing ability STABILZYME ® SELECT product applied in Dengue Virus antigen (DENV-2) produced in suckling mouse brain BALB / C CNPB for a period of 12 weeks was evaluated. These antigens were evaluated in an ELISA system according to the time capture. To assess the stability of dengue viruses (DEN-2) antigen produced in suckling mice brain during a period of 12 weeks were used ELISA Ig M. The Virus inoculation, Suckling mice of 1-2 days old BALB/C CNPB were intracerebrally inoculated with 20 ml of DENV-2 reference strain S168203 from the NIBSC. The purity of the strain was confirmed by reverse transcription polymerase chain reaction (RT-PCR) Production of DENV-2 antigen, For the production of antigen from suckling mouse brain, the methodology described by Clarke and Casals in 1958 was used. ELISA antigen titer IgM capture (INS), Serial dilutions of the antigen were performed and compared the optical densities (O.D.) obtained at each concentration was performed. The stability evaluation of the antigens produced was performed using the STABILZYME® SELECT stabilizer preserved at temperatures 2-8 ° C and compared to antigens without applying stabilizer and maintained at temperatures of 2-8 ° C and -20 ° C. All antigens were

evaluated for 12 weeks by ELISA capture IgM (INS) using a validated panel of sera. A higher titer of DENV-2 antigen produced in the brain of suckling mouse concentration 1:20 with an average OD of 1.625 for the positive control was observed. A significant difference at 12 weeks of assessment where the antigen with STABILZYME ® SELECT preserved at a temperature of 2-8 ° C maintained their initial O.D. readings unlike antigen without applying stabilizing preserved at 2-8 ° C. O.D. readings between antigens with STABILZYME ® SELECT at temperatures of 2-8 ° C and the antigen without stabilizer kept at -20 ° C showed no significant differences. Using suckling mice for antigen production of DENV is a widely used method. The antigen production technique more used is with sucrose-acetone (Clarke and Casals, 1958) (IPK, 2005). The antigen production method with suckling mouse brains is a tedious technique. We conclude that the application of STABILZYME ® SELECT product maintains the stability of DENV-2 antigen produced in suckling mouse brains 2-8 ° C during the evaluation time of 12 weeks.

PLANT AND INVERTEBRATE VIROLOGY - PIV


Plant and Invertebrate Virology: PIV 207

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Plant and Invertebrate Virology: PIV

3. LVA/UNICAMP - Laboratório de Virologia Animal/ Universidade Estadual de Campinas, Rua Monteiro Lobato - n° 255, Campinas -SP, 13083-970

4. LTP/UNICAMP - Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão Geraldo, Campinas - SP, 13083-970

This study was aimed to investigate the effect of high hydrostatic pressure on Tobacco Mosaic Virus (TMV), a virus model for immunology and one of the most studied virus to date. When subjected to treatment with pressures it was observed a significant change in the recognized epitopes in comparison to sera from immunized mice with the native form of the virus. These alterations were further studied by combining the high pressure treatment with urea or low temperatures and inoculation of these altered virions in Balb-C mice, the collected sera titers were determined by ELISA and cross referenced between the groups tested. The titter obtained showed that the antigenicity of viral particles was maintained after treatments using monoclonal antibodies against the native form. The epitope prediction algorithms could not infer the some observed changes in the epitope profile suggesting conformational changes in the protein structure. The antigenicity of canonical epitopes was maintained, although binding intensities were different among the treatments used. Patterns of recognition from the epitope mapping were then cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations might have occurred. Our findings suggest that different cleavages sites were exposed after the treatments; this was verified via epitope mapping using sera from mice immunized with the virus after the high pressure condition was imposed.

PIV151 - A BEGOMOVIRUS EXISTING AS A COMPLEX OF WELL DEFINED SUBPOPULATIONS IN A NON-CULTIVATED HOSTGodinho, M.T.; Xavier, C.A.D.; Lima, A.T.M.; Zerbini, F.M.

DFP/UFV - Departamento de Fitopatologia da Universidade Federal de Viçosa, Campus Universitário, Viçosa - MG, 36570-000

DNA plant viruses, particularly the begomoviruses, cause serious epidemics in economically important crops worldwide. In Brazil several indigenous begomoviruses have been described infecting tomatoes following the

PIV38 - EXPRESSION OF PARASPORIN PROTEINS FROM BACILLUS THURINGIENSIS IN INSECT CELLSLamar, D.C.; Correa, R.F.T.; Ribeiro, B.M.


Bacillus thuringiensis (Bt) is a pore-forming bacteria able to produce a crystalline protein inclusions during sporulation. Some strains exhibit entomopathogenic activity which makes Bt a largely-used bioinsecticide to control both agricultural insect pests and human disease vectors. Interestingly, somo Bts produce non-insecticidal crystalline inclusion-forming proteins called parasporins which are preferentially toxic to tumor cells. Bt proteins are efficiently expressed in insect cells using the baculovirus/insect cell-based expression system. Therefore, in this report, we have successfully expressed two Bt-derived parasporin genes using recombinant baculoviruses and insect cells. For this, both parasporin-2 gene and a newly discovered parasporin-3-related gene (41%) derived from a Brazilian Bt strain were inserted into a commercial vector pFastBac1™ (Invitrogen) and recombinant baculoviruses were constructed using the commercially available Bac-to-Bac system (Invitrogen). Recombinant viruses were used to infect cells and the recombinant proteins were detected by SDS-PAGE and immunobloting. Heterologous proteins will be analyzed by transmission and scanning electron microscopies to verify possible crystalline formations in the cytoplasm of insect cells. Moreover, the recombinant proteins are being purified by affinity chromatography based on a hexahistidine-tag for further toxicity tests against tumor cells.

PIV82 - HIGH HYDROSTATIC PRESSURE EFFECT ON THE EPITOPE MAPPING OF THE TOBACCO MOSAIC VIRUS COAT PROTEINLima Neto, D.F.1,2; Barnabé, A.C.3; Caserta, L.C.3; Martini, M.C.3; Rabelo, A.4; Bonafé, C.F.S.4; Arns, C.W.3

1. LNBio – Laboratório Nacional de Biociências, Rua Giuseppe Máximo Scolfaro, 10000 Pólo II de Alta Tecnologia de Campinas, Bairro Guará Campinas – SP, 13083-100

2. CNPEM - Centro Nacional de Pesquisa em Energia e Materiais, Rua Giuseppe Máximo Scolfaro, 10.000 - Polo II de Alta Tecnologia, Campinas - SP, 13083-970




introduction of a novel species of the whitefly vector in the mid-1990s. Non-cultivated plants harbor many begomoviruses, and it is believed that these hosts may act as reservoirs and as mixing vessels where recombination may occur. Begomoviruses also display nucleotide substitution rates equivalent to those of RNA viruses. In this work we sampled Sida acuta plants showing typical viral symptoms in a small area (aprox. 1 ha) in the municipality of Viçosa, MG, Brazil. Total DNA was extracted from fifty samples and the viral genome was amplified by RCA, cloned and sequenced. A total of 65 full-length genomes (33 DNA-A and 32 DNA-B components, from 26 samples) were obtained and the 89% DNA-A identity threshold established by the ICTV was used for taxonomic placements. Sequence analysis indicated that the clones correspond to a novel species for which the name Sida acuta mosaic virus (SAMV) is proposed. Additionally, the analyses indicated the coexistence of three well-defined SAMV strains, with mixed infections and pseudorecombination among them. We reconstructed the phylogenetic relationships for full-length genomic components, CP and Rep genes using Bayesian inference (BI). Well-supported clades (posterior probabilities higher than 0.99) were observed in all phylogenetic trees representing each distinct SAMV strain. Our results indicate a complex evolutionary interplay amongst begomovirus isolates even in small populations. FINANCIAL SUPPORT: FAPEMIG, CAPES AND CNPQ

PIV156 - EVALUATION OF BRAZILIAN SUGARCANE GENOTYPES RESISTANCE TO SUGARCANE MOSAIC VIRUS UNDER GREENHOUSE AND FIELD CONDITIONSGonçalves, M.C.; Silva, M.F.; Perecin, D.; Xavier, M.A.; Landell, M.G.A.; Pinto, L.R.

1. Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002


3. IAC - Instituto Agronômico, Avenida Barão de Itapura, 1.481, Botafogo Campinas - SP, 13020-902

Sugarcane mosaic virus (SCMV), the causal agent of mosaic, is one of the main viruses infecting sugarcane in Brazil, which is mainly controlled by the use of resistant cultivars. Favorable epidemiological conditions to mosaic dissemination and recent descriptions of

new isolates reinforce the current importance of the disease. In addition, there is little information about genetic parameters and heritability associated to mosaic resistance in sugarcane. In this regard, the present study aimed to evaluate the resistance of 79 sugarcane genotypes (varieties and elite clones) to a severe SCMV strain and also to estimate genetic parameters associated with mosaic resistance. Buds were collected from sugarcane stools without mosaic symptoms, maintained at the IAC Sugarcane Breeding Station, Ribeirão Preto, from which leaf samples of each stool were tested by PTA-ELISA (Plate Trapped Antibody-ELISA) in order to verify the virus sanity. Three weeks old seedlings, planted in stiff plastic tubes (volume of 50 cm3) in a two complete block design, were artificially inoculated with SCMV in aphid proof greenhouse conditions. Symptoms were evaluated by a grade scale and the confirmation of infection by the serological test PTA-ELISA were performed, being six months later validated under field conditions, adopting the same experimental design. Symptom data were submitted to variance analysis, adopting the Split plot temporal arrangement. The mean incidence of mosaic was low under greenhouse conditions, with higher values in the field assay. Based on field results, variance analysis revealed significant genotype and genotype x environmental effects. The interaction of sugarcane genotypes with days of evaluation revealed a differential behavior in mosaic symptom expression, including the recovery in some of them. The broad-sense heritability at individual level and means based were 19.37% and 62.18%, respectively, indicating that the resistance to mosaic tends to be a quantitative trait. The combination of symptom evaluation by grade scale with serological test ELISA for SCMV detection proved to be efficient for selection of sources of resistance to mosaic, detecting the virus in symptomless genotypes, and pointing out twenty two genotypes as resistant to SCMV strain in study. Financial support: FAPESP (BIOEN 2008/56146-5), IAC (Instituto Agronômico de Campinas) and CAPES.




PIV159 - SEQUENCING AND PHYLOGENETIC ANALYSIS OF PROTEINS P1 AND HC-PRO OF ISOLATES OF SUGARCANE MOSAIC VIRUSGonçalves, M.C.; Bueno, F.C.; Harakava, R.; Gonçalves, M.C.

Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002

In Brazil, sugarcane, maize and sorghum are quite affected by Sugarcane mosaic virus (SCMV), responsible for the mosaic disease in these crops. The introduction of new maize hybrids and its cultivation in off-season near sugarcane fields has resulted in further spread of virus strains in the country. The P1 and HCPro SCMV proteins play an important role in the virus infection, replication and transmission. The objective of this work was to better understand the genomic organization and variability of sugarcane and corn isolates of SCMV by means of sequencing and sequence analysis of these proteins. The infected plant material of sugarcane and maize were maintained in dehydrated and frozen at -20oC plant tissue. Isolates SCMV “PIR-2”, “RIB-1” and “M-Campinas” were transmitted by mechanical inoculation to sorghum “Rio” seedlings using sodium phosphate buffer 0.01 M, pH 7.2, 0.1 % of sodium sulphite; symptoms of infection became evident about six weeks after inoculation, and total RNA was extracted using Trizol reagent. Amplification of the viral genome, was firstly targeted by synthesizing a cDNA template with a oligo dT and reverse transcriptase (RT), followed by a regular PCR. Fragments of 1210, 1274 nt corresponding to the target proteins were amplified with primer pairs 5UTR+HCP1R and HCP1R+HCP3R respectively, designed in this work. The products were properly identified, aliquot and analyzed by electrophoresis on 1.2% agarose gel. After identification of the target fragments under UV light, respective bands were excised from the gel, purified and sequenced. Nucleotide sequences were analyzed and the consensuses were submitted to BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) algorithm for comparison to homologous sequences. The generated sequences and the obtained from GenBank were aligned using the

BioEdit program. Analyses of the sequences confirmed that the designed primers allowed the amplification of the viral genome corresponding to proteins P1 and HC-Pro. Sequences were deposited in “GenBank” providing information generated in the project to the scientific community. Multiple alignments and phylogenetic analysis by Neighbor Joining, based on P1 and HCPro sequences of different SCMV isolates, showed that Brazilian isolates studied in this work showed higher sequence identity and common origin with Australian and Argentinean isolates. Financial support: FAPESP (BIOEN 2008/56146-5). FCB was recipient of a CNPq PIBIC fellowship.

PIV190 - MOLECULAR CHARACTERIZATION OF A NOVEL SIDA-INFECTING BEGOMOVIRUS FROM SOUTHERN BRAZILFerro, C.G.1; Silva, J.P.1; Pinto, V.B.1; Mar, T.B.1; Godinho, M.T.1; Lima, A.T.M.1; Lau, D.2; Zerbini, F.M.1


2. EMBRAPA Centro Nacional de Pesquisa de Trigo, Rodovia BR-285, 3081, Passo Fundo - RS, 99001-970

Begomoviruses (whitefly-transmitted, single-stranded DNA plant viruses) are among the most damaging pathogens causing epidemics in economically important crops worldwide. The incidence of begomovirus infections in crops increased in Brazil during the 1990s following the introduction of Bemisia tabaci Middle East-Asia Minor 1 (MEAM1, previously Bemisia tabaci biotype B). It is believed that MEAM1 transmitted begomoviruses from non-cultivated plants to crops with greater efficiency than the indigenous B. tabaci species. Non-cultivated plants harbor many begomoviruses, and it is believed that these hosts may act as mixing vessels where recombination may occur resulting in novel species adapted to new hosts. In this study we molecularly characterized a novel begomovirus infecting Sida sp. plants showing typical symptoms of begomovirus infection, collected in the state of Rio Grande do Sul in March 2010. The viral genome was amplified by rolling-circle amplification (RCA), cloned and sequenced. The DNA-A is 2601 nucleotides (nt) long and has a genomic organization similar to those of other begomoviruses, except for the presence of an additional




open reading frame (ORF) in the complementary strand (AC5). Pairwise sequence comparisons indicated a maximum nucleotide sequence identity of 81.5% with Tomato dwarf leaf virus (ToDLV, GenBank access number JN564749). Phylogenetic analysis placed this isolate in a monophyletic cluster with other begomoviruses from the Americas. No reliable recombination events were detected by the RDP4 program. Therefore, based on the criteria established by the International Committee on Taxonomy of Viruses, this isolate represents a novel begomovirus species for which the name Sida chlorotic mottle virus (SiCMoV) is proposed. The continuing detection of new begomovirus species highlights the remarkable genetic diversity of this group of viruses, as well as the power of the RCA technique in assessing this diversity. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG

PIV215 - EXPRESSION OF REPLICATION COMPLEX DOMAINS OF TOMATO BLISTERING MOSAIC VIRUS (TOBMV) IN ESCHERICHIA COLIJunqueira, B.R.T.1; Vasconcelos, K. de O.1; Oliveira, S.2; Dantas, G.1; Nagata, T.1


2. UniCEUB - Centro Universitário de Brasília, Campus do UniCEUB - Asa Norte - Brasília - DF, 70790-075

Tomato blistering mosaic virus is a tymovirus member and possesses a single-stranded RNA genome in positive polarity. Tymovirus infection causes small vesicles along the chloroplast periphery. These vesicles in chloroplast were shown to be the replication site, forming viral replication complex (VRC). Despite knowing the replication site, little is known about the tymovirus replication mechanism. In order to elucidate the replication process, the overall aim of this project is antibody production for the major replication complex proteins as metyltransferase, helicase and RNA dependent RNA polymerase (RdRp). For this purpose, the expression of domains of these three proteins was performed using Escherichia coli system via Gateway technology (Invitrogen). The three domains were cloned in the entry plasmid pENTR2B and recombined to destination plasmid pDEST17. E. coli BL21AI was transformed with selected clones confirmed by sequencing. The protein expression of RdRp, Helicase and Metyltransferase at 2 and 4 hours post-induction

was confirmed by Western blotting using antibody against HisTag. These results confirmed that the system chosen for protein expression is effective. The improved solubility of these proteins is ongoing interest at moment. Financial support: CNPq.

PIV232 - MOLECULAR CHARACTERIZATION OF BACULOVIRUS IDENTIFIED FROM CULEX QUINQUEFASCIATUS LARVAE IN SÃO PAULO STATE, BRAZILde Carvalho, I.M.V.G.1; Benites, H.G.2; Bernardino, J. de S.T.3; Queiroz, A.T.L.4; Oliveira, I.B.R.2; Araújo Coutinho, C.J.P. da C.2


2. SUCEN - Superintendência de Controle de Endemias, R Paula Sousa, 166 - Centro, São Paulo, SP, 01027-000


4. Centro de Pesquisas Gonçalo Moniz, R. Waldemar Falcão, 121 - Candeal, Salvador - BA, 40296-710

Some mosquito species are humans and animals pathogens vectors, such malaria, encephalitis, dengue, yellow fever, and heartworm disease. A broad spectrum larvicides and adulticides have been used to combat these insects. However, the environmental harmful effect and the drug-resistance rate increasing in mosquitoes population forces new vector-control approach development. The baculovirus use could be an alternative to chemical methods. Culex quinquefasciatus baculoviruses were identified in São Paulo, Brazil. The objective was to perform parcial molecular characterization of helicase, polymerase, odv-e27, vlf-1 and lef-8 baculovirus genes from São Paulo (Brazil). The Samples DNA was amplified, the sequences were aligned, using ClustalX 2.0, with partial sequences of helicase, polymerase, odv-e27, vlf-1, and lef-8 from reference baculoviruses genes of other insect orders (Lepidoptera and Hymenoptera) deposited in GenBank database. Phylogenetic analysis was performed. Previous genes analysis results showed that the viruses found in the six larvae in this study showed 98% similarity with Culex nigripalpus NPV, the only species from Deltabaculovirus genus previously described. This specie shows dipteran




infection and high mortality, showing remarkable potential as biological control. FINANCIAL SUPPORT: 2012/23947-0

PIV299 - BOMBYX MORI COLON RESISTANCE TO ALPHABACULOVIRUSBaggio, M.P.D.1; Vessaro Silva, S.A.2; Ribeiro, L.F.C.3; Brancalhão, R.M.C.3


2. UEM - Universidade Estadual de Maringá, Avenida Colombo, 5790 - Jardim Universitário, Maringá - PR, 87020-900

3. UNIOESTE - Universidade Estadual do Oeste do Paraná, R. Universitária, 1619 - Universitário, Cascavel - PR, 85819-110

Caterpillars of Bombyx mori (Lepidoptera, Bombycidae) build a cocoon of silk, from which is extracted yarn, used in the production of various tissues. This productive activity, known as sericulture, presents a major economic, social and environmental impact in the state of Paraná. However, the same can be affected by nuclear polyhedrosis disease, caused by Bombyx mori nucleopolyhedrovirus (BmNPV), an entomopathogenic virus (Baculoviridae, Alphabaculovirus), which infects B. mori, compromising the production of cocoons and causing damage to the productive chain. Studies have demonstrated that BmNPV is poliorganotrophic and various are the target-organs; however, some have shown to be resilient to pathogens and the present study aims to analyze the behavior of the colon region, in the hindgut, of B. mori to BmNPV, providing support to the establishment of the viral infectious cycle. The colon is an important region of the hindgut that acts in the absorption of water and salts and in the formation and elimination of fecal pellets in the insect. Caterpillars of B. mori 5th instar were inoculated with a viral suspension of BmNPV, and 2̊ to 9̊ day post inoculation (dpi) colon segments were processed for light microscopy. The sections obtained were stained by the cytochemical technique Azan, for viral identification. The analysis revealed that colon cells were not susceptible to BmNPV, at all times examined. However, viral occlusion bodies were observed in adipose, tracheal tissue and into the extracellular medium, indicating the viability of the inoculum used. Thus, despite the resistance to BmNPV,

infection of other targets potentially compromises the metabolic function of the hindgut, on absorption of water and minerals and in the formation and elimination of feces.

PIV300 - CYTOPATHOLOGY OF THE TRACHEAL SYSTEM OF BOMBYX MORI(LEPIDOPTERA:BOMBYCIDAE)CAUSED BY ALPHABACULOVIRUSBaggio, M.P.D.1; Senem, J.V.2; Ribeiro, L.F.C.2; Torquato, E.B.2; Brancalhão, R.M.C.2



Bombyx mori is an insect of the order Lepidoptera that is only found in germplasm banks; it is used in scientific research and for commercial purposes. In this case, the silk cocoon, which is produced at the end of the 5th larval instar, is used in the production of various yarns and fabrics, constituting the sericicola industry. One factor that affects the national sericulture, compromising the commercial production of cocoon, is a nuclear polyhedrosis disease caused by the virus from the Baculoviridae family, Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), genus Alphabaculovirus (AlphaBV. Studies have proved that BmMNPV is polyorganotropic and there are several target organs, such as the tracheal system; however, details of its cytopathology aren´t known. The tracheal system is responsible for the aeration of the tissues of the insect. The study described the cytopathology of the tracheas of hybrid larvae of B. mori, infected experimentally with BmMNPV, isolated geographically in the state of Paraná. Fifth instar hybrid larvae were divided into two groups; one control, and the other inoculated. After ingestion, and on different days post-inoculation (dpi), from the 2nd to the 9th dpi, the larvae were anesthetized and dissected. Segments of organs containing branches of the trachea, were collected and fixed in Karnovsky modified for transmission electron microscopy. On the 2 st dpi, fresh hemolymph analysis was conducted to determine the susceptibility of the hemocytes. The results revealed that the hemocytes were infected from the 2 nd dpi and the epithelial cells of the trachea were infected from the 4th dpi. The cytopathology of the tracheal cells showed




hypertrophic nucleus, containing the viroplasm, the site of the synthesis of the nucleocapsids. Subsequently, the formation and development of the polyhedra occured, accentuating the nuclear hypertrophy and culminating in cell lysis. Virions were also observed, immersed in the basal lamina of the trachea, which appeared to be disorganized. Thus, the cytopathology of the trachea was consistent with the infection caused by AlphaBV, and the data that was obtained provides a better understanding of the infectious cycle of BmMNPV in the body of the insect. The time of infection, later for the hemocytes, and the presence of virions in the basal lamina of the trachea, indicated that this system is a secondary target for infection, and also that the hemolymph is an important dispersant of viral infection.

PIV338 - GENOME ANALYSIS OF AN ATYPICAL ISOLATE OF LETTUCE MOSAIC VIRUS (LMV)Duarte, P. de S.G.; Lucas, M.A.; Figueira, A. dos R.; Costa, S.B.F.G.

UFLA - Universidade Federal de Lavras, Câmpus Universitário, Lavras - MG, 37200-000

Virus diseases are considered a key challenge for the production of lettuce (Lactuca sativa L.) in Brazil, where the losses in infected plants can reach 100%, depending on weather conditions and cultivars. The Lettuce mosaic virus (LMV) is currently considered the most important virus infecting lettuce in Brazil, and the symptoms observed in infected plants are mosaic, leaf distortion and even death of the more susceptible cultivars. In this study, an isolate of LMV, named LMV-Cf, was partially sequenced and analyzed in order to investigate the genomic variations which could potentially be linked to the induction of atypical symptoms in infected lettuce plants cv. Regina 579, characterized by closure of the head. For genome amplification by RT-PCR, the primers were designed based on the nucleotide sequences of LMV isolates, available in the GenBank, and the amplified genome fragments were purified and sent for sequencing. The 5’UTR, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa VPg, CP and 3’UTR regions were completely sequenced and analyzed. Among those sequences, two regions showed significant changes: the first one was found in the LMV-Cf P3 protein, which showed a deletion of three nucleotides, resulting in the exclusion of a glutamic acid in the C-terminal of the protein. The comparison of the

LMV-Cf P3 nucleotide sequence with the similar genomic region of other LMV isolates, available in the GenBank, ranged from 93% to 97%, which was very similar to those observed among the LMV isolates used for comparison. However, there was a deletion of one amino acid in the LMV-CF sequence, positioning it in a separate branch of the phylogenetic tree. Another difference was found in the nucleotide sequence of 6K2 protein coding-gene, which usually is highly conserved among the LMV isolates from the database, showing 100% identity. The 6K2 nucleotide sequence of LMV-Cf showed only 92% identity when compared with those LMV isolates. Since there are several evidences of the involvement of P3 and 6K2 proteins in the symptoms expression by host plants, they are considered candidates to be investigated regarding their probable correlation with the atypical symptoms of closed head showed by the infected plant. Site-directed mutagenesis and protein expression in host plants are required to get this information. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG

PIV342 - CIRCULAR SSDNA SATELLITES ASSOCIATED WITH BEGOMOVIRUSES IN THE AMERICASCastillo, J.N.1; Olive, E.F.1; Mar, T.B.2; Ferro, C.G.2; Silva, J.P.2; Lau, D.3; Moriones, E.1; Zubiaur, Y.M.4; Zerbini, F.M.2

1. IHSM - Instituto de Hortofruticultura Subtropical y Mediterránea, 29750 Algarrobo-Costa, Málaga - ESPAÑA

2. DFP/BIOAGRO/UFV - Departamento de Fitopatologia e Instituto de Biotecnologia Aplicada à Agropecuária da Universidade Federal de Viçosa, Campus Universitário, Viçosa - MG, 36570-000

3. EMBRAPA/Centro Nacional de Pesquisa de Trigo, Rodovia BR-285, 3081, Passo Fundo - RS, 99001-970

4. CENSA - Centro Nacional de Sanidad Agropecuaria, San José de Las Lajas, Apdo Postal 10, Mayabeque, Cuba

Begomoviruses (genus Begomovirus, family Geminiviridae) are plant ssDNA viruses that are transmitted by the whitefly Bemisia tabaci. Begomoviruses cause serious diseases in economically important crops in tropical and subtropical regions. Two types of circular ssDNA satellites half the size of the helper virus components have been described: betasatellites and alphasatellites. Betasatellites are associated with monopartite begomoviruses from the




Old World and are dependent on them for replication, movement in plants and transmission. Betasatellites consist of an A-rich region, a non-coding satellite conserved region and a single ORF coding for a multifunctional protein. Alphasatellites, also typically associated with Old World begomoviruses, contain a single ORF coding for a replication-associated protein with similarity to those of nanoviruses and an A-rich region. Unlike typical satellites, alphasatellites are capable of self-replication in host plants but require a begomovirus for movement within the plant and for insect transmission. In screening symptomatic non-cultivated plants collected in Brazil and Cuba for the presence of begomoviruses by rolling circle amplification (RCA), we identified two alphasatellites and a novel class of ssDNA satellites half the size of betasatellites and alphasatellites. The alphasatellites were amplified from Euphorbia heterophylla and Sida sp. plants sampled in Chapada (state of Rio Grande do Sul, Brazil) infected by the bipartite begomovirus Euphorbia yellow mosaic virus and were found to be phylogenetically related to alphasatellites recently reported from the state of Mato Grosso do Sul (Brazil), Cuba and Venezuela. The small ssDNA satellites were found associated with bipartite begomoviruses infecting malvaceous species in Cuba. They do not possess any ORFs, contain an A-rich region, and share a short conserved region with betasatellites. Our results extend the diversity and geographical range of ssDNA satellites associated to bipartite begomoviruses in the Americas, suggesting that they may be widespread in the continent. Financial support: CAPES, CNPq (fellowship Pesquisador Visitante Especial to JNC) and FAPEMIG (fellowship Pesquisador Visitante to EFO).

PIV344 - FAUNA STUDY AND VIRUS ISOLATION ATTEMPTS IN CULICIDAE CAUGHT IN THE MOCAMBO AREA, EMBRAPA, BELEM, PARÁ, BRAZILNeto, J.P.N.1; Castro, K. da S.1; Monteiro, H.A. de O.1; Castro, F.C.1; Saraiva, H.A.C.1; Segura, M. de N.O.1; Júnior, J.W.R.1; Nascimento, B.L.S.1; Carvalho, V.L.1; Tesh, R.B.2; Vasconcelos, P.F. da C.1


2. UTMB - Department of Pathology, The University of Texas Medical Branch at Galveston,301 University Boulevard, Galveston, Texas, 77555-0144

The Culicidae family insects are flies belonging to the Insecta class, Diptera order, suborder Nematocera and Culicomorpha infraorder, also known as mosquitoes. These insects transmit disease agents in humans, some of which can cause high mortality and morbidity of endemic or epidemic form.Material and Methods: Four excursions were conducted in the Mocambo forest, EMBRAPA, Belém city, Pará state, Brazil (August and October 2013, January and March 2014), lasting five days each. The arthropods collection was conducted by protected and enlightened human attraction in soil and canopy modalities. Arthropods were identified and separated in inoculation groups and stored at -70 ° C freezer. The groups were inoculated in newborns albino Swiss mice, and alternatively in VERO and C6/36 cells. Results: In the excursions it was collected 2.772 culicidae, divided into 185 groups for virus isolation attempted. In total 12 genera with 36 species of Mosquitoes were identified. Among the identified species were considered,as follows. Constants during the excursion time: Aedes hortator and Aedes serratus, among others; some of the Accessory: Johnbelkinia longipes, Limatus durhamii; Accidental: Aedes fulvus, Aedes taeniorrhynchus, Aedes oligopistus, among others. The species caught, Coquillettidea venezuelensis were classified as Eudominant; as Dominant: Coquillettidea arribalzagae, as Subdominant had Aedes serratus; Eventually: Mansonia titillans species; Finally had some Rare species like Sabethes belisarioi, Coquillettidea albicosta and Aedes fulvus. Groups of inoculated insects into newborn mice tested shows negative. However there was viral isolation of a new arbovirus of the Bunyaviridae family, Orthobunyavirus genus, a group of Aedes fulvus (strain AR800584) inoculated into C6/36 cells. Conclusion: Important arboviruses vectors were collected as Sabethes belisarioi, Coquillettidea venezuelensis, Haemagogus janthinomys, Sabethes glaucodaemon, Sabethes cloropterus, among others. A new arbovírus was isolated belonging to the Bunyaviridae family, Orthobunyavirus genus. The study demonstrates the need to conduct further research into the Mocambo forest, emphasizing the arboviruses main vector species, entomological monitoring and performing viral isolation attempts. Financial support: PIBIC/CNPQ/IEC




PIV360 - NEXT-GENERATION SEQUENCING APPLIED FOR IDENTIFICATION OF VIRUSES IN WATERMELON (CITRULLUS LANATUS) PLANTSQuirino, M.S.1; Ribeiro, B.M.1; Aguiar, R.W. de S.2; Orílio, A.F.1; Melo, F.L.1


2. UFT - Universidade Federal do Tocantins, Avenida NS 15, 109 Norte - Plano Diretor Norte - Palmas - TO, 77001-090

The culture of watermelon (Citrullus lanatus Thunb) belongs to the Cucurbitaceae family and it is susceptible to several viral pathogens. These viral infections do not cause obvious symptoms in its host plants and, frequently, the viral particles occurs in low concentrations. However, the capacity to analyze asymptomatic viral infections or coinfections increased with the appearance of high throughput sequencing. Thus, this study was designed to identify viruses present in symptomatic watermelon plants. The watermelon leaves with viral symptoms were collected from September to December 2013 in three producing regions of Tocantins state. The plant material was subjected to semi-purification of the viruses using centrifugation through a sucrose cushion. Total RNA was extracted from this viral enriched fraction using “RNeasy Plant Mini Kit” (Qiagen). Next, the RNAseq library was prepared and sequenced by Illumina HiSeq 2000 platform. Forty million reads were generated, and after quality trimming and de novo assembly using CLC Genomics Workbench software, nearly fifty thousand contigs were obtained. All contigs were submitted to blastx against the viral RefSeq database and 4,912 contigs produced hits with viral sequences. To further confirm these results, we submitted these selected contigs to blastx against the GenBank non-reduntant database (nr database), and only the plant viruses were selected. We were able to identify viral genomes belonging to the families Potyviridae, Partitiviridae and Bunyaviridae. The largest contig (10,371 bp) showed 91% identity with Papaya ringspot virus. One contig of 1,704 bp contig presented 98% identity with Zucchini yellow mosaic virus. We also found two contigs of 1,575 and 1,606 bp related to the segments 1 and 2 of Partitiviridae. Interestingly, these contigs presented an overall identity of 60%, suggesting these contigs represent a new viral species belonging to the Partitiviridae family. Moreover,

we found highly divergent contigs (6,596 and 2,674 bp) related to Bunyaviridae L and S segment, respectively. Overall, we were able to identify viruses already described infecting watermelon as well as novel viral species that are currently been characterized in our laboratory.

PIV366 - DETECTION OF POTATO VIRUS Y (PVY) STRAINS IN PLANTS WITH SINGLE AND MIXED INFECTIONCosta, S.B.F.G.; Santos, B.A.; Figueira, A. dos R.; Duarte, P. de S.G.

UFLA - Universidade Federal de Lavras, Câmpus Universitário, Lavras - MG, 37200-000

Potato virus Y (PVY) is currently one of the most important viruses in potato worldwide, mainly due to its high field dissemination capacity and the constant appearance of new genetic variants. The detection and field management of PVY has been increasingly complex. Among the mechanisms responsible for the genomic variability, the recombination is the main one, probably due to the occurrence of mixed infections in the field. In this study, the DAS and TAS-ELISA and RT-PCR multiplex technique were compared, aiming at determining its efficiency for diagnosing and discriminating the main strains present in Brazil (PVYO, PVYN-Wi and PVYNTN), in single and mixed infections. In order to get plants with single and mixed infections, Nicotiana tabacum cv. Turkish plants were inoculated with these strains, individually and in combination. In addition, probes were developed for detection and quantification of these strains by real time PCR (qPCR). Willing to detect mixed infections in the field, ninety potato tubers PVY infected were collected and analyzed by DAS and TAS-Elisa and RT-PCR multiplex. The N. tabacum plants inoculated with the different strains reacted with the expected symptoms, ranging from light mosaic to necrosis, according to the strain. However, in all combinations in which the PVYO was inoculated with the necrotic strains, the plants did not present the expected vein necrosis, indicating a predominance of the symptoms induced by the common strain. The serological tests revealed a tendency of higher concentration of strains with O serology (PVYO and PVYN-Wi) when N. tabacum presented mixed infections. The RT-PCR was capable of discriminating the tree strains, presenting the patterns




of bands specific to each of them. Among the 90 tubers tested 5 were negative, 11 were positive for the PVYNTN, and 74 were positive for the common and/or Wilga strain, with 8 being positive for mixed infections. The occurrence of mixed infections and highly favorable climate for the multiplication and dissemination of the PVY could explain the high genomic variability of this virus derived from recombination among virus isolates. The probes and primers designed were effective in discriminating each strain by qPCR, either in single or mixed infections. As qPCR presents a higher sensitivity, the designed probes revealed a good potential to be employed for the detection and quantification of these strains in infected potato plants.FINANCIAL SUPPORT: Capes, CNPq, Fapemig

PIV377 - DEVELOPMENT OF MOLECULAR TOOLS TO STUDY TOSPOVIRUS REVERSE GENETICSLeite, F. de S.; Lopes, A.P. de M.; Bertran, A.G.M.; Resende, R. de O.; Orílio, A.F.


The use of methods based on recombinant DNA technology such as reverse genetics (RG) to study the role of viral gene products in inducing disease has increased substantially our knowledge of the complex steps and interactions involved in the infectious cycle of plant viruses. Tospovirus [(-) ssRNA], are worldwide spread pathogens mainly of vegetable, causing severe economical losses, to which a RG system has not been developed yet. Currently, our research line focuses on the development of both a mini-genome approach and a classical full-length infectious clone strategy towards tospovirus RG. This work presents the development of highly specific and sensitive ribo-probes generated to support our RG research. First, primers for RT-PCR amplification were synthesized for specific RNA regions of the L, M (NSm gene) and S (NSs and N genes) segments of tomato spotted wilt virus (TSWV). Subsequently, total RNA was obtained from Datura stramonium plants infected with TSWV (BR-01 isolate) using TRIzol reagent (Invitrogen) followed by RT reaction with SuperScriptIII (Invitrogen) and standard PCR. The amplicons were cloned in the vector pGEM-T easy (Promega) and positive colonies were selected by standard restriction enzyme digestion of extracted

plasmid DNA and automated Sanger sequencing. Ribo-probes were labeled with digoxigenin (Dig RNA labeling mix, Roche) by in vitro transcription with T7 and SP6 RNA polymerases according to manufacturer’s specifications. The detection sensitivity of the probes was measured by dot-blot analysis of serial dilutions of total RNA extracted from leaf tissue of Nicotiana benthamiana and D. stramonium infected with TSWV and GRSV (Groundnut ringspot virus) and healthy plants. Probes showed high specificity to TSWV and in general limit of detection was 20 ng of total RNA. Therefore, molecular ribo-probes were produced for each segment and strand orientation of TSWV which will support to distinguish between introduced RNA transcription and that derived from viral replication complex of RG system. This work is essential in our RG research strategies and can provide important data regarding strand and segment specific accumulation and replication of the viral genome. This information is necessary to place in time the many events that are part of the infectious process, such as cell-to-cell infection, systemic infection, accumulation and activity of defective-interfering RNAs and their effects over the other viral genomic segments.

PIV385 - MOLECULAR ANALYSIS OF SQUASH MOSAIC VIRUS (SQMV) ISOLATES FROM TOCANTINS - BRAZILEdreira, N.1; Figueira, A.1; Nascimento, I.2; Balani, D.; Duarte, P.S.G.1


2. UFT - Universidade Federal do Tocantins, Avenida NS 15, 109 Norte - Plano Diretor Norte - Palmas - TO, 77001-090

Several viruses can affect cucurbits causing considerable losses in quality and quantity of production. High incidences of the mosaic caused by Squash mosaic virus (SqMV) have been detected in the field of some Brazilian regions, and those incidences depend on numerous factors such as temperature, field location and vectors. In this study the coat protein gene of fourteen Brazilian isolates of Squash mosaic virus (SqMV) collected in Tocantins state, named PTY1, PTY2, PTY4, PTY5, PTY10, PTY12, PTY14, PTY15, FA, GR2, LC2, LC3, PN1 and PN2, were amplified by RT-PCR, analyzed and compared with other isolates available in the GenBank. The identity between the nucleotides of Brazilian SqMV isolates




ranged from 86% to 100%, while the identity of these isolates with SqMV isolates from the GenBank varied between 86% and 88%. The lowest identity (87%) was observed among the isolates AB054689 from Japan and PTY1 PTY10, PTY12, PTY14, PTY15, FA1, GR2 and LC2, and also among the PN2 and the SqMV isolates from the database. The amino acid identities of the Brazilian isolates ranged from 95%, among the isolate PN2 and all the other isolates, to 100% among PTY1 and PTY10, PTY12, PTY14, PTY15, FA1, GR2 and LC2 isolates; PTY2 and PTY5; LC3 and PN1. When compared with isolates from the GenBank, identities ranged from 91% (between isolated PN2 and DQ868881.1 and EU421060.1, both Chinese isolates, and the American isolate AF059533.1) to 97% (among several isolates). The phylogenetic tree based on the nucleotide sequence showed two major clusters, one containing the Brazilian isolates PTY2, LC3, PTY5, PN1, PN2 and PTY4 and the SqMV isolates from the database, and the second group composed by the remaining Brazilian isolates. However it changed when the phylogenetic tree was constructed based on the sequence of amino acids. Several Brazilian isolates showing nucleotide substitutions resulted in amino acid changes, characterizing the nom synonymous substitutions type and confirming the large variability existing among Brazilian SqMV isolates. To confirm this variability, genomic studies of SqMV isolates should also include their RNA2 fragment, in order to provide the necessary support for breeding programs which aim to develop SqMV resistant cucurbits plants. FINANCIAL SUPPORT: CAPES, CNPQ, FAPEMIG

PIV398 - NOVEL VIRAL GENUS AND VIRAL SPECIES INFECTING FORAGE PLANTS IN BRASILSilva, K.N.1; Nicolini, C.1; Melo F.L.1; Silva, M.S.2; Fernandes, C.D.3; Nagata, T.1; Resende, R.1


2. EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901

3. EMBRAPA GADO DE CORTE, Parque Estação Biológica - PqEB s/nº. Brasília - DF, 70770-901

Panicum sp., Brachiaria sp and Stylosanthes sp. showing mosaic symptoms on leaves and growth reduction were collected in the State of Mato Grosso do Sul in the

experimental field of Embrapa Beef Cattle. To obtain a viral enriched fraction, the leaves were ground in PBS buffer, filtred and centrifuged through a sucrose cushion. Viral RNA was extracted using RNeasy Mini Kit following the manufacturer’s instructions. The RNA samples were pooled and sequenced at Macrogen INc. (Korea) using Illumina HiSeq 2000 technology. We obtained approximately 20,299,626 of reads, which were trimmed and assembled de novo using CLC Genomics Workbench 7.0. The assembled contigs (3,254) were submitted to blastx against the RefSeq Viral database and the contigs related to plant viruses were selected. We were able to identify complete genomes of viruses from some plant virus families: Potyviridae, Secoviridae and Tymoviridae. Among Potyviridae, we identified contigs related to Johnsongrass mosaic virus and two highly divergent genomes related to Rose yellow mosaic and Blackberry virus Y, which probably constitute two novel genera within Potiviridae family. Moreover, we found genomes related to Maize chlorotic dwarf virus (Seconviridae) and to a novel species/genus in the family Tymoviridade. We design specific primers to detect each virus in the surveyed hosts Panicum sp., Brachiaria sp and Stylosanthes sp. Johnsongrass mosaic virus, Rose yellow mosaic virus, Blackberry virus Y and Maize chlorotic dwarf virus were already amplified back by RT-PCR Host range and phylogenetic analysis are being conducted for further characterization of these viruses. FINANCIAL SUPPORT: CNPQ/REDE PRO-CENTRO-OESTE, FAP-DF, CAPES

PIV406 - PARTIAL CHARACTERIZATION OF BEGOMOVIRUS STRAIN FROM CLITORIA FAIRCHILDIANA ON RIO DE JANEIRO STATE, BRAZILBrioso, P.S.T.

UFRRJ - Universidade Federal Rural do Rio de Janeiro, Rodovia BR 465 - Km 7 - Campus Universitário, Seropédica - RJ, 23851-970

The Sombreiro (Clitoria fairchildiana) is a plant used in urban landscaping and for medicinal purposes (by extracting rotenoids from seeds that has anti-inflammatory activity). The objective of this study was to identify the viral genus associated with mosaic in leaves of C. fairchildiana, existing in Rio de Janeiro. Mechanical inoculation for Chenopodium amaranticolor were performed in sodium phosphate buffer pH 7.5




containing 0.1% sodium sulfite at dilutions 1:5, 1:10 and 1:20; transmission by grafting cuttings of diseased to healthy plant; transmission by seed originating from diseased plant; PCR test with primers for Begomovirus. Negative results were obtained in the mechanical inoculation and transmission by seed, however, positive results have occurred in transmission by grafting and in the PCR test. This is the first occurrence in the world of Begomovirus on this plant species.

PIV435 - IDENTIFICATION OF THE P26 GENE AND ITS EVOLUTION IN THE BACULOVIRIDAE FAMILYCraveiro, S.R.1; Inglis, P.W.2; Grynberg, P.2; Togawa, R.C.2; Ribeiro, Z.M.A.2; Castro, M.E.B.2



The family Baculoviridae comprises a group of arthropods-specific viruses with circular double-stranded DNA. Baculoviruses exhibit rod-shaped nucleocapsids embedded in a crystalline protein matrix composed of polyhedrin in nucleopolyhedroviruses (NPVs) and granulin in granuloviruses (GVs). The Baculoviridae family is composed of genera Alphabaculovirus (lepidopteran-specific NPVs), Betabaculovirus (lepidopteran-specific GVs), Gammabaculovirus (hymenopteran-specific NPVs) and Deltabaculovirus (dipteran-specific NPVs). Alphabaculoviruses are also divided into Groups I and II based on their envelope fusion proteins GP64 and F, respectively. The p26 gene is present in all Group I and II NPVs. However, the majority of the virus with more than one p26 copy belongs to Group II NPVs. The function of the p26 gene is not well-defined, but p26 is thought to be required for optimal virion occlusion in the polyhedron. The p26 gene was identified within NPVs genomes so far sequenced and was used bioinformatics tools in order to characterize the P26 protein and elucidate the evolution this gene in Baculoviridae family. The p26 copies found in baculoviruses are conserved in position, adjacent to the p10 gene (P1) in all the NPVs containing a single copy, adjacent to iap-2 gene (P2) in all Group II NPVs containing the second p26 copy and adjacent to the ptp1 and ptp2 genes (P3) in Group I NPVs with the second p26 copy. The isoelectric point and molecular

weight of the deduced P26 protein was calculated and is presented in the context of their genomic positioning. The Bayesian phylogenetic tree obtained from the p26 copies found in NPVs genomes showed four clearly defined clades (IA, IB, IIA and IIB) supporting the hypothesis for the occurrence of three independent capture events of the p26 gene by baculoviruses. The presence and location of signal peptide cleavage sites in P26 amino acid sequences was analysed and only in the clade IB was found signal peptide. Although the function of P26 is not well understood, the signal peptide may lead to differences in activity of the clade IB proteins indicating a possible distinct function from other classes of P26. However, further investigations are needed for a better understanding of this protein in baculoviruses. FINANCIAL SUPPORT: Capes/Embrapa

PIV446 - GENOME SEQUENCING OF TWO POTEXVIRUS INFECTING PRICKLY PEAR (OPUNTIA COCHENILLIFERA)Lamas, N.S.2; Sanches, M.M.2; Freitas, D.M.T.A.2; Reis, M.B.A.2; Sousa, J.G.A.2; Romano, E.2; Melo, F.L.1; Ribeiro, S.G.2



Viruses from the genus Potexvirus are widely spread in different genera of the family Cactaceae. Prickly Pear (Opuntia cochenillifera) plants collected in Pernambuco State were propagated and cultivated in the green house. RNA from symptomless plants was extracted from 45 plants and used for whole transcriptome shotgun sequencing, using an Illumina Hi-seq 2000 platform, aiming to study water stress related genes. De novo assembly of the reads was made with VELVET program and several contigs were found to be potexvirus-derived. To further characterize these virus sequences, we used Geneious software to perform Blastn searches. Comparisons of the nucleotide (nt) and the predicted amino acids (aa) sequences with other potexvirus members were made with SDT and CLUSTALW programs. Two distinct potexviruses genomes were identified. One genome comprises 6.636 bp and was identified as Schlumbergera virus X (SchVX), with a nt identity of




94% for the entire genome sequence. The identities for the nt sequences of the polymerase and CP genes are 93% and 94%, respectively. The predicted aa sequences for the polymerase and CP genes share 97% identity with SchVX. The other assembled genome (OcPotex) is 6.664 bp. The nt sequence of the entire genome has highest identities of 73% to 74% with different strains of Cactus virus X (CVX) and Zygocactus virus X (ZyVX). The nt and aa sequences of OcPotex polymerase and CP genes also share similar identities with those of CVX and ZyVX, requiring additional studies for a conclusive taxonomic assignment of OcPotex. FINANCIAL SUPPORT: EMBRAPA, CNPq, FAP-DF

PIV465 - INVOLVEMENT OF TMV RESPONSE RELATED-PROTEIN IN THE DEFENSE OF TOMATO TO TOMATO CHLOROTIC MOTTLE VIRUSVilleth, G.R.C.1; Lacerda, A.L.1; Lacorte, C.1; Brasileiro, A.C.M.1; Boiteux, L.S.2; Ribeiro, S.G.1


2. EMBRAPA HORTALIÇAS, Parque Estação Biológica - PqEB s/nº.Brasília - DF, 70770-901

Begomoviruses currently represent one of the most serious problems for the tomato cultivation worldwide. In Brazil, breeding for resistance to bipartite tomato begomoviruses allowed to the development of the resistant line ‘LAM-157’ (carrying tcm-1 locus) which is a near-isogenic line to the susceptible Santa Clara cultivar. To study the interaction and the differential genotype response to Tomato chlorotic mottle virus (ToCMoV), a transcriptomic analysis using RNA sequencing (RNAseq) was carried out. Several genes showed differential expression in resistant ‘LAM-157’ plants inoculated with ToCMoV. One of these genes, the TMV response-related protein (TMV-RRP) gene, putatively involved in plant response to Tobacco mosaic virus infection, showed significant up-regulation (log2 fold change > 2.5). RNAseq results were confirmed by qPCR over a time course of virus infection in ‘LAM-157’, highlighting the possible involvement of TMV-RRP in the process of tomato defense/resistance to ToCMoV. To validate this hypothesis in planta, LAM-157- derived TMV-RRP gene was cloned and transferred to Nicotiana benthamiana plants by Agrobacterium transformation. Transgenic

plants will be inoculated with ToCMoV to check for the influence of TMV-RRP super-expression in the disease phenotype and virus accumulation. FINANCIAL SUPPORT: EMBRAPA, CNPQ, INCTIPP, FAPDF.

PIV466 - IN SILICO ANALYSIS OF BACULOVIRUS CORE GENE PROMOTERSAndrade, M. de S.; Pacheco, T.J.A.; Ribeiro, B.M.; Melo, F.L.

LABORATÓRIO DE VIROLOGIA/UnB - Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília - DF, 70910-900

The baculoviruses are large dsDNA insect viruses with a high number of described species infecting several hosts, classified in four genus, the Alphabaculovirus, Betabaculovirus, Gammabaculovirus and Deltabaculovirus. To date, 61 species have been completely sequenced and are publicly available in GenBank. The analysis of these genomes revealed 37 core genes shared by all of them. These core genes participate in basic biological functions, such as RNA transcription, DNA replication and the formation of the virion structure. Moreover, most baculovirus genes can be classified in two groups: those transcribed by cellular RNApol II (early genes) and those transcribed by viral RNApol (late genes). Recently, the AcMNPV transcriptome was published and the transcription start sites (TSS) for its ORFs were mapped and putative motifs were described. However, it remains to be determined if the observed pattern is conserved through baculovirus evolutionary lineages. Therefore, we performed an in silico analysis of baculovirus core gene putative promoters. Firstly, the core genes (+ 1000 bp upstream of ATG) from all Alphabaculovirus and Betabaculovirus (57) was extracted and classified in early, early/late and late (6,4 and 27, respectively) according to AcMNPV data. The predicted TSS were manually annotated for all late genes (TAAG motif). Subsequently, we calculated the distance of TSS to ATG and the variation was higher for Alphabaculovirus than for Betabaculovirus. Moreover, the distance variation was higher for those genes with TSS that does not overlap with another ORF. We screened the early promoter for known insect transcription bactor binding sites (TFBS). We found two recurrent TFBS: HSF and Dfd, both TFBS described in insects. Finally, we analyzed the early data set in MEME and CentriMo, and




we found different motif for each early core gene, thus, we suggest that the early promoters are more complex and variable than late promoters and other news motifs play an important role in transcription regulation in addition to TATAA box motif. Overall, the late promoter was more conserved than early promoters, suggesting that the promoters of genes transcribed by host RNA polymerase are adapted to the host gene regulatory network. Financial suport: CNPq

PIV471 - SEQUENCING OF NEW POLEROVIRUS COTTON VIRUS FROM BRAZIL USING SMALL RNA DATASET OBTAINED BY DEEP-SEQUENCINGSilva, D.C.P.S.; Silva, A.K.F.; Silva, D.C.P.S.; Vaslin, M.F.S.


In a previous work, our group identified three recombinants virus isolates infecting cotton plants in Brazil which symptoms and transmission mechanism are closely related with the Cotton blue disease (CBD). CBD is an important cotton crop pathology present in America, Africa and Asia that causes significant economic losses. In Brazil it obligate the cotton farmers to plant only CBD resistant cultivars now days. The disease is transmitted by Aphis gossypii and is caused by a Luteoviridae virus, genus Polerovirus, the Cotton leaf roll dwarf virus (CLRDV). Typical symptoms include stunting due to internodal shortening, leaf rolling, intense green foliage. The isolates identified in this previous works, using CLRDV molecular diagnosis, were recovered from plants showing CBD atypical symptoms. Analyses of a partial polymerase sequence of these virus isolates showed that they presented low identity with CLRDV and can be considered as representatives of a new species of the genus Polerovirus. In orther to better characterize this new species, named Cotton red leaf virus (CoRLV), we submitted the isolate CoRLV-P01 to a deep sequencing by “Illumina”. All small RNAs of an infected plant were sequenced and “contigs” were constructed. However, the contigs weren’t able to reconstruct the viral genome. So, a new analysis of the small RNA data set libraries was performed using the software SearchSmallRNA (Andrade & Vaslin, 2014). Using CLRDV Brazilian and Argentine in independent analysis as reference genome, we were able to obtain 69% of the complete virus genome.

With this new analysis, the 3’ block of this putative polerovirus genome was almost completely recovered. Only a small portion of the 3’UTR region is absent. The complete CP, MP and RT proteins sequences are almost identical of those of the CLRDV isolates. However, the intergenic region and the almost complete polymerase (P2) show identities ranging from 73-82% with CLRDV. P2 amino acid sequence share similar ID levels with Brassica yellow virus, Pepper yellow leaf curl virus and Beet western yellows virus. Primers were design based in the genome reconstruct and were used to RT-PCRs using three virus independent isolates. The nucleotide sequence comparison of the amplicons showed the maximum identity of 73% with CLRDV and 71% with Pepper yellow leaf curl virus. In order to obtain the complete genome sequence of this virus, more primers combinations are been tested. FINANCIAL SUPPORT: FAPERJ AND CAPES

PIV474 - THE PHYLODINAMIC AND PHYLOGEOGRAPHIC OF TOMATO CHLOROTIC SPOT VIRUS: VIRAL SPREAD IN THE AMERICASde Almeida, M.M.S.1; Lucas, F.M.1; Martinez, R.T.2; Oliveira, R.1


2. IDIAF - Instituto Dominicano de Investigaciones Agropecuarias y Forestales, Evaristo Morales, Santo Domingo, República Dominicana

Viruses in the genus Tospovirus (family Bunyaviridae) are plant pathogens transmitted by insects known as thrips, with a trisegmented single-stranded ambisense RNA (S, M and L) genome and the ability to replicate in vector to. The diversity of the genus comprises 8 recognized species with a variable host range and geographic distribution. The genus Tospovirus are widespread in the Americas, where Tomato chlorotic spot virus (TCSV), Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), Chrysanthemum stem necrosis virus (CSNV), Zucchini lethal chlorosis virus (ZLCV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), Alstroemeria necrotic streak virus (ANSV), Melon severe mosaic virus (MeSMV) and Bean necrotic mosaic virus (BeNMV) have been reported. TCSV was first noticed in Brazil in the early 1990’s, with GRSV. Nowadays, GRSV is present in both




new world and the old world, whereas TCSV is find only in the Americas. A phylodinamic study was conducted to infer the TCSV spread. Thirty-three sequences corresponding to RNA S with coding (N protein) and no-coding regions were downloaded from GenBank. The alignment was carried out using MUSCLE software. The phylogenetic and phylodinamic relationships were inferred by BEAST package using Bayesian Evolutionary Analysis. According to the sampling year, the spatial phylogenetic reconstruction and the dynamic evolution were determined by SPREAD program. Probably, TCSV has two different origins in South America, one in Brazil (S45325) and the other in Argentina (U49709, U49707), in 1996. Brazilian lineage spread to the country to North America and Caribe. Once in Caribe, it was introduced back in North America. The first introduced in North America most likely in 1994, led to the first reassortant intra-species isolates. This new isolate has the S and L RNAs from GRSV and the M RNA from TCSV. The isolates originated from the second introduction in the North America around the year 2006 are so close related to Caribbean isolates, at least 97% identical. Comparing the first report of TCSV in Brazil with the last (JQ034525) sampled in 2009, the nucleotide sequence accumulated modifications resulting in 93% identity each other. That isolate is more related to Argentine lineage sharing 99% identity, with a clearly well supported phylogenetic reconstruction. There is not an updated information about the strain that are present nowadays in Brazil.

PIV479 - CHARACTERIZATION OF SOYBEAN-INFECTING SIDA MICRANTHA MOSAIC VIRUSFreitas, D.M.T.A.2; Fusaro, A.2; Lacorte, C.2; Boiteux, L.S.1; Ribeiro, S.G.2



The occurrence of begomoviruses in soybean (Glycine max) in Brazil has been sporadically reported and has not been associated with yield losses. However, the list of soybean-infecting begomoviruses it is growing, and there is a concern that yield losses may increase, since soybean infecting begomoviruses causing moderate to severe losses has been recently reported

in Argentina. This potential threat has intensified the surveys for begomovirus infection in soybean-producing areas. Samples obtained from soybean plants collected at Federal District, showing typical symptoms of begomovirus infection were analyzed. Viral DNA from infected plants was subjected to rolling-circle amplification (RCA), cloning and sequencing. Phylogenetic analyses and sequencing comparisons of the DNA-A and DNA-B clones, confirmed infection by Sida micrantha mosaic virus (SimMV). Cloned DNA-A and DNA-B components were introduced into seedlings of Phaseolus vulgaris ‘Pérola’, Glycine max ‘Conquista’ and ‘Williams 82’, Solanum lycopersicum ‘Santa Clara’, Nicotiana benthamiana and Sida rhombifolia by a biolistic method to confirm their infective capacity. Four weeks after inoculation, symptoms of begomivrus infection appeared in almost all plants species inoculated, only S. lycopersicum and Phaseolus vulgaris ‘Pérola’ showed no symptoms and no infection. Leaves from soybean and Sida rhombifolia exhibited yellow and golden mosaic, yellow vein, chlorotic mottling, necrosis, blistering, leaf distortion and dwarfing symptoms; symptoms such as dwarfing, leaf distortion and blistering were observed in N. benthamiana plants. The infection was confirmed by PCR amplification using begomovirus universal primers and sequencing of the obtained amplicon. This is the first report of infectious clones of soybean-infecting SimMV and these clones will be a valuable tool to study soybean-begomovirus interaction.FINANCIAL SUPPORT: EMBRAPA, CNPQ, INCTIPP, FAPDF.

PIV485 - HIGH GEMINIVIRUS-INFECTION ON A POTATO FIELD GROWN FOR SEED PRODUCTION IN BRAZIL CENTRALLima, M.F.1; Barriolli, C.M.C.2; Santos, D.I.3


2. UNIP - Universidade Paulista, Av Paulista, 900 São Paulo - SP, 13720-000

3. FACULDADE ANHANGUERA, Rua Alameda Maria Tereza, 2000 Valinhos, São Paulo - SP, 13278-181

Virus diseases are one of the main causes of yield losses in potato (Solanum tuberosum L.) production worldwide. The main viruses associated with this crop belong to the genera Potyvirus, Polerovirus, Carlavirus and Potexvirus;




however, emerging diseases caused by geminivirus (Family Geminiviridae) transmitted by whitefly (Bemisia tabaci) had been detected affecting this crop over the last years. In July, 2014, a 50-day-old potato field, cvs. Agata, Cupido, Manitou, Faluka, Mustang and Ambition of ca. 50ha showing geminivirus-like symptoms such as strong yellow mosaic, leaf reduction size with the edges turned upwards and stunting of plants, was observed in the State of Goiás, Brazil, one of the most important potato growers in the country. A variable range (2%-40%) of symptoms incidence was observed depending on the cultivar planted and its position in the field. Forty-eight leaf samples were collected from symptomatic plants and subjected to total DNA extraction for polymerase chain reaction (PCR) using universal primers and rolling circle amplification (RCA) for geminivirus detection, besides restriction fragment length polymorphism (RFLP) to access diversity of the isolates. In addition, sample were also tested by using serological methods (DAS-ELISA) for detection of Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS) and Potato leafroll virus (PLRV) using polyclonal antibodies. All samples did not react with neither of the polyclonal antibodies tested, indicating that none of these viruses was involved in the cause of the symptoms observed in potato plants. However, a band of ca. 1,2 kb was visualized on a 1,2% agarose gel stained with ethidium bromide from the majority of the potato samples collected confirming they were geminivirus-infected. These data are of great concern considering that that potato field was grown for seed production. Also, PCR-positive samples produced an RCA product, indicating the high sensitiveness of the technique. Restriction enzyme profiles suggest the existence of low variability among these geminivirus isolates. Cloning and sequencing of these isolates are in progress. These data indicate that despite the existence of some reports of occurrence of geminivirus in potato fields, however, is it necessary to keep the monitoring of these viruses in the crop, considering the presence of virus sources as well as high whitefly populations in the field and its ability to colonize many plant species.

PIV520 - COTTON MICRORNAS GHR-MIR2910 AND GHR-MIRNA162 ARE UPREGULATED DURING COTTON LEAFROLL DWARF VIRUS (CLRDV) INFECTIONVaslin, M.F.S.1; Romanel, E.2; Silva,T.F.2; Silva, D.C.P.S.1; Corrêa, R.L.1



Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new conserved miRNAs. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin- associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks. Ghr miR2910 was 7 to 7.5 times more expressed during infection. His role is still unknown, and its possible target would be the 18S ribosomal RNA. Besides him, the GHr miR-171, 157, 172 and 162 expression were also increased in the infection. On the other hand, the miRNA miR319, 393, 3476, 2111, 355, 159 have been drastically reduced in the infection. RT-PCR reactions in real time were carried out and confirmed the differential expression of these different miRNAs. MiRNAs exclusive




cotton, as the 2110 and the 3476 are now being studied to try to understand its function. Dysregulation of metabolic pathways regulated by these miRNAs might explain the symptoms observed in plants showing the disease in cotton blue. FINANCIAL SUPPORT: CAPES, CNPQ AND FAPERJ

PIV521 - DETECTION OF TOSPOVIRUSES IN PEANUT ON MAIN PRODUCER AREAS OF THE STATE OF SÃO PAULO, BRAZILAndrade, G.P.1; Carvalho, R.C.P.2; Ribeiro, G.P.1; Pantoja, M.B.1; Costa, P.M.G.1; Ferreira, P.G.A.1; Godoy, I.J.3; Santiago, J.C.L.1

1. UFRPE - Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife - PE, 52171-900


3. IAC - Instituto Agronômico, Avenida Barão de Itapura, 1.481, Botafogo Campinas - SP, 13020-902

The peanut (Arachis hypogaea) is a leguminous plant with a high socioeconomic importance. It constitute a food source rich on protein, vitamin, and minerals, used as cooked or toasted grains and in industrialized products. Furthermore, it has been used for producing cosmetics and biofuels. Worldwide, peanut is produced on tropical and subtropical regions and is considered the fifth leguminous in yield. In Brazil, it is very important, generating income and contributing for sustainability of small, medium and big farms. Despite the high economic relevance, several factors as pests and diseases affect this crop. The diseases caused by viruses, especially those belonging to the genus Tospovirus, may induce drastic yield losses, nearly 100 per cent, and low quality of the product when highly susceptible cultivars are used in presence of high population of thrips vectors. The tospoviruses in peanut induce a range of symptoms from small reduction on plant height to severe stunting, top necrosis, mild to strong mottling, ring spot, as well as plant death in severe cases. The virus infection may also affect the peanut pods and kernels that become stunted, deformed, and discolored. The first report of a tospovirus infecting peanut in the world was done in 1915, in Australia. In Brazil, it was initially found in 1941. The objective of this work was to analyze the occurrence of tospoviruses in the peanut producing areas of the

State of São Paulo. Symptomatic and asymptomatic samples in 10 counties were collected and submitted to biological, serological and molecular tests. Virus transmission was obtained by mechanical inoculation and by grafting, being observed typical symptoms on peanut and other indicator hosts. The enzyme-linked immunosorbent assay (ELISA) and ImmunoStrip tests showed positive reaction to three tospovirus species and the RT-PCR determined the presence of the Tomato spotted wilt virus (TSWV), Groundnut ring spot virus (GRSV) e Tomato chlorotic spot virus (TCSV).

VETERINARY VIROLOGY - VV


Veterinary Virology: VV 224

September/October 2014 Volume 19 – Supplement 2 - Abstracts/Posters - Veterinary Virology: VV

between 37 and 38), since birds are apparently healthy. All samples were negative by qPCR NDV L gene class I. Suspicious samples were also tested by qPCR F gene specific for velogenic strains in order to evaluate the pathogenicity and both were negative. Despite the low sampling, we deem that, in the studied locations, there is no class I NDV circulation and there is a low prevalence of class II low pathogenicity NDV. Financial support: FAPESP 2013/12112-8

VV25 - ABSENCE OF CLASS I NEWCASTLE DISEASE VIRUS IN WILD BIRDS FROM ANTARCTIC REGIONThomazelli, L.M.1; Seixas, M.1; Araujo, J.1; Ometto, T.1; Meneguin, C.1; Petersen, E.2; Barboza, J.D.B.1; Petry, M.V.2; Durigon, E.L.3


2. LABORATÓRIO DE ORNITOLOGIA E ANIMAIS MARINHOS/UNISINOS - Laboratório de Ornitologia e Animais Marinhos da Universidade do Vale do Rio dos Sinos, Avenida Unisinos, 950 - Cristo Rei, São Leopoldo - RS, 93022-000

3. IB/UFMT - Instituto de Biociências da Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - 78060-900

The Antarctic region is the largest natural wilderness left on the planet, and nature, the freer of human influence in all regions of the earth. Therefore, it is also the most remote, unknown and most preserved of all continent. Between 48 Antarctic and sub-Antarctic seabirds species observed in coastal countries of South America, 38 were registered in Brazil. Migratory birds, especially seabirds, may carry the Newcastle disease virus (NDV) from endemic areas, acting as transmitting agents in their long routes between wintering and breeding areas. Serological evidence and virus isolation indicate that the NDV occasionally circulates in Antarctica, however, our knowledge about the global distribution of this virus, especially class I, in wild bird populations is limited. Thus, the aim of the study was to analyze by real-time PCR (qPCR), the presence of both classes I and II NDV, in samples of seabirds, captured in Elephant Island, on Antarctic region. Five-hundred tracheal - cloacal swabs samples from five different species (petrel, skua, dove

VV24 - ABSENCE OF CLASS I NEWCASTLE DISEASE VIRUS IN WILD BIRDS FROM PANTANALBarboza, J.D.B.1; Ometto,T.1; Araujo, J.1; Seixas, M.1; Meneguin, C.1; Colmanetti, T.1; Pinto, L.B.2; Sabino, U.3; Pinho, J.B.3; de Aguiar, D.M.2; Durigon, E.L.1; Thomazelli, L.M.1


2. HV/UFMT - Hospital Veterinário da Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - 78060-900

3. IB/UFMT - Instituto de Biociências da Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT - 78060-900

The Newcastle disease is a highly contagious acute viral illness, notifiable, affecting wild and commercial birds. Due to potential risk that the disease represents to the economy of countries producers and exporters of chicken, the pathogens responsible are well known and studied. However, there are two major types of lineages termed class I and II. The first (class I) is more commonly associated with wild birds and do not usually have high pathogenicity in poultry, so it is scarcely studied worldwide and no literary records in Brazil. The class II viruses are the most important in the pathology of poultry and therefore becomes essential to have an active epidemiological surveillance, since Brazil is the largest exporter of chicken meat in the world. Wild birds, especially migratory, may carry the Newcastle disease virus (NDV) from endemic areas, acting as transmitting agents in their long routes between wintering and breeding areas. Therefore, the objectives were to analyze by real-time PCR (qPCR), the presence of both classes NDV in samples of wild birds, migratory or resident, in different regions of Brazilian wet lands, Pantanal biome, Mato Grosso. Three hundred thirty tracheal - cloacal swabs samples from wild birds were tested, of about 80 different species, all without any clinical signs of the disease. By qPCR M gene class II NDV, two samples (0.6%) were suspected positive. These samples deemed suspicious because they not were confirmed by DNA sequencing due to a low viral load observed (ct




cable, Antarctic and Papua penguin), were tested, all without any clinical signs of disease. By qPCR M gene class II NDV, two samples (0.4%) were positive. DNA sequencing of these samples was not possible due to a low viral load (ct between 35 and 38), since birds are apparently healthy. All samples were negative by qPCR L gene class I NDV. Positive samples were also tested by qPCR F gene specific for velogenic strains in order to evaluate the pathogenicity and both were negative. Therefore, we believe that there is no class I NDV circulation and there is a low prevalence of class II low pathogenicity NDV, in the studied location. However, more studies are needed to confirm these data. Financial support: FAPESP 2013/05485-2

VV26 - CASE REPORT: BOVINE CORONAVIRUS ASSOCIATED WITH OTHER PATHOGENS IN DAIRY FARM IN STATE OF SAO PAULO, BRAZILOkuda, L.H.; Tunin, A.; Catroxo, M.H.B.; Nassar, A.F.C.; Souza, S.F.; Brandão, P.E.; Fernandes, A.M.; Ribeiro, C.P.; Nogueira, A.H.C.; de Stefano, E.; Pituco, E.M.


2. Zoetis, Rua Luiz Fernando Rodrigues, 1701, Vila Boa Vista, Campinas - SP, 13064798

3. FMVZ/USP - Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Av. Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900

Enteritis constitutes a major problem in cattle, affecting mainly young animals. Several pathogens are involved such as bacteria, virus and parasites and commonly there is an association of agents causing diarrhea in animals. The bovine corononavirus (BCoV) belongs to order Nidovirales, subfamily Coronavirinae, genus Betacoronavirus of Coronaviridae family is considered one of the most important pathogens in neonatal diarrhea in calves, also associated with respiratory diseases and winter disentery in adult cattle, causing major economic losses in dairy farming and cutting. The BCoV belongs to Betacoronavirus genus, Nidovirales order, Coronavirinae sub-family of Coronaviridae family. The transmission route is fecal-oral by ingestion of water and food contaminated with feces or respiratory secretions and aerosols. The inter-species transmission has been the subject of several studies, which wild and domestic ruminants are characterized as reservoirs of BCoV. The

zoonotic potential has been proven experimentally. In Brazil, the first description occurred in 16 dairy herds located at State of Sao Paulo. Since then, several reports have demonstrated the presence of BCoV in other states such as Rio Grande do Sul, Paraná, Minas Gerais and Mato Grosso do Sul. The diagnosis is based on detection of antibodies or virological methods, which have already identified different BCoV genotypes. Considering the importance to determine the causative agent in diarrhea in cattle, the aim of present study was identify the cause of diarrhea in a dairy farm located at Araçatuba region of Sao Paulo state, Brazil with association of different tests. For that, four feces samples from one cow, one heifer and two calves were sent to Instituto Biologico to perform the differential diagnosis associating several techniques such as electron microscopy, bacteriological and virological tests. The bacteriological result was negative to salmonelosis, but one calf presented opportunists bacteria of Enterobacter genus while the parasitological test detected worms of Strongylidae family and the heifer, Eimeria spp. The electron microscopy revealed particles with morphology consistent with Coronavirus genus in four animals. The PCR results to BoHV-1 and BVDV are both negative. The viral isolation in MDBK cells was observed cytopathic effect in Holstein’s cow which was submitted to RT-PCR with positive result and confirmed by sequencing as BCoV. This study showed mixed infections in the herd, i

VV28 - SEROLOGICAL AND MOLECULAR EVIDENCE OF ORTHOPOXVIRUS IN URUGUAYLuiz, A.P.M.F.; Pereira, A.F.; Alves, P.A.; Trindade, G.T.; Abrahao, J.A.; Kroon, E.G.


Bovine vaccinia (BV) is a viral disease that affects dairy cattle and milkers, being classified as an occupational zoonosis. This disease has as etiologic agent the Vaccinia virus (VACV), which belongs to the Poxviridae family, Orthopoxvirus (OPV) genus. Infected dairy cattle usually present ulcerative lesions on their teats and udders. Rural workers, when infected, presented mainly lesions on their hands. Therefore, BV has a great economic impact, compromising the milk industry and public health services. The VACV exhibits serologic crossreactivity




with other OPV species and was used during the World Health Organization smallpox eradication campaign. The origin of VACV in Brazil is unknown, although some studies have suggested that VACV strains used during the campaign may be related to outbreaks of bovine vaccinia and other studies suggest an autochthonous origin. In the last 14 years, outbreaks of BV have been detected across Brazil. Despite BV impact in Brazil, the VACV circulation in Uruguay was not described. We investigated for the presence of OPV-neutralizing antibodies and VACV DNA in 125 sera samples from Uruguay. Of the 125 sera, neutralizing antibodies against OPV were detected in 28 (22,4%); of these, 10 (35,7%) had titers of 100 NU/ml, 11 (39,3%) had titers of 200 NU/ml, 5 (17,9%) had titers of 400 NU/ml, and 2 (7,1%) had a titer of >800 NU/ml. To detect viral genome, we conducted PCR to amplify the viral growth factor gene (vgf) and hemaglutinin gene (HA) DNA. Two of the 125 sera were positive in PCR assays for vgf gene and one serum was positive for HA gene. Although no exanthematous VACV outbreaks have been described in bovines in Uruguay, our results suggest that herds may be exposed to VACV in areas surrounding the Brazilian border and therefore may be at risk for VACV infection.

VV30 - MUTATION IN THE CAPSID PROTEIN OF PORCINE CIRCOVIRUS GENOTYPE 2B (PCV2B) DETERMINE CYTOPATHOGENICITY IN SWINE TESTICLE CELLSCruz, T.F.1; Ordonez, F.J.P.3; Castro, A.M.M.G.2; Richtzenhain, L.J.2; Araújo Jr, J.P.1



3. UNIVERSIDAD DE CALDAS, Manizales, Caldas, Colômbia

Porcine circovirus type 2 (PCV2) is a DNA virus characterized by absence of cytopathic effects (CPE) in cell culture. Thus, the objective of the present study was to describe the occurrence of CPE in swine testicle (ST) cells infected with PCV2 presenting mutations in the viral capsid. Four isolates of PCV2 (B, C, D and E) with amino acid mutations in the ORF2 were used in this study. The isolates were obtained after viral isolation in ST cells. The isolated B has one mutation (T200I), isolated C has

two mutations (T200I; M72I), isolated D and E has three mutations (T200I; M72I; N77D). To compare the CPE, ST cells with semiconfluent monolayer were inoculated with lymph node suspension (original PCV2b; group A), isolate B (group B), C (group C), D (group D) and E (group E). ST cells (control flask) were inoculated with only minimum essential medium. The total of 17 passages was performed, but for some groups of cells, number of passages was lower due to viral action. The PCV2 viruses from last passage of groups A, B, C, D and E were genetically characterized by sequencing. Each flask was stained with Giemsa to assess cell morphology. The cell counting was performed on ten different fields randomly selected, and the magnification of 400X was used for evaluation. One mutation (T200I) was observed in the sequence of virus from 17th passage of group A. Three mutations (T200I, M72I and N77D) were found in the last passages of groups B (13 passage), C (11 passage), D (4 passage) and E (6 passage). The cells were fusiform to rounded, becoming necrotic with eosinophilic cytoplasm, condensed chromatin, karyorrhexis and karyolysis. Finally, a decrease was observed in the number of cells that were more elongated and the morphologic characteristics were maintained. The CPE was more severe in the 9th passage of group C and B, 3rd passage of group D and 4th passage of group E. The control group and A did not show CPE. This is the first report that describes the occurrence of CPE caused by PCV2 in ST cells. The two (T200I, M72I) and three (T200I, M72I, N77D) amino acid mutations in the viral capsid were observed in groups that shown CPE. The occurrence of one mutation (T200I) did not cause CPE in ST cell. The CPE observed in group B could be due to the occurrence of three mutations (T200I, M72I and N77D) detected in the 13th passage by sequencing. The presence of mutations in the viral capsid likely favoreced the viral replication, wich caused lower cell survival.




VV32 - PORCINE CIRCOVIRUS GENOTYPE 2B PRESENTING AMINO ACID MUTATIONS IN THE CAPSID DETERMINES ENHANCED REPLICATION IN SWINE TESTICLE CELLSCruz, T.F.1; de Castro, F.L.2; Ordoñez, F.J.P.3; Richtzenhain, L.J.2; Araujo Jr, J.P.1

1. IB/UNESP - Instituto de Biociências da Universidade Estadual Paulista, Avenida 24 A, 1515 Bela Vista, Rio Claro - SP, 13506-900


3. UNIVERSIDAD DE CALDAS, Manizales, Caldas, Colômbia

PCV2 is a small, single-stranded DNA virus that belongs to the family Circoviridae. PCV2 is characterized by absence of cytopathic effects (CPE) and lower viral titer in cell culture. Thus, the aim of this study was to describe the increased of viral replication in isolates of PCV2 that have amino acid mutations in the capsid protein associated with the occurrence of CPE. Four isolates of PCV2 (B, C, D and E) with amino acid mutations in the ORF2 were used in this study. The isolates were obtained after viral isolation in swine testicle (ST) cells. The isolated B has one mutation (T200I), isolated C has two mutations (T200I; M72I), isolated D and E has three mutations (T200I; M72I; N77D). ST cells with semiconfluent monolayer were inoculated with lymph node suspension (original PCV2b; group A), isolated B (group B), C (group C), D (group D) and E (group E). ST cells (control flask) were inoculated with only minimum essential medium. To evaluate viral replication, aliquots (intracellular viruses) were separated from the infected and control cell suspensions after trypsinization. Extracted DNAs were analyzed by quantitative PCR (qPCR) for ORF2-PCV2. The total of 17 passages was performed, but for some groups of cells, number of passage was lower due to occurrence of CPE. The viral loads determined for isolates B, C, D and E were higher when compared to original virus (group A). All passages from the control group were negative for PCV2 by qPCR. The occurrence of CPE more severe coincided with the peak of viral load (1011 DNA copies/ml ST cell suspensions) in groups B, C, D and E. The CPE resulted in cell death. Thus, the number of passages was lower for isolates (D and E) presenting three amino acid mutations

in the capsid protein. For isolated B (one mutation), C (two mutations) and original PCV2b, the number of passages performed was higher compared to isolates D and E. After the occurrence of CPE, due to cell death, the viral load was lower in the following passages. These results from this study suggested that the amino acid mutations alone or collectively were likely responsible for the enhanced PCV2 replication in ST cells. This is the first report that describes the occurrence of CPE caused by PCV2 in ST cells associated with increased viral load. The CPE observed in group B could be due to the occurrence of three mutations (T200I, M72I and N77D) detected in the thirteenth passage by sequencing (data not shown). FINANCIAL SUPPORT: FAPESP 06/59002-9

VV45 - PROSPECTIVE STUDY OF CORONAVIRUSES IN RODENTS IN A RURAL AREA OF MINAS GERAISSacchetto, L.1; Miranda, J.B.2; Amaral, C.D.2; Borges, I.A.2; Vieira, F.N.2; Ambrósio, L.L.D.2; Alves, P.A.2; de Siqueira, T.R.1; Paglia, A.P.2; Trindade, G. de S.2; Drumond, B.P.1



Emerging viruses are an important public health problem worldwide. Some animals, including small mammals, have an important role in the maintenance and transmission of viruses by acting as natural host reservoirs. Coronaviruses (CoVs) (order Nidovirales, family Coronaviridae, subfamily Coronavirinae) are large enveloped positive-stranded RNA viruses that infect a broad range of mammals. Bats and rodents are rich sources of viruses, such as coronaviruses, that can be transmitted to humans. Therefore, the aim of this work was to perform a prospective study of coronaviruses. The capture of small mammals was performed on a rural property (forest, pasture and domiciliary areas) located in the city of Rio Pomba, Minas Gerais, from September 2012 to September 2013. Lung samples were obtained from 59 rodents and preserved in RNAlater. The samples were macerated and total RNA was extracted using RNeasy® Minikit (QIAGEN). For cDNA synthesis, RNA was submitted to RT-PCR with random primers fallowed




by real time PCR (qPCR) assay to detect coronaviruses (a larger region of the RdRp) using primers that targeted a conserved region of the virus genome using SYBR® Green PCR Master Mix (Applied Biosystems). Plasmids containing the genes of interest were used as positive controls. Up to date, RNA extraction was performed for all samples and qPCR was performed for 18 lung samples. So far, none of the tested samples was positive for coronavirus. The serum and other organs of the collected rodents are going to be tested in the future. Surveillance studies of animal species, like rodents, are essential to understand the circulation, maintenance and transmission of these viruses as well as their emerging potential. Financial support: FAPEMIG, CNPq, CAPES, UFJF, PROPESQ/UFJF.

VV62 - DETECTION OF BOVINE HERPESVIRUS 1 IN SEMEN OF BULLS FROM BEEF CATLE FARMS IN RIO GRANDE DO SULHenzel, A.1; Mascitti, A.K.2; Salla, P. de F.2; Lunge, V.R.2; Spilki, F.R.1


2. ULBRA - Universidade Luterana do Brasil, Rua Martinho Lutero - 301 - Universitário, Cachoeira do Sul - RS, 96501-595

Brazilian cattle herd is the second largest in the world, with more than 200 million animals. The state of Rio Grande do Sul (RS) is responsible for approximately 5% of this total with the majority of the beef cattle herds located in the Center-West, Southeast and Southwest regions of the state. Some of the most important respiratory and reproductive cattle diseases are caused by bovine herpesvirus type 1 (BoHV-1), an alphaherpesvirus belong of the Varicellovirus genus, Herpesviridade family. Viral transmission occurs by respiratory, ocular and genital secretions. BoHV-1 may also be present in the semen of infected bulls, leading to dissemination by natural mating or artificial insemination. BoHV-1 has been detected in association with clinical cases, but the effective occurrence of this virus is still unknown under conditions of extensive cattle farming. The aim of this study was to investigate the occurrence of BoHV-1 in bulls from beef cattle farm located in the main producing regions of the RS. Three

hundred and ninety-one (n=391) samples of serum e semen from bulls were collected. All samples were obtained from eighteen different farms from Center-West, Southeast and Southwest regions of RS. The serum samples were submitted to serum neutralization test, while the semen were used for detection of viral DNA by polymerase chain reaction (PCR) targeting the viral DNA polymerase gene. The results of the serum neutralization showed the presence of 44 animals positive for BoHV-1 (17.6% of total) corresponding to twelve (66.7%) of the studied farms. Five of the farms had a higher frequency of animals positive for BoHV-1 (above 15%), with several cases of animals with antibody titers above 1:64. BoHV-1 PCR detection in semen showed the occurrence of two positive samples, precisely in one of the farms with higher BoHV-1 antibody titers. This particular has a history of nutritional and health management deficiencies, possibly being related to the BoHV-1 shedding in semen. FINANCIAL SUPPORT: FAPERGS, ULBRA, FEEVALE

VV63 - SEROLOGICAL EVIDENCE OF HEPATITIS E VIRUS (HEV) IN SWINES FROM SANTA CATARINA STATE, BRAZILLopes, K.G.S.1; Silva Júniro, J.V.J.1; Kramer.B.2; Bordin, L.C.2; Pandolfi, J.R.2; Bertani, G.R.3; Silva, V.S.2; Gil, L.H.V.1

1. CPQAM/FIOCRUZ - Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz, Av. Professor Moraes Rego, s/n - Campus da UFPE - Cidade Universitária, Recife - PE, 50.740-465



The hepatitis E virus (HEV) is the only member of the family Hepeviridae, its viral genome (vRNA) consists of a single strand RNA (ssRNA), positive-sense, surrounded by a non-enveloped capsid. The HEV has four genotypes (genotypes 1-4), the genotypes 1 and 2 are found exclusively in humans and only genotype 3 and 4 have zoonotic potential with the pigs being the primary animal, although the presence of vRNA has also been reported in other animal species. The transmission by direct contact with infected animals or consumption of contaminated food by zoonotic genotypes has increased the concern




of public health agencies with this infection. To evaluate the presence of HEV in swines, serum samples from confined pigs and wild boars from Santa Catarina state, Brazil, were submitted to the immunoassay (ELISA) for detection of anti-HEV IgG. The test was performed in duplicate and samples with results very close to the cutoff were retested. About the overall results, 100% of wild boars samples (68 samples) were negative for anti-HEV IgG, whereas 50% of domestic pigs samples (16 samples) were positive. These results confirm the circulation and absence of HEV in Brazilian herds and wild boars studied, respectively. Additionally, this work is the first serologic report about the HEV in wild boars from Brazil. Thus, these data highlight the risk of zoonotic transmission and contributes to epidemiology studies about HEV in Brazil.

VV83 - DETECTION OF BOHV-4 IN FOLLICULAR FLUID AND BOVINE SEMENAlbuquerque, N.R.M.1; Finoketti, F.1; Campos, F.S.1; Brito, M.E.D.1; Paulo, W.M.1; Franco, A.C.1; Firpo, R.M.2


2. UFCSPA - Universidade Federal de Ciências da Saúde de Porto Alegre, R. Sarmento Leite, 245 - Centro Histórico, Porto Alegre - RS, 90050-170

Bovine herpesvirus 4 (BoHV-4), a member of the family Herpesviridae, subfamily Gammaherpesvirinae, genus Rhadinovirus is widely distributed in the cattle population, and has been recovered from various tissues of reproductive organs as well as from aborted fetuses. In this study, a search was made to detect the presence of BoHV-4 genomes in follicular fluids and semen of cattle. One hundred and twenty follicular fluid samples obtained from ovaries of females in slaughterhouses and 164 semen samples were acquired from different distributors. Samples were examined for the presence BoHV-4 genome fragments with a nested PCR targeting the viral thymidine kinase gene, designed to amplify a fragment of 315 base pairs. Six out of 120 (5%) follicular fluid samples and 15 out of 164 (9%) semen samples contained amplifiable BoHV-4 genome segments. The amplicons obtained were cloned and subjected to sequencing, what confirmed that the amplified products in fact corresponded to the expected BoHV-4 genomic

region. This study provides evidence for the occurrence of BoHV-4 genomes in samples of follicular fluid and semen of cattle in Brazil. In addition, these findings highlight the need for epidemiological surveillance for BoHV-4 in such specimens in order to prevent possible spreading of such agent through techniques of artificial reproduction.

VV84 - PREVALENCE OF BLUETONGUE VIRUS ANTIBODIES IN BOVINE FROM ARAPONGAS MUNICIPALITY, STATE OF PARANA, BRAZILBronkhorst, D.E.1,2; Negri Filho, L.C.3; Moraes, E.M.S.1; Katto, S.4; Affonso, M.Z.4; Neto, D.B.4; Headley, S.A.5; Alfieri, A.A.6; Silva, L.C.4; Bogado, A.L.G.1; Barca Junior, F.A.1; Okano, W.4

1. MV/UNOPAR - Medicina Veterinária da Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140

2. IC/FUNADESP - Iniciação Científica da Fundação Nacional de Desenvolvimento do Ensino Superior Particular, SCS Quadra 7 - Bloco “A”, Salas 726/728 Torre do Pátio Brasil Shopping, Brasília - DF, 70307-901

3. BOLSISTA PIBIT CNPQ/UNOPAR - Programa Institucional de Bolsas de Iniciação em Desenvolvimento Tecnológico e Inovação / Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140

4. Mestrado em Saúde e Produção de Ruminantes/UNOPAR - Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140

5. PATOLÓGICA VETERINÁRIA/UEL- Universidade Estadual de Londrina, Rodovia Celso Garcia Cid | Pr 445 Km 380 | Campus Universitário, Cx. Postal 10011, Londrina - PR, 86057-970

6. Mestrado em Saúde e Produção de Ruminante/UEL- Universidade Estadual de Londrina, Rodovia Celso Garcia Cid | Pr 445 Km 380 | Campus Universitário, Cx. Postal 10011, Londrina - PR, 86057-970

Bluetongue (BT) is an important viral disease of domestic and wild ruminant that is transmitted by hematophagous Culicoides midges, that is caused by the bluetongue virus (BTV), member of the Orbivirus genus in the family Reoviridae, included in the OIE diseases list. Cattle and wild ruminants are frequently infected but fatality rates from BT in cattle are low, it has important consequences for the dairy and beef cattle sector because it can reduce milk yields as well as cow




fertility. Malignant catarrhal fever is the main differential diagnosis in cattle. This study aimed to evaluate the prevalence of BTV infection in adult dairy cattle. A total of 218 serum samples from healthy bovines from the 32 herds from rural settlement “Dorcelina Folador” in the region of Arapongas, Paraná state,were submitted to the agar-gel immunodiffusion (AGID) test for bluetongue virus antibodies as recommended by the OIE. The antigen used was commercial BTV antibody test Kit VMRD Inc. This study was approved by the Ethical Committee for the use of Experimental Animals of the Universidade Norte do Parana (UNOPAR), Brazil (Protocol 015/13). The prevalence of seropositive animals for BT found was 33.5% (73/218). The percents of seropositive herds in agar gel immunodiffusion test for bluetongue virus were 65% (21/32). These data indicate a wide BTV circulation in bovine and show that infection is widespread among the properties of the Arapongas. In 2001 and 2002, four outbreaks of this disease in Paraná State were reported to the OIE, clinical cases occurring in goats and sheep. The prevalence of seropositive bovine for BT found in the Minas Gerais was 59.1.%, São Paulo 53.7%, Mato Grosso do Sul 42%, Rio de Janeiro 40.9%, Santa Catarina 37.8% and Rio Grande do Sul 89.7%. The prevalence of infection by the BTV may result from the annual average temperature of Arapongas which is 18°C, reaching 22°C in some months, which may have contributed to the vector propagation and consequent spread of infection. Financial support: This work was supported by PIBIT-CNPq, FUNADESP, UNOPAR e KROTON.

VV87 - STUDY OF THE CIRCULATION OF VACCINIA VIRUS IN SMALL RODENTS OF URBAN AREAS OF THE STATE OF MINAS GERAISMiranda, J.B.1; Apolinário, I.B.1; Müller, U.B.2; Ferreira, P.C.P.1; Bonjardim, C.A.1; Kroon, E.G.1; Howard, J.C.3; Trindade, G. de S.1


2. UNIVERSE OF COLOGNE3. INSTITUTO GULBENKIAN DE CIÊNCIA,

Rua Quinta Grande 6, 2780-156 Oeiras, Portugal

Vaccinia virus (VACV) is the prototype virus of the family Poxviridae, which comprises many viruses that can infect vertebrates and insects. VACV was used as vaccine

during smallpox vaccination campaigns worldwide. After the eradication of smallpox, the vaccination was suspended due side effects of the infection with VACV. Nowadays, outbreaks of Bovine vaccinia (BV), a disease caused by VACV, have been reported in Brazil. This disease mainly occurs at rural environments affecting milk cattle and their handlers, who become infected after contact with lesions placed in the cow’s teats and udders. Infected humans present as a major symptom ulcerative lesions in the hands. Little is known about VACV origin and which hosts participate at its transmission cycle. There are many evidences of the participation of small rodents. VACV strains have been isolated from sylvatic rodents and the same virus was isolated from humans, cattle and mice during an outbreak of BV. It has also been shown that intranasal infected mice can shed viral particles in their feces, and these particles remain viable for some time. Also, viruses belonging to the genus Orthopoxvirus, the same in which VACV is placed have rodents as reservoirs, like Monkeypox virus and Cowpox virus. To better understand the role of rodents in the circulation of VACV, 14 Mus musculus were captured at urban areas close to Belo Horizonte, in the state of Minas Gerais. The animals were trapped in size selective cages and baits of hazelnut cream were used. After anesthesia, mice were measured and had their sera collected. The organs were also sampled and stored properly. Sera were used for DNA extraction via PCI and this DNA used as sample for PCR reactions. The targets for PCR reactions were the genes A56R, A26L and C11R. Two samples were positive for at least one of these genes. Sequencing of the sample positive in the A26L reaction revealed a similarity between the sequence analyzed and the sequences of Brazilian isolates, such as Passatempo virus and Araçatuba virus. These findings reinforce the participation of small rodents in the VACV cycle, at the same time as it raises questions about how there are not documented outbreaks of VACV in urban areas if the virus is present at these places. Further experiments are necessary to better understand the relationship of the virus with other brazilian isolates.




VV114 - MICROSCOPIC LESIONS AFTER CHALLENGE WITH TWO BRAZILIAN AVIAN INFECTIOUS BRONCHITIS VARIANTSMores, M.A.Z.1; Okino, C.H.1; Montassier, H.J.2; Mattos, G.L.M.1; Brentano, L.1; Coldebella, A.1; Esteves, P.A.1; Trevisol, I.M.1



Avian Infectious Bronchitis (IBV) is one of the greatest health challenges on poultry industry. The emergence of new variants has been frequent recently, therefore studies reporting pathogenesis are important to characterize the most representative IBV strains. The aim of this study was to evaluate microscopic lesions in SPF chickens challenged with two Brazilian IBV field isolates previously genotyped as IBV variants. Sixty two one-day old SPF chickens were divided into three groups and housed in positive pressure isolators. Groups A and B were challenged at 28 days of life with 104.0IED50 of 3735 and 3736 IBV variants, respectively. Group C was kept as mock infected negative control group. Nine birds from each group were euthanized at 1 day-post infection (dpi) and at 5 dpi, remaining six birds at 8 dpi. All birds were necropsied and trachea, sinuses, Harderian gland, lung, kidney and gonads were collected, fixed in buffered formalin and processed by routine histology. At 1 dpi acute tracheitis was observed in few birds of groups A and B. Exudation of mucus and heterophils; degeneration, necrosis and sloughing of epithelial cells and heterophilic infiltrate in the mucosa were predominant lesions. At 5 dpi, all birds of groups A and B had chronic tracheitis, with complete ciliary loss, lymphocytes and plasma cells infiltrating the mucosa and epithelial hyperplasia. In sinuses, fifty percent of the birds from groups A and B had mild chronic inflammation, the same trend was observed at 8 dpi in this tissue. At 8 dpi tracheal mucosa was regenerating, showing glandular degeneration, lymphocytic infiltration and mild epithelial hyperplasia. Chronic interstitial nephritis was observed in few birds of groups A and B at 8 dpi. Mild lymphocytic infiltration was observed in the Harderian gland of all birds of challenged groups. No lesions were observed in the tissues of group C and in lungs and gonads of all groups. Significant positive correlation (P

< 0.0001) was reported between scores of microscopic lesions and ciliary activity (gold standard method) on tracheal samples at 5dpi, by Pearson test. No significant microscopic differences were found between these two IBV variants tested. The results indicate that, besides the genotypic variation, lesions produced by the IBV variants here studied were similar to those described for standard IBV strains with respiratory tropism, producing main lesions in the upper respiratory tract and, renal damage in few birds. FINANCIAL SUPPORT: PROJETO EMBRAPA 03.12.03.012.0.00

VV119 - OCCURRENCE OF CANINE HERPESVIRUS INFECTION IN DOGS OF BREEDING KENNELS IN TROPICAL CLIMATE ZONEde Souza, F.F.; Tavares, D.C.; Kurissio, J.K.; Araujo Jr, J.P.; Toniollo, G.H.


The canine herpesvirus (CHV) is a cause of abortion in dogs, but can be detected in healthy animals. This virus is associated to different clinical abnormalities as death of newborns and lesions in respiratory and reproductive tracts in adult dogs. There is no vaccine against CHV in Brazil due to scarcity of studies confirming the importance of virus in breeding kennels. Thus, this study aimed to correlate the abortion incidence with percentage of positive dogs to CHV at Ribeirão Preto region. Adult (1 to 5 years-old) dogs (39 males and 209 females) from different breeds (Schnauzzer, Pug, Poodle, Yorkshire Terrier, Lhasa Apso, Shih-Tzu, Maltese, German Spitz and French Bulldog) from 9 commercial kennels were used. Blood samples were obtained by jugular vein puncture and placed into vials containing 10% EDTA. The DNA extractions were made with Wizard® Genomic Purification Kit (Promega, Madison, WI, USA). The extracted DNA was subjected to qPCR using primers directed to the thymidine kinase CHV3 (CGTGGTGAATTAAGCCTAA) and CHV4 (ATGCTATTGGGTGTCTATC), and GoTaq™ qPCR master mix kit (Promega, Madison, WI, USA) in the ABI 7300 (Life Technologies™, SP, Brazil) equipment. As a negative control reaction, nuclease-free water was used. The reaction conditions for amplification of the gene fragment for thymidine kinase CHV were initial denaturation at 95°C/2 min, followed by 40 cycles of




denaturation at 95°C/30 sec, annealing at 57°C/30 sec, and extension at 72°C/30 sec. Although the kennels evaluated in this study presented cases of abortion (2.1 to 42.9%/female number) and neonatal death (1.8 to 32.7%/9 kennels), none sample showed positive results to CHV. We suggest as cause of these alterations the presence of other pathogens (Neospora caninum, Leptospira spp, Toxoplasma gondii, Ehrlichia canis and Brucella canis) or latent of herpervirus infection. The stud dogs from kennels are constantly submitted to stress, immunosuppression or pregnancy, which can recidivate the virus. Moreover, the herpesvirus is not stable in the environment, with optimal replication at temperatures less than 37ºC, and in tropical climate zone the media annual temperature is high (mean 26ºC). This hypothesis has been raised in other studies, with a higher incidence of cases in regions with milder temperatures. Furthermore, we used the qPCR that identifies the CHV-DNA in the blood of host, which may not have been found due to the infection latency, which can be resolved with serological test. FINANCIAL SUPPORT: FAPESP - PROCESSO NO 13/06993-1.

VV126 - ROTAVIRUS FREQUENCY IN DIFFERENT RAISING SYSTEMS OF DAIRY HEIFERS IN THE WEST OF PARANAGallego, J.1; Macedo, R.1; Ribeiro, A.S.1; Kunz, A.F.1; Mello, J.L.1; Grolli, P.1; Taffarel, L.E.2; Takiuchi, E.1



Currently, family dairy properties represent 38.8% of the properties in the state of Parana and they are responsible for 44% of the milk production. In order to reduce the production costs and improvement of zootechnical and health rates, many familiar producers have run the raising of their heifers off-site the dairy farm, transferring them to outsourced rearing units. The raising phase of heifers is the second largest cost in the dairy business and the occurrence of diarrhea during this period can cause significant economic losses to producers. Rotavirus A (RV-A) is recognized as the major viral etiologic agent in cases of neonatal diarrhea.

This paper aims to analyze the frequency of rotavirus in heifers kept under two different production systems: (i) conventional breeding system in a family basis and (ii) outsourced rearing of heifers and calves system. In the period from 2012 to 2013, 150 and 95 stool samples were collected (diarrheal and non-diarrheal) from heifers up to 30 days old from a unit of rearing calves and heifers and a family based dairy property (respectively, both located in the western region of the state of Paraná, Brazil).The RV diagnosis was carried out in fecal samples by silver-stained polyacrylamide gel electrophoresis (SS-PAGE). In outsourced rearing units the RV-A was detected in 11.3% (17/150) of samples of all animals with clinical signs of diarrhea, which was easily controlled. The family based dairy property showed positivity of 47.3% (45/95) which 93% of it was with diarrhea and mortality of 20% (9/45). The installation and the sanitary management adopted may explain the discrepancy in the frequency of RV found in different systems. In outsourced rearing units, heifers were kept in individual masonry stalls and suspended floor, while in the family based dairy property, animals of different ages occupied stalls and collective plots with poor hygiene, which favored the transmission cycle RV. Furthermore, the proposal of a exclusive manpower in the work of creating heifers results in greater attention to the promotion of animal health, which is usually neglected in most of the dairy farms where lactating animals receive most attention and investment in management. Already established in the U.S. and EU, outsourced rearing units systems has been performing promisingly in dairy farming of Paraná, and the expectation of control of neonatal diarrhea, particularly RV, is significant advantage to the producer.

VV130 - PARTIAL “M” PROTEIN SEQUENCING OF CANINE CORONAVIRUS STRAINS CIRCULATING IN THE STATE OF RIO DE JANEIRO, BRAZILBottino, F. de O.; Domingues, C.F.; Costa, E.M.; Pimentel, F.B.; de Castro, T.X.; Garcia, R. de C.N.C.


Canine coronavirus (CCoV) is a single-stranded RNA virus belonging to the Coronaviridae family and is responsible for mild to severe gastroenteritis in dogs. To date, CCoVs can be classified in two genotypes, CCoV-I and CCoV-II according to the genetic identity observed




between these viruses and Feline Coronavirus (FCoV) type I and II, respectively. The aim of this study was to characterize the CCoV genotypes detected in fecal samples from diarrheic puppies in Rio de Janeiro, by partial amplification of the “M” protein gene. A total of 41 fecal samples collected from diarrheic puppies (2006-2014) that tested positive for CCoV by using RT-PCR assay for M gene (CCV1/CCV2) were used in this study. The 409bp amplicons were purified and then subjected to direct sequencing with the Big Dye Terminator® kit and an ABI Prism® 3730 DNA analyzer (Applied Biosystems CA). Sequence editing and multiple alignments were performed with BioEdit Sequence Editor 7.0 software. Nucleotide similarity with sequences deposited in the GenBank database was assessed using the BLAST tool. Sequence analyses were performed with the MEGA 5.1 software program. For 27/41 strains, the sequences presented enough quality for analysis. Based on the AA changes found in residues 127, 173, 193, 200 and 201 of the M protein, eight strains were characterized as CCoV-I while another 19 as CCoV-II. Alignment of deduced amino acid sequences showed 10 nonsynonymous substitutions. Among the 10 AA changes, five were found in all CCoV-I strains: 127 (Ile/Val→Ala), 173 (Val→Thr), 193 (Ile→Met), 200 (Asp→Glu) and 201 (Asn→His). These AA changes were present in the strains of this study as well in the reference strains. Moreover, six of the Brazilian strains analyzed contained non-synonymous substitutions not previously described. AA changes at residues 119 (Ile→Thr) and 121 (Gly→Cys) were found in strain RJ1133/12. Another two strains (RJ915/08 and RJ916/08) contained a non-synonymous substitution at residue 120 (Ala→Thr), while RJ999/10 was substituted at residue 144 (Lys→Arg). An AA change at residue 137 (Ile→Phe) was found in two CCoV-II strains from 2011(RJ1084/11 and RJ1091/11). In this study, the AA changes found in residues 127, 173, 193, 200 and 201 of the M protein could distinguish between CCoV-I and CCoV-II strains. These non-synonymous substitutions had already been described in European samples, but the exact consequence of these mutations is not yet clear. FINANCIAL SUPPORT: FAPERJ, CNPQ, PROPPI-UFF, PROGRAD-UFF

VV138 - HIGH FREQUENCY OF BOVINE VIRAL DIARRHEA VIRUS 2 IN RIO GRANDE DO SUL, BRAZILWeber, M.N.1; Silveira, S.1; Machado, G.1; Groff, F.H.S.2; Mósena, A.C.S.1; Silva, M.S.1; Budaszewski, R.F.1; Duppont, P.M.1; Corbellini, L.G.1; Canal, C.W.1


2. SEAPA - ecretaria da Agricultura, Pecuária e Agronegócio, Avenida Getúlio Vargas, 1384. Menino Deus, Porto Alegre - RS, 90150-004

Ruminant pestiviruses can infect cattle populations worldwide and cause significant economic losses due to their impact on productivity and health. Knowledge of pestivirus diversity is important for control programs and vaccine development and for determining probable sources of infection. In this work, we describe a search for ruminant pestiviruses in the sera of 9,078 calves from six to 12 months of age that were sampled for foot and mouth disease monitoring. The calves were first analyzed in pools and the positive samples analyzed individually. Thirty-three RT-PCR positive animals were detected (0.36%) from 6.64% (23) of the 346 herds tested. Sequencing analysis of the 5’ non-coding region showed the presence of BVDV-1a (15 strains), -1b (3), -1d (1) and -2b (14), with a higher frequency (42.4%) of BVDV-2 in comparison with other countries. This study may also be the reference for future control programs in Southern Brazil because it reports the prevalence of cattle with active infections and the genetic background of the circulating strains. Financial support: CAPES, CNPq, FAPERGS and Propesq/UFRGS.




VV143 - A QPCR PLATFORM FOR ZOONOTIC VIRAL AGENTS DIAGNOSTIC: APPLICATION ON RODENTS EMERGING VIRUSES DETECTION AT THE MINAS GERAIS STATE, BRAZILAlves, P.A.1; Borges, I.A.1; Amaral, C.D.1; Rezende, I.M.2; Drumond, B.P.2; Magalhães, C.L. de B.3; Almeida, G.M.F.1; Ferreira, P.C.P.1; Abrahão, J.S.1; Kroon, E.G.1; Trindade, G. de S.1


2. UFJF - Universidade Federal de Juiz de Fora, R. José Lourenço Kelmer - Martelos, Juiz de Fora - MG, 36036-

3. UFOP - Universidade Federal de Ouro Preto, Campos Morros do Cruzeiro, s/n - Bauxita, Ouro Preto - MG, 35400-000

The emerging infectious diseases play an important role in public health, and zoonoses have a great part in this scenario. In the context of emerging viruses, it is believed that with environmental degradation, some species of small mammals are now approaching anthropic disturbed areas, increasing contact with both human populations with domestic animals and herds, which could increase the flow of zoonoses between natural and anthropic system. Among the viral agents associated with these zoonoses, the following families should be highlighted: Bunyaviridae, Flaviviridae and Poxviridae. The aim of this study is to develop and validate a qPCR platform to detect potential zoonotic viral agents circulating in rodents distributed in the geographical extension of the state of Minas Gerais by qPCR. This technique was chosen for its high sensitivity and robustness considering the possibility of numerous samples. For positive controls specific sequences were chosen and assembled in gene blocks (gBlocks™), cloned into pGEM-T easy vector (Promega), and used for the validation of the designed primer pairs. Specific primers were designed for all viruses of interest including Apeu virus (APEU), Juquitiba virus (JUQV), Yellow Fever virus (YFV), St. Louis encephalitis virus (SLEV), Vaccinia virus (VACV) and Pseudocowpox virus (PCPV). Standard curves ranging from 1.0ng to 1.0fg were prepared, reactions were performed using the SYBR® Green PCR Master Mix (Applied Biosystems), and the detected efficiency for each primer pair were: 94% (APEU), 108% (JUQV), 103% (YFV), 99% (SLEV), 96% (VACV) and

96% (PCPV). The linearity coefficients (R2) were close to 1. These initial results indicate that the platform can be used for detection of the targeted viruses in field samples, which will be prepared and used as template for our reactions in the near future.

VV145 - MUTATIONS IN THE CAPSID PROTEIN OF PORCINE CIRCOVIRUS TYPE 2 INDICATE A VIRAL IMMUNE EVASIONGava, D.1; Schaefer, R.1; Serrão, V.H.B.2; Cantão, M.E.1; Silva, M.C.3; Zanella, J.R.C.1

1. Embrapa Swine and Poultry Research Center, Animal Health Laboratory, EMBRAPA

2. Institute of Physics of São Carlos – University of São Paulo-USP

3. Diagnostic Center For Animal Health (CEDISA)

Postweaning multisystemic wasting syndrome, the most common clinical manifestation of porcine circovirus type 2 (PCV2) diseases (PCVD), was first described in 1996 in Canada and in 2000 in Brazil. PCVD has caused economic losses with high mortality rate in Brazilian pig farms until the introduction of PCV2 vaccine in 2008. During December of 2012, a suspect PCVD case was reported in 57-67 days-old pigs in a vaccinated wean-to-finish farm in Southern Brazil. The affected pigs showed coughing, dyspnea, enlargement of inguinal lymph nodes, wasting and diarrhea. The objective of this study was to diagnose and characterize the PCV2 infection in a PCV2 vaccinated pig herd. Lymph node, kidney and lung tissue samples were collected and processed according to conventional methods for histopathology (H&E), real-time PCR and immunohistochemistry (IHC). DNA sequencing was performed by Sanger method. The obtained sequences were analyzed and assembled with Phred/Phrap/Consed softwares. Phylogenetic analyses of the whole genome and the ORF2 gene (capsid protein) were performed by Neighbor-Joining method in MEGA 5.2 software. Using molecular modeling methodology (I-TASSER) a structural model of the capsid protein was obtained. The structural model was validated and the mutated residues were identified. PCV2 infection was demonstrated by H&E and confirmed by IHC. A high viral load was detected by real-time PCR (5.67x1011 DNA copies/uL). Sequence analysis revealed a PCV2-b genotype. The comparison among the ORF2 amino acid sequence with other PCV2 sequences




revealed three amino acids substitutions, in domains F57I, N178S and A190T. The results of the structural analysis showed a substantial change on the physical-chemical characteristics of one residue. Moreover, the analysis also indicates a possible disruption in the secondary structure of the capsid protein around the 178 residue which is an important region for antibodies recognition. Therefore, modifications in the viral protein conformation could have caused an inefficient antibody binding that was observed by the lack of vaccine efficacy. This mechanism could explain the recent vaccine failures detected in swine in Brazil. However, additional studies using in vivo and in vitro models are needed to confirm this hypothesis.

VV155 - PESTIVIRUS CONTAMINATION IN CELL CULTURESda Silva, M.S.; Mósena, A.C.S.; Weber, M.N.; Silveira, S.; Torikachvili, M.; Dupont, P.M.; Budaszewski, R.F.; Streck, A.F.; Canal, C.W.


The genus Pestivirus of the family Flaviviridae compriseS RNA viruses that often cause diseases in ruminants and pigs. Pestiviruses contamination in cell cultures and fetal calf serum (FCS), widely used as a nutrient in cell culture, is frequently reported in the literature. Viral contaminations may negatively affect diagnostic tests, vaccine production and research. In this study, 41 cell lines of different animal origins were analyzed by the reverse transcription-polymerase chain reaction (RT-PCR) targeting the 5′ untranslated region (UTR). Eighteen (43.9%) samples were positive and the amplification products from eight were sequenced. Phylogenetic analyses revealed that five strains are closely related to BVDV-1, two to BVDV-2 and one to ‘HoBi’-like viruses. The current findings reinforce the need for a constant pestivirus control in FCS and cell cultures to keep the biological safety and correct diagnostic results. Financial support: CAPES, CNPq, FAPERGS and Propesq/UFRGS.

VV166 - BETACORONAVIRUS LINEAGE 2A DETECTION IN YOUNG ALPACAS (VICUGNA PACOS) FROM FARMS IN ANDEAN REGION OF SOUTHERN PERULuna, L.; Gregori, F.

LABMAS/USP - Laboratório de Biologia Molecular Aplicada e Sorologia. Departamento de Medicina Veterinária Preventiva e Saúde Animal da Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo – SP, 05508-270

Coronavirus is a large family of viruses that may cause a diversity of symptoms (e.g. respiratory, enteric, nervous), infecting many animal and human hosts. Considering the high mortality rate in young alpacas due to occurrence of diarrhea and data regarding the etiology is scarce, the objective of this work was to detect coronavirus in 50 stool samples of young alpacas from 3 farms located at the Andean region of southern Peru by a RT-nested PCR test targeting a fragment of RpRd gene (RNA dependent RNA polymerase) followed by nucleotide sequencing. A total of 11 samples resulted positive and the nucleotide phylogenetic analysis of sequences demonstrates that all were characterized as Betacoronavirus Lineage 2a, a group frequently found in both domestic and wild ruminants. These results confirm coronavirus circulation in the sampled area and may be implicated with the diarrhea in young alpacas.

VV167 - ANALYSIS OF ANTIBODY LIBRARY VARIABILITY FROM CHICKEN IMMUNIZED WITH BOVINE HERPESVIUS TYPE 1Japolla, G.; Almeida, R.G.; Penido, B.A.F.; Brito, D.E.M.W.; Souza, L.R.G.


The bovine herpesvirus type 1 (BoHV-1) is a member of the family Hesperviridae and is recognized as an important agent of economic loss in cattle. The economic impact caused by the BoHV-1 is expressed, among other features, by embryonic death, abortion, birth of debilitated animals, reduced reproductive efficiency of cows and bulls, poor feed conversion and growth delay in calves. In order to produce and analyze the variability of antibody library, two chickens were immunized with BoHV1 (105.5 DICC/50mL). Then the chickens were euthanized and their spleens were quickly removed




for total RNA extraction and cDNA synthesis. The light and heavy chains of antibodies genes were amplified to produce the single chain Fv fragments (scFv). The whole scFv repertoire were cloned into phagemid vectors, expressed as fusion proteins in filamentous bacteriophages and amplified by the E. coli bacteria infection. Plasmidial DNA from 15 clones was extracted and the product was analyzed in agarose gel 2%. The DNA was digested with the restriction enzyme BstNI in order to evaluate the variability of the library through the analysis of the restriction fragments. The results showed that only two of the 15 clones share the same fragment size characterizing the other 13 as unique clones with specificities theoretically distinct. In another study, this variability of antibodies generated by chickens has not been verified, probably due to immunodominânce of some antigen. In this work, the chickens didn’t live in sterile environment, getting in touch with a wide range of micro-organisms present in the environment, food and water. This contact could generate an immune response against these organisms and consequently amplification of gene fragments of antibodies without specificity to BoHV-1. In the future these clones will be used for a variety of selection and screening strategies for other purposes, such as diagnosis or for vaccine production. FINANCIAL SUPPORT: CNPQ EDITAL: MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PROCENTRO-OESTE Nº 031/2010.

VV176 - DETECTION AND CHARACTERIZATION OF PARVOVIRUSES IN STOOL SAMPLES FROM DISEASED AND HEALTHY CATSChiappetta, M.C.; Mósena, A.C.S.; Soares, M.S.; Weber, M.N.; Budaszewski, R.F.; Streck, A.F.; Canal, C.W.

LABORATÓRIO DE VIROLOGIA, FACULDADE DE VETERINÁRIA/UFRGS - Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama, 110 - Farropilhas, Porto Alegre - RS, 90040-060

The Protoparvovirus genus (formerly known as Parvovirus), from the Parvoviridae family, consists of several species of viruses extremely resistant in the environment that cause important diseases in domestic animals. Cats can be infected with feline parvovirus (FPV) that causes high mortality in kittens, leukopenia, gastroenteritis and nervous signs. Canine parvovirus type 2 (CPV-2) infection of cats was already reported

displaying similar clinical signs. However, recent studies in cats infected with CPV-2c reported marked clinical signs of hemorrhagic gastroenteritis. The aim of the study was to detect and genotype parvoviruses in fecal samples of cats with distinct clinics symptoms. Stool samples were collected from 75 cats with and without diarrhea from different veterinary clinics in Porto Alegre, Brazil. The total extraction of DNA was performed using a silica based protocol and the PCR targeted a 583 bp fragment of viral protein 2 (VP2) gene. Eight out of 75 samples (10.16%) were positive in the PCR, and two (25%) of those eight were from animals presenting diarrhea. Four positive samples were selected for DNA sequencing to determine the species and the whole VP1/2 genes were analyzed. Using BLAST analysis, two samples were similar to FPV, and the other two were closer to CPV-2c. The results showed the presence of CPV-2c in feces of cats, reinforcing that canine parvovirus can also infect cats, and that there is the possibility of transmission between dogs and cats. FINANCIAL SUPPORT: CAPES, CNPQ, FAPERGS AND PROPESQ/UFRGS.

VV180 - SEARCH FOR STUDY OF CORONAVIRUS IN WILD AND DOMESTIC BIRDS FROM ILHA DE MARAJÓBarbosa, C.M.; Araújo, J.; Góes, L.G.; Ometto, T.; Seixas, M.; Thomazelli, L.; Durigon, E.L.

Laboratory of Clinical and Molecular Virology, Instituto de Ciências Biomédicas, USP

Recently, new more virulent strains of human CoVs (HCoVs) emerged, including the betacoronavirus cause of Severe Acute Respiratory Syndrome (SARS-CoV) epidemic in Asia and a new CoV-MERS reported in the Middle East. Besides human infections, species of wild and domestic birds can also carry respiratory and enteric viruses, including coronaviruses (COVs). Recently, Infectious Bronchitis Virus- like viruses and some new CoVs genetically distinct from IBV were identified in families of birds and mammals both wild and domestic. Due to their high mutation and recombination rate during replication, CoVs are able to generate extensive genotypic variation, which facilitates adaptation to new host species so CoVs are considered not only pathogens of veterinary importance but also a threat with zoonotic potential, enhancing the surveillance for possible animal reservoirs. Therefore, this study aims to identify the species of coronavirus that are affecting




birds and establish its zoonotic potential . Initially, were analyzed 93 bird samples, taken by the field staff from the Laboratory of Clinical and Molecular Virology, in an expedition to Ilha de Marajó in the state of Pará in 2006. Those samples were collected from cloacal and orotracheal swabs, placed in cryotubes containing 500μL of VTM and kept in ultra-freezer (-80°C).at the laboratory in USP. Using Chu et al.(2011)’s methodology, the nucleic acids were extracted using “Magmax ™ -96 Total RNA Isolation Kit” and subjected to reverse transcription reaction (RT). Samples were tested by conventional PCR (Polymerase Chain Reaction) using Coronavirus protocols followed by Nested-PCR. As positive controls, samples of bovine coronavirus and avian coronavirus were used. At the moment, two samples were detected by Nested -PCR to coronavirus, considered suspicious. These samples will be further sequenced to confirm the diagnosis and correlation of findings with the known sequences of coronavirus. FINANCIAL SUPPORT: CAPES

VV186 - EPIDEMIOLOGICAL STUDY OF BOVINE ROTAVIRUS AFTER DAM VACCINATION IN AN ENDEMICALLY INFECTED DAIRY HERD IN NORTHWEST PARANAMello, J.L.; Ribeiro, A.S.; Macedo, R.; Gallego, J.C.; Kunz, A.F.; Grolli, P.; Takiuchi, E.

CAMPUS PALOTINA/UFPR - Universidade Federal do Paraná - Campus Palotina, R. Pioneiro, 2153, Palotina - PR, 85950-000

Rotavirus A (RVA) is a major cause of diarrhea in young calves. A prophylactic strategy for RVA is based on immunization of pregnant cows in order to increase the concentration of neutralizing antibodies in colostrum and milk. The aim of this work was to evaluate RVA infection in newborn calves after dam vaccination in an endemically infected dairy herd in Mariluz, northwest region of Paraná state (March 2013 to February 2014). Before introduction of dam vaccination (seven months prior the study) the selected farm presented recurrent episodes of neonatal diarrhea due RVA, showing morbidity and specific mortality and lethality rates of 39%, 13% and 33% respectively. The dams were primo-vaccinated with a commercial inactivated vaccine containing G6 and G10 RVA serotypes according to manufacturer’s instructions. After calving, 40 fecal samples were collected from calves which ages ranged from seven to 21 days. The RVA

dsRNA was extracted using a combination of phenol/chloroform/isoamyl alcohol and silica/guanidinium isothiocyanate methods and detected by silver-staining polyacrylamide gel electrophoresis (ss-PAGE) method. Of the 40 animals evaluated, the presence of RVA in feces was demonstrated in 47.5% of the samples (19/40). In addition, four animals died by dehydration and secondary infections, which two of it were attributed to the RVA. Results indicate that maternal vaccination does not prevent infection by RVA. Surprisingly, the morbidity rate increased to 48% when compared to the population of calves before the introduction of vaccination. However, there was reductions of 9, 8 and 22% in mortality rates by diarrhea, specific mortality by RVA and lethality, respectively, but with no statistical significance (p>0, 05). Morbidity rate by RVA after vaccination, have also been described by other authors and the interpretation must be cautious, because the co-infection by other pathogens was not investigated, neither the possibility of RVA infection with distinct genotypes of those contained in the vaccine. However, severe mistakes of health management of the dairy property, regarding cleaning and disinfection, animal stocking of different ages in the same stalls and not separating the animals with clinical signs of diarrhea, may have caused the constant circulation and propagation of RV, suggesting that prophylactic vaccination has low efficacy when stipulated without the right management corrections in a endemically infected dairy herd by RVA.

VV189 - CONJUGATION OF COLLOIDAL GOLD PARTICLES WITH ANTI-BOVINE IGG FOR THE DETECTION OF ANTIBODIES AGAINST BOVINE HERPESVIRUS TYPE 1Japolla, G.1; Almeida, R.G.1; Junior, C.P.J.2; Souza, L.R.G.1

1. UFG - Universidade Federal de Goiás, Av. Esperança, s/n - Setor Itatiaia, Goiânia - GO, 74001-970


The bovine herpesvirus type 1 (BoHV-1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae and genus Varicellovirus. Viruses of this subfamily are characterized by a short replication cycle, relatively large number of hosts and the ability to establish latent




infections in neurons. The bovine species is primary host for this herpesvirus, what is widely acknowledged due to the damage caused. BoHV-1 is associated with a variety of clinical disorders, including rhinotracheitis (IBR), conjunctivitis, genital infections, miscarriages and neurological disorders. For an effective control of diseases caused by this virus a correct diagnosis of the same is necessary, and this, several diagnostic techniques are being used, but none of them allows the quick diagnosis to be made in the field. In this study a conjugation of colloidal gold particles with bovine IgG was performed as a precondition for the development of a rapid diagnostic test. For this, the antibody anti bovine trade done in goat was conjugated to colloidal gold using classical methods for reduction of gold ions through the action of sodium citrate reducing agents, applying tetracloroauric acid as sources of gold ions. Next, the slot-blot test was performed in duplicate coating a nitrocellulose membrane with different dilutions of BoHV-1 (107 to 102 DICC/50ml). As negative control reaction the membrane was coated with BSA, while the positive control was purified immunoglobulin of serum of animals proved positive for BoHV-1. After coating, the membranes were inoculated with a solution containing the same immunoglobulins used as positive control (6.8mg/ml) overnigt at 19° C and washes with PBST 0.05% were carried out shortly after inoculation of bovine IgG conjugated with colloidal gold. A strong mark was observed on the test with the lowest dilution (107) and in form of decreasing intensity, visualized to the dilution of 104. At the negative control lines no staining was observed, while in the positive control lines a strong coloring was visualized, thus validating the test performed. The results obtained demonstrate the feasibility of developing a quick test for lateral flow diagnosis of infected animals for the detection of antigens or antibodies, aiding in the diagnosis of infected or vaccinated animals. FINANCIAL SUPPORT: CNPQ EDITAL: MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PROCENTRO-OESTE Nº 031/2010.

VV194 - CELLULAR IMMUNE RESPONSE OF AVIAN INFECTIOUS BRONCHITIS VARIANT BY FLOW CYTOMETRYMattos, G.L.M.1; Okino, C.H.2; Trevisol, I.M.2; Mores, M.A.Z.2; Brentano, L.2; Esteves, P.A.2; Lopes, L. dos S.2; Bastos, A.P. de A.2; Caron, L.F.3; Filho, T.F.3; Duarte, I.3; Montassier, H.J.

1. Embrapa Suínos e Aves e Aluno de Doutorado no Programa de Pós Graduação em Medicina Veterinária. Área de Concentração Patologia Animal, da UNESP - Universidade Estadual Paulista, FCAV

2. Embrapa Suínos E Aves3. Imunova Análises Biológicas e Universidade

Federal do Paraná4. UNESP - Universidade Estadual Paulista,

FCAV, Departamento de Patologia Veterinária, Programa de Pós Graduação em Medicina Veterinária

Avian infectious bronchitis virus (IBV) is an avian coronavirus that causes a highly infectious disease characterized by respiratory, reproductive and renal signs, depending on the viral tropism and results in a significant economic impact to the worldwide poultry industry. Failures of vaccine protection on the field, may be related to incomplete cross-protection between the vaccine strain and the variant strains. The present work aimed to evaluate, immune-related markers (cluster of differentiation) in the peripheral blood of birds challenged with a Brazilian field isolate of IBV (F3735 strain), by flow cytometry. SPF birds divided into three groups of 9 animals were housed in positive pressure isolators. At one day old group A was vaccinated with attenuated vaccine strain (H120) of IBV. At 28 days of age, birds from both vaccinated group A and unvaccinated group B were challenged with 10 4.0 EID50/bird of the variant strain F3735. Birds belonging to group C were not vaccinated nor challenged (negative control group). Blood samples were collected from nine birds of each group on 5 days post-infection. Leukocyte surface antigens were assessed with specific monoclonal antibodies combined (CD4/TCRαβ-Vβ1/CD45; Kul-1/MHCII/CD45; CD8α/CD28/CD45; and Bu-1a/CD45). Data were analyzed by analysis of variance using the MIXED procedure of SAS (2008) by testing the fixed effects of treatment. Comparison between means was made by Student’s t test (p ≤ 0.05). No significant differences were observed between groups A and C, and group




B presented a significantly higher immune response of, CD4+TCRvβ1+, CD4+TCRvβ1- and CD8α+CD28+ cells. The subpopulations CD4-TCRvβ1+, CD8α-CD28+, CD8α+CD28-,Kul-1+MHCII-, Kul-1+MHCII+, Kul-1-MHCII+ and Bu-1a+ did not present significant differences between all treatments. As expected, these results showed an increased population of naïve and activated T helper (CD4+TCRvβ1- and CD4+TCRvβ1+) cells and naïve or memory T cytotoxic (CD8α+CD28+) cells in blood of unvaccinated and IBV challenged birds, whereas the immune cells dynamics in the vaccinated/challenged group compared to the negative control group (not unvaccinated/not challenged) showed no significant differences, suggesting immune protection against challenge conferred by vaccination, and a systemic exacerbated response mediated by both naïve and activated T helper cells and naive T cytotoxic cells.

VV205 - FREQUENCY OF ARENAVIRUS ANTIBODY IN WILD RODENTS CAPTURED IN MIDWEST, SOUTHEAST AND SOUTH REGIONS OF BRAZILOliveira, A.L.R.1; Bisordi, I.1; Souza, R.P.1; Levis, S.2; Pereira, L.E.1; Montoro, M.G.1; Barbosa, V.M.1; Suzuki, A.1; Timenetsky, M.C.S.T.3

1. Center for Disease Vector Transmission, Adolfo Lutz Institute

2. Dr. Julio Maiztegui3. Center Of Virology, Adolfo Lutz Institute

Viral hemorrhagic fevers caused by arenavirus are emerging and severe illnesses. Humans are accidentally infected mainly by mucosal exposure to excretions from chronically infected wild rodents. The presence of antibodies in reservoirs species may indicate viral circulation in a population of a specific region. The natural reservoir of the Sabia virus, the etiologic agent of Brazilian hemorrhagic fever, remains unknown even after the occurrence of human cases in Cotia and Espírito Santo do Pinhal counties in São Paulo State. This study aims to search for wild rodents possibly associated with arenavirus transmission. We randomly selected 1.242 rodents blood samples taken from a bank of biological samples formed during epidemiological surveillance for hantavirus and analyzed by ELISA for the detection of specific IgG antibodies to arenaviruses. The selection was performed using a randomizing algorithm available at www.randon.org. The results show that

1.234 (99.4%) were not reagent, but 8 (0.6%) samples tested were positive for IgG antibodies for arenavirus. Positive results were detected in the following rodent species: 3 Necromys lasiurus and 2 Calomys callosus from Bodoquena county of Mato Grosso do Sul State, 1 Necromys lasiurus from Campo Alegre de Goiás from Goiás State and 2 Calomys tener from Espírito Santo do Pinhal in São Paulo State. Hemorragic fevers caused by arenaviruses are considered endemic diseases, occurring in restricted geographical areas, but the species of rodent reservoirs may have wider geographic distribution. Search for the circulation of these viruses in rodents captured in natural habitats is important for better understanding of the relationship between the virus and its host, as well as to support the epidemiological surveillance and control of this zoonosis.

VV212 - ACTIVE AND PASSIVE IMMUNE RESPONSE INDUCED BY COMERCIAL VACCINE AGAISNT BOVINE ROTAVIRUSESGodoy, H.P.1; Mazer, L.C.1; Gonçalves, A.C.S.1; Godoy, H.P.1; Sepulveda, L.2; Samara, S.I.1; Buzinaro, M.G.1


2. Ouro Fino

The aim of this study was evaluated the active and passive immunity against rotavirus induced by vaccination with an inactive commercial vaccine in cows and their respective calves from a farm of Cravinhos, state of São Paulo. The analysis of IgG, IgG1, IgG2 , IgM and IgA was done in serum 60 days before calving (before vaccination); 30 days before calving (before revaccination) and in the day of calving, with indirect ELISA. The levels of IgG, IgG1, IgG2, IgM and IgA in colostrum were evaluated in the day of calving, with the same technique. The levels of calves’ immunoglobulin were evaluated in the day of born and 1, 7, 21 and 28 days of age. The excretion of rotavirus was evaluated in fecal samples of calves by SDS-PAGE. In this study, levels of IgM and IgA were increased in cows that were not vaccinated, and their calves, fed their colostrums, have levels of IgM increased one day after born. Vaccinated cows have serum IgG1 and colostral IgG2 higher than non vaccinated cows but statistics differences in levels of immunoglobulin were not found




in calves. In both groups, was detected rotavirus in feces of calves, and besides the low occurrence in this study, one calf of vaccinated cow excreted rotavirus in Day 21, while two calves born of non vaccinated cows excreted rotavirus with 7 and 14 days. In conclusion, the vaccination of cows with a commercial inactivated vaccine did not prevent the rotaviruses in calves.

VV227 - OCCURENCE AND GEOGRAPHIC DISTRIBUTION OF BOVINE ENZOOTIC LEUKOSIS (BEL) POSITIVE HERDS IN SÃO PAULO STATEOliveira, L.G.; Almeida, H.M. de S.; Borges, L.; Gatto, I.R.H.; Medeiros, A.S.R.; Oliveira, L.G.; Samara, S.I.

FCAV/UNESP - Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, SP, 14884-900

Bovine Enzootic Leukosis (BEL) is an infecto-contagious disease caused by a virus of Retroviridae family. The disease, which has chronical evolution, causes malignant lymphoma in cattle and also the proliferation of lymphoid cells. This study focused on analyzing the geographic distribution and prevalence of positive BEL cases in São Paulo state. A total of 5,916 bovine serum samples received between the years of 2011 and 2013 from 20 different municipalities of São Paulo state were tested for BEL using the immunodiffusion in agar gel test at Universidade Estadual Paulista – UNESP Jaboticabal. The results were analyzed using the software Terraview® version 4. 2. 2. Statistical analysis such as Moran index and Local Moran Index (LISA) were used to determine if there was spatial autocorrelation between positive cases and the region they occur. In the years of 2011, 2012 and 2013 were detected 123 (7.48%) positive samples out of 1,645 tested samples, 107 (3.22%) out of 3,324 and in 16 (1.69%) out of 947, respectively. In general, out of 5,916 tested samples, 246 were positive, showing a prevalence of 4.16%. Only 62 or 1.05% of total samples were considered suspect at the immunodifusion agar gel test. All positive animals were female, except in one case, with age ranging from 12 months to 8 years; this occurs mainly because female animals are kept for a longer period in the farm due to reproductive reasons. Living longer increases the chances of chronic manifestation of the disease. The Moran Index value of 0.17615 with p value of 0.01 showed no spatial autocorrelation among positives cases. However the LISA analysis showed that

there could be an evidence of spatial autocorrelation between positive cases and the geographic region. The disagreement between the statistical tests is probably caused because of the lack of data from other more cities of the neighborhood, so further regional studies are required. In conclusion, it was possible to show that BEL is present in bovine herds of São Paulo state, and affects mostly females. The central west area of the state is where the majority of positive herds are, which is very concerning once bovine breeding is one of the region’s main economical activity. FINANCIAL SUPPORT: CNPQ

VV230 - RABIES VIRUS PHOSPHOPROTEIN MRNA IS AN EFFECTIVE TARGET TO SIRNA-MEDIATED RNAI ANTIVIRAL TREATMENT IN VITRODurymanova Ono, E.A.; Brandão, P.E.

FMVZ/VPS/USP - Faculdade de Medicina Veterinária e Zootecnia, Departamento de Medicina Veterinária Preventiva e Saúde Anima da Universidade de São Paulo, Av. Duque de Caxias Norte, 225. Pirassununga - SP, 13635-900

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus: Rabies Virus). The search for antivirals against rabies is one of the frontiers on the field but, despite a protocol (the Milwaukee Protocol) based on ketamin, ribavirin, midazolam and amantadin was successful after the treatment of a human patient, it was shown as not reproducible. RNA interference is an alternative as an antiviral technology against RABV already shown as effective in vitro in cell cultures and in vivo in the mouse model. Most of the researchers on this field have used siRNAs targeting nucleoprotein, glycoprotein or RNA polymerase (large) protein mRNAs, but from until now there are no reports on RNA interference for knock down of RABV phosphoprotein P, which is a multifunctional protein in its interaction with N and a key component of the virion-associated RNA polymerase complex as a regulatory protein in viral genome replication. The aim of this study was to assess the decrease in the titer of rabies virus in vitro using stealth short-interfering RNAs against P mRNA. To this end, three siRNAs were used with antisense strands complementary to rabies virus P mRNA. BHK-21 cells monolayers were infected with 10 to 0.01 TCID50% of PV and AgV3 (Desmodus Rotundus, antigenic variant




3); after 2 hours of virus adsorption, the cells were transfected with each of tree stealth siRNAs (in separate assays each) using Lipofectamine-2000™ and RABV direct immunofluorescence assay was performed after 24h of transfection. All three siRNAs reduced the titer of PV and AgV3 strain, with the higher drop in titer regarding siRNA 360 treatment (0,4log drop for Pasteur Virus and 1,0log drop for AgV3). No cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000™. These results show that the phosphoprotein of rabies virus can be use like an effective target for antiviral strategy for rabies.

VV246 - SEROLOGICAL SURVEILLANCE FOR AVIAN INFLUENZA AND INFECTIOUS LARYNGOTRACHEITIS IN GENETIC MATERIAL FROM BRAZILIAN POULTRY EXPORTS DURING 2013Luciano, R.L.; Cardoso, A.L.S.P.; Tessari, E.N.C.; Stoppa, G.F.Z.; Kanashiro, A.M.I.; Castro, A.G.M.

CAPTAA/INSTITUTO BIOLÓGICO - Centro Avançado de Pesquisa Tecnológica do Agronegócio Avícola do Instituto Biologico, Avenida Conselheiro Rodrigues Alves, 1.252, São Paulo - SP, 04014-002

Brazilian poultry industry ranks important position in the international scene. Currently, the country kept its position as largest exporter and the third principal producer of poultry meat. In respect this, Brazil has exported genetic material from poultry (breeders, grandparent breeders and great grandparent breeders) to many countries, so that is extremely important to certify that these material obey the sanitary requirements ordered by the International Zoo-sanitary Code (IZC). All exporting products must having the international zoosanitary certificate (IZC) according with the establishment of requirements by the importing country. Avian influenza (AI) and Infectious laringotracheitis virus (ILT) are notifiable diseases to the World Organization for Animal Health (OIE) and have been causing great losses to industrial poultry breeding in many countries around the world. One of techniques adopted by OIE to diagnosis of avian influenza (AI) and infectious laryngotracheitis (ILT) is agar gel immunodiffusion test (AGID). The Centro Avançado de Pesquisa Tecnológica do Agronegócio Avícola (CAPTAA), Instituto Biológico (IB), located in Descalvado, SP, Brazil is a laboratory accredited by the Ministry of Agriculture,

Livestock and Food Supply (MAPA) to perform the serological diagnostic to AI and ILT. During 2013, 14.728 serum samples from breeders, grandparent breeders and great grandparent breeders destined for exportation were forwarded to CAPTAA and submitted to the serological diagnosis to AI and ILT by IDGA tests. The tests were perform according the OIE procedures. These samples were from three Brazilian states: Sao Paulo, Minas Gerais e Rio Grande do Sul, and corresponding to 381 flocks: 57 flocks from great grandparent breeders (11.61% of the total samples analyzed), 187 flocks from grandparent breeders (60.48%) and 137 flocks from breeders (27.91%), respectively. There was no detection of antibodies against AI and ILT in any of the flocks, which contributes to attest that the health status of these samples, intended to exporting of Brazilian poultry genetic material was considerate free to AI and ILT. These results are important to epidemiological point of view to demonstrate the high quality level in the Brazilian avian genetic production.

VV252 - STANDARDIZATION OF A CONVENTIONAL PCR FOR DETECCION OF BOHV-1 AND 5 IN SEMENde Carvalho, I.L.Q.1; Gasparini, M.R.2; Leite, R.C.2; Stancioli, E.F.B.1

1. ICB/UFMG - Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte - MG, 31270-901

2. VET/UFMG - Escola de Veterinária da Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte - MG, 31270-901

Bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) are important animal pathogens, causing sanitarian and economic losses in cattle herds. The BoHV-1 is responsible for various clinical manifestations, including respiratory and reproductive tract diseases and generalized infection in young cattle. Beyond the symptoms already mentioned, the BoHV-5 causes non supurative meningoencephalitis in bovines. These viruses are transmitted via body fluids like semen, being the artificial insemination an important contagion form for the epidemiology of these pathogens. It is estimated that in 2013 about 14 million doses of semen were commercialized in Brazil, which justifies that the World Organization for Animal Health (OIE) recommends testing the semen production for the presence of these




viruses. This work aimed the development of a PCR test for the rapid and specific detection of BoHV-1 and 5 in bovine semen samples. The assay was done using the genetic regions which codify the glycoproteins B (gB) and G (gG). The detection sensitivity was analyzed in culture of kidney bovine cells CRIB and in semen, both experimentally infected with the viruses. In the amplification of the gene which codifies the gG (~500 bp) the sensitivity of detection of the virus in cell culture was 5,128 TCID50 for BoHV-1 and 5,75 TCID50 for BoHV-5. In experimentally infected semen the sensitivity was 51,28 TCID50 and 57,5 TCID50 for BoHV-1 and BoHV-5, respectively. The analytical sensitivity using recombinant plasmids was 300 ag for BoHV-1 and 3 fg for BoHV-5. In the amplification of the gene which codifies the gB (96 bp) the detection sensitivity of the virus in cell culture was 5,128 TCID50 and 0,575 TCID50, and in experimentally infected semen was 51,28 TCID50 and 5,75 TCID50 for BoHV-1 and BoHV-5, respectively. The reaction specificity standardized was tested using Equid herpesvirus 1 (EHV-1), Equid herpesvirus 4 (EHV-4), Suid herpevirus 2 (SuHV-2), Ovine herpesvirus 2 (OvHV-2) e Human herpesvirus 1 (HHV-1) which shows its high specificity and efficiency. After an initial standardization, 54 clinical semen samples were tested and the verified positivities were 54.4% (gG) and 92,6% (gB). The developed tests presented excellent performance, which shows its possible utilization for semen screening in control programs.

VV256 - MOLECULAR DIAGNOSIS OF PORCINE CIRCOVIRUS TYPE 2 (PCV-2) AND TORQUE TENO SUS VIRUS TYPE 1 AND 2 (TTSUV1 AND TTSUV2) IN SWINE HERDS FROM RIO DE JANEIRO STATE, BRAZILCastro, T.; Cruz, A.C.; Silveira, R.; Baez, C.; Silva, S.; Dias, J.; Varella, R.; Castro, T.


Porcine Circovirus type 2 (PCV-2) and Torque teno sus virus (TTSuV) are emergent in swine herds and endemic in many swine-producing countries. These agents were already described in Brazil, causing severe economic impact on swine production although the information concerning to these viruses circulation in Rio de Janeiro state is absent. The aim of this study was to perform the molecular diagnosis of PCV-2, TTSuV-1 and TTSuV-2

from commercial herds in Rio de Janeiro State, Brazil. The clinical signs presented by the infected animals were also reported. A total of 201 pigs from 21 farrow to finish herds located in Rio de Janeiro State were studied. A Clinical examination and weighing were performed before blood collection. DNA was extracted from serum samples by High Pure Viral Nucleic Acid Kit, ROCHE® and submitted to polymerase chain reaction (PCR) and Nested PCR using primer sets targeting the VP2 gene of PCV-2 and non-coding region gene of TTSuV1 and TTSuV2. All the PCR amplicons were sequenced and confirmed using the BLAST tool. PCV-2 DNA was detected in 28/201 serum samples; TTSuV-1 in 28/201; and TTSuV2 in 25/201 samples. Co-infection was reported in 50/201 animals: TTSuV1 and TTSuV2 in 37/201 pigs; TTSuV1 and PCV-2 in 4/201 pigs; TTSuV2 and PCV-2 in one pig and triple infection (TTSuV1, TTSuV2 and PCV-2) in eight pigs. The majority of the co-infected animals (27/50) did not showed any clinical signs. Eleven showed respiratory signs (sneezing and respiratory sounds); five showed enteric signs (diarrhea and presence of blood in feces) and seven showed systemic signs (sneezing, blood in feces and arthritis). PCV-2 is the primary agente causing postweaning multisystemic wasting syndrome (PMWS). Nevertheless, the literature suggests that the co-infections with other vírus as TTSuVs can be associated with more severe clinical signs. Furthermore, TTSuV1 was recently associated with clinical porcine respiratory disease complex (PRDC), which suggests that this virus might play a role in the development of this complex. Our data indicates that PCV-2, TTSuV1 and TTSuV2 are circulating in swine herds from Rio de Janeiro and cases of co-infection with PCV-2 and TTSuV may be common in clinically normal pigs, hindering the adoption of biosecurity measures in such populations. FINANCIAL SUPPORT: FAPERJ

VV257 - MOLECULAR CHARACTERIZATION OF FELINE HERPESVIRUS TYPE 1 (FHV-1) AND FELINE CALICIVIRUS (FCV) INFECTING DOMESTIC CATS FROM RIO DE JANEIRO, BRAZILCastro, T.; Baumworcel, N.; Soares, A.; Silva, S.; Almeida, N.; Castro, T.





Feline conjunctivitis can be caused by several pathogens, leading to similar clinical signs of disease. Two viruses in particular, feline calicivirus (FCV) and feline herpesvirus 1 (FHV-1), are responsible for at least 76% of the conjunctivitis cases. These agents are widespread in the domestic cat population, despite the use of the vaccines. Both viruses are related to persistent infections due to their mechanism of evasion of the immunologic system (latency and high genetic variability). In this study, 108 ocular discharge samples were obtained from unvaccinated kittens. The genome extraction was performed immediately after collection using the PureLink™ Spin Column-Based Kit (Invitrogen®). For FHV-1 detection, a fragment of 287 bp of the thymidine kinase enzyme (TK) gene was amplified by PCR. For FCV detection, reverse transcriptase PCR (RT-PCR) using random primer (Invitrogen®) was performed. These products were subjected to a Nested-PCR reaction, using internal primers able to amplify a fragment of 467 bp. A commercial vaccine Felocell CVR-C (Pfizer Animal Health, USA), was used as a positive control. FHV-1 DNA was detected in 62/108 (57,4%) samples. FCV genome was detected in 40/108 (37,0%) samples. Co-infection of FHV-1 and FCV infection was detected in 20/108 samples (18,5%). All the PCR amplicons were sequenced and nucleotide (nt) similarity with sequences deposited in the GenBank database was assessed using the BLAST tool. The phylogenetic analysis of TK FHV-1 gene revealed an unique cluster comprising the FHV-1 strains from this study with others FHV-1 from Brazil. The phylogenetic analysis of RdRp FCV gene showed two clusters: one comprising only strains from this study and another one grouping strains from this study with strains from around the world. This is the first molecular characterization of FCV and FHV-1 in specimens from cats in the State of Rio de Janeiro and revealed that these viruses are circulating in the domestic feline population. FINANCIAL SUPPORT: FAPERJ

VV259 - IMPLEMENTATION OF NEXT GENERATION SEQUENCING TECHNIQUES FOR PATHOGEN DISCOVERY IN CEREBROSPINAL FLUID OF PATIENTS WITH ENCEPHALITIS AND MENINGITISNunes, C.F.; Soares, A.C.; Urbano, P.R.; Gerhardt, D.; Romano, C.M.

IMTSP - Instituto de Medicina Tropical de São Paulo,

Av. Dr. Enéas de Carvalho Aguiar, 470, Jardim América, São Paulo - SP, 05403-000

The central nervous system (CNS) may be affected by several agents, including viral bacterial or fungal. CNS infections can trigger severe symptoms and, according to the site of infection, maybe designated as encephalitis or meningitis. Viruses are the most common cause of these diseases, followed by bacteria and fungus. Agents such as polyomavirus, Herpesvirus (Simplex, 6 and varicella-zoster) influenza A, enterovirus, mumps, flavivirus, M. tuberculosis and C. neoformans, are responsible for most of the CNS infections, and have a high incidence worldwide. However, prevalence of different agents varies according to population, individual’s immune status, age and region of study. In fact, the prevalence of these infectious agents is well established in many countries. However, there is a lack of information regarding the prevalence of these agents in the Brazilian population. Although there are diagnostic methods available for identifying most of the etiologic agents that cause encephalitis and meningitis (EM), to obtain results in short period is essential for targeting the most appropriate treatment of the disease. Here we use next generation technology (Ion Torrent) to investigate putative microbial agents in CNS infections through metagenomic approaches in liquor. The advantage of this technique over traditional ones is the capability to detect not only known agents, but also identify pathogens not commonly associated to these pathologies in a fast way. Samples (n=4) from patients with suspected viral EM is were spiked before extraction with baculovirus and bovine viral diarrhea viruses as a control for acid nucleic extraction. After a set of centrifugation steps to exclude human cells, genomic DNA/RNA was obtained using Macherey Nagel commercial kit. Reverse transcription was performed with random primers using High Capacity (Applied Biosystems) kit. Double-stranded DNA was synthesized using Klenow enzyme Fragment (3’>5 ‘exo-) (NEB), followed by NGS sequencing. Filtering, trimming and assemble of the reads were performed in CLC genomic workbench software. Our methods appear to be efficient since small reads of viruses used to spike the samples as internal control were recovered during analysis. In one sample investigated, around 150 contigs of Burkolderia (bacterial agent) were assembled. In conclusion, we demonstrated that the metagenomic




methods implemented in the present work are capable to detect viral and bacterial agents affecting the CNS.

VV261 - ANTI-HCV ACTIVITY OF THE TOXIN PLA2-CB FROM VENOM OF CROTALUS DURISSUS TERRIFICUSShimizu, J.F.; Russo, R.; Cintra, A.C.O.; Sampaio, S.V.; Aquino, V.H.; Rahal, P.; Jardim, A.C.G.


Hepatitis C is a disease caused by the Hepatitis C virus (HCV) infection and represents a public health problem worldwide. There is no vaccine for HCV and the current therapy is not effective for all treated patients, is expensive and presents many side effects. Thus, there is an evident need to develop more efficient therapeutic approaches. In this context, natural compounds can provide an alternative source for the identification of products with therapeutic potential. toxins extracted from animal venoms have shown antiviral activity for other viruses such as dengue virus, yellow fever, measles, among others. Therefore, this study aims to evaluate the effects of PLA2-CB, a subunit of crotoxin complex extracted from Crotalus durissus terrificus, on HCV replication in vitro. Huh 7.5 cells line stably expressing subgenomic replicon SGR-FEO of HCV were treated with different concentrations of each compound for 48 hours, and viral replication and cell viability was measured by MTT and luciferase assays, respectively. The results showed that the treatment of the cells with PLA2-CB concentrations of 6 µg / mL and 4 µg / mL showed a reduction of approximately 58% in replication, and presented an EC50 of 5.7 µg / mL. Western blot analysis also demonstrated a reduction of virus NS5A protein expression in treated cells as a result of replication inhibition. Our data showed that the PLA2-CB presents promising results in inhibiting viral replication. However, more studies are needed for a better understanding of how these compounds act in the machinery of HCV. FINANCIAL SUPPORT: FINANCIAL SUPPORT: FAPESP (2011/11753-4) E (2013/03897-1)

VV274 - EVALUATION OF AN INDIRECT ELISA FOR FELINE CORONAVIRUS (FCOV) USING A RECOMBINANT NUCLEOCAPSID (N) PROTEIN BY COMPARISON WITH A COMMERCIALLY AVAILABLE IMMUNOASSAYAlmeida, A.C. da S.1; Filoni, C.1; Hora, A.S.2; Brandâo, P.E.2; Araújo Jr., J.P.1

1. IBB/UNESP - Instituto de Biociências da Universidade Estadual Paulista, Bairro: Distrito de Rubião Junior S/N, Botucatu - SP, 18618-970


Feline coronavirus (FCoV) belongs to the Coronaviridae family, Nidovirales order and seroprevalence range up to 90% in domestic cats and their wild counterparts. Approximately 5 to 10% of FCoV exposed felids develop the feline infectious peritonitis, an immune-mediated, systemic, progressive, and fatal disease that is considered one of the most important infectious diseases that affects felids. The aim of this study was to evaluate the indirect ELISA (previously standardized with the use of recombinant FCoV nucleoprotein) based on the comparison with the commercial ImmunoComb® FCoV Antibody Test Kit for anti-FCoV antibodies detection in sera from naturally infected cats. Serum samples from 111 cats were tested by the commercial immunoassay and ELISA. The accuracy of the indirect ELISA relative to commercial kit was 94.7%, the relative sensitivity 85.9% and the relative specificity 75% at the cut-off of calculated as two standard deviations higher than the arithmetic mean of the absorbance of negative samples by the reference test (0.41 OD). The indirect ELISA using the recombinant FCoV nucleoprotein is a new, safe, and rapid diagnostic quantitative method that can be used at reasonable low costs for measuring the precise amounts of specific antibodies against FCoV.

VV275 - MOLECULAR DIAGNOSIS OF VIRAL AGENTS IN DIARRHEIC PUPPIES IN RIO DE JANEIRO, BRAZILCosta, E.M.; Pimentel, F.B.; Domingues, C.F.; Bottino, F. de O.; Castro, T.X.; Garcia, R. de C.C.


Canine parvovirus (CPV) and canine coronavirus (CCoV) are considered to be the main pathogens responsible




for acute gastroenteritis in young dogs. Recently, canine calicivirus (CaCV), canine astroviruses (CaAstV) and Group A-Rotavirus (RV-A) have also been reported as potential viral pathogens. This study was carried out to identify the viral agents associated to gastroenteritis in puppies in Rio de Janeiro,Brazil using molecular methods. Fecal samples collected between January 2008 and March 2014 from 254 privately owned puppies with diarrhea were tested. Genomic RNA/DNA was extracted from 10% fecal suspensions using the PureLink™ Spin Column-Based Kit (Invitrogen®). For CPV detection, PCR was performed with primers 555F/555R (4003-4585) which amplify a 593bp fragment of the VP2 capsid gene. For RNA-viruses, the reverse transcription was performed with random primer (Invitrogen®) and Superscript III enzyme (Invitrogen®). Differential primers were used in PCR assays for detection of CCoV (gene M), CaCV (RdRp gene), AstV (RdRp gene) and RV-A (NSP-4 gene). Sequencing was performed in PCR-positive samples for molecular characterization. A total of 91 samples were negative for all agents. Single infection was detected in 135 puppies (CPV=120, CCoV= 10, AstV=2, RV-A=2, CaCV=1). Mixed infections were detected in 31 puppies: 20 cases of CPV/CCoV, three of CPV/RV-A, one of CCoV/CaCV, one of CCoV/AstV, one of CCoV/RV-A, four of CPV/CCoV/AstV and one of CPV/CCoV/RV-A. Among 166 positive puppies 52 had received at least one doses of polyvalent vaccines. Based on clinical reports, 76/135 puppies with single infection and 10/31 puppies with mixed infection presented more severe clinical signs (vomiting, hemorrhagic diarrhea). For the remaining puppies the association of clinic signs was variable (vomiting and/or nonhemorrhagic diarrhea). Genotyping results confirmed that CPV-2b is still the most prevalent type circulating in Rio de Janeiro. Based on partial sequencing of M gene, eight strains were characterized as CCoV-I while another 16 as CCoV-II. The CaCV positive sample shared higher nucleotide identity with the Vesivirus genus. In conclusion, the results of this study suggest that puppies with acute gastroenteritis should be screened for AstV, CaCV and RV-A as well as the established pathogens CPV and CCoV. Financial support: FAPERJ, CNPq, PROPPI-UFF, PROGRAD-UFF

VV294 - CLINICAL SIGNS, TRACHEAL CILIOSTASIS AND DETECTION BY QRT-PCR OF BRAZILIAN IBV VARIANTS OF CHICKEN AND PIGEON ORIGIN IN CHICKENSMartini, M.C.1; Caserta, L.C.1; Santos, M.M.A.B.2; Barnabé, A.C.S.1; Padilla, M.A.1; Neto, D.F.L.1; Ferreira, H.L.3; Arns, C.W.1




In a previous study, a pigeon coronavirus grouped with the H120 (Massachusetts) IBV strain and a chicken coronavirus characterized as BR-I IBV strain were detected and characterized by our group. With the present study we aimed to detect by qRT-PRC and analyzee the clinical signs and tracheal ciliostasis lesions induced by these viruses in experimentally infected chickens. To do so, three groups named Corona-PigeonMass (67T), Corona-ChickenBR-I (801) and Corona-ChickenBR-I (810), containing 25 animals per group, were inoculated by intranasal route with 100mL containing 105.5 EID50 of the respective strain. A negative control group inoculated with PBS solution was evaluated. The clinical signs were observed during 21 days. Samples and secretions were collected from five inoculated chickens per group at 4, 7, 9, 11, 14 and 21 days post inoculation (dpi) at necropsy. Viral RNA was investigated by qRT-PCR assay for the UTR gene of IBV. The results showed that coronaviruses were detect at most around 5th dpi in sinus, trachea, ileum, kidney and tonsil; in cloaca and testicle, coronaviruses were detected a bit earlier (2th and 4th dpi, respectively) and in pro-ventricle and lung a bit latterly (7th and 11th dpi respectively) by qRT-PCR in all inoculated groups. Clinical signs such as respiratory problems, sneezing, lethargy and nasal discharge were observed mainly at 5th dpi in all inoculated groups. A complete ciliostasis was observed (0% of active cilia) at 4, 7 and 9 dpi in the 67T group; whereas 50% of the cilia activity was recovered at 14 dpi. As for the 801 and 810 groups a complete ciliostasis was observed slight later at 9 and 11 dpi; while the 50% of cilia activity was also




recovery at 14 dpi. Our results suggests that Coronavirus strains of chicken and pigeon origins can induce a complete ciliostasis in chickens, supporting the findings for clinical signs and reiterate the pigeons’ importance in the dissemination and evolution of avian coronavirus. Financial Support: CAPES, CNPq (475571/2012-6) and FAPESP (Grant 2013/02058-6).

VV308 - DETECTION OF CYNOMOLGUS CYTOMEGALOVIRUS DNA IN BRAZILIAN MACACA FASCICULARISStangherlin L.M.1; Santos, E.S.1; Pinto, M.A.2; Silva, M.C.C.1

1. UFABC - Universidade Federal do ABC, Rua Catequese, 242 - Jardim, Santo André - SP, 09090-400


Cytomegaloviruses (CMV) are highly specie-specific members of the Betaherpesvirinae sub-family which have been isolated from various mammalian species. The Human Cytomegalovirus (HCMV) is associated with serious diseases in immunocompromised individuals, such as transplant recipients and AIDS patients. HCMV is also a cause of severe neurologic abnormalities and hearing loss in infected newborns. One of the limitations of studying CMV is the fact that HCMV does not infect animals, due to the characteristic specie specificity of Beta-herpesviruses. Consequently, studies in vivo are performed in CMVs of the respective mammal species. Due to their close relationship to humans, nonhuman primates are considered the ideal research model for these studies. The Rhesus Cytomegalovirus CMV (RhCMV) is being characterized regarding to its genomic similarity to HCMV and pathogenicity in vivo. Thus, Rhesus macaques (Macaca mulatta), are primarily used in the CMV studies. However due to the high demand and the short the supply of Rhesus monkeys other monkey species are becoming of interest for these studies. Cynomolgus Monkeys (Macaca fascicularis) are infected with cynomolgous cytomegalovirus (cyCMV), as shown by serological studies. Recently a cyCMV has been isolated and completely sequenced, providing a novel model for studies to address fundamental questions of HCMV biology, pathogenesis and immunology. This study aims to investigate the presence and natural prevalence of cyCMV infection in the cynomolgous monkey colony

of the Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro. Sera from cynomolgus monkeys are being tested for the presence of cyCMV DNA by real time PCR (qPCR) and by nested PCR using primers for the presence of cyCMV gB gene. Our results show that a large percentage (77%) of the animals are positive for HCMV DNA. The determination of the presence and incidence of cyCMV infection in cynomolgous will lead to further studies of the molecular epidemiology, characterization of CMV infection in vivo and virus evolution, as well for the identification of CMV free animals that can be kept separated from the rest of the animals and used in studies of CMV infection, pathogenesis, interaction with other pathogens and vaccine testing. Funding: UFABC

VV312 - SUSCEPTIBILITY OF HAMSTERS TO EQUINE HERPESVIRUS TYPE 1 INFECTION CAUSING ENCEPHALITIS AND RESPIRATORY DISEASEArévalo, A.F.1; Mori, E.3; Mori, C.M.C.; Miyashiro, S.I.; Zanatto, D.A.; Cunha, E.M.S.; Lara, M. do C.C. de S.H.; Viillalobos, E.M.C.; Tonietti, P. de O.; Gamon, T.H.M.; Maiorka, P.C.


2. INSTITUTO BIOLÓGICO, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002


The mouse has proved to be an excellent model for studying the pathogenesis of encephalitis caused by neuropathogenic strains of equine herpesvirus type 1 (EHV-1). However, recent studies showed that intranasal infection in hamsters resulted in more acute and severe symptoms than those observed in mice, suggesting greater susceptibility of the specie to the agent. To evaluate respiratory and neurological changes resulted from EHV-1 infection in hamsters. Material and Methods: Male Syrian hamsters, 3-wk-old, were infected via intranasal route with EHV-1 strains obtained from infected aborted foal fetuses (A4/72, A9/92, A3/97 and Iso/72). The animals were weighed and examined daily for clinical and neurological signs. According to the symptoms, groups of five hamsters were euthanized by overdose of isoflurane or intraperitoneal ketamine/




xylazine injection. During necropsy, CNS, lungs, liver, spleen and thymus were collected for virus isolation in E-dermal cells culture. In addition, bronchoalveolar lavage (BAL) was performed to determine total and differential white blood cell (WBC) count. Results: Similar to the experiments with mice, hamsters challenged with A4/72 and A9/92 strains showed severe clinical manifestations in the 3rd d.p.i., such as weight loss, dyspnea, dehydration, recumbency and death. It was also observed neurological signs such as hyperexcitability, spastic paralysis, loss of proprioception, circling and seizures. However, unlike the mice, which did not developed the disease; hamsters inoculated with A3/97 and Iso/72 strains showed clinical and neurological symptoms in the 4th d.p.i., in which the most evident were respiratory changes, mainly epistaxis. Virus isolation from the CNS was positive in all animals; however, lungs were positive only in the groups infected by strains A9/92 and A4/72. BAL showed that WBC count was higher in A4/72 infected hamster when compared with A9/92, A3/97 and Iso/72 and control group. Activated macrophages with large vacuolated cytoplasm, some of them containing granules within, and a great amount of erythrocytes were observed in all inoculated animals’s smears unlike the control smears, which had only inactivated cells and rare erythrocytes. Conclusion: The results indicate that hamster is the most susceptible specie to infection caused by EHV-1, serving as an experimental model for studies of respiratory and neurological disorders caused by this agent.

VV315 - GENETIC STABILITY OF RABIES VIRUS ISOLATED FROM VAMPIRE BAT DESMODUS ROTUNDUSPereira, P.M.C.1; Fernandes, M.E.S.1; Achkar, S.M.1; Oliveira, R.N.1; Castilho, J.G.1; Roehe, P.M.2,3,4; Carnieli Jr, P.1; Batista, H.B.C.R.1


2. IPVDF - Instituto de Pesquisas Veterinárias Desidério Finamor, Estrada Municipal do Conde, 6000. Eldorado do Sul - RS, 92990-000

3. FEPAGRO - Fundação Estadual de Pesquisa Agropecuária, Fundação Estadual de Pesquisa Agropecuária, R. Gonçalves Dias, 570, Porto Alegre - RS, 90130-060


Rabies is a fatal disease that infects the Central Nervous System (CNS) of mammals. Rabies virus (RABV) is a RNA non-segmented and single-stranded virus, member of the Lyssavirus genus within the Rhabdoviridae family. Despite the recognized stability of RABV, differences among isolates from different species have been found. Members of the Chiroptera order are reservoirs of many lissaviruses and the vampire bat Desmodus rotundus became the most important reservoirs of RABV in Latin America. Rabies transmitted by bites from D. rotundus, affects cattle and sporadically humans causing economic losses in livestock and in public health. This work was carried out with the aim to identify the stability of genetic lineage of RABV from vampire bat D. rotundus. For this, a group of six Swiss Webster mice was inoculated with RABV strain isolated from vampire bat D. rotundus (first passage). The CNS of mice were collected, a new suspension was prepared and inoculated in another group of mice (second passage) this procedure was repeated by ten successive passages. The inoculation was made by intracerebral route and the animals were maintained with water and food at libidum. The CNS strains of first, fifth and tenth passages were submitted to RNA extraction, RT-PCR and sequencing of the nucleoprotein gene (N) of the virus. None difference in clinical signs of rabies after ten successive passages was identified in inoculated mice. In addition was not found significant differences in the genetic sequence of N gene. Our results show that genetic lineage of RABV from vampire bat D. rotundus is stable after 10 successive passages in mice. In conclusion high genetic stability of RABV recovered from vampire bat D. rotundus, was confirmed and other genes will be analyzed to confirm this hypothesis. FINANCIAL SUPPORT: FAPESP




VV317 - MOLECULAR ANALYSES OF VP6, VP7, VP4, AND NSP4 GENES OF PORCINE ROTAVIRUS GROUP H DETECTED IN BRAZILPossatti, F.1; Molinari, B.L.D.1; Possatti, F.1; Lorenzetti, E.1; Otonel, R.A.A.1; Leme, R.A.1; Freitas, L.A.1; Bronkhorst, D.E.2; Alfieri, A.F.1; Alfieri, A.A.1


2. UNOPAR - Universidade Norte do Paraná, Av. Paris, 675 - Jd. Piza, Londrina - Paraná, 86041-140

Rotaviruses (RV) are a common cause of viral gastroenteritis in humans and animals. Despite the seven groups/species of RV (A-G), recently it was proposed the creation of a new RV group/specie H (RVH) based on VP6 sequence analysis. This species includes the following: the novel adult diarrhea RV strain (ADRV-N) isolated from specimens collected during an outbreak of gastroenteritis among adults during 1997 in China; strain J19, identified during the same outbreak in China in 1997; the human RV strain B219, detected in a sporadic case of diarrhea in Bangladesh during 2002; the porcine RV strain SKA-1 that was isolated from a pig with diarrhea in Japan; 3 Brazilian RVH-positive stool specimens (BR59, BR60, and BR63), and 30 US RVH-positive stool specimens detected in swine herds in 2014. In this study we determined the VP6, VP7, VP4, and NSP4 nucleotide and deduced amino acid sequences of 6 (BR59, BR60, BR61, BR62, BR63, and BR64) RVH-positive stool specimens obtained from piglets with diarrhea in Mato Grosso do Sul, Central-West region of Brazil in 2012, using RT-PCR assay. Based on the high sequence identities (˃ 99%) of the VP6, VP4, VP7, and NSP4 genes among 5 of the studied fecal specimens (BR59 to BR63), they are considered the same local rotavirus strain denominated RVH/BRA-1. In contrast, since the fecal sample BR64 showed a relatively high difference (81.6% nt identity and 83.4% aa identity) in the VP7 sequence when compared to the other 5 specimens it was named RVH/BRA-2 strain. Comparative phylogenetic analysis showed that the 6 RVH strains do not cluster together with any available sequences of the members from the established RV groups (RVA-RVG), however, it seems to be related to RVB and RVG. These results confirm the presence of RVH in Brazil, demonstrate their genetic diversity, and provide new data that will assist in

understanding the viral phylogeny and epidemiology, as well as the explanation of patterns of viral evolution and biological properties of RVH. Financial Support: FINEP, CAPES, CNPq, and Fundação Araucária/PR.

VV318 - AN OLD VIRUS IN A NEW SPOT: DETECTION OF HANTAVIRUS IN WILD RODENTS FROM CENTRAL REGION OF MINAS GERAIS STATEAlves, P.A.1; Amaral, C.D.1; Borges, I.A.1; Alves, P.A.1; Miranda, J.B.1; Sacchetto, L.2; Costa, G.B.1; Abrahão, J.S.1; Paglia, A.1; Figueiredo, L.T.M.3; Drumond, B.P.2; Trindade, G. de S.1




Hantaviruses are important zoonotic pathogens related to rodents and small mammals. In nature, these viruses cause asymptomatic and persistent infections, and may be transmitted to humans via inhalations of aerosols from urine, feces. Also, the virus can be transmitted through saliva or direct contact with skin lesions or rat bites. The Hantavirus Cardiopulmonary syndrome (HPS) is considered one of de most important emerging diseases in Brazil, with high mortality rate (~40%) in humans. The disease has been reported in several regions of the country, including the western part of Minas Gerais state with a high incidence. This work aimed to detect Hantavirus in small mammals trapped on rural areas of Serro city, central region, State of Minas Gerais. An effort of 4800 traps was performed between 2012-2013 and 56 animals were caught (1,1%). Forest, pasture and domiciliary areas were covered. A greater number of captures are seen in rain forest (52 %), followed by domiciliary areas (32 %) and pasture (16 %), which demonstrates a high incidence of these rodents in disturbed areas. Sera from all rodents were tested by IgG-ELISA using the N recombinant protein of Araraquara virus as antigen. Two positive samples were found. RNA was extracted from RNA Latter (Life Technologies) preserved lungs and Real time PCR was also performed on 18 samples. One Olygorizomys sp




was found positive for the hantavirus S segment. The Olygorizomys sp, was trapped in domiciliary area indicating a close contact among wild and domestic life. Indeed, this genera has already been associated to the Juquitiba virus, which is of the most widely distributed hantavirus found in south Brazil. This is the first description of a Hantavirus circulating in this area of the Minas Gerais State. FINANCIAL SUPPORT: FAPEMIG, CAPES, CNPQ, MAPA

VV320 - DIAGNOSIS AND MOLECULAR CHARACTERIZATION OF CANINE PARVOVIRUS IN DOGS TREATED AT THE VETERINARY HOSPITAL OF FEDERAL UNIVERSITY OF PARANA - CAMPUS PALOTINAKunz, A.F.1; de Mello, J.L.1; Gallego, J.C.1; Macedo, R.1; Steffens, R.1; Oyafuso, M.K.1; Possati, F.2; Alfieri, A.2; Takiuchi, E.1



Canine parvovirus is a highly contagious enteric disease caused by canine parvovirus type 2 (CPV2). Currently three antigenic variants of CPV-2 are circulating, called CPV 2a, 2b and 2c. The frequency distribution of these variants vary according to geographical location and the time reference period of the studies. Recent studies have reported the type 2c as the most prevalent in certain regions of Brazil. However, the types of CPV2 involved in cases of canine gastroenteritis in Paraná state in recent yearsare unknown. The objective of this study was to detect and identify types of CPV-2 circulating in the dog population residing in the city and around Palotina, Paraná between 2013 and 2014. Twenty-four fecal samples from dogs with gastroenteritis treated at the Veterinary Hospital of UFPR campus Palotina were evaluated. The animals were between one month and 14 years old and had various historical of vaccination. The viral DNA extraction was performedusing a combination of phenol/chloroform/isoamyl alcohol and silica/guanidiniumisothiocyanatenucleic acid methods, and was amplified by PCR a 583 bp fragment (nt 4003-4585) of the VP2 gene. The sequencing reactions were

performed with the same primers as those used for PCR on an automated DNA sequencer (ABI3500). Nucleotide and amino acid sequence alignment was performed with the Clustal method using the BioEdit software. The CPV-2 was detected by PCR in 83.33% (20/24) of the samples tested. Of these, one animal had incomplete vaccination schedule (3 doses), five received one or two doses of vaccine, six were not vaccinated and the vaccination history of eight animals was unknown. CPV-2 was detected in the feces of animals of different ages, with higher incidence (80%) on the animals between 1 and 12 months old. Surprisingly, CPV-2 was also detected in the animalof 14 years old, which received only one dose of vaccine when a puppy and whose death recorded. Of the 20 positive samples by PCR, seven (35%) were sequenced to determine the type of CPV-2. In the sequence analysis, all were classified as CPV-2c, revealing the presence of the codon GAA (Glu) at position 426 of the viral protein VP2. The wide detection of CPV-2c in the canine population of the state of Paraná, even in adult animals with vaccination history, reinforces the importance of investigations into the virulence of this variant and the need for its incorporation into vaccines for effective disease prevention.

VV322 - SEROLOGIC EVALUATION AND DETECTION OF CANINE PARVOVIRUS TYPE 2C IN DOGS KEPT IN AN ANIMAL PROTECTION SHELTER IN THE CITY OF MEDIANEIRA, PARANAKunz, A.F.1; Grolli, P.1; Gallego, J.C.1; Macedo, R.1; de Mello, J.L.1; Possati, F.2; Alfieri, A.2; Takiuchi, E.1



Canine parvovirus type 2 (CPV-2) is the main agent of acute viral gastroenteritis, affecting predominantly young animals until six months old. Another clinical syndrome associated to CPV-2 is myocarditis in newborn dogs undergoing intrauterine infection or in the first weeks of life, culminating in sudden death. Currently three CPV-2 antigenic variants are circulating called CPV-2a, 2b and 2c, which only 2c is not consisted in the vaccines available. The detection of antibodies (Ab)




anti-CPV-2 in non-vaccinated animals indicates natural exposure to the virus and determining the Ab title reveals the level of protection after natural infection or vaccination. The aim of this study was to determine the title of Ab anti-CPV-2 and CPV-2c detection in dogs kept in a shelter runned by a non-governmental organization in the city of Medianeira, PR. Forty-five serum samples from adult dogs with unknown vaccination history were evaluated. To measure Ab title, sera were subjected to hemagglutination inhibition technique being considered protective titles exceeding 80. In addition, samples were collected from feces of normal consistency of an adult bitch with a history of recent sudden death of her litter. DNA was extracted using the combination of the techniques phenol/chloroform/isoamyl alcohol and silica/guanidiniumisothiocyanate, being amplified by PCR technique a fragment of 583 bp VP2 gene and subsequent sequencing. Forty-five of the animal sera tested, 43 (95.5%) had protective antibody titles against infection by CPV-2 (above 80), titration ranged from 128 to 2048. CPV-2 was detected by PCR in the fecal sample of the adult bitch and sequence analysis of the amplified fragment revealed the CPV-2c variant. The presence of Ab can be the result of the exposure to remnants of the virus or vaccine response resulting from previous exposure to shelter life. The confirmation of the detection of CPV-2c in the fecal sample of the adult bitch can justify the sudden death of the litter from the infection. The results of this study highlight the importance of titration of Ab to evaluate the susceptibility of these animals to disease and consequently the need to institute prophylactic vaccination. Also proves the circulation of CPV-2c in the canine population in our region, pointing the need for further studies of its virulence and antigenicity, as well as to define the effectiveness of vaccines in heterologous protection against this variant.

VV330 - OCCORRENCE OF BOVINE ROTAVIRUS IN DAIRY AND BEEF HERDS IN MINAS GERAIS STATE, BRAZIL BETWEEN 2006-2010Godoy, H.P.; Gonçalves, A.C.S.; Camargo, T.; Mazer, L.; Samara, S.I.; Buzinaro, M. da G.

FCAV/UNESP - Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Via de Acesso Prof.Paulo Donato Castellane s/n, Jaboticabal, SP, 14884-900

Neonatal diarrhea is a major health problem affecting calves in the first weeks of life, causing huge economic losses. Among the various agents involved in this syndrome, rotaviruses have been reported as the most frequently detected during disease outbreaks. Belonging to the family Reoviridae, rotavirus presents 75 nm in diameter, triple capsid protein, and has the genetic material as dsRNA, with 11 genomic segments. The external capsid proteins, VP4 and VP7, induce the production of neutralizing antibodies and are useful for viral classification. The objective of this study was to determine the frequency of rotavirus infection in calves of dairy and beef herds between the years 2006 to 2010, on properties of Minas Gerais State, Brazil. For this, survey data of 19 municipalities with a total of 36 herds and 304 samples, of which 31 were obtained from animals with diarrhea and 273 clinically normal animals were studied. Employing the PAGE technique the observed frequency of infection was 11.1% (4/36) and 2.6% (7/273) found in animals with and without diarrhea, respectively. Genotyping of positive samples for rotavirus was performed by the method of reverse transcription-polymerase chain reaction (RT-PCR) and highlighted the presence of infection by genotype G6P[5] and G10P[11] in dairy herds and the genotypes G6P[5] and G6P[5]P[11] in the beef herds. The results indicated a significant circulation of rotavirus in the beef herds. Hygiene measures, management and immunoprophylaxis are important to reduce the rate of infection and the persistence of the agent in the herds. FINANCIAL SUPPORT: FAPESP

VV336 - DETECTION OF FOUR PUTATIVE NEW BOVINE PAPILLOMAVIRUS TYPES FROM THE BRAZILIAN AMAZONChiappetta, C.M.; Daudt, C.; da Silva, F.R.C.; Chiappetta, C.M.; Streck, A.F.; Canal, C.W.

LABORATÓRIO DE VIROLOGIA VETERINÁRIA/UFRGS - Laboratório de Virologia Veterinária da Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090, Agronomia, Porto Alegre - RS, 91540-000

Papillomaviruses (PV) are small and complex viruses that belong to the Papillomaviridae family, composed by at least 29 genera. They have a circular double-stranded DNA containing approximately 8,000 base pairs (bp). The bovine papillomavirus (BPV) cause an infectious disease




that is characterized by chronic benign proliferative tumors that affect cattle worldwide. Additionally, these viruses can also cause tumor and neoplasia at different body regions, such eyes, skin, urinary bladder, upper gastrointestinal and genital tract. This report aimed to develop a prospective study of BPV infection in cattle from Northern Brazil based on nucleotide sequences of the L1 ORF that is a highly conserved region between all papillomavirus. Twenty-one samples of skin warts clinically diagnosed as papillomatosis from Acre and Rondônia States were analyzed. The partial amplification of L1 gene was performed with the degenerated primer pair FAP59/FAP64 and the amplification products were sequenced. The phylogeny was estimated with a Bayesian MCMC method, using BEAST version 1.7.2 and run using the GTR substitution model and the resulting data were analyzed using Tracer software. The DNA segment of the BPV L1 gene enabled the identification of four BPVs types (BPV1, 2, 11 and 13) already reported, four putative new BPV types (05RO10, 09RO11, 02AC12 and 06AC14) and three putative new BPV subtypes. To our knowledge, this is the first record of BPV from the Brazilian Amazon and these findings point to the great genetic diversity of BPV types that can be present in this region. Finally, the discovery and characterization of novel BPV types will increase the understanding of viral evolution and provide support for further studies about therapeutic treatment. FINANCIAL SUPPORT: CAPES, CNPQ, FAPERGS AND PROPESQ/UFRGS.

VV343 - IDENTIFICATION OF POTENTIAL MULTI-RECOMBINANT STRAINS OF CANINE DISTEMPER VIRUSStreck, A.F.; Budaszewski, R. da F.; Weber, M.N.; Mósena, A.C.S.; da Silva, M.S.; Dupont, P.M.; Borchardt, A.C.; Streck, A.F.; Canal, C.W.

LABVIR/UFGRS - Laboratório de Virologia do Instituto de Ciências Básicas da Saúde da Universidade Federal do Rio Grande do Sul, Prédio do ICBS/UFRGS - Sala 208 Rua Sarmento Leite, 500 - Bairro: Cidade Baixa, Porto Alegre - RS, 90050-170

Canine distemper virus (CDV) is a highly contagious viral pathogen that can cause a lethal systemic disease in dogs and other carnivores. CDV belongs to the genus Morbillivirus within the family Paramyxoviridae and possesses a non-segmented single-stranded

negative RNA genome encoding eight proteins. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine if recombination plays a role in CDV evolution. In this study, we analyzed 30 complete CDV strains isolated from dogs and non-dog hosts using seven distinct algorithms to detect genetic conversions and incongruent phylogenies. Fourteen putative recombination events were detected, being most of them potential multi-recombinants. Interestingly, most of the genome mosaics were produced by recombination events between strains from different genotypes, including classical vaccine strains, and strains isolated from different host species, mainly dogs and raccoons. These results suggest that recombination plays an important role in CDV evolution and that attenuated vaccines can recombine with circulating wild-type virus. FINANCIAL SUPPORT: CNPQ, FAPERGS, CAPES AND PROPESQ/UFRGS

VV349 - UNDERESTIMATION AND TEMPORAL TREND OF INCREASING COMPLEXITY OF HIV-1 BF1 RECOMBINANTS IN RIO DE JANEIRO, BRAZILMarques, B.C.L.; Morgado, M.G.; Guimarães, M.L.


HIV has extreme genetic variability due to high mutation and recombination rates caused by the lack of proofreading activity of the reverse transcriptase, associated to the high rate of viral replication. Notwithstanding, molecular epidemiological studies are mostly based in the characterization of just one genomic region, resulting in an underestimation of recombinant forms. Previous studies conducted in Rio de Janeiro depicted a prevalence of 5% to 28% of HIV-1 sub-subtype F1 and 2% to 15% of BF1 recombinants, depending on the genomic region and/or from the studied population group. The present study aimed to characterize five HIV genomic regions, including the most recombinogenic ones, to estimate the real prevalence of sub-subtype F1 and to understand the complexity of the recombination profiles. From a total of 635 HIV-1 positive samples collected from 1998 to 2013 and previously characterized based on the C2-V3 env region, 74 were classified as F1. From those, 56 had biological samples and were included in this study. The DNA samples were extracted, amplified and sequenced in gag, PR/RT, INT, env and nef regions.




The sequences were aligned with reference samples by ClustalW. Phylogenetic analyzes were performed using Neighbor Joining with Tamura-Nei substitution method by MEGA 5.1. The recombination analysis was performed by bootscan in Simplot 3.5.1 program. The prevalence of sub-subtype F1 sequences varied according to the analyzed genomic region from 46% to 71%, as well as BF1 recombinants represented 14% to 25% of the forms. All samples were amplified at least in three genomic regions and 39% of them confirmed the previous F1 classification, whereas 61% were reclassified as BF1. Based in the studied regions most of the BF1 recombinants were unique recombinant forms and only two were a second generation of a Circulation Recombinant Form (CRF_12BF and CRF_39BF). Based in our analysis, samples collected from 1998-2003 presented only intragenic recombination (14%), whereas samples from 2004-08 exhibited intergenic (6%) and intragenic (21%) profile and in those from 2009-13 all profiles (intragenic-15%; intergenic-18%; inter/intragenic-26%) were verified. In conclusion, our results confirmed that recombinant forms were underestimated when just one genomic region was used for classification and a temporal trend of increasing complexity of the BF1 recombinant forms was observed over time.

VV358 - CONCURRENT IRIDOVIRAL AND PRESUMPTIVE HERPESVIRAL INFECTON ASSOCIATED WITH MORTALITY IN TILÁPIA (OREOCHROMIS NILOTICUS) FROM PAULO AFONSO, BAHIA, BRAZILMaganha, S.R. de L.1; Navarro, J. de O.1; Almeida Queiroz, S.R. de1; de Pádua, S.B.2; de Menezes Filho, R.N.2; Araújo, A.P.3; Alencar, A.L.F.1; Arruda, E. de P.1; Rosa, M.P.1; Michelin, E.C.1; Fernandes, A.M.1; de Sousa, R.L.M.1



3. ACQUAPISCIS - Acquapiscis Consultoria e

Medicina Veterinária em Aquicultura, Rua Vieira de Morais, 1201, Campo Belo, São Paulo - SP, 04617-014

Ranavirus (RV), members of genus Ranavirus, family Iridoviridae, are emerging pathogens responsible for outbreaks of great ecological and economic impacts on fish, amphibians and reptiles of importance in aquaculture around the world, including Brazil. Cyprinid herpesvirus 3 (CyHV-3), also known as Koi herpesvirus, is a double-stranded DNA virus and belongs to genus Cyprinivirus and family Alloherpesviridae. CyHV-3 is very pathogenic and is responsible for a high rate of morbidity and mortality in common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio Koi) causing large-scale losses in worldwide aquaculture. Additionally, it has been suggested that other species of fish can be asymptomatic carriers of CyHV-3. The main goal of this study was to investigate cause of death of tilapias (Oreochromis niloticus) from one nursery farm located in Paulo Afonso, Bahia. For this purpose, 14 fishes with a range of 30 to 50g body weight were collected. Fragments of gill, eye, liver, intestine, kidney and spleen were sampled and fixed in buffered formalin solution 10%. Clinical signs included exophthalmia, ocular opacity, swimming in whirling, skin darkening and apathy, resulting in mortality rate of 30%. Concurrent infections by different bacterial agents were observed as Streptococcus agalactiae, Francisella sp. and Flavobacterium sp. Infestations by monogeneas and trichodinids were also detected. Histologically, the core of renal epithelial cells exhibited chromatin margination, consistent with herpesvirus infection. Basophilic cytoplasmic inclusions were abundant in pancreas, in a pattern typical of iridovirus infection. Aliquots of 50mg (pool of tissues) were submitted to DNA extraction followed by PCR (polymerase chain reaction) for sequential amplification of 409- and 348bp-fragments of the TK gene of CyHV-3. In addition, the same samples were subjected to PCR amplification with primers to the iridoviral MCP gene followed by nucleotide sequencing of the correspondent amplicons. All samples were positive for iridovirus and the PCR products showed a high percentage of similarity to the corresponding sequences deposited in GenBank. Although no amplicons of MCP gene could be obtained with the primers set used in this study, concomitant infection of RV and CyHV-3 is not a rare event, indicating that different molecular diagnostic approaches can be




further used to better understanding the etiological nature of these infections. FINANCIAL SUPPORT: FAPESP (PROC. Nº 2012/08846-3, 2014/04327-7)

VV364 - DETECTION AND CHARACTERIZATION OF MAMASTROVIRUS 5 IN DOGS FROM BRAZILAlves, C.D.B.T.; Souza, C.K.; Streck, A.F.; Budaszewki, R. da F.; Granados, O.F.O.; Torikachvili, M.; Dupont, P.M.; Weber, M.N.; Canal, C.W.


Mamastroviruses (MamV) have been described in many animal species associated with diarrhea and digestive disorders. There are few studies about the epidemiology of MamVs in dogs; however, astrovirus-like particles and viral genomes have been sporadically described. Currently, the canine astrovirus was accepted as a distinct species in the Astroviridae family, named Mamastrovirus 5 (MamV5). MamV5 was detected in symptomatic puppies occasionally suffering from diarrhea in association with other enteric viruses, such as rotavirus and coronavirus and/or parvo-like viruses. In addition, recent studies reported that around 10% of puppies showing clinical signs of diarrhea have also the MamV genome. Therefore, the aim of the present study was to verify the presence and characterize the genome of MamV5 from dogs of different Brazilian regions. Rectal swab samples were collected from 107 dogs with or without diarrhea, from five States of Brazil, with different ages and breeds and both genders. Total RNA was extracted and RT-PCR was performed using two primer pairs: pan-Mamastrovirus primers for screening, and specific primers to identify MamV5. Additionally, specific primers for 16S ribosomal RNA of Escherichia coli were used as an internal control. The results showed that the pan-Mamastrovirus primers detected 39 positive samples and fifteen were positive using the specific primer pair for MamV5. Three out of these samples were selected for full-genome sequencing and the capsid protein phylogenetic analyses showed a high similarity between Brazilian samples and MamV5 sequences from other countries, such as China, Korea, Italy and France. We hypothesize that one main MamV5 clonal population was efficiently spread worldwide. These findings also indicate that MamV5 is present

in dogs from Brazil and suggests their role as a canine enteric pathogen.

VV369 - FIRST DOCUMENTED EVIDENCE OF BOVINE VIRAL DIARRHEA VIRUS GENOTYPE 2 (BVDV 2) IN COLOMBIAVillamil, V.; Vera, V.; Ramirez, G.; Jaime, J.

UNAL - Universidade Nacional da Colômbia, Avenida Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia

The Bovine Viral Diarrhea Virus (BVDV) has emerged as one of the economically important pathogens in cattle populations, generating different clinical presentations that can range from asymptomatic to respiratory disorders, reproductive failure, congenital abnormalities and general immunodepression. In Colombia, the presence of the virus was first reported in 1975 in a batch of heifers imported from Holland. The virus has been classified into two biotypes (cytopathic and noncytopathic) and three genotypes (1, 2, 3) according to their nucleotide sequence. Genotype 1 is widely distributed in Colombia causing reproductive problems in cattle. Genotypes 2 and 3 have lower incidence and have not yet been detected in the country. Both genotypes are differentiated based on the 5 ‘non-coding region (5’NCR). Depending on the infecting strains, can be recognized at least three different clinical syndromes associated with BVDV infection: an acute disease in cattle, the mucosal disease and persistently infected animals (PI). The purpose of this study was to determine the presence of BVDV genotype 2 in Colombia evaluating four representative areas of cattle production using RT-PCR on serum and ear notches. For this, 375 prepartum cattle’s sera, 245 precolostral sera from calves born to these cows and 103 calve’s (25 post-birth days) sera were collected. Additionally, 178 ear notches of these calves were taken. Subsequently, extraction of viral RNA from samples was performed. RT-PCR was due using specific primers to amplify a 288 bp segment to discriminate the presence of BVDV and those who tested positive underwent new RT-PCR using primers to amplify a 221 bp specific sequence for BVDV genotype 2 on the 5’NCR region. 16 bovine females samples were positive for BVDV, two of which corresponding to BVDV-2; five precolostral samples of serum were positive to BVDV and two sera samples taken at 25 after birth days were positive for BVDV. Finally, 17 ear notches were




positive, of which 16 corresponded to BVDV-2. This study confirms the presence of BVDV in the areas of major livestock production in our country and represents the first documented evidence of the presence of genotype 2 BVDV in Colombia. This constitutes a new element to be considered to controlling the disease in the country. SOPORTE FINANCIERO: UNIVERSIDAD NACIONAL DE COLOMBIA, COLCIENCIAS, UNIDAD DE INVESTIGACIÓN FMVZ, UNIVERSIDAD NACIONAL DE COLOMBIA

VV370 - DETECTION OF PRRSV RNA FROM PORCINE COLOSTRUM – DERIVED MICROVESICLESCano, L.; Vera, V.; Ramírez, G.; Jaime, J.

UNAL - Universidade Nacional da Colômbia, Avenida Carrera 30 # 45, Bogotá, Cundinamarca 111321, Colômbia

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an important disease for the swine industry due to economic losses. In Colombia, the presence of the virus was reported in 1997 and produces recurring outbreaks. It has been determined that several viruses may be transmitted through released by cells. The extracellular vesicles, as microvesicles and exosomes, are released by different types of cells and have been detected in body fluids including milk and colostrum. These vesicles are a mechanism of intercellular communication employed by proteins, lipids and RNAs transportation. The association between the extracellular vesicles with molecules involved in viral cell entry, RNAs and proteins of viral envelope, suggested that these vesicles can act as a possible transmission route and as well as mechanism of PRRSV immune evasion. This hypothesis is called mechanism of Trojan Horse, which proposes that the virus or viral genome, using the exosome´s formation and transportation processes is able to hide away from the immune system, can infect other cells and generate new virus particles. This mechanism represents an important infection way and would have applications in antiviral therapies and vaccines. In this study, the possibility of PRRSV transmission from sow to piglets through colostrum extracellular vesicles was proposed using the Trojan Horse strategy. A hundred and fifty milliliters of colostrum were collected from eight sows from a positive PRRSV farm located in Valle del Cauca, Colombia. Each colostrum was processed by differential ultracentrifugation and sucrose density gradient to purify extracellular vesicles. In order to identify the

vesicles, electron microscopy of the final pellet was performed. After that, RNA was extracted from cell fractions, extracellular vesicle fraction and serum. RT-PCR was performed to amplify a fragment of 197bp, corresponding to ORF6 and ORF7 PRRSV genes. Vesicles of different sizes, morphologically characterized as microvesicles or exosomes, were evidenced. All samples were positive for PRRSV by RT-PCR. This study shows the identification of PRRSV in cell excretion vesicles present in porcine colostrum. The viral transmission by colostrum was also confirmed by PRRSV-RNA detection in serum and cell fraction. This contribution is the first step to demonstrate that PRRSV is transmitted from sow to suckling pigs using the Trojan Horse strategy and contributes to understand the disease´s pathogenesis. FINANCIAL SUPPORT: UNIVERSIDAD NACIONAL DE COLOMBIA, PROGRAMA NACIONAL DE SEMILLEROS DE INVESTIGACIÓN, CREACIÓN E INNOVACIÓN DE LA UNIVERSIDAD NACIONAL DE COLOMBIA. UNIDAD DE INVESTIGACION FMVZ, UNIVERSIDAD NACIONAL DE COLOMBIA.

VV371 - DETECTION OF PORCINE CIRCOVIRUS TYPE 1 (PCV1) IN PIGS FROM COMMERCIAL FARMS IN BRAZILCruz, T.F.; Yamatogi, R.S.; Araujo Jr, J.P.


Porcine circoviruses (PCV) are non-enveloped viruses, with single-stranded DNA genome that belong to the family Circoviridae. PCV2 is associated with several diseases, while PCV1 is considered a non-pathogenic virus detected in vaccines, cell cultures or products used for cell culture preparations. PCV1 is also found in domestic pigs but not much is known about its epidemiology and worldwide distribution. Thus, the aim of this study was to detect PCV1 in blood samples collected from pigs in several farms in Brazil. Eight hundred and fifty-eight whole blood samples were collected from pigs (65-160 days-old) in the period from March 2013 to June 2014. These samples were collected for monitoring of vaccination against PCV2, which were analyzed by PCR quantitative (qPCR). Eighteen farms located in eight states of Brazil were used in the study. The number of samples per state was: São Paulo (n=40), Minas Gerais (n=30), Paraná (n=130), Santa Catarina (n=168), Rio




Grande do Sul (n=170), Distrito Federal (n=200), Mato Grosso (n=90) and Ceará (n=30). Extracted DNAs were analyzed by qPCR for PCV (ORF1, both PCV1 and PCV2). One hundred and thirty-eight samples were positive for PCV by qPCR. Thus, seventy PCV positive samples (1 x 105 to 6.2 x 1010 copies of PCV DNA/ml) were selected to be tested for PCV1. Only eight samples were positive for PCV1. For the total of 858 samples, PCV1-positive samples corresponded to 0.9%. For the 70 samples positive for PCV, PCV1-positive samples corresponded to 11.4%. The eight samples positive for PCV1 were from the states of São Paulo (n=2, 160 days-old), Minas Gerais (n=1, 150 days-old), Rio Grande do Sul (n=2, 90 and 145 days-old), Santa Catarina (n=2) and Ceará (n=1). Five animals were vaccinated against PCV2, one animal was not vaccinated and two were not informed. The levels of viremia were 9.6 x 103 to 7.8 x 104 copies of PCV1 DNA/ml. The results of this study suggest that PCV1 has a low prevalence in Brazil. Therefore, PCV1 infection is a rare event that can be associated with a lower transmission capacity. Moreover, as the frequency of PCV1 is very low, detection of PCV in commercial farms vaccinated against PCV2 enables monitoring of the presence of PCV2 and variants of the virus that may arise due to mutations in ORF2. The ORF2 is widely used to design primers for PCV2, but this region has a high mutation rate. While primers for PCV were designed to a more conserved region (ORF1) and with lower mutation rate

VV374 - IDENTIFICATION OF FIBROPAPILLOMA-ASSOCIATED HERPESVIRUS IN GREEN TURTLE, CHELONIA MYDAS, ON THE COAST OF PARANÁ STATE, BRAZILde Alcantara, B.K.1; Domiciano, I.G.1; Domit, C.3; Claus, M.P.2; Saporiti, V.1; Alfieri, A.F.1; Bracarense, A.P.F.R.L.1; Alfieri, A.A.1


2. IFSC - Instituto Federal de Educação, Ciência e Tecnologia de Santa Catarina, Rua 14 de Julho, 150 - Coqueiros, Florianópolis - SC, 88075-010


Fibropapillomatosis is a neoplastic disease of the skin and internal organs that affects green turtles, Chelonia mydas. The etiology and pathogenesis is partly understood and it has been associated with papillomavirus and mainly herpesvirus, environmental and genetic factors. The aim of this study was to identify papillomavirus and herpesvirus in skin fibropapillomas of green turtles on the coast of Paraná, southern Brazil. Coastal (40 Km) and bays monitoring of Paranaguá Estuary Complex were performed from 2009 to 2012 and sea turtles carcass were rescued. In total, 22 fibropapillomas were collected from six juveniles green turtles. The number of sampled fibropapillomas varied from one to seven of each animal (average=3.7±3.01). DNA from all samples was extracted using commercial kit, according to the manufacturer’s instructions. Two consensus primers set were used to amplify the papillomavirus (PV) DNA, FAP59/FAP64 that target a fragment of 480 bp of PVs L1 ORF, and AR-E1F2/E1R9 that target a fragment of E1 ORF. For herpesvirus DNA identification, a nested-PCR was performed using specific Chelonia mydas HV primers set GTHV1/GTHV2 (165 bp) and GTHPR1/GTHPR2 (110 bp). The primers AR-E1F2/AR-E1R9 allowed the amplification of approximately 580 bp in one sample, which was negative in herpesvirus identification. However, the low quality of the sequencing product failed to identify the PV type. No sample was amplified by PV detection primers FAP59/FAP64. In the nested-PCR, the amplicons of the expected length of herpesvirus were obtained in 21 DNA samples. Five positive nested-PCR amplicons were sequenced to confirm the presence of herpesvirus associated with fibropapilloma of green turtle. The nucleotide sequence showed 100% identity with Chelonia Herpesvirus 5 (ChHV-5) (GenBrank accession number AF299108.1). The present study represents the first DNA characterization of ChHV-5 in green turtles on the coast of Paraná. The results reinforce the herpesvirus association with fibropapillomatosis in green turtles, and the participation of papillomavirus infection was not excluded. The ChHV-5 variant is possibly widespread on southern Brazil, but more studies are necessary to understand the dissemination of the viral strain between population stocks on the Brazilian coast, and the role of PV infection in fibropapilloma pathogenesis. Financial support: CNPq, FINEP, Fundação Araucária and CAPES.




VV380 - RETROSPECTIVE STUDY SHOWED THAT PPV4 IS CIRCULATING INTO THE BRAZILIAN SWINE HERDS SINCE 1999Castro, A.M.M.G.2; Bersano, J.G.1; Ogata, R.A.1; Nara, J.M.1

1. LABORATÓRIO DE DOENÇAS DE SUÍNOS “WASHINGTON SUGAY/INSTITUTO BIOLÓGICO - Laboratório de Doenças de Suínos “Washington Sugay” Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002

2. FMU - Faculdades Metropolitanas Unidas, Avenida Brigadeiro Luís Antônio, 1089 - Bela Vista, São Paulo - SP, 01317-002


Parvoviruses are small, non-enveloped viruses with a linear, non-segmented single-stranded DNA of 4 to 6.3 kb that infect a wide range of species of animals. Porcine parvovirus 1 (PPV1) is considered one of the major causes of reproductive failure in swine. Nowadays, five different groups of porcine parvoviruses (PPV) have been identified, being PPV4 one of them. The aim of this study was to perform a PPV4 DNA retrospective analyses in fifty pig tissue samples collected at maintained at -80ºC over the period of 1999-2005. These were randomly selected from the archive of the Laboratório de Doenças de Suínos “Washington Sugay”, Instituto Biológico and tested for PPV4. DNA extraction was performed using the QIamp DNA/RNA Kit (Qiagen). PCR for PPV4 detection was standardized in our laboratory using the pairs of primers PPV4a (5’ TCA TAG CAC TAT GGC GAG C 3’)/PPV4b (5’AGC ATT CTG CGT TGG ACA 3’) that amplifies a fragment of 284 pb. The PCR products were analyzed on 1.5 % agarose gel stained with GelRed (Uniscience). A PCR control was performed by testing the extracted DNA in parallel to β-actin amplification to avoid false-negative PCR results. Positive and negative controls reconstituted with sterilized Milli Q water were used in all reactions. All samples (50) were positive for β-actin and 9 of them (8 from São Paulo and 1 from Minas Gerais state) corresponding to 18 % were positive for PPV4. These positive samples were distributed into the years of their collect as n=1 (1999); n=1 (2000); n=4 (2002); n=2 (2003) and n=1 (2004). These results proved that PPV4 was circulating into the swine herds before 2000

in Brazil. Even though, the impact of this virus for the pig industry remains unknown. FINANCIAL SUPPORT: SIGA NRP 4568/20013

VV381 - FIRST REPORT OF PPV4 DETECTION IN RODENTS CAPTURED IN BRAZILIAN SWINE HERDSCastro, A.M.M.G.2; Bersano, J.G.1; Silva, S.O.S.3; Ferrari, K.L.3; Ogata, R.A.1; Nara, J.M.1; Richtzenhain, L.J.3

1. LABORATÓRIO DE DOENÇAS DE SUÍNOS “WASHINGTON SUGAY/INSTITUTO BIOLÓGICO - Laboratório de Doenças de Suínos “Washington Sugay” Instituto Biológico, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002

2. FMU - Faculdades Metropolitanas Unidas, Avenida Brigadeiro Luís Antônio, 1089 - Bela Vista, São Paulo - SP, 01317-002


Porcine parvovirus 4 (PPV4) is a small, non-enveloped virus with a linear, non-segmented single-stranded DNA that was found in 2010 in lungs from a disease pig that was also infected with porcine circovirus 2 (PCV2). Since then, the studies showed that PPV4 infect pigs with different ages and that the overall prevalence in pigs herds varied from 1.5 % to 39.7 %. However, the impact of this virus for the pig industry and the factors of the epidemiological chain remain unknown. The aim of this study was to investigate the presence of PPV4 in rodents captured in Brazilian swine herds. Twelve samples of tissue pool of Rattus norvegicus were collected from a pig farm in the Sao Paulo State. The samples were homogenized using a StomacherH 80 Biomaster (Seward) in 20% (v/w) TE buffer (pH 8.0). DNA was extracted using phenol-chloroform and proteinase K protocols and stored at 22⎕C. A PCR for PPV4 detection was performed using the pairs of primers PPV4a (5’ TCA TAG CAC TAT GGC GAG C 3’)/PPV4b (5’AGC ATT CTG CGT TGG ACA 3’) that amplifies a fragment of 284 pb. The PCR products were analyzed on 1.5 % agarose gel stained with GelRed (Uniscience). The amplified fragments from positive samples were submitted to bidirectional sequencing reactions that were performed using the BigDyeTM Terminator v3.1 (Applied Biosystems Life Technologies). Consensus sequences were assembled using the Phred/Phrap and




CAP3 programs with an analysis quality point of 20. One out twelve (8.3 %) of the tested Rattus norvegicus were positive for PPV4. Because this is the first report of PPV4 in rodents, to confirm the positive result, this sample was sequenced. The fragment generated was submitted to BLAST/n and confirmed the PPV4 identity relatedness. These results proved the detection of PPV4 in rodents and further phylogenetic studies are necessary to show the importance of these findings for the pig industry. FINANCIAL SUPPORT: SIGA NRP 4568/20013

VV382 - BRAZILIAN VARIANT OF THE INFECTIOUS BRONCHITIS VIRUS: RESPIRATORY AND RENAL PATHOGENICITYCaserta, L.C.1; Martini, M.C.1; Barnabé, A.C.S.1; Ferreira, H.L.2; Arns, C.W.1

1. IB/UNICAMP - Instituto de Biologia Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, Rua Monteiro Lobato, 255, Campinas - SP, 13083-862


Infectious bronchitis virus (IBV) belongs to order Nidovirales, family Coronaviridae, Gammacoronavirus genus. A Brazilian variant (BR) of IBV is causing great economics losses in Brazilian poultry industry. Nonetheless, experimental studies to evaluate the clinical signs and viral tropism to different organs are scarce. The present study aimed to evaluate clinical signs, gross lesion, and viral RNA after an in-vivo experiment with IBV Brazilian variant genotype BR-I in day-old chicks during 42 days. Tissue samples (sinus swab, trachea, lungs, proventriculus, ileum, cecal tonsil, cloacal swab, kidney and testis) were collected and viral RNA was purified and used for real time RT-PCR (qRT-PCR) targeting the untranslated region (UTR) gene. Clinical signs were mainly respiratory and a growth reduce was noticed as well. Macroscopic changes were hyperemia and mucous exudate in trachea but the most remarkable lesion were pale kidneys and ureters filled with urates. Higher RNA viral levels were detected in organs of digestive system, except in proventriculus. In this system, viral RNA was detected until 42 days post inoculation (dpi). In respiratory system, high viral RNA levels were detected

between the 2nd and 5th dpi, in accordance with observed clinical signs. Gross lesions were observed in kidney at 4th and 5th dpi; whereas high viral RNA levels were detected two days later by qRT-PCR. Quantities of viral RNA were detected in testis samples, but similarly to the proventriculus detection level, due to the absence of macroscopic lesions, it is still not possible to affirm that this quantity was possible to cause microscopic lesions. Histopathology analysis will be performed in order to relate microscopic findings, viral presence in tissues and clinical signs, and also, ELISA analysis will be performed to evaluate the presence of antibodies against IBV. FINANCIAL SUPPORT: FAPESP – BIOTA PROJECT 2911/50919-5 FAPESP 2013/02058-6, CNPQ (475571/2012-6) AND FAPESP (GRANT 2013/02058-6).

VV394 - PRELIMINARY STUDY ON MOLECULAR AND HISTOPATHOLOGICAL DETECTION OF BETANODAVIRUS IN FISH FROM SOUTHERN AND NORTHEASTERN REGIONS OF BRAZILNavarro, J. de O.1; Maganha, S.R. de L.1; Almeida Queiroz, S.R. de.1; de Pádua, S.B.3; de Menezes Filho, R.N.3; Araújo, A.P.2; Alencar, A.L.F.1; Arruda, E. de P.1; Michelin, E.C.1; Munin, F.S.1; Fernandes, A.M.1; de Sousa, R.L.M.1


2. ACQUAPISCIS - Acquapiscis Consultoria e Medicina Veterinária em Aquicultura, Rua Vieira de Morais, 1201, Campo Belo, São Paulo - SP, 04617-014


Betanodavirus (BNDV) is a non-enveloped, 25nm icosahedral virus with a single-stranded positive-sense RNA of approximately 4.6 kilobases containing two segments (RNA1 and RNA2). BNDV is the etiologic agent of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) that affects fish of different species, causing mass mortality worldwide. So far there are no reports of circulation of the BNDV in Brazil. The aim of this study was to investigate the presence of BNDV




in fish from southern and northeastern regions of Brazil. A total of 150 fish samples from diseased (n = 87) and apparently healthy (n = 63) specimens were collected in fish farms and rivers in the states of São Paulo (Pardo and Mogi-Guaçu Rivers watersheds), Ceará (Jaguaribara and Horizonte cities), and Bahia (Paulo Afonso city). Fresh tissue fragments of the central nervous system, eye, liver and kidney were sampled and stored at -20ºC. Additionally, buffered formalin-fixed tissue samples from Oreochromis niloticus (tilápia) specimens collected from fish farms with outbreaks characterized by mass mortality (Ceará and Bahia states). Aliquots of approximately 50 mg (pool of tissues) were subjected to RNA extraction followed by reverse transcription-polymerase chain reaction (RT-PCR) and nestedPCR targeting a 420bp-fragment of the BNDV RNA2 segment. A tissue pool from a tilápia specimen collected in Jaguaribara (Ceará state) tested positive. When each tissue type was tested separately, liver was positive. Histopathological changes as basophilic inclusions bodies were identified in liver and pancreas of this specimen and others from the same area, suggesting a typical BNDV infection. Interesting, no clinical signs consistent with VNN or VER were observed in these animals, indicating a subclinical infection. A broader prospection for BNDV in fish farms in Jaguaribara as well as nucleotide sequencing and virus isolation in cell culture for the positive samples are ongoing. These findings corroborate the assumption that BNDV infection has a worldwide distribution and call attention for potential interactions between fish and BNDV other than mortality. Nevertheless, this study points out the presence of a viral agent associated with significant economic losses in aquaculture, one the most promising area in Brazilian animal production.

VV400 - DIARRHEA IN PIGLETS BY AN UNCOMMON GENOTYPE COMBINATION (G26P[13]) OF PORCINE GROUP A ROTAVIRUSLorenzetti, E.; Medeiros, T.N. da S.; Molinari, B.L.D.; Ribeiro, J.; Possatti, F.; Leme, R. de A.; Pereira, F.L.; Crespo, S.E.I.; Alfieri, A.F.; Alfieri, A.A.

LVA/DMVP/UEL - Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva da Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Pr 445 Km 380, Campus Universitário, Londrina - PR, 86057-97

Porcine group A rotavirus (RVA) is the main viral cause of pigs neonatal diarrhea in worldwide. The most common combinations of G and P genotypes in RVA strains in piglets are G5P[7] (OSU strain), G4P[6] (Gottfried strain), G3P[7] (CRW8 strain), and G11P[7] (YM strain). However, several other combinations of G and P genotypes have been detected in piglets. The aim of this study was to describe the identification of the G26 genotype in wild-type porcine RVA strains from Brazil and its combination with the P[13] genotype. Two diarrheic fecal samples (BRA381 and BRA382) from suckling (14 to 28-day-old) piglets collected in July 2012 from a pig farm located in the state of Rio Grande do Sul that were RVA-positive by polyacrylamide gel electrophoresis (PAGE) were submitted for RT-PCR analyses of the VP7 (G genotype) and VP4 (VP8* - P genotype) genes to amplify 1,062 bp and 876 bp products, respectively. The RT-PCR products were sequenced and submitted for nucleotide (nt) sequence analysis to identify the G and P genotypes. The VP7 gene of both porcine RVA field strains displayed high nt identity (94.6% to 94.7%) with the G26 porcine strains. The VP4 (VP8*) gene of one strain exhibited 83.5% to 96.3% nt identity with the P[13] porcine strains, and the another porcine RVA strain carried an untypeable P genotype. Based on the sequence analysis of the VP7 and VP4 (VP8*) genes, the two field porcine strains (BRA381 and BRA382) belong to the genotypes G26P[13] and G26P[X], respectively. The G26 genotype was recently described in a five-day-old piglet with diarrhea in the Miyazaki prefecture, Japan (TJ4-1 strain/G26P[7]) and also in a pig from Hebei Lulong, China (Z650 strain/G26P[X]). Thus far, the G26 genotype of porcine RVA was only detected in piglets from Asia and in combination with the P[7] genotype. This is the first description of the porcine G26 genotype in diarrheic piglets outside Asia and is also the first detection of the uncommon combination G26P[13] genotype in porcine RVA strains. FINANCIAL SUPPORT: FINEP, CAPES, CNPq, and Fundação Araucária/PR.




VV401 - MOLECULAR DETECTION AND CHARACTERIZATION OF BOVINE ASTROVIRUS IN CENTRAL-SOUTH REGION OF BRAZILde Almeida Queiroz, S.R.1; Candido, M.1; Alencar, A.L.F.1; Batinga, C.A.1; de Godoy, S.H.S.1; Munin, F.S.1; de Almeida Queiroz, S.R.1; Buzzinaro, M. da G.2; Livonesi, M.C.3; Fernandes, A.M.1; de Sousa, R.L.M.1



3. FCF/UNIFAL - Faculdade de Ciências Farmacêuticas da Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700. Alfenas - MG, 37130-000

Enteric diseases associated with diarrhea, dehydration and weight loss is one of the major problems of cattle worldwide, contributing to significant morbidity and mortality, especially among newborns. In this context, demands for improving the laboratory diagnosis of infectious enteric diseases became constant, given the economic losses involved. Among the major viral enteropathogens of worldwide distribution and recent history, highlight the bovine astrovirus (BoAstV), whose first mention dates from 1978. Enteric BoAstV induces diarrhea especially among newborns and immunocompromised animals, having a prevalence above 60% in the first 5 weeks of life. Due to lack of data concerning the occurrence of this virus in Brazil, the present study was aimed at molecular detection and characterization of BoAstV strains in fecal samples from cattle with and without diarrhea of different ages. The study was conducted on 272 animals from different states of Brazil, the fecal samples were tested by RT-PCR with primers specific for the pol gene of astroviruses, obtaining positivity of 14.3%. Eleven samples from the states of São Paulo, Minas Gerais and Rio Grande do Sul were subjected to nucleotide sequencing and phylogenetic analysis. The similarity between the deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and was between 86.2 to 94.8% compared with sequences of other BoAstV described. In phylogenetic reconstructions,

9 samples were grouped together in a separate clade from other BoAstV, a sample (BoAstV-155-BRA) formed a paraphyletic branch to a clade that grouped both other BoAstV and deer astrovirus and the remaining sample (BoAstV-267-BRA) formed a basal branch to bovine and porcine astrovirus. However, the positioning of the two different samples (BoAstV-155-BRA and BoAstV-267-BRA) did not derive from episodes of positive selection or recombination. In summary, the results indicate, in an unprecedented manner, the circulation of enteric BoAstV variants in herds of cattle from states of the South-Central region of Brazil. This work was financially supported by FAPESP (Proc. nº 2012/18441-0) and CNPq (Proc. nº 472509/2010-1).

VV402 - IDENTIFICATION OF A NOVEL EQUINE INFECTIOUS ANEMIA VIRUS STRAIN BY MASSIVE SEQUENCING IN AN ENDEMIC ZONE IN PANTANAL, BRAZILMalossi, C.D.1; Cavalcante, R.V.2; Ullmann, L.S.2; Nogueira, M.F.T. de L.3; de Aguiar, D.M.5; Kroon, E.G.4; Araujo Jr, J.P.1

1. IBB/UNESP - Instituto de Biociências da Universidade Estadual Paulista, Bairro: Distrito de Rubião Junior S/N, Botucatu - SP, 18618-970

2. FMVZ/UNESP - Faculdade de Medicina Veterinária e Zootecnia da Universidade Estadual Paulista, Distrito de Rubião Junior, s/n Caixa Postal 560, Botucatu - SP, 18618-970



5. FAMEV/UFMT - Faculdade de Agronomia, Medicina Veterinária e Zootecnia da Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, nº 2367 - Bairro Boa Esperança. Cuiabá - MT, 78060-900

Equine infectious anemia (EIA) is a persistent lentiviral disease with mandatory notification. Although the disease was identified more than 150 years ago, it has a limited diagnosis with false-negative results and no available cure or vaccine. Complete proviral genomic sequences have only been obtained from three field strains of equine infectious anemia virus (EIAV), EIAV Wyoming, EIAV Liaoning and EIAV Miyazaki2011-A, from USA, China, and Japan, respectively. There is no




complete genomic sequence of viral RNA from field sample. In Brazil, EIAV is endemic in Pantanal region. This study aimed to sequence EIAV’s genomic RNA at the first time in naturally infected horses. Plasma of infected horses from Corumba, MS, was collected. The sample was clarified at 2000xg/10 minutes and supernatant centrifuged at 16500 xg/30 minutes. Total RNA was extracted of pellet and mRNA purified. cDNA was synthesized with random primers or oligodT primer for each sample and EIAV was detected by PCR. The dsDNA library was prepared using RNAse H, T4 polymerase and, T4 ligase following by Nextera-XT kit (Illumina Inc.) and sequenced with the MiSeq System (Illumina Inc.). Geneious R6 was used to analyze the sequences, using map to reference with complete EIAV genome (accession no. AF247394). A coding sequence of 7528bp in length (without 5`and 3`UTR) presenting 96.7% of coverage showing 120 nucleotides of gaps and 30 nucleotides of insertions. Among the field strains, this isolate has just 81%, 81% and 80% nucleotide sequence identity with the EIAV Liaoning, Wyoming, and Miyazaki2011-A strains, respectively. Furthermore, phylogenetic studies using Corumba EIAV sequence against known viral strains of EIAV strongly suggests this isolate comprise a separate monophyletic group. With these results it is possible to do a better characterization of the virus circulating in Brazil and may improve the molecular diagnostic of AIE using primers specific for Brazilian strain.FINANCIAL SUPPORT: FAPESP, FUNDIBIO

VV403 - MOLECULAR DETECTION OF THE BOVINE IMMUNODEFICIENCY VIRUS (BIV) IN CATTLE OF THE MINAS GERAIS STATE, BRAZILRodrigues, A.P. de S.1; Fonseca Jr, A.A.2; Lima, G.K.1; Gasparini, M.R.1; Naves, J.H.F. de F.1; Bicalho, J.M.1; Wood,C.3; Leite, R. de C.1; dos Reis, J.K.P.1


2. LANAGRO/MG - Laboratório Nacional Agropecuário de Minas Gerais, Av. Rômulo Joviano, s/nº Caixa Postal 35, 50, Pedro Leopoldo - MG, 33600-000

3. NEBRASKA CENTER FOR VIROLOGY UNIVERSITY OF NEBRASKA, 4240 Fair St, Lincoln, NE 68583, Estados Unidos

The bovine immunodeficiency virus (BIV) is the causative agent of bovine immunodeficiency which is known to infect cattle worldwide. As in other retrovirus infections, hosts develop a long term infection and most infected animals remains asymptomatic. Some animals can present variable disease signs such as lymphocytosis, lymphadenopathy, weight loss, weakness, decreased milk production and some secondary infections. BIV is present both in dairy and cutting herds and can be transmitted vertically in utero via placenta and colostrum, or horizontally through the exchange of bodily fluids and blood. Although BIV has been reported in several countries, the prevalence of this infection in Brazil is still unknown. The aim of this study was to detect BIV proviral DNA in blood samples of cattle and estimate the occurrence of infection in the state of Minas Gerais, Brazil. Blood samples from 391 cattle were collected from two regions of the state, Zona da Mata and Central. Blood was centrifuged at 1609g to isolate the buffy coat which was subjected to DNA extraction. Proviral DNA was detected by semi nested polymerase chain reaction (SN-PCR) using primers to amplify the conserved region in the pol gene of BIV. The product of SN-PCR was subjected to electrophoresis on 1.5% agarose gel and stained with ethidium bromide. The SN-PCR results indicate a BIV occurrence of 12,5% in the state. The amplified sequences were confirmed by cloning and nucleotide sequencing. The similarity of the nucleotide sequence of the BIV from Minas Gerais isolates with the reference strain (R-29) was 99%. This is the first study reporting the presence of BIV in the Minas Gerais, Brazil. The results indicate the need to conduct a detailed study on the prevalence of BIV infection in Brazil, and more particularly its association with various diseases that are prevalent in cattle, which may contribute negatively to the cattle industry. Keywords: Bovine immunodeficiency virus, semi-nested PCR, occurrence, Minas Gerais. FINANCIAL SUPPORT: FAPEMIG, CNPq ; INCT-Pecuária.




VV410 - SEARCH FOR CANINE CIRCOVIRUS (CACV-1) IN DOGSStefanello, F.1; Daros, F.1; Zanella, E.L.1; Costa, M.M.1; Zanella, J.R.C.2

1. UPF - Universidade de Passo Fundo, BR 285, São José, Passo Fundo - RS, 99052-900


Canine circovirus type 1 or CaCV-1 was recently characterized in dogs and is associated with disease or death in these animals. The CaCV-1 belongs to the Circoviridae family, which members have single strand DNA, circular and non-enveloped. Circoviruses are known to infect animals and are associated with serious diseases in swine and poultry, causing wasting in animals, reduction in production, malformations and tegument necrosis, lymphoid depletion and immunosuppression. The CaCV-1 isolated for the first time in 2012 from routine serological tests and was similar to the one found in swine, but not showing clinical signs. In this study, 100 canine blood samples collected from February to July 2014 at the Veterinary Hospital from the University of Passo Fundo were tested for CaCV-1 at the Animal Health and Genetics Laboratory from Embrapa Swine and Poultry research center. Viral DNA was extracted using MagMAX (Ambiom) isolation kit, and real time PCR (qPCR) using specific primers and probes for CaCV-1 were used for detection of the pathogen. The qPCR test detected sequences for the capsid and replicate genes of the CaCV-1, but all 100 samples tested were negative for the presence of the CaCV-1. This work allowed implementing a real time diagnostic test for CaCV-1, allowing an early diagnostic in dogs with different clinical signs or even different species, like swine. This diagnostic tool is also important to be accessible for studies of the pathogenesis and epidemiology of ssDNA viruses co-infections in economically important species such as swine. Financial support: The authors thank Neide Simon for the technical support. F Daros and JRCiacci Zanella is a fellow of the National Council for the Scientific and Technological Development. F Stefanello is a fellow of FAPERGS.

VV411 - ANALYSES OF HUMAN HERPESVIRUS 1 ISOLATED FROM NATURALLY INFECTED MARMOSET (CALLITHRIX SP)Kurissio, J.K.; Ulmann, L.S.; Rodrigues, M.V.; Araújo Jr., J.P.


Human Herpesvirus type 1 (HHV-1) is a virus transmissible from human to non-human primates. Primate from Callithrichidae family may be highly susceptible to infection by human herpesvirus with fatal cases.The use of molecular techniques may help the detection of the agent in animals with clinical suspicion.The present study aimed to conduct viral isolation and genomic analysis of herpesvirus from an infected non-human primate.Thus, the detection of HHV-1 was performed by nested PCR on samples of DNA extracted from brain fragments of 8 marmosets clinically suspected after death. Six samples showed amplification of viral DNA that were purified and sequenced. The sequencing was performed by Sanger’s method and the sequence analysis obtained 100% identity to DNA polymerase region of HHV-1 (GenBank KF498959, JQ352184 and JQ780693).Viral isolation of HHV-1 was done in Vero cell culture, and the inoculum was a brain fragment from the positive on nested PCR. After 24 hours p.i. (post inoculation) cytopathic changes characteristic of herpesviral infection were observed on the infected cell monolayer. An aliquot of the culture supernatant of infected cells 30 hours p.i. was collected and DNA was extracted for the whole genome sequencing. Deep sequencing with Miseq® (Illumina, Inc.) was used.Genome analysis was performed with Geneious program obtaining a sequence of 149,667pb.The sequence was assembled using as reference the genome of human herpesvirus type 1 (GenBank HM558507).The sequence showed 93.1% identity and 65.5% GC, with 62 divergent amino acid from coding regions: UL (23) US (37), IRS (1), RS1 (1), and inserting the sequence ACCGCC the UL26 region.The consensus sequence generated was submitted to phylogenetic analysis using MEGA 6.0 program with Tamura-Ney model and the 500 rounds of Bootstrap.In phylogenetic analysis, the clade marmoset herpesvirus was considering highly reliable (bootstrap 81%). The result showed that the phylogeny obtained from marmoset herpesvirus has the ancestral human




herpesvirus type 1 (GenBank AF67648), but the structure of the assembly showed possible modifications as observed in the genomic characterization of molecular sequence analysis. Therefore, the molecular analyses confirmed that infection with herpesvirus detected and isolated from the marmoset originated from human herpesvirus type 1. Nevertheless, variations in relation to its ancestor can be attributed to interspecies jumps or changes or adaptive virus in this species

VV415 - SPATIAL DISTRIBUTION OF CASES OF RABIES IN HERBIVORES OF PERNAMBUCO STATE IN THE PERIOD OF 2010 TO 2011Santos, G.R.1; - Silva, G.C.P.1; Santos, R.F.1; Godoy, H.P.1; Brandespim, D.F.3; Carvalho, A.A.B.1


2. UFRPE - Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife - PE, 52171-900

Rabie is considered one of the world’s largest antropozoonoses importance because it represents a strong economic impact of agribusiness and a high risk to public health, not only by the drastic and lethal outcome, but also by the high social and economic cost. In Brazil, more than 23,000 suspected cases of rabies in herbivores have been reported in 10 years; however, due to sub-reports, it is almost impossible to determine the actual losses caused by the disease. In Pernambuco, the creations of large herbivores are on growth and employed at national ranking eighth in milk production in the country. These data emphasize the importance of conducting epidemiological studies on the rabies situation in that State. The objective was to analyze the spatial distribution of positive cases of rabies in herbivores in the years 2010 and 2011 through the GIS, a tool that allows better identification of risk areas through spatial and visualization of data. Mapping of cases reported in the state of Pernambuco, Brazil was conducted, comprising the five meso (Metropolitana, Agreste, Mata, São Francisco and Sertão), composed of 185 municipalities, using MapInfo Professional 7.0 software. The laboratory diagnosis of rabies LANAGRO Recife / PE received from the State of Pernambuco, 67

samples in 2010 and 74 in 2011. A total of 141 samples received, 68 were confirmed positive, 39 (58% ) in 2010 and 29 (39%) in 2011. outbreaks of rabies in the period proved to be distributed in the five meso State. Most of them occurred in the middle region of the Wasteland, in two consecutive years, probably due to its topography and because the milk basin State is located there. The spatial distribution shows that some municipalities that had outbreaks in 2010, showed no positive cases in 2011. This decrease suggests a reflection of preventive measures more effective actions of the surveillance system after the focus. It was observed that the rabies virus is present in all mesoregions the State of Pernambuco, which emphasizes the need for effective and continuous actions of Agricultural Surveillance to control the disease. Epidemiological studies are an important support for health surveillance in animal health.

VV426 - FIRST DESCRIPTION OF PORCINE ENTERIC PICORNAVIRUSES FROM WILD BOARS IN BRAZILLeme, R. de A.1; Donin, D.G.1; Ratti, D.1; Molinari, B.L.D.1; Possati, F.1; Lorenzetti, E.1; Otonel, R.A.A.1; Pereira, F.L.1; Massi, R.P.1; Claus, M.P.2; Alfieri, A.F.1; Alfieri, A.A.1


2. IFSC - Instituto Federal de Educação, Ciência e Tecnologia de Santa Catarina, Rua 14 de Julho, 150 - Coqueiros, Florianópolis - SC, 88075-010

Porcine teschovirus (PTV), porcine sapelovirus (PSV), and enterovirus G (EV-G) are porcine enteric picornaviruses that circulate worldwide in asymptomatic domestic pigs. However, depending on the virus serotype and infection conditions, these picornaviruses may be important etiological agents of enteric, respiratory, reproductive, or neurological disorders in young and adult animals. Wild boars (Sus scrofa scrofa, Suidae family) are susceptible to infections by PTV, PSV, and EV-G. Pecari tajacu and Tayassu pecari are wild animals earlier believed to be related to the genus Sus and reclassified as genera Pecari and Tayassu into the Tayassuidae family. Sus scrofa, Pecari tajacu, and Tayassu pecari belong to the same order (Artiodactyla), are phenotypically and behaviorally similar, and largely distributed in distinct geographical




regions of Latin America. The aim of this study was to evaluate the natural infection by these porcine enteric picornaviruses from wild boars and peccaries in Brazil. Fecal samples (n=36) from wild animals of Paraná state were evaluated. Young (2 to 7-months-old, n=14) and adult (2 to 4-years-old, n=8) asymptomatic wild boars living on a captive farm were sampled in June, 2013. Pecari tajacu (n=5) and Tayassu pecari (n=9) aged 6 to 8-months-old were sampled on a zoo of Cascavel city in March, 2014. The animals had no contact with domestic pigs. Nucleic acid extraction was performed using a combination of phenol/chloroform/isoamyl alcohol and silica/guanidinium isothiocyanate methods. Polymerase chain reaction (PCR) and nested-PCR (n-PCR) assays were performed using primers targeting the 5’-non-translated region of the porcine enteric picornavirus genomes. Porcine enteric picornaviruses were detected in 12 of the 22 wild boar fecal samples. Enterovirus G was most frequently (11/22) detected, followed by PTV (10/22) and PSV (4/22). Young wild boars were more frequently (9/14) infected with PTV, PSV, and EV-G than adult animals (3/8). Sequencing analysis showed similarities varying from 97.7% to 100% for PTV, 92.4% to 96.2% for PSV, and 87.1% to 100% for EV-G. Fecal samples from Pecari tajacu and Tayassu pecari tested negative for the three viruses and these animals did not represent infection reservoirs. To the authors’ knowledge, this study represents the first molecular screening for PTV, PSV, and EV-G from wild boars of Latin America and the first to screen peccaries as alternative host species for porcine enteric picornavirus.FINANCIAL SUPPORT: FINEP, CAPES, CNPQ, AND FUNDAçãO ARAUCáRIA/PR.

VV427 - COMPARISON OF DIFFERENT SOROLOGICAL TESTS FOR DIAGNOSTIC OF FELINE IMMUNODEFICIENCY VIRUS (FIV) INFECTIONFerreria, M.C.1; Heleno, N.1; Gonçalves, S.1; Martins, N.2; Miyashiro, S.3; Reis, J.1; Hagiwara, M.3; Hosie, M.4; Heinemann, M.3; Teixeira, B.1

1. DMVP/UFMG - Departamento de Medicina Veterinária Preventiva da Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte - MG, 30161-970

2. UEMA - Universidade Estadual do Maranhão, Tirirical - Cidade Universitária Paulo VI, São Luís -MA, 65055-000



The infection of domestic cats (Felis catus) by feline immunodeficiency virus (FIV) results in the impairment of the immune system involving mainly the depletion of CD4+ T cells, increasing the susceptibility of opportunistic infections and even death. The need to perform diagnostic tests and identify positive animals comes from the indigence to know the data of prevalence of diseases, to set the profile prophylactic, therapeutic and control for infected animals. Veterinarians have few alternatives for the detection of FIV infection and the sensitivity and specificity of the diagnostic tests still not known face to Brazilian samples. Another relevant point is the high cost of the tests, factor that often deters the owners to request the exam to the vet. Analyzing two hundred and one (201) serum samples from domestic cats this study compared the ability to identify cats naturally infected by FIV of three different tests: ECOVET – FIV Ab + FeLV Ag test; SNAP COMBO® - Ab-FIV/Ag-FeLV, IDEXX and a recombinant p24 antigen ELISA. The sensitivity and specificity of the all tests carried out showed agreement.

VV431 - CHARACTERIZATION OF CIRCULATING STRAINS OF FELINE IMMUNODEFICIENCY VIRUS IN BRAZIL: PRELIMINARY DATATeixeira, B.1; Ferreria, M.C.1; Heleno, N.1; Boni, T.2; D’elia, M.1; Cruz, J.3; Miyashiro, S.2; Pereira, P.L.1; Reis, J.1; Hosie, M.4; Hagiwara, M.2; Heinemann, M.2

1. DMVP/UFMG - Departamento de Medicina Veterinária Preventiva da Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, campus Pampulha, Belo Horizonte - MG, 30161-970


3. INTA - Instituto Superior de Teologia Aplicada, Rua Coronel Antônio Rodrigues Magalhães, 359, Bairro Dom Expedito Lopes, Sobral - CE, 62050-100





The rapid emergence of AIDS in humans during the period between 1980 and 2000 has led to extensive efforts to understand more fully similar etiologic agents of chronic and progressive acquired immunodeficiency disease in several mammalian species. Lentiviruses that have gene sequence homology with human immunodeficiency virus (HIV) have been found in different species (including sheep, goats, horses, cattle, cats, and several Old World monkey species). Feline immunodeficiency virus (FIV) causes in domestic cats a progressive decline of the number of circulating CD4+ T cells, leaving the animals susceptible to opportunistic infections, and sometimes death. Viruses related to domestic cat FIV occur also in a variety of nondomestic felids. The objective of our study is to perform a better characterization of circulating strains of this large and ancient group of viruses (FIVs) in the country. In this work, we provide our preliminary data: the frequency of FIV antibodies in domestic cats was 17,2% (43/250 animals) and in wild felids was 0% (0/76 animals), by ELISA. In addition, to date, all samples from this study (isolates from Belo Horizonte and São Paulo) identified B as the only subtype circulating in FIV positive domestic cats in Brazil. Anyway, more widespread surveys of Brazilian isolates are required to determine whether a single subtype of FIV predominates in Brazil. This preliminary data on FIV infection paves the way for future studies characterizing circulating strains of feline immunodeficiency virus in Brazil. Such characterization will allow us to better understand the significance of lentiviral infection in Brazilian felids.

VV441 - CPV-2B AND CPV-2C CO-CIRCULATION IN DOGS IN BRAZILSilva, A.P.1; Freitas, L. de A.1; Spera, C.G.1; Miyabe, F.M.1; Diniz, J.A.1; Crespo, S.E.I.1; Grecco, S.2; Barcellos, M.2; Pérez, R.2; Alfieri, A.F.1; Alfieri, A.A.1


2. Universidade da República, Avenida 18 de Julio 1824, Montevideo 11100, Uruguai

Canine parvovirus (CPV) is a pathogen responsible for hemorrhagic enteritis in pups. The single-stranded DNA genome has two ORFs that encode nonstructural (NS) and structural (VP) viral proteins. VP2 sequence

is completely contained within VP1 and it mainly comprises the capsid of CPV; amino acid substitutions in its sequence can alter biological characteristics of the virus. CPV-2 emerged as a new pathogen of dogs from a variant of feline panleucopenia virus in the end of 1970s and spread rapidly. A few years later, CPV-2a replaced CPV-2. In 1984, a new type called CPV-2b emerged and co-circulated with CPV-2a. In 2000, a new variant was discovered, and at present it co-circulates with the other two types, but it seems to be replacing the other variants worldwide. Monitoring the circulation of CPV is important in order to establish control measures and improve the quality of existing vaccines. The aim of this study was to evaluate fecal samples of dogs from four different geographical Brazilian regions to determine the CPV subtypes currently circulating in dogs population. Forty-three stool samples of dogs aged 2 to 6 months were selected. All dogs presented clinical signs of hemorrhagic gastroenteritis and their feces were sent to CPV detection. The samples were collected in the states of Paraná (n=13), Minas Gerais (n=3), Paraíba (n=24), and Mato Grosso (n=3). All samples were tested by conventional PCR assay using a primer pair that amplifies a 583 bp fragment of CPV VP2 gene. The PCR products were subjected to RFLP using the restriction enzyme MboII, which recognizes GAAGA sites, present only in the residue 426 of CPV-2c, making it possible to differentiate CPV-2c from other types. From the 43 samples analyzed, 14 (32.6%) were positive for CPV-2c and 24 (55.8%) for CPV-2/2a/2b. Five amplicons did not show digestion in RFLP. All CPV-2c strains were from the states of Paraná and Mato Grosso. Two amplicons of each state were selected and subjected to sequence analysis. The amplicons from Paraná and Mato Grosso confirmed to be CPV-2c. The amplicons from Minas Gerais and Paraíba showed to be CPV-2b. Although some reports claim that CPV-2c has been replacing other types of CPV, this study demonstrates that there is still CPV-2b circulating in some Brazilian geographical regions. Financial support: CNPq, CAPES, and Fundação Araucária/PR




VV443 - STANDARDIZATION AND VALIDATION OF MOLECULAR METHOD DIAGNOSTIC FOR MALIGNANT CATARRHAL FEVERMartins, M. de S.N.; Lima, M. dos S.; Silva, T.G.; Pinto, V. da S.C.; de Stefano, E.; Okuda, L.H.; Pituco, E.M.


Malignant catarrhal fever (MCF) is an acute viral disease, affects multiple systems, is reported worldwide, usually fatal and affects mainly ruminants. Ten species of viruses belonging to the MCF group are known, all Herpesvirus, Family Gammaherpesvirinae, genus Macavirus. The main in Brazil is sheep-associated MCF, induced by ovine herpesvirus 2 (OvHV-2) strain .Due to the similarity of lesions, with encephalitis/encephalopathy, foot and mouth disease bluetongue and vesicular stomatitis, it must be considered as potential differential diagnoses where MCF is suspected. The use of molecular methods allow confirmation of the presence of MCF viruses in infected animals and may also be useful for phylogenetic and epidemiological studies in both natural and MCF-susceptible hosts. However, these are not yet routinely available in laboratories. Therefore, the objective was to standardize and validate a quantitative PCR (qPCR) SYBR®Green system for diagnosis of OvHV-2. The primers used detect the region ORF75 that encodes a protein highly conserved of the viral tegument. The standard sample used was from both Laboratory of Bovine Viral Diseases and Laboratory of Pathology of the Biological Institute, São Paulo, with characteristic lesions for FCM. This sample was sequenced and in the BLAST obtained 97-100% identity with various OvHV-2 isolates from different parts of the world. To determine the analytical sensitivity of the reaction, the concentration of the purified viral DNA was checked and serial dilutions were made from 105 to 100 copies of DNA/uL. Concentrations of primers from 200 to 1000nM were tested and the best concentration was 500nM, either for the reverse as the forward primers. The threshold detection was 100 copies of DNA/uL, dilution at which all samples in triplicate in three reactions made on different days, showed positive results. The Melting curve showed a single peak flowering at approximately 86 °C, demonstrating the specificity of the reaction. As the threshold detection was satisfactory it is indicated for routine laboratory. Although it was not found in the

literature studies of MCF performed with SYBR®Green system, the results suggest that this is a reliable and safe method for detecting OvHV-2, it showed good analytical sensitivity and specificity, reproducibility and linearity of the reaction. Therefore, it can and should be used as a tool for diagnosis and effective surveillance in order to avoid socioeconomic consequences.

VV444 - COINFECTION OF VACCINIA BEEF AND DAIRY CATTLE PSEUDOVARIOLA IN PROPERTY OF A MATO GROSSO STATE, BRAZILMartins, M. de S.N.1; Silva, T.G.1; Martins, M. de S.N.1; Pinto, V. da S.C.1; Lima, M. dos S.1; Okuda, L.H.1; Santos, S.P.2; Pituco, E.M.1


2. Defesa Agropecuária

Bovine vaccinia and pseudovaríola are vesicular disease of cattle caused by two species of viral family Poxviridae: vaccinia virus (orthopoxvirus) and pseudocowpoxvirus (gender Parapoxvirus). They are enveloped viruses and their genome consists of double-stranded DNA and its site of replication occurs in the cytoplasm. It is characterized by vesicular lesions cause teats of cows and calves in the oral cavity, compromising the production chain of milk, as it hampers milking and can also infect humans, becoming a public health problem. Brazil in cases of coinfection caused by poxviruses of different genera have also been described in outbreaks of vesicular disease in cattle and humans. This report describes an outbreak of vesicular disease in dairy cattle in São José dos Quatro Marcos in the state of Mato Grosso, where the animals had vesicular lesions on the ceilings, with approximate travel 8-13 days and the officers also had vesicular lesions on the hands. After confirmation of negative diagnosis for FMD and vesicular stomatitis, bovine epithelium three samples from this herd were sent to the Laboratory of Viral Diseases of bovine Biological Institute for differential diagnosis. After extraction with Trizol ® DNA, the samples were subjected to semi-nested PCR reaction using primers that amplify, respectively, fragments of 890 bp for the HA gene of vaccinia virus and 256 bp for pseudocowpoxvirus B2L gene. All samples were positive in both semi-nested PCR and subjected to sequencing, which showed high identity between the Brazilian samples with vaccinia




virus (98%) and pseudocowpoxvirus (99%) deposited in GenBank. This study corroborates the findings of the literature has found that vaccinia virus coinfection and pseudocowpoxvirus in cattle and demonstrates the circulation of these viruses in the country.

VV447 - CASE REPORT: DETECTION OF BVDV-3 IN A BOVINE WITH ENCEPHALITIS FROM THE STATE OF SÃO PAULOPinto, V. da S.C.; Martins, M. de S.N.; Lima, M. dos S.; Silva, T.G.; Okuda, L.H.; Pituco, E.M.


The Bovine Viral Diarrhea Virus (BVDV) belongs to the family Flaviviridae, genus Pestivirus. Its genome consists of single-stranded RNA of positive polarity. Presents high level of antigenic variability and causes great losses to the cattle, milk and meat industries. There are several subtypes between genotypes of BVDV-1 and BVDV-2, however, more recently it was reported the existence of what could become a new genotype, the BVDV-3, also called Hobi-like Virus, isolated from samples Fetal Bovine Serum (FBS) probable origin in South America, especially the Brazil. The BVDV is responsible for a wide variety of clinical manifestations varying from inapparent, acute and even fatal infections. The most important are gastroenteric, respiratory, and especially the reproductive system changes. Cases of encephalitis caused by BVDV are not very reported and for this reason we describe this study. The Biological Institute of São Paulo received for differential diagnosis of encephalitis, a sample of central nervous system (CNS) from a bovine with neurological signs belonging to a property of Patrocínio Paulista, São Paulo, Brazil, negative for rabies. Nucleic acid extraction was performed using Trizol® according to manufacturer’s instructions. Then was subjected to qualitative RT-PCR using primers that amplify the gene 5´UTR, highly conserved, capable of detecting all genotypes of BVDV. The CNS sample was positive for BVDV in comparison to the positive control, and subjected to sequencing to identify the genotype. The result revealed that it was BVDV-3 and showed high identity and genetic similarity to several sequences deposited in GenBank (HoBi_D32/00 access: AH013732.2; LV01/12 access: KC465388.1, Italy-68/13 access: KJ627180.1, among others). The animal was a

bovine of four months old, male, had incoordination, paddling movements, flaccid paralysis of the hind limbs and behavioral changes, and come to death in a few days. Diagnostics for Herpesvirus type 1 and 5, and Neospora caninum were conducted with negative results. This is the first report of BVDV-3 in animal with neurological symptom in Brazil being detected mainly in batches of FBS. It also indicates the need to mapping the existing BVDV genotypes in Brazil and contribute to surveillance actions in cases of encephalitis.

VV459 - A SEMI-NESTED PCR TO DETECT PROVIRAL EQUINE INFECTIOUS ANEMIA VIRUS DNA IN NATURALLY INFECTED HORSESVilela, A.P.P.1; Cursino, A.E.1; Ferreira, P.C.P.1; de Lima, M.F.N.T.2; Kroon, E.G.1



The Equine Infectious Anemia (EIA) is caused by Equine infectious anemia virus (EIAV), a retrovirus that infects all members of the family Equidae, and clinical cases of disease are reported in horses and ponies (Equus caballus) and also in donkeys (Equus asinus). The major mode of EIAV transmission is through mechanical transmission by biting insects or by contaminated fomites. The EIA is a worldwide disseminated disease, and it is also broadly disseminated in Brazil. It exhibits a high prevalence in Pantanal, affecting horses performance and, indirectly, the livestock farming. Given its burden, EIA is one of the eleven equine diseases listed as notifiable by OIE (World Organization for Animal Health). Currently, EIA diagnosis is based on the serological assays, such as agar gel immunodiffusion (AGID), considered gold standard test. However, there is a need for the development of more sensivite and specific assays for EIA diagnosis, since the serological tests show some limitations. This study aims to standardize a semi-nested PCR for EIAV. DNA extracted from the peripheral blood leucocytes of naturally infected horses, from Corumbá, Mato Grosso do Sul, Brazil, were used for this study. For semi-nested PCR EIAVltr-reverse1 and EIAVltr-reverse2 primers were used (Dong et al., 2012), in combination with the EIAVltr-forward 4 primer (designed in this study),




targeting the highly conserved 5’ end of the EIAV genome, extending from the long terminal repeat (LTR) to the trans-activator (tat) gene. The PCR product was fractionated by polyacrylamide gel electrophoresis, and the amplified DNA was excised, purified, and sequenced. The sequences obtained showed nucleotide identity with EIAV sequences deposited in the GenBank database. In summary, our preliminary results showed that the semi-nested PCR was effective for EIAV detection in field samples from Pantanal, and may be useful as a alternative for EIA diagnosis. Experiments to determine the sensivity and specificity of this semi-nested PCR assay are underway. FINANCIAL SUPPORT: CNPQ, CAPES, EMBRAPA

VV461 - VALIDATION OF THE SEROLOGICAL METHOD OF DIAGNOSIS OF BOVINE VIRAL DIARRHEA (BVD) BY ELISA AND VERIFICATION OF THE NEUTRALIZATION METHOD FOR DETECTION OF ANTIBODIES FOR BVDVRodrigues, A.P. de S.1; Oliveira, T.F.P.2; Azevedo, I.C.2; Rivetti Jr, A.V.2; Thomaz, M.M.2; Rodrigues, A.P. de S.2; Oliveira, A.M.2; Camargos, M.F.2


2. LANAGRO/MG - Laboratório Nacional Agropecuário de Minas Gerais, Av. Rômulo Joviano, s/nº Caixa Postal 35, 50, Pedro Leopoldo - MG, 33600-000

The methods of viral neutralization and ELISA are commonly used in the diagnosis of viral diseases in animal laboratories, such as Bovine Viral Diarrhea (BVD), which is caused by a virus from the family Flaviviridae, genus Pestivirus that affects cattle, sheep, goats, swine and wild animals. Currently, validation and verification of analytical methods are usually required to official laboratories from the Brazilian Agriculture Ministry as a measure of quality of these tests, besides being a criteria checked in national and international auditing. In this way, the aim of this work was the validation of the ELlSA test and verification of viral neutralization tests used to detect antibodies against Bovine Viral Diarrhea Virus (BVDV). The following parameters were evaluated in the validation of ELISA: analytical sensitivity, repeatability and reproducibility, uncertainty of measurement and participation in proficiency tests. For the verification

of the method of virus neutralization, the repeatability, reproducibility and uncertainty of measurement were also evaluated. In addition, a comparison was made between the results obtained by ELISA and by virus neutralization for BVDV (two different strains) in 64 serum samples, before and after inactivation in a water bath for 30 minutes at 56° C, besides the evaluation of the selectivity of the methods. In the results, the analytical sensitivity of ELISA varied according to the sample, and the titers obtained varied from 2 to 16. In five of the eight samples tested the titer was 8. Repeatability and reproducibility of the ELISA obtained a CV (coefficient of variation) ≤ 10% and the expanded uncertainty of measurement was 0.213. Furthermore, ELISA showed satisfactory results in proficiency tests organized by the Veterinary Laboratory Agency. The virus neutralization obtained CV values <2.3% in samples of higher titer and around 13% in samples of lower titer and the expanded uncertainty of measurement was 0.652. The results of the comparison between ELISA and virus neutralization (NADL strain) reached a concordance of 63.3% and using Singer strain it was 86.7% and the results also indicate that the methods are selective. Thus, the techniques of viral neutralization and ELISA for BVDV are suitable for the routine use of the official laboratories of Brazilian Agriculture Department. Keywords: ELISA, virus neutralization, bovine viral diarrhea virus, validation, verification. FINANCIAL SUPPORT: LANAGRO/MG

VV462 - DETECTION OF PICOBIRNAVIRUS ASSOCIATED WITH CRYPTOSPORIDIUM IN THE DIARRHEAL FECES OF CALVES IN THE WESTERN REGION OF PARANAMacedo, R.1; Garcia, F.G.1; Snak, A.1; Ribeiro, A. de S.1; Gallego, J.1; Kunz, A.F.1; Lustosa, J.1; Otonel, R.A.A.2; Alfieri, A.A.2; Osaki, S.C.1; Takiuchi, E.1



The Picobirnavirus (PBV) is an emerging group of non-enveloped viruses with bisegmented RNA genome whose detection has been reported in both samples of diarrheic feces and non-diarrheal feces in different hosts




including man, birds and reptiles. Cryptosporidium are opportunistic protozoa that affect all animal classes, especially in the first weeks of life, can lead from self-limiting diarrhea episodes to death. The etiology of diarrhea outbreaks attributed to protozoa Crypotosporidium, is well proven. However, the participation of the PBV as the causal agent of diarrhea is still controversial. The intent of this study was to report the simultaneous occurrence of Cryptosporidium spp. and PBV in diarrheal feces of naturally infected calves. 24 diarrheic fecal samples with less than 60 days old were selected, collected between 2012 and 2013, in the Western Region of Paraná. The samples were positive for PBV by electrophoresis on polyacrylamide gel (PAGE) stained with silver nitrate and confirmed by RT-PCR for Genogroup I. For detection of Cryptosporidium spp. The samples were analyzed using Ziehl-Neelsen coloring modified. The 28 positive samples PBV, (28.57%) contained Cryptosporidium spp. These results are quite promising, since few studies of detection of PBV by PAGE in cattle are reported in the literature, and frequency (0.69% to 3.67%) lower than that observed in our findings. Furthermore, the detection of PBV and Cryptosporidium spp. so far, has been described only in samples of human feces, suggesting the association of these enteropathogens. Due to the lack of knowledge about the epidemiology of PBV, mainly in cattle, and their involvement in cases of diarrhea in natural or mixed infections, more detailed studies are needed to complete the diagnosis and understanding of the association between PBV and Cryptosporidium spp. of neonatal diarrhea episodes in calves.

VV481 - DETECTION OF VIRUS BLUETONGUE IN SHEEP IN THE STATE OF SÃO PAULOPinto, V. da S.C.; Lima, M. dos S.; Silva, T.G.; Martins, M. de S.N.; Pinto, V. da S.C.; Souza, S.F.; Venditti, L.L.R.; Okuda, L.H.; Pituco, E.M.

INSTITUTO BIOLÓGICO, Av. Cons. Rodrigues Alves, 1252 - Vila Mariana, São Paulo - SP, 04014-002

Bluetongue is an infectious, non-contagious disease transmitted by Culicoides sp. The bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. So far 26 BTV serotypes have been identified in tropical, subtropical and some temperate regions of the world. Sheep, cattle, goats and various species of wild ruminants

are affected, but clinical signs are usually observed in sheep and include a high rate of mortality, reproductive problems, weight loss and indirect losses as a result of export restrictions. The aim of this study was to verify the presence of BTV in sheep of different breed, from 14 properties in State of São Paulo. A total of 538 animals, which age ranged from 37 months, were examined from 2007 to 2013. Were collected blood samples for direct detection of the virus by the methods of RT-PCR Real-Time (RT-qPCR), isolation in embryonated chicken eggs (ECE) and cell culture of BHK-21 strain, and sequencing of the positive samples. The sheep herds, 61.54% (8/14) had viremic animals, which represented 12% (64/538) of occurrence of viremic animals, detected by RT-qPCR. Samples positive in RT-qPCR were subjected to virus isolation in ECE and subsequently adapted in cell culture BHK-21 resulted in 75% (48/64) adapted to sample this isolation system. The positive samples were sequenced be confirmed strain of BTV-4. The more sensitive and specific, early diagnosis is necessary to avoid the damage caused by the spread of the disease. Also, allows analysis on a large scale in a short period of time, for certification of animals for domestic and international trade. Molecular studies for typing of BTV isolates in Brazil are very important to evaluate of epidemiological situation thus contributing to sanitary measures of control; reducing the risk of introduction of new BTV strains to minimize the negative impact of the disease and to improve the exchange of products of animal origin.

VV486 - SYNTHETIC NAPHTHOQUINONES INHIBIT IN VITRO REPLICATION OF BOVINE HERPESVIRUS-5Pinto, A.M.V.1; Leite J.G.P.2; Ferreira, V.F.1; Ferreira, S.B.1; Gonzaga, D.T.1; da Rocha, D.R.1; Paixão, I.C.N.P.1



Bovine herpesvirus type 5 is an important agent of meningoencephalitis in cattle. It has been identified in outbreaks of neurological disease in bovine in several Brazilian States. Furthermore, BoHV-5 has also been isolated from respiratory and genital tracts of animals without neurological disease symptoms, from cryopreserved semen, oocytes, embryos, and inside




spermatozoids. These observations have stimulated the search for compounds that can be used as means of controlling bovine herpesvirus infections or preventing recrudescent. In order to investigate cytotoxicity and anti BoHV-5 activity three synthetic naphthoquinones (Dimero, Lapachol and Quins 28) obtained by chemical synthesis were assayed “in vitro” and showed antiviral activity on BoHV-5RJ42/01 replication. Acyclovir was assayed in all experiments as compound standard. Cytotoxic effect were measured in MDBK cells treated with different compounds concentrations (25, 50, 250, 500 and 1000 µM) using the tetrazolium salt (3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide and the concentration of compounds required to reduce the number of viable cells by 50 % were for Dimero (58 µM ± 3.5); Lapachol (49.7 µM ± 9.2); Quins 28 (396 µM ± 7.3) and acyclovir (989 ± 2). Antiviral analyses of compounds against BoHV-5RJ42/01 were measured by inhibition of cytopathic effect on infected cells. The naphthoquinones derivatives EC50 (Dimero 2.3 µM ± 0.8; QUINS-28 = 2.8 µM ± 1.7); Lapachol = 10 µM ± 1.2); and ACV = 166 µM ± 2). All compounds showed low virucidal ability but Dimero, Lapachol and Quins 28 blocked BoHV-5RJ42/01 attachment and penetration in MDBK cell. For the time of addition studies monolayers were infected with 1x104 PFU BoHV-5RJ42/01 and treated with different compounds at time 0 h or at intervals of 1, 2, 3, 4, 5, and 6 h post-infection. Time of addition studies revealed Dimero, Lapachol and Quins 28 compounds suppressed all stages of the BoHV-5RJ42/01 replication cycle. On the other hand ACV showed slighter inhibitory activity on BoHV-5RJ42/01 replication before 3 h postinfection but reduction of 50% and 85% was observed after 3 and 6 h postinfection respectively. Investigations about specific mechanisms of the antiviral activity of these naphthoquinones are underway. Key words: BoHV-5, naphthoquinones and antiviral Financial support: CNPQ/UFF/ FIOCRUZ

VV489 - FREQUENCY OF BOVINE ROTAVIRUS IN DAIRY AND BEEF CATTLE IN MINAS GERAIS STATE BETWEEN THE YEARS 2006-2010Godoy, H.P.; Gonçalves, A.C.S.; Samara, S.I.; Buzinaro, M.G.


Neonatal diarrhea is a major health problem affecting calves in the first weeks of life, causing huge economic losses. Among the various agents involved in this syndrome, rotaviruses have been reported as the most frequently detected during disease outbreaks. Belonging to the family Reoviridae, rotavirus present 75 nm in diameter, triple capsid protein and has dsRNA as the genetic material with 11 genomic segments. The external capsid proteins, VP4 and VP7, induce the production of neutralizing antibodies, setting the basis for viral classification. The objective of this study was to determine the frequency of rotavirus infection in calves of dairy and beef cattle, detected between the years 2006 to 2010, on properties of Minas Gerais state / Brazil. For this, survey data were included from 19 municipalities with a total of 36 herds and 304 samples, of which 31 were obtained from animals with diarrhea and 273 clinically normal animals. By the PAGE technique, frequencies of infection were 11.1% (4/36) and 2.6% (7/273) found in animals with and without diarrhea, respectively. Genotyping of samples positive for rotavirus was performed by the method of reverse transcription-polymerase chain reaction (RT-PCR) and highlighted the presence of infection by genotype G6P[5] and G10P[11] in dairy herds and the genotypes G6P[5] and G6P[5]P[11] in beef herds. The results indicated a significant circulation of rotavirus in the herds studied. Hygiene measures, management and immunoprophylaxis are important to reduce the rate of infection and the persistence of the agent in herds. FINANCIAL SUPPORT: FAPESP

VV493 - AVIAN METAPNEUMOVIRUS (AMPV)) AND INFECTIOUS BRONQUITES VIRUS (IBV) IN WILDLIFE PSITTACIDAESimas, P.V.M.1; Barnabé, A.C.S.1; Caserta, L.1; Martini, M.C.1; Lima Neto, D.F.1; Durães Magalhães, R.1; Moraes, A.P.1; Nagel, N.E.2; Lierz, M.2; Hafez, H.M.2; Felippe, P.A.N.1; Arns, C.W.1


2. FREIE UNIVERSITÄT BERLIN, Kaiserswerther Straße 16-18, 14195 Berlin, Alemanha

Psittacidae are some of the most intelligent birds. This family consists of one of the groups most affected by trafficking in wildlife, for its great diversity of colors and




ability to imitate human speech arouses the interest of people around the world. Besides hunting for marketing, suffer from the continued destruction of their habitat. Long-lived animals, whose larger species can live over 50 years. Brazil is the country with the largest number of representatives of this family. The FAPESP-BIOTA Project has shown keen interest to know the real situation concerning these bird diseases in order to preserve these animals at Sao Paulo State. Viral diseases of psittacine birds are presently detected by PCR. For all these reason, the aim of this study is to identify IBV and aMPV using the RT-PCR technique. 289 samples were collected from 13 birds’ species (119 oropharyngeal – O; 170 cloacal swabs – C), of which 2.77% (2 O and 6 C) were positive for IBV and 2.77% (6 O and 2 C), for aMPV. Interesting evidence was the fact that there is a greater positivity for aMPV in samples from the cloaca. Thus, we can conclude that the epidemiological surveillance in birds is necessary since they can serve as a reservoir for viral evolution and may cause outbreaks in wild birds, animals of commercial interest and even in humans, given its proximity to the humans and also for the degradation of their natural habitat.

VV498 - DISTRIBUTION OF ANTIBODIES AGAINST INFLUENZA VIRUS IN PIGS FROM FARROW-TO-FINISH FARMS IN MINAS GERAIS STATE, BRAZILDias, A.S.1; Costa, É.A.1; Guedes, M.I.M.C.1; Rajão, D.S.1; Guedes, R.M.C.1; Zanella, J.R.C.2; Lobato, Z.I.P.1



Swine influenza virus (SIV) is the cause of an acute respiratory disease affecting swine worldwide. In Brazil, SIV has been identified in pigs since 1978 and after emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds. The objective of this study was to evaluate the serological profile for influenza virus in pigs from farrow-to-finish farms in Minas Gerais state, Brazil. Thirty farms with no SIV vaccination history from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/South- west and Belo Horizonte metropolitan area) were

selected. In each farm, blood samples were randomly collected from 20 animals per category of the production cycle: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), for a total of 100 samples per farm and a total of 3.000 animals in the study. Samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A ⁄ swine ⁄ Iowa ⁄ 8548-2 ⁄ 98) reference strain. The percentages of animals with antibodies against H1N1pdm09 and H3N2 SIV were 26.23% and 1.57%, respectively, considering all samples tested. The percentages of seropositive herds for both viruses were 96.6% and 13.2%, respectively. Only four farms had antibodies anti-H3N2, suggesting lower circulation of this virus subtype in the State. Means of antibodies titers for H1N1pdm09 were higher in sows and gilts and decreased along the other categories. In general, serology followed a characterized pattern of antibodies profile, in which antibodies titers are higher in females group, decreasing through eight to 14 weeks of age when seroconversion by natural infection normally occurs. However, farms from South/South- west area showed very variable serological profiles, with seroconversion normally occurring earlier (4-7 weeks) in the production system. In this area, some farms kept detectable antibodies levels throughout the ages and means of antibodies titers did not range negative values, suggesting virus circulation in all groups. In the other hand, one farm showed means of antibodies titers reasonably constant throughout the ages in the production cycle, suggesting virus circulation in all categories. FINANCIAL SUPPORT: CNPQ AND FAPEMIG

VV504 - SCREENNING AND DETECTION OF ALPHA- AND BETACORONAVIRUS IN BATS FROM SÃO PAULO AND PARANA STATEGoes, L.G.B.1; Campos, A.C.1; Ambar, G.2; Carvalho, C.3; Favaro, A.B.3; Crispin, L.A.1; Queiroz, L.H.3; Neto, A.C.2; Durigon, E.L.3


2. DEPARTAMENTO DE ZOOLOGIA/IB/UNESP - Departamento de Zoologia do Instituto de Biociências




da Universidade Estadual Paulista, Bairro: Distrito de Rubião Junior S/N, Botucatu - SP, 18618-970

3. DEPARTAMENTO DE APOIO PRODUÇÃO E SAÚDE ANIMAL/FMVA/UNESP - Departamento de Apoio Produção e Saúde Animal da Faculdade de Medicina Veterinária da Universidade Estadual de São Paulo, R. Clóvis Pestana, 793 - Dona Amelia, Araçatuba - SP, 16050-680

Coronaviruses are enveloped positive-sense single strand RNA viruses capable to infect birds and mammals including humans and associated with respiratory, enteric, neurological or hepatic disease in a great diversity of species. The Chiroptera order harbor the biggest number and genetic diversity of Alpha- and Betacoronavirus including virus phylogenetically related to emergent coronavirus afflicting humans, CoV-SARS (Severe Acute Respiratory Syndrome) and the CoV-MERS (Middle East Respiratory Syndrome). Despite the large number of bat species in Brazil, and the great diversity of CoV in bats, studies about the occurrence and diversity of CoV in Brazilian bats are scarce. We analyzed the presence of CoV RNA in intestinal samples of 189 bats specimens of 10 different bat species (Artibeus lituratus, Artibeus planirostris, Carollia perspicillata, Eptesicus furinalis, Eumops glaucinus, Lasiurus cinereus, Molossus molossus, Molossus rufus, Myotis nigricans and Sturnira lilum) captured in cities from northwest region of São Paulo and two different sites from Parana State, between 2010 and 2013. Total RNA was extracted from 30mg of tissue in NucliSENS® easyMAG® automatic extractor (Biomérieux) and the complementary DNA were obtained by RT-PCR using random primers. The screening of coronavirus were performed as described by Chu et al., 2011, a Nested PCR assay that amplify 440bp of the gene RNA dependent RNA polymerase. Amplicons were sequenced for BatCoV genotype identification. Coronavirus RNA were detected in four samples obtained from bats captured in Parana State, two Alphacoronavirus (α-CoV) in Carollia perspicillata, one Betacoronavirus (β-CoV) and one α-CoV in specimens of Artibeus lituratus. The β-CoV was most closely related to coronaviruses detected in Carollia perspicillata from Costa Rica, and grouped with others Betacoronavirus lineage C, same lineage from MERS CoV. The two α-CoV detected in C. perspicillata was most related to the BatCoV detected in same species of bat from Bahia state in a previously study. Finally, the

α-CoV detected in A. lituratus was genetic related with BatCoV detected in same genera bat from Panama and Costa Rica. To our knowledge, this is the first report of the possible detection of a β-CoV lineage C in Brazil. Continued surveillance of New World bats for novel CoVs and further research efforts to comprehend the genetic diversity of CoV in different bat species and biomes from Brazil are needed. FINANCIAL SUPPORT: FAPESP

VV506 - AVIAN CORONAVIRUSES SCREENING AND DETECTION IN ANTARCTIC SEABIRDSGoes, L.G.B.1; Seixas, M.1; Campos, A.C.1; Araujo, J.1; Ometto, T.1; Hurtado, R.1; Thomazelli, L.1; Kruger, L.2; Petry, M.V.3; Durigon, E.L.1


2. CENTRO DE CIÊNCIAS DA SAÚDE/UNISINOS - Universidade do Vale do Rio dos Sinos, Avenida Unisinos, 950 - Cristo Rei, São Leopoldo - RS, 93022-000

3. Laboratório de Ornitologia e Animais Marinhos/UNISINOS - Laboratório de Ornitologia e Animais Marinhos da Universidade do Vale do Rio dos Sinos, Avenida Unisinos, 950 - Cristo Rei, São Leopoldo - RS, 93022-000

Coronaviruses are RNA single-stranded positive polarity virus capable to infect different species of birds and mammals including man. Coronaviruses are classified into four different genera: Alpha- and Betacoronavirus present in mammals and Gamma- and Deltacoronavirus mainly present in different species of birds. Infections by coronavirus (CoV) are associated to pathologies of the digestive, respiratory, hepatic, renal tract and the central nervous system depending on the host. Over the past 10 years, two new highly pathogenic CoV were detected in humans, the SARS-CoV (Severe Acute Respiratory Syndrome) and the newly described CoV-MERS (Middle East Respiratory Syndrome), both with a high mortality rate and probably originated from “spill-over” events from bats and camels to humans respectively. In contrast of the great number of studies and the discovery of a significant number of novel coronavirus in bats, relatively little is known about the biology of avian coronaviruses in wildlife, and even less in Antarctic seabirds. The diversity of coronaviruses in avian hosts, the potential of mutation and recombination of these viruses and the detection




of Gamma- and Deltacoronavirus in few mammals make essential the knowledge about the diversity of coronavirus in different avian species. The present study aims to analyze the presence and molecularly characterize CoV in five avian species from the Antarctic Region including Southern giant petrel, Chinstrap penguin, Gentoo penguin, Cape petrel and Brown skua birds. Endotracheal and cloacal swabs samples were collected from animals captured at the Elephant Island in Subantarctic region, between December of 2010 and February of 2011. Total RNA was extracted from the samples with MagMaxTM RNA Isolation Kit and the randomic complementary DNA generated by RT-PCR with Superscript Reverse Transcriptase enzyme. The detection of CoV was performed by Nested PCR assay for amplification of 440pb of the RNA-dependent RNA-polymerase, enabling the detection and genotype identification after sequencing. One hundred and eighty-eight samples were screened and one Gammacoronavirus RNA was detected in a Southern giant petrel sample. The phylogenetic analysis demonstrate a close relation with a CoV detected previously in a Glaucous-winged gull (Charadriiforme Order) from Commander Island (Russia). This is the first report about the circulation of coronavirus in migratory seabirds of the Antarctic region. FINANCIAL SUPPORT: FAPESP (12/14255-8)

VV514 - COMPARISON OF PCR, HEMAGGLUTINATION TEST (HA), AND IMMUNOCHROMATOGRAPHY ASSAY (IC) TO DETECT CANINE PARVOVIRUS IN STOOL SAMPLES OF DOGSBalbo, L. de C.; Silvia, A.P.; Beuttemuller, E.A.; Freitas,L. de A.; Spera, C.G.; Massi, R.P.; Miyabe, F.M.; Alfieri, A.F.; Alfieri, A.A.

UEL - Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380 - Campus Universitário, Londrina - PR, 86057-970

Canine parvovirus (CPV) is one of the most common viruses responsible for acute hemorrhagic enteritis in dogs and is an important cause of morbidity and mortality in 6 to 12 week-old pups. Clinical signs in puppies include hemorrhagic or mucoid diarrhea, vomiting, and dehydration, and secondary infections can develop rapidly. The clinical diagnosis of CPV infection is inconclusive, since clinical signs found in CPV are very similar to symptoms caused by other pathogens.

Therefore, as the clinical diagnosis and identification of the agent is desirable for treatment and control of the infection, a clinical diagnosis should always be confirmed by laboratory tests. Several methods have been developed for the laboratory diagnosis of CPV. This study aimed to identify CPV in fecal samples of dogs examined at the Veterinary Teaching Hospital that had hemorrhagic enteritis using hemagglutination test (HA), immunochromatography assay, and PCR, and compare the use of different techniques to detect CPV. Twenty samples were selected; all of them were positive for CPV in a PCR assay that target a 583 pb fragment of the VP2 gene. Eight (40%) samples obtained titles considered positive (≥ 512) in HA, and twelve (60%) samples were considered positive in a fast test based on immunochromatography. Five positive PCR products were sequenced, all of them were identified as CPV-2c, showing that this variant is circulating in Brazil. Methods based on detection of CPV by PCR have been shown to be highly sensitive. However, immunochromatography is an optimal diagnostic tool when taking into consideration that it has low cost and is more practical in routine clinical medicine, assisting veterinarian in situations where needs to make decisions and take immediate measures safely, given that it has a higher sensitivity than HA. Also, it is easier to perform and has shown better results than the HA. FINANCIAL SUPPORT: CNPq, CAPES, and Fundação Araucária/PR

VV516 - IDENTIFICATION OF PERSISTENTLY INFECTED CALVES WITH BOVINE VIRAL DIARRHEA VIRUS BY RT-PCRFinoketti, F.; Firpo, R.; Campos, F.S.; Immig, J.; Torres, F.D.; Franco, A.C.; Roehe, P.M.


Bovine Viral diarrhea Virus (BVDV) is a member of the genus Pestivirus within the Flaviviridae family. The virus can be transmitted horizontally to embryos and fetuses, what may result in fetal absorption, abortion, cerebellar hypoplasia, teratogenicity or the birth of persistently infected animals (PI) Persistent infections play a key role on virus perpetuation, since PI calves may continually shed the virus in secretions and excretions and are major disseminators of the infection.




Consequently, the identification of the PI animals is one of the main objectives in attempting to control BVDV infections in endemically infected cattle. In the present study, a method for identification of PI animals using reverse transcription-polymerase chain reaction (RT-PCR) is described. Ear tissue fragments of 560 animals of different ages were collected in a farm on Bacia de Castro, Paraná, Brazil. The samples were screening using a commercial enzyme-linked immunossorbent assay (HerdChek BVDV Ag/Serum Plus Test Kit, IDEXX). Ten samples were ELISA positive and 21 samples - positive animals and their –progenitors were submitted to RT-PCR. RNA from ear tissues was extracted with a commercial kit (Purelink, Invitrogen®). The RNA was submitted to reverse transcription using random primers and to PCR with primers OPES 13A and OPES 14A (Elvander et al., 1998), expected to amplify a 296 bp region on the non-coding 5’ portion of the viral genome . Out of 21 samples, 10 (47,6%) were positive to RT-PCR and then submitted to molecular characterization through nucleotide sequencing. As reported by the result, we may affirm that the correlation between the ELISA and the RT-PCR was 100%. Therefore, the RT-PCR was effective on the identification and confirmation of PI animals. FINANCIAL SUPPORT: CAPES; CNPq

VV517 - CORTISOL PRODUCED IN PARTURITION PERIOD OF DAIRY COWS INFECTED NATURALLY BY BOVINE HERPESVIRUS 1 STIMULATE VIRAL SHEDDING AND ANTIBODY RESPONSEJunior, A.S.1; Ferreira, H.C.C.1; Granda, M.C.1; Silva, A.A.B.2; Ribeiro, M.G.2; Stancioli, E.F.B.2; Rebouças, M.S.1; Valério, O.C.1; Almeida, M.R.1



Bovine herpesvirus 1 (BoHV-1) are related to respiratory and reproductive diseases in young and adults animals. In dairy cattle, the impact is extremely harmful. The ability of bovine herpesvirus to establish latent infection makes them perpetually attached to their hosts. So, they may return to the infection productive phase in the presence of high levels of corticosteroid hormones. Pregnancy

is the most physiological phenomenon associated with corticosteroid increased levels, especially during parturition. There are still no reports comparing cortisol levels in cows during parturition with shedding of BoHV-1 and antibody response to that virus. Thus, the aim of this study was to compare levels of cortisol with viral shedding in nasal secretions and milk in two different phases of the dairy cows naturally infected: four months of gestation and parturition day. For this purpose, 35 pregnant dairy cows naturally infected by the virus were subjected to collect of blood serum, nasal secretions and milk and the cows were divided into two groups according to the production phase as previously mentioned. Doses of the hormone cortisol by chemiluminescence assay, serum levels of immunoglobulin M and G by indirect ELISA and assessment of viral load in nasal secretions and milk by real time PCR assays were performed. We had observed increased levels of cortisol and viral load in milk postpartum (P <0,05). In addition, in the same period, there was a positive correlation between blood IgG levels and viral load in milk (P = 0.0088, r = 0.7939), and between blood IgM levels and viral load in nasal secretions (P = 0.0249 , r = 0,5750). From these results, it is concluded that, in naturally infected dairy cows, parturition is a favorable situation to increase viral shedding and antibody response against BoHV-1. High levels of cortisol in the postpartum period are related to the reactivation of productive infection by BoHV-1, promoting its largest shedding in secretions. The viral load in the milk is directly related to the blood IgG level as well as IgM response is also proportional to shedding viral load in nasal secretions. FINANCIAL SUPPORT: CAPES, FAPEMIG, CNPq

VV518 - NEW HEPACIVIRUS IDENTIFIED IN CAPTIVE NON-HUMAN PRIMATES FROM SOUTHERN BRAZILUllmann, L.S.1; Li, L.2; Cubas, Z.S.3; Morais, W.3; Fantinatti, B.1; Deng, X.2; Pusterla, N.4; Leutenegger, C.5; Biondo, A.W.6; Delmart, E.2; Araujo Jr, J.P.1


2. BSRISF - BLOOD SYSTEMS RESEARCH INSTITUTE, 270 Masonic Ave, San Francisco, CA 94118, Estados Unidos

3. RBBV4. UC-DAVIS - University of California, Davis,

7551 Madison Ave, Citrus Heights, CA 95610, Estados Unidos




5. IDEXX LABORATORIES - Idexx Laboratories Innovative Diagnostics and Technologies, cp 132. Ponte Preta - Louveira - SP, 13.290-000


The genera Flavivirus, Pestivirus, Hepacivirus, and Pegivirus belong to the Flaviviridae family. Hepatitis C virus and Hepatitis G virus (GBV-B) are within the Hepacivirus genus, with variants identified in non-human primates, wild rodents, dogs, and bats. Brazil is considered a sanctuary of flaviviruses and important diseases such as dengue and yellow fever occur throughout the country. As non-human primates contribute to important human diseases, the study of the viruses present in these animals represents an important tool for detection of possible emergent viruses. Thus, viral metagenomics was performed with plasma samples from 10 non-human primates (Alouatta guariba and Cebus apella) from Southern Brazil. Viral enrichment was made by centrifugation, filtration (0.45 µm membranes), and treatment with nucleases. Viral nucleic acid was purified, cDNA was obtained with random primers and the SuperScript III Reverse Transcriptase kit, and dsDNA was obtained with the Klenow enzyme. An amplification step was performed with 5 µL of dsDNA, random primer and AmpliTaq Gold DNA Polymerase. After two purifications with magnetic beads, dsDNA was used for library preparation with TruSeqTM Sample Preparation v2 LT kit. Library quality was checked with 2100 Bioanalyzer and the quantification was estimated with KAPA Library Quant Kit. Libraries were pooled and sequencing with MiSeq® System with 2x250 cycles reagent kit. Geneious R7 and Mega 6.0 were used for analysis. Out of 2,181,312 generated reads, 3,819 had hit to GBV-B (NC001655) and were used for de novo assembly, resulting in 10 contigs. One contig had 8,885 bp and was used as reference for concatenation with the whole raw data from the pool sequenced, resulting in a consensus sequence of 9,708 bp (HQ 99.1%). Amino acid analysis resulted on the viral polyprotein with a CDS of 2,830 aa. The sequence showed 63% identity with 39 gaps compared with the deposited sequence (BAK24073). Phylogenetic analysis with neighbor-joining tree (1,000 rounds of bootstrap) resulted in a monophyletic group that shares ancestry

with GBV-B. Results suggest a new species of Hepacivirus similar to GBV-V. FINANCIAL SUPPORT: FAPESP, CAPES, AND IDEXX LABORATORIES.

VV519 - DETECTION OF INFLUENZA A VIRUS IN MIGRATORY BIRDSSantos, M.C.1; Ferreira, D. de L.1,2; Lima, S.T.1; Santos, M.C.1; Mello, W.A.1; Medeiros, R.1,2


2. NÚCLEO DE MEDICINA TROPICAL/UFPA - Núcleo de Medicina Tropical da Universidade Federal do Pará, Rua Augusto Corrêa, 01 - Guamá, Belém - Pará, 66075-110

Influenza virus is known for its ability to infect a wide variety of animals, such as mammals (humans, pigs, horses), poultry (chicken, goose, turkey) and even wild birds of Anseriformes (duck, wild goose and swan) and Charadriiformes (gulls, terns, waterfowl and kingfishers) orders. Wild birds are the natural hosts of avian Influenza virus, favoring possibility of spreading to other birds and aquatic animals when considering the migration factor. The Brazilian territory is crossed by distinct migratory routes of wild birds from the hemispheres north and south of the continent. Therefore, it is acknowledged the need for monitoring this virus in these birds considering its implications for the health of the human population. To investigate the occurrence of the Influenza virus in migratory birds captured in the coastal regions of Bahia, Pernambuco and Pará states, Brazil. The samples were analyzed using techniques of molecular biology, comprising two main steps: a) extraction of DNA / RNA from biological specimen; b) amplification of the gene encoding manipulator of citocromooxidase by the technical control chain reaction mediated by (PCR) amplification and RNAv using technique of reverse transcription followed by real-time PCR (RT-qPCR). The results showed that, from the total number of samples tested, 7.2% (n = 158) were positive for Influenza A virus by RT-qPCR. There was a difference of positivity for the virus among the analyzed bird species, being 35.4% to the Charadriiformes order, 44.9% of the Anseriformes order, 1.2% for the birds belonging to the Pelecaniformes order and 18.3% for those of the Suliformes order. Between samples of the Passeriformes and Columbiformes orders, no sample was positive for Influenza virus.




The data suggest variation between sampling sites, as Pará state showing the lowest percentage of positivity (11.3%), Bahia with the higher rate (65.8%), followed by Pernambuco with the second higher value of positivity (22.7%). This study shows that although the presence of rare investigations in Brazilian territory, there has been circulating Influenza A viruses among several species of migratory birds using the Pará, Bahia and Pernambuco states as stopping and reproducing places for their species. These reports justify further investigations to understand the dynamics of avian Influenza viruses in the population of circulating wild birds in Brazil, and its role as a potential source of infection for other animals, including man. FINANCIAL SUPPORT: CNPQ, SVS/MS

VV527 - IDENTIFICATION OF AVIAN PARAMYXOVIRUSES BY A CONVENTIONAL RT-PCR IN OROPHARYNGEAL AND CLOACAL SWABS FROM WILD BIRDSScagion, G.P.1; Silva, R.K.2; Sebastiani, M.C.2; Ciconello, F.N.2; Caserta, L.C.1; Martini, M.C.1; Felippe, P.A.N.3; Arns, C.W.1; Ferreira, H.L.2

1. DGEB/IB/UNICAMP - Departamento de Genética, Evolução e Bioagentes do Instituto de Biologia da Universidade Estadual de Campinas, Rua Bertrand Russel, s/n Caixa Postal 6109, Campinas - SP, 13083-970

2. DEPARTAMENTO DE MEDICINA VETERINÁRIA/FZEA/USP - Departamento de Medicina Veterinária da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Av. Duque de Caxias Norte, 225 - Campus da USP, Pirassununga - SP, 13635-900

3. ICS/UNIP - Instituto de Ciências da Saúde da Universidade Paulista, Av. Paulista, 900 - Cerqueira César, São Paulo - SP, 01310-100

Avian paramyxoviruses belong to genus Avulavirus of Paramyxoviridae family. These viruses are classified into 12 serotypes (AMPV-1 to APMV-12). They have already been identified in wild birds and are responsible for economic losses in poultry industry. AMPV-1 was already detected in different regions of Brazil. Nonetheless, only recently other serotypes, such as APMV-2 and APMV-10, were detected in samples from wild birds on the coast of Espirito Santo and Rio de Janeiro States by virological tests. Studies in other regions of nearby Brazilian poultry industry are, thus, still scarce. The present study aimed to evaluate the circulation of those viruses in wild birds by RT-PCR targeting a conserved gene of this viral family. To

do so, A RT-PCR test targeting polymerase gene previously standardized was tested in our lab with different vaccine viruses. Then, 100 samples (50 oropharyngeal and 50 cloacal swabs) from wild birds were collected and tested by the test. RT-PCR test was able to detect different viruses representing four different genera of the viral family. Nonetheless, this test was up to 1000 times less sensitive when compared to a specific test targeting each virus. Two out of tested samples were detected by this assay. This test seems to be a potential tool to detect new viruses despite its low sensitivity. Therefore, we expect to increase the knowledge about the epidemiology of avian paramyxoviruses in Brazil. FINANCIAL SUPPORT: FAPESP (NUMBER: 2011/09019-0).

VV528 - CHALLENGES IN MONITORING AVIAN INFLUENZA VIRUSFerreira, H.L.

DEPARTAMENTO DE MEDICINA VETERINÁRIA/FZEA/USP - Departamento de Medicina Veterinária da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Av. Duque de Caxias Norte, 225 - Campus da USP, Pirassununga - SP, 13635-900

Wild waterbirds and particularly Anatidae are the main reservoir of avian influenza viruses (AIV, family Orthomyxoviridae) for most of the known 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes in birds. Active surveillance in wild birds has increased worldwide after the emergence of the H5N1 subtype; nonetheless, nucleotide sequence data are still limited in comparison to other regions of the world. This information remains important to better understand the AIV ecology as influenza viruses circulating in animals pose threats to human health. In Brazil, AIV surveillance is realized by RRT-PCR, virus isolation, and genetic characterization of cleavage site in HA after a suspicion based on clinical signs and mortality or routinely. Village chickens are also sampled for AIV surveillance. The importance, challenges in monitoring AIV will be discussed. FINANCIAL SUPPORT: EC (NUMBER: 2011/09019-0).


Index278

September/October 2014 Volume 19 – Supplement 2 - Index

AAbdelhaleem, K.R. 136Abrahão, J. 122, 123Abrahao, J.A. 225Abrahão, J.S. 10, 12, 13, 17, 18, 21, 54, 66,

68, 69, 74, 101, 102, 103, 108, 149, 233, 248

Abrão, E. 184Abrão, E.P. 183Abreu, C.M. 15, 43Achkar, S.M. 13, 55, 247Acosta, P.O.A. 180, 184Acrani, G.O. 12, 21, 88, 91Affonso, M.Z. 229Afonso, L.A. 118Aguiar, A. 162Aguiar, R.W. de S. 213Aguirre, A.A. 14, 60Aita, C. 103Akinaga, M.M. 72, 73Albanese, J.M. 12, 30Albuquerque, A.C.C. 143Albuquerque, N.R.M. 229Alencar, A.L.F. 252, 257, 258Alessandra, V.B.T.G. 12, 22Alfieri, A. 248, 249Alfieri, A.A. 14, 57, 229, 247, 255, 258,

262, 264, 267, 272Alfieri, A.F. 14, 57, 247, 255, 258, 262,

264, 272Alfonso, H.L. 78, 145Almeida, A.C. da S. 244Almeida, G. 122, 123Almeida, G.G. 13, 54Almeida, G.M. de F. 74Almeida, G.M.F. 10, 18, 102, 233Almeida, H.M. de S. 239Almeida, J.F. 80Almeida, L.L. 13, 55Almeida, M.G.B. 170Almeida, M.R. 273Almeida, M.T.R. 147Almeida, N. 242Almeida Queiroz, S.R. de 252, 257Almeida, R.G. 235, 237Almeida, S.E. de M. 11, 12, 29, 50, 111Almeida, S.M.V.S. 129Almeida, T.N.V. 125, 134, 136Alvarenga, P. 122Alves, A. de A. 147Alves, C.D.B.T. 52, 252Alves, F.A.G. dos S. 173, 194Alves, M.R. de M. 142Alves, M.R.M. 159

Alves, P.A. 64, 225, 227, 233, 248Alves, P.T. 15, 41Alves, R. 86, 122, 124Alves, T.M. de A. 86Alvim, L.B. 69Amaral, C.D. 64, 227, 233, 248Amarilla, A. 134Amarilla, A.A. 78Ambar, G. 270Ambrosini, V.A. 197Ambrósio, L.L.D. 227Amorim, J.H. 137Amorim, L. 12, 28, 107Amorim, L. dos S.C. 90Amorim, L.M.F. 110, 111Amorim, N.A. 14, 27Amorim, R. 10, 14, 18, 24, 80Andrade, A.C. dos S.P. 68, 69Andrade, A. de S. 146, 202Andrade, G.P. 222Andrade, J. 124Andrade, J.S.R. 133Andrade, K.R. 102, 103Andrade, L.O. 116, 117Andrade, M. de S. 218Andrade, S.T.Q. de 83Andriguetti, N.B. 12, 29, 111Antão, K.L. 95, 140Anthony, S.J. 14, 60Apolinário, I.B. 230Aquino, V.H. 78, 134, 186, 243Arantes, A. de M. 11, 34, 134Arantes, T. 12, 31Arantes, T.S. 66, 102, 103Araújo, A.P. 252, 257Araújo, A.P. de 14, 58Araujo, B.R. 178Araújo Coutinho, C.J.P. da C. 210Araujo, J. 224, 271Araújo, J. 236Araújo, J.M.G. 127Araujo Jr, J.P. 13, 14, 56, 57, 227, 231, 254,

259, 273Araújo Jr, J.P. 120, 137, 226Araújo Jr., J.P. 244, 261Araujo, L. 150Araújo, S.B. 70Ardisson, A.D.M.P. 11, 46Arévalo, A.F. 246Arns, C.W. 11, 51, 98, 207, 245, 257, 269,

275Arruda, E. 11, 14, 33, 38, 88, 89, 91, 175,

181Arruda, E. de P. 252, 257

Assis, F.L. 12, 21, 108Assis, M. 124Augusto, L.T.S. 149Azevedo, I.C. 266Azevedo, R. 172Azevedo, S.S.D. 154

BBaccarin, A.M. 14, 57Badial, R.M. 169Badra, S.J. 82, 134, 145, 156Badr, K. 65, 120Badr, K.R.A. 145Baez, C. 242Baffini, V.R. 173, 194Baggio, M.L. 154Baggio, M.P.D. 210, 211Balani, D. 215Balbo, L. de C. 272Baldi, C. 158Bandeira, R. da S. 112, 161Barardi, C.R.M. 12, 28, 104, 105, 109Barbagelata, L. 195Barbagelata, L.S. 171, 174Barbosa, C.M. 236Barbosa, E. de C. 86Barbosa, J.E.F. 111Barbosa,M.L. 198Barbosa, M.R. 112Barbosa, M.RF. 12, 29Barbosa, V.M. 239Barboza, J.D.B. 224Barca Junior, F.A. 229Barcellos, M. 264Barnabé, A.C. 207Barnabé, A.C.S. 11, 51, 245, 257, 269Barrela, K.M. 112Barreto, D.M.V.S. 129Barreto, E.S. 167Barriolli, C.M.C. 220Barros, C. 71Barros, C.A. 85Barros, C.S. 64, 77Barros, H.L.S. 62Barros, I.C. 150, 177Barros, J. de A. 180Barth, O.M. 170Barth, O.M.S. 193Bastos, A.P. de A. 238Batinga, C.A. 258Batista H.B.C.R. 55Batista, H.B.C.R. 247Batista, I.C.A. 137Batista, M.N. 72, 73, 83


Index279


Batista, T.N. 14, 57Battisti, M.A. 149Baumworcel, N. 242Bellei, N. 14, 38, 94, 179, 188Belley, N. 204Bello, G. 168, 182, 189Belo, C.A.D. 14, 59Bélot, J.L. 11, 44Benites, H.G. 210Bergmann, M.R. 11, 46Bergo, E.S. 152Bergter, E.B. 11, 44Bernardino, J. de S.T. 86, 122, 210Bersano, J.G. 255, 256Bertani, G.R. 15, 42, 192, 228Bertol, J.W. 176Bertran, A.G.M. 215Beuttemuller, E.A. 272Bezerra, D.A.M. 12, 53, 182Bhatt, N. 175Bicalho, J.M. 260Bila, D. 175Biondo, A.W. 273Biselli, J. 135Biselli, J.M. 137Bisordi, I. 11, 31, 239Bittar, C. 12, 13, 23, 36, 82Bittencourt, L.H.F.B. 166Bittetti, M. 64Bloom, D.C. 98Boas, L.V. 87Boff, L. 81, 147Bogado, A.L.G. 229Boiteux, L.S. 218, 220Boldrini, N.A.T. 139, 153Bolpetti, A. 163Bomfim, W.A. 67Bonafé, C.F.S. 207Bonanno, V.M. dos S. 112Bonanno, V.M.S. 12, 29Bonfim, C.M. do 79Boni, T. 263Bonjardim, C.A. 13, 54, 74, 149, 230Bonon, S. 186Bonon, S.H.A. 139, 158, 193Boratto, P.V. de M. 66Boratto, P.V.M. 10, 12, 18, 21, 101, 102,

103Borchardt, A. 13, 56Borchardt, A.C. 251Bordignon, J. 202Bordin, L.C. 228Borges, A.A. 93, 94, 185, 186Borges, A.S. 80

Borges, I.A. 64, 227, 233, 248Borges, L. 239Borges, L.G. dos A. 99Botelho, J. 121Botelho, J.A. 186Botelho, L. 10, 18, 102Botosso, V.F. 203Bottino, F. de O. 232, 244Brabo, M.V.V. 171Bracarense, A.P.F.R.L. 255Braga, A.C.S. 72, 73, 83Braga, R.M. 170Brancalhão, R.M.C. 210, 211Brandão, C. 202Brandão, C.J. de F. 146Brandâo, P.E. 244Brandão, P.E. 225, 240Brandespim, D.F. 261Brasileiro, A.C.M. 218Brentano, L. 12, 52, 231, 238Brioso, P.S.T. 216Brito, C.J.B. 90Brito, D.E.M.W. 235Brito, M.E.D. 229Brito, V. de L. 87Bronkhorst, D.E. 14, 57, 229, 247Bronzoni, R.V. de M. 162, 167Brown, D.T. 84Budaszewki, R. da F. 252Budaszewski, R. da F. 251Budaszewski, R.F. 52, 233, 235, 236Bueno, F.C. 208Bueno, V.D.C. 157, 158Burbano, R.M.R. 150Burlandy, F.M. 12, 29Burth, P. 87Burutarán, L. 101Bustamante, F.C. 198Buzinaro, M. da G. 250Buzinaro, M.G. 191, 239, 269Buzzato, G.P. 181Buzzinaro, M. da G. 258

CCabello, M. 155Cabrera, E. 135Caetano, D.G. 182, 189Cahu, G.G. de O.M. 143, 180Cahú, G.G.O.M. 156Calastri, L.P. 116Caldas, L.A. 73Caldas, S. 72, 84Calegari, L.P. 78Calixto, R.S. 68, 69, 149

Calmon, M. de F. 75, 169Calzavara Silva, C.E. 138Calzavara-Silva, C.E. 86Camargo, C. 188Camargo, C.N. 179Camargos, M.F. 266Camargo, T. 250Camilo, H.P. 169Campbell, C.M.P. 154Campos, A.C. 270, 271Campos, A.D. 176Campos, D. 14, 39Campos, D.A. 179, 197Campos, F.C. 12, 31Campos, F.S. 11, 50, 75, 99, 229, 272Campos, G.R.F. 12, 23, 82Campos, G.S. 146, 202Campos, M. 123Campos, R.K. 10, 17, 101Campos, R.M. 73, 84, 143Canal, C.W. 13, 52, 56, 233, 235, 236, 250,

251, 252Candeias, J.M.G. 163Candiani, T. 122Candido, M. 258Cano, L. 253Cantão, M.E. 13, 53, 234Cardoso, A.L.S.P. 241Cardoso, B.F. 130, 132, 166Cardoso, D. 65, 120Cardoso, D. das D. de P. 11, 34, 125, 126,

127, 131, 134, 136, 163Cardoso, D.D.P.C. 145Cardoso, E.S. de C. 129Cardoso Jr, C.A.M. 14, 26Cardoso, R.S. 91Carenzi, L. 175Carenzi, L.R. 181Carestiato, F.N. 118, 159Carlos, L.Z. 86Carmona, R. de C.C. 142, 159Carneiro, A.C.A. 69Carneiro, A.P. 14, 26Carneiro, B.M. 72, 73, 83Carneiro, R. dos S. 131, 163Carnieli Jr, P. 247Carnieli, Jr. P. 55Caron, L.F. 238Carraro, E. 176, 179, 197Caruzo, T.A.R. 176Carvalho, A.A.B. 261Carvalho, B.K.S. 14, 40Carvalho, C. 270Carvalho, C.A.M. 85, 92


Index280


Carvalho, D.G. 87Carvalho-Filho, J.R. 86Carvalho, L.D. 63Carvalho, L.R. 167Carvalho, M.O. de O. 193Carvalho, M.O.O. 170Carvalho, R.C.P. 222Carvalho, V.L. 213Carvalho, W.J. 14, 26Caserta, L. 11, 51, 269Caserta, L.C. 207, 245, 257, 275Castellanos, L. 95Castilho, J.G. 13, 55, 247Castrignano, S.B. 112Castro, A.G.M. 241Castro, A.M.M.G. 226, 255, 256Castro, A.R. 187Castro, F. 122Castro, F.C. 213Castro, Í. de A. 126, 127Castro, K. da S. 213Castro, L.F.F. 194Castro,L.F.F. 164Castro, M.E.B. 217Castro, T. 242Castro, T.X. 244Catroxo, M.H.B. 225Cavalcante, M. 64, 71, 77Cavalcante, R.V. 259Cavalcanti, S.M.B. 118, 159Cavaleiro, N. 10, 18Cecílio, A.B. 72, 84Chagas, C. 64Chanasit, J.S. 143Chávez, J.H. 62Chiappetta, C.M. 250Chiappetta, M.C. 236Cibulski, S.P. 113Ciconello, F.N. 275Cilli, A. 142Cintra, A.C.O.; 243Cirne-Santos, C.C. 95Cirqueira, C. dos S. 162Claus, M.P. 255, 262Clemente, W.T. 103Coelho, A. 14, 39Coelho, L.F.L. 12, 15, 22, 40, 69, 119, 128,

130, 131Coelho, M.R.C.D. 143Coêlho, M.R.C.D. 156, 180Coimbra, R. 122Coimbra, T.L.M. 11, 31Colares, J.K.B. 146Coldebella, A. 12, 52, 231

Colina, R. 101Colmanetti, T. 224Colombo, A.C.F. 133Colombo, T.E. 135, 137, 196Coltro, V.P. 77Comone, P. 203Conceição, A.O. 70Corbellini, L.G. 233Cordeiro, T.I. 159Correa, A. 113Correa, A. de A. 113Correa, R.A. 12, 31, 99, 113Correa, R.F.T. 207Corrêa, R.L. 221Correa, T. dos S. 11, 34Corrêa, T. dos S. 136Corrêa, V.M. 123Côrtes, F.H. 182, 189Cortines, J. 12, 21Costa, A.G. 14, 58Costa, A.O. 10, 18, 102Costa, A.P.F. 127, 150, 152, 201Costa, C.S. 75Costa, D.T.M. 147Costa, É.A. 270Costa, E.M. 232, 244Costa, E.P. 13, 15, 34, 41Costa, E.V. 12, 29Costa, G.B. 13, 54, 129, 149, 248Costa, I.B. 150, 177Costa, L.C.P. das N. 160Costa, L.D.C. 126Costa, L.F. de M. 146Costa, L.F.M. 202Costa, L.J. 97Costa, L.J. da 14, 24, 80Costa, M.A. 150Costa, M.C.M. 150Costa, M.M. 260Costa, P.M.G. 222Costa, S. 186Costa, S.B.F.G. 212Costa, S.C.B. 139, 158, 193Costenaro, J.G. 11, 50, 75Covas, D.T. 10, 13, 19, 36, 172Crapez, M.A. 108, 110, 111Craveiro, S.R. 217Crespo, S.E.I. 14, 57, 258, 264Criado, M. 175Criado, M.F. 14, 38, 88, 89, 91Crispin, L.A. 270Cruz, A.C. 242Cruz, C.A. 191Cruz, F. da S.P. 15, 42, 192

Cruz, J. 263Cruz, O.G. 178Cruz, T.F. 14, 57, 226, 227, 254Cubas, Z.S. 273Cunha, A.C.C. 90Cunha, E.M.S. 246Cunha, M. dos P. 125, 127, 131, 163Cunha, M.S. 11, 31Cursino, A.E. 266

DDabelic, R. 14, 25da Costa, L.C.F. 15, 40da Costa, L.J. 10, 14, 18, 27da Costa, S.M. 10, 18da Costa, V.G. 125da Fonseca, F.G. 13, 35Dalla Vecchia, A. 105, 106Damaso, C. 70Damaso, C.R.A. 107Damazo, N.A. 109Dantas, G. 210da Paixão, C.G.S. 133da Rocha, D.R. 268da Rosa, R.D. 77Daros, F. 260da Silva, A.A.S. 186da Silva, D. 21da Silva, D.J.F. 167da Silva, D.M. 180da Silva, E.E. 10, 12, 18, 29da Silva, É.E. 173da Silva, E.M.L. 14, 27da Silva, E.R.M. 164da Silva, F.C. 80da Silva, F.E.R. 147da Silva, F.G. 11, 31da Silva, F.R.C. 250da Silva, G.P.D. 97da Silva, J.L. 143da Silva Jr, H.C. 201da Silva, L.D. 161da Silva, L.L.P. 14, 27, 91da Silva, L.P. 11, 34da Silva, L.S. 191, 196da Silva, M.C.C. 88da Silva, M.G. 189, 190da Silva, M.R.S. 73, 84da Silva, M.S. 52, 235, 251da Silva, M.V. 159da Silva, T.R. 11, 50da Silva, V.A.G.G. 98da Silveira, N.J.F. 131Daszak, P. 14, 60


Index281


Daudt, C. 13, 56, 250de Aguiar, D.M. 224, 259de Albuquerque, A.C.C. 180de Albuquerque, N.R.M. 12, 31de Alcantara, B.K. 255de Alencar, A.L.F. 14, 58de Almeida, D.V. 185de Almeida, G.M. 10, 17de Almeida Jr, R.F. 92de Almeida, M. da G.B. 193de Almeida Queiroz, S.R. 258de Amorim, L.M. da F. 98de Andrade, M.A.A.M. 89de Araujo, J. 11, 50de Araújo, J.M.G. 92de Azevedo, K.M.L. 11, 32de Azevedo, M.L.B. 201de Barros, L.H.R. 140de Campos, A.M. 149de Carvalho, I.L.Q. 241de Carvalho, I.M.V.G. 86, 122, 210de Castro, C.A.V. 168de Castro, F.L. 11, 50, 227de Castro, I.R.D. 86de Castro, R.O. 14, 27de Castro, T.X. 232de Faccin-Galhardi, L.C. 66, 67de Fatima, A. 81de Figueiredo, M.L.G. 134de Freitas, G.V. 117de Godoy, S.H.S. 258de Jesus, B.L.S. 14, 38, 89Delatorre, E. 168D’elia, M. 263de Lima, L.M.P. 87, 172de Lima, M.C. 95, 140de Lima, M.F.N.T. 266de Lima, M.M. 130de Lima, P.C. de S. 180Dellariva, T.C. 139Delmart, E. 273de Lucena, W.A.T. 180de Macedo, M.D. 10, 19de Magalhães, D.A.R. 10, 19de Medeiros, R.M. 11, 50de Mello, J.L. 248, 249de Melo, W.A. 174de Meneses, M.D.F. 84de Menezes Filho, R.N. 252, 257de Moraes, A.P. 98de Morais, V.M.S. 180Denani, C.B. 85Deng, X. 273de Oliveira, A. do S.L. 182

De Oliveira, F. 62de Oliveira, J.G. 86de Oliveira, J.M. 178de Oliveira, P.S.L. 94de Oliveira, T.S. 163de Pádua, S.B. 252, 257de Paula, F.E. 14, 38de Paula, S.O. 12, 30, 78, 117de Pinho, R.A.P. 171Dergovics, F.L. 112de Sá, J.P.O. 95, 140de Sena, A.A.S. 80de Siqueira, T.R. 227de Sousa, D.D. 180de Sousa, E. 89de Sousa, M.S. 196de Sousa, R.L.M. 14, 58, 252, 257, 258de Souza, E.M.A. 174de Souza, F.F. 231de Souza, K.M.C. 126, 127de Souza, M.C.B.V. 90de Souza, M.R 14, 58de Souza, R.P. 11, 31de Souza, V.C. 180de Souza, W.M. 135de Stefano, E. 225, 264Dias, A.S. 270Dias, C.M.G. 143Dias, J. 242Dias, M.C. 169Dias, R.S. 12, 30, 117Dibo, M.R. 155Di Felice, E. 63Diminitrova, Z.E. 13, 36Diniz, J.A. 264Dobao, E. 118do Carmo, A.P. 13, 35Dolabella, S.S. 189, 190Domiciano, I.G. 255Domingues, C.F. 232, 244Domit, C. 255Domitrovic, T. 12, 22Donin, D.G. 262Dorea, A. 146Dornas, F.P. 10, 12, 17, 21, 66, 101, 102,

103do Rosario, J.S.C. 198dos Anjos, K. 76dos Reis, A.S. 143dos Reis, J.K.P. 260dos Santos, A. de O. 173, 197dos Santos, A.O. 194dos Santos, C.N.D. 202dos Santos, C.P. 12, 31

dos Santos, F.A.L. 132, 166dos Santos-Junior, N.N. 78dos Santos, M.A.M. 130dos Santos, M.C. da S.G. 15, 40dos Santos, R.N. 12, 31dos Santos, T.A. 198dos Santos, V.R. 106Drummond, B.P. 131Drumond, B. 135Drumond, B.P. 64, 119, 120, 121, 123,

137, 196, 227, 233, 248Duarte, I. 238Duarte, P. de S.G. 13, 47, 212, 214Duarte, P.S.G. 13, 47, 215Dupont, P.M. 235, 251, 252Duppont, P.M. 52, 233Durães Magalhães, R. 269Durigon, E.L. 11, 14, 50, 60, 198, 224, 236,

270, 271Durymanova Ono, E.A. 240Dutra, A.G.S. 84Dutra, K.R. 123

EEbani, E.C. 116Edreira, N. 215Eduardo, M.B.P. 12, 29Eiras, A.E. 155Éleouët, J.F. 12, 24Eleutério Jr, J. 201Elias, T.C. 131Ellott, R.M. 12, 21Elmahdy, E.M. 104Escremim, F. 175Espada, S.F. 66, 67Esposito, D.L.A. 14, 25, 183Esteves, P.A. 12, 52, 231, 238Evers, F. 166

FFabres, R.B. 12, 29Faccin-Galhardi, L.C 118Fagotti, A. 153Fagundes, E.M.S. 63, 81Fagundes, J.M. 76Fagundes, L.G. 131Fantinatti, B. 273Faria, M.G. 13, 15, 34, 41Faria, N.A. 111Farias, K.J.S. 92, 127, 150, 152Farias, L.M. 76Farman, M. 13, 47Fausto, A.K. da S. 11, 44


Index282


Favarin, M. do C. 13, 36Favaro, A.B. 270Favaro, E.A. 120, 155Favero, L.M. 14, 57Fecury, P.C.M.S. 182Feldner, A.C. de C.A. 86, 122Felippe, P.A.N. 11, 51, 269, 275Fernandes, A.M. 14, 58, 225, 252, 257,

258Fernandes, A.T.G. 170, 193Fernandes, C.D. 216Fernandes, G.C. 119, 121Fernandes, J.E.F. 13, 48Fernandes Jr, P.C. 15, 41Fernandes, M.E.S. 13, 55, 247Fernandes, M.J.B. 70Fernandes, O.A. 13, 49Fernandez, J.C. 154Fernandez, J.C.C. 175Ferrari, K.L. 256Ferraz, A. de B.F. 81Ferraz, M.L. 165Ferraz, M.L.C.G. 86, 122Ferreira, A.C. 126, 151, 152, 153Ferreira, C.G.M. 119Ferreira, D. de L. 274Ferreira, D.F. 84, 92, 107, 110, 111, 143Ferreira, F.D. 73Ferreira, H.C.C. 273Ferreira, H.L. 245, 257, 275Ferreira, J. 195Ferreira, J.C. 13, 55Ferreira, J. de A. 171, 174Ferreira, J.G.G. 138Ferreira, J.M.S. 12, 22, 68, 123, 128Ferreira, L.C. de S. 137Ferreira, L.S.S. 133, 141Ferreira, M.U. 13, 37Ferreira P.C.P. 138Ferreira, P.C.P. 10, 13, 17, 18, 54, 74, 149,

230, 233, 266Ferreira, P.G.A. 222Ferreira, S. 154, 163Ferreira, S.B. 268Ferreira, V.F. 80, 268Ferreira, V.F.F. 90Ferreira, V.N. dos S. 92Ferreria, M.C. 263Ferro, C.G. 209, 212Fiaccadori, F.S. 11, 34, 125, 126, 127, 131,

134, 136, 145, 163Figueira, A. 215Figueira, A. dos R. 13, 47, 212, 214Figueira, A.R. 13, 47

Figueiredo, C. 71Figueiredo, L.T. 78Figueiredo, L.T.M. 82, 120, 134, 135, 145,

156, 162, 248Figueiredo, M.L.G. 135Figueiredo, P. de O. 13, 54Filho, A.A. da S. 84Filho, E.F. de O. 63Filho, F.A.X.M. 146Filho, J.R.C. 122Filho, R.N. de M. 14, 58Filho, S.L. de M. 128Filho, T.F. 238Filizzola, E.M.A. 171Filoni, C. 244Finoketti, F. 229, 272Firpo, R. 272Firpo, R.M. 229Florencio, C.M.G.D. 147Florêncio, C.M.G.D. 148Floriano, V.G. 125Fongaro, G. 12, 28, 104, 105Fonseca, B.A.L. 183, 184Fonseca Jr, A.A. 260Fonseca, L.A.B.V. 12, 30Fonseca, L.P. 116Fraiha, A.L.S. 14, 58Francisco, F.L. de M. 86Franco, A.C. 11, 12, 31, 50, 75, 99, 229,

272Franco, G.M. 81Franco, I.R. 15, 40, 131Franco, O.L. 87Fregolente, M.C.D. 176Freitas, D.M.T.A. 217, 220Freitas, G.M. 12, 21Freitas, G.R.O. 14, 26, 65, 68, 135Freitas, J.C. de O.C. 127, 152, 201Freitas, L.A. 247Freitas, L.B. 139, 153Freitas, L. de A. 264Freitas,L. de A. 272Freixo, H. de O. 178Froelich, L. 65, 135Fujimura, P.T. 14, 26Fumagalli, M.J. 15, 40, 131Fumian, T.M. 112, 124, 133Fusaro, A. 220

GGabbay, Y.B. 112, 138, 144, 160, 161, 191Gadelha, S.R. 129Gagliardi, T. 175, 181Gagliardi, T.B. 14, 25

Gaidet, N. 11, 50Galan, L. 154Galbieri, R. 11, 44Galindo, D.B. 11, 50Gallego, J. 166, 232, 267Gallego, J.C. 237, 248, 249Galler, R. 201Gama, L. 15, 43Gamon, T.H.M. 246Ganime, A.C. 113Garboggini, F.F. 98Garcia, D.G. 87Garcia, F.G. 267Garcia, L.A.T. 109, 114Garcia, L.A.T.; Nascimento, M.A.; Barardi,

C.R.M. 114García, M. 101Garcia, P. 205Garcia, R. de C.C. 244Garcia, R. de C.N.C. 232Garcia, S.C. 12, 29, 112Garrafa, P. 112Garrido, V. 64, 71, 77Gasparini, M.R. 241, 260Gatti, M.S.V. 176Gatto, I.R.H. 239Gauger, C.P. 15, 41Gava, D. 13, 53, 234Gerhardt, D. 14, 37, 243Gheit, T. 154Gibrail, M.M. 145Giehl, I. 105Giehl, I.C. 96, 106Gilio, A.E. 142Gil, L.H.V. 228Gil, L.H.V.G. 15, 42, 192Giongo, A. 99Giongo, V. 12, 28, 71, 77, 107, 108Giongo, V.A. 110, 111Giraldo, P.C. 201Giuliano, A.R. 154Godinho, M.T. 11, 43, 207, 209Godoi, A.M. 66, 118Godoi, A.M. de 67Godoy, H.P. 191, 239, 250, 261, 269Godoy, I.J. 222Góes, L.G. 236Goes, L.G.B. 270, 271Goes, T. 93, 94Gomes, A.C.C. 116Gomes, A.M. de O. 92Gomes, A.M.O. 85Gomes, A.V.B.T. 69, 128, 130Gomes, E. 195


Index283


Gomes, E.R. 174Gomes, É.R. 171Gomes, L.G. da S. 129Gomes, M. 147Gomes, R.A. 63Gomes, Y.M. 141Gonçalves, A.C.S. 239, 250, 269Gonçalves, A.K. da S. 201Gonçalves, G. dos S. 15, 43Gonçalves, M.C. 208Gonçalves, M. dos S. 171, 174Gonçalves, R.B. 85Gonçalves, S. 263Gonzaga, D.T. 268Gonzalo, B. 155Goodin, M.M. 13, 47Gotardi, A.H.B. 188Goulart, L.R. 13, 14, 15, 26, 34, 41, 80Gouvea, M.N. 203Gouvea, T.D. 118Gradiz, J.J. 14, 57Granados, O.F.O. 252Granato, C. 94, 157, 158, 165, 179, 188Granato, C.F.H. 164, 204Granda, M.C. 273Granja, F. 180, 184Grecco, S. 264Gregianini, T.S. 99Gregori, F. 235Gregório, D. de S. 142Grinsztejn, B. 182, 189Groff, F.H.S. 233Grolli, P. 232, 237, 249Grotto, R.M.T. 167Grynberg, P. 217Gudo, E. S. 175Guedes, M.I.M.C. 14, 58, 270Guedes, R.M.C. 270Guerra, J.M. 194Guerra, O.G. 188Guerra, S. de F. 191, 196Guerra, S. de F. dos S. 144, 182Guimarães, M. 14, 39, 187Guimarães, M.L. 185, 251Guimarães, T.E. 73Guimarães, V.N. 125, 127, 131, 163Guioti, F. 126, 152, 153Guissoni, A.C. 65, 120Guissoni, A.C.P. 145Gurjão, T.C.M. 112Gusmao, A.F. 152Gusmão, A.F. 126

H

Hachich, E.M. 12, 29Hafez, H.M. 11, 51, 269Hagiwara, M. 263Harakava, R. 208Harsi, C.M. 148, 204Hassan, R. 152Headley, S.A. 229Heck, T.M. da S. 12, 29, 111Heinemann, M. 263Heinen, L.B. da S. 130, 132, 166Heleno, N. 263Henzel, A. 12, 29, 96, 103, 105, 106, 111,

113, 228Hernandez, J. das M. 161Hora, A.S. 244Hosie, M. 263Howard, J.C. 230Huatuco, E.M.M. 198, 205Hurtado, R. 14, 60, 271Hurtado, R.F. 11, 50

IIbidi, S.M. 142Iglezias, S.D.A. 162Igreja, R.R. 146Immig, J. 272Inglis, P.W.; 217Ismael, N. 175ivonesi, M.C. 258

JJaime, J. 253Jain, S.A. 189, 190Jani, I. 175Januário, M.E. da S. 14, 27Japolla, G. 235, 237Jardim, A.C.G. 13, 36, 243Jesus, L.F. de 96Johnson, J.E. 12, 22Joventino, K.M. de S. 127, 150, 152Julião, J.A.S. 13, 34Junior, A.S. 273Junior, C.P.J. 237Júnior, E.C.S. 171Junior, J.C.R. 14, 57Junior., J.P.A. 196Júnior, J.W.R. 213Junior, L,B.D. 133Junior, L.B.D. 141Junior, M.C.R. 172Junior, P.C.F. 13, 34Junior, R.B.M. 175Júnior, S.M. de A. 11, 50

Júnior, W.P.L. 180Junqueira, B.R.T. 210Junqueira, D.M. 11, 50Junqueira, R.M. 185Justino, M.C.A. 182

KKamikawa, J. 188Kanamura, C.T. 162Kanashiro, A.M.I. 241Kashima, S. 10, 13, 19, 36, 172Katto, S. 229Khan, M.J. 145Khudyakov, Y. 13, 36Kimura, L.M. 194Kisielius, J.J. 11, 31, 112Klein, T.M. 184Kluge, M. 12, 31Knak, M.B. 11, 50, 75Kobayashi, M.T. 169Kohn, L.K. 98Kramer.B. 228Kratz, J.M. 81, 147Kroon, E. 122, 123Kroon, E.G. 10, 12, 13, 15, 17, 18, 21, 40,

54, 66, 68, 69, 74, 86, 101, 102, 103, 108, 121, 149, 155, 225, 230, 233, 259, 266

Kruger, L. 271Kunz, A. 12, 28, 105Kunz, A.F. 166, 232, 237, 248, 249, 267Kurissio, J.K. 13, 14, 56, 57, 163, 231, 261Kury, C.M.H. 178Kuster, R.M. 73

LLacerda, A.L. 218Lacorte, C. 218, 220Lamar, D.C. 207Lamas, N.S. 217Landell, M.G.A. 208La Scola, B. 10, 12, 17, 18, 21, 66, 101,

102, 108Lau, D. 209, 212Laurindo, M.F.L. 157Lazari, C. dos S. 157, 158Lazari, C.S. 164Leastro, M.O. 10, 13, 17, 49Lei, G. 11, 46Leite, F. de S. 215Leite J.G.P. 268Leite, J.P.G. 14, 59, 101, 124, 141Leite, J.P.V. 76


Index284


Leite, R.C. 241Leite, R. de C. 260Leite, T. 14, 39, 187Leme, R.A. 247Leme, R. de A. 258, 262Lemes, L.N. 11, 34Lemos, T.M.A.M. 152Leutenegger, C. 273Levis, S. 239Lewis, M. 15, 43Lierz, M. 11, 51, 269Li, L. 273Lima, A. 10, 18, 102Lima, A.S.N. 86Lima, A.T.M. 11, 43, 207, 209Lima, D.M. 146Lima, G.A. 178Lima, G.K. 260Lima, G.M. 87Lima, M. dos S. 264, 265, 268Lima, M.F. 220Lima, M.I.S. 15, 41Lima, M.P. 14, 58Lima, M.T. 68, 69Lima, N.C. da S. 194Lima Neto, D.F. 11, 51, 207, 269Lima, P.C. de S. 143Lima, R.G. 139Lima, R.N. 96Lima, S,F. 141Lima, S.F. 133Lima, S.T. 274Lima, T.L.C. 92Lima, W.A. 175Lima, W.T.A. 11, 14, 33, 38, 181Linhares, A. da C. 144, 160, 182, 191, 196Linhares, A.G. 13, 56Linhares, L.S.A. 13, 56Linhares, R.E.C. 66, 67, 118Lira, E.L. 186Livonesi, M.C. 15, 40Lizasoain, A. 101Lobato, Z.I.P. 14, 58, 270Lobo, P. dos S. 144Lo, D.S. 142Loh, E. 14, 60Lopes, A.P. de M. 215Lopes, B.P.T. 119Lopes, D.O. 123Lopes, E. 165Lopes, K.G.S. 228Lopes, L. dos S. 238Lopes, L.V.A. 116Lopes, L.V. de A. 116, 117

Lopes, N. 66, 67, 118Lopes, T.R.R. 143Lopes,T.R.R. 156Lorenzetti, E. 14, 57, 247, 258, 262Loureiro, E.C.B. 138Loving, L.C. 15, 41Lucas, F. 96Lucas, F.M. 219Lucas, M.A. 212Lucena, M.S.S. de 161Lucena, W.A.T. 143Luchs, A. 142Luciano, R.L. 241Lucinda, N. 76Luiz, A.P.F. 122Luiz, A.P.M.F. 225Luna, L. 235Luna, L.K. de S. 11, 33Lunel, N.L.T. 12, 21Lunge, V.R. 228Lustosa, J. 267Lustosa, J.M. 166Luz, C.R.N.E. da 160Luz, I. 113Luz, R.B. 12, 29Lyra, J.S.P.O. 90

MMabunda, N. 175Macedo, M.D. de 13, 36Macedo, R. 166, 232, 237, 248, 249, 267Machado, A. de M.V. 15, 42, 192Machado, A.M. 188Machado, A.M.V. 76Machado, A.R.S.R. 188Machado, B.C. 142, 159Machado, D. 93Machado, G. 123, 233Machado, P.R.L. 92, 127, 150, 152, 201Maeda, A.Y. 11, 31Magalhães, C.L. de B. 233Maganha, S.R. de L. 14, 58, 252, 257Magri, M.E. 12, 28, 105Maiorka, P.C. 246Malaquias, L.C.C. 12, 15, 22, 40, 69, 128,

130, 131Malossi, C.D. 259Mamani, P.R. 82, 156Marcio, M.S. 11, 45Marcolino, L.D. 189, 190Maria, F.H. de O.S. 95, 140Mariano, A.P.M. 129Marinheiro, J.C. 148Marinho, R.S.S. 14, 59

Marin, L.J. 129Marques, B.C.L. 251Mar, T.B. 209, 212Martinelli, A. de L.C. 172Martines, R.B. 162Martinez, R.T. 219Martini, M.C. 11, 51, 98, 207, 245, 257,

269, 275Martins, C.P.S. 63Martins, D. de L. 87Martins, F. dos S. 68, 69Martins, M. de S.N. 264, 265, 268Martins, M.L. 13, 35, 63, 81Martins, N. 263Mascarenhas, D.O.J.D’A.P. 196Mascarenhas, J.D’A.P. 161, 182, 191Mascarenhas, J.D.P. 12, 53, 112, 138, 144Mascitti, A.K. 228Massi, R.P. 262, 272Mathijs, E. 63Matias, B.F. 13, 15, 34, 41Matos, A.C.D. 14, 58Matos, N.B. 194Matos, R.P.A. de 75Matsui, T. 12, 22Mattos, B.B.B. 11, 44Mattos, G.L.M. 12, 52, 231, 238Mauroy, A. 63Mazer, L. 250Mazer, L.C. 239Mazzarotto, G.A.C.A. 202M.B. de L.D. 131Medeiros, A.S.R. 239Medeiros, M.A. 201Medeiros, N.P.T. 185Medeiros, R. 171, 174, 195, 274Medeiros, R.S.S. 194Medeiros, T.N. da S. 14, 57, 258Mees, L. 75Mehnert, D.U. 112Meira, G.L.S. 73Meire, G.L.S. 143Melchiades, V. 64, 71, 77Melgaço, F.G. 113Mello, F.C. do A. 159Mello, I.M.V.G.C. 13, 36Mello, I.O. 141Mello, J.L. 232, 237Mello, W. 195Mello, W.A. 119, 133, 141, 171, 274Melo, D.M. 94Melo, D.M. de 185, 186Melo F.L. 216Melo, F.L. 13, 48, 49, 213, 217, 218


Index285


Mendes, G. 87Mendonça, L.A. 119, 121Mendonça, L.M. 97Mendoza, Y.Y. 155Meneguin, C. 224Meneses, M.D.F. 73Menezes, E. de F.C. 138Menezes, I. de Q. 178Menezes, W. da R. 118Menoni, S. 186Menoni, S.M.F. 193Merlone, M.P. 13, 37, 178Miagostovich, M. 101Miagostovich, M.P. 113, 124, 133, 141Michelin, E.C. 252, 257Migliolo, L. 87Milanelo, L. 13, 56Minnicelli, C. 152Miquelitto, F. 184Miranda, A.E. 139, 153Miranda, E.A. 189, 190Miranda, F.J.A. 150Miranda, J.B. 13, 54, 64, 227, 230, 248Mirandola, P.H. 188Miura, V.C. 167Miyabe, F.M. 264, 272Miyaki, C. 203Miyashiro, S. 263Miyashiro, S.I. 246Miyazaki, R.D. 166Modena, J.L.P. 14, 38, 181Modis, Y. 14, 25Molinari, B.L.D. 247, 258, 262Mondini, A. 126, 151, 152, 153, 155Monezi, T.A. 112Monta, M.J. 146Montassier, H.J. 12, 52, 231, 238Montebianco, C.B. 11, 44Monteiro, D.C.S. 14, 40Monteiro, H.A. de O. 213Monteiro, J.M. de C. 78, 117Monteiro, T.A.F. 150, 177Montoro, M.G. 239Moraes, A.P. 269Moraes, E.M.S. 229Moraes, F.M. 183Morais, L.D. da S. 80Morais, L.L.C. de S. 112Morais, S.M. de S. 12, 22, 128Morais, V.M.S. 143, 156Morais, W. 273Morandi, B.C. 98Moreira, C.M. 158Moreira, L. 94, 188

Moreira, L.P. 179Moreira, M.S.A. 93Moreira-Vieira, F. de F. 68Moreli, M.L. 125, 167Moreno, E.C. 149Moresco, V. 109Mores, M.A.Z. 12, 52, 231, 238Morgado, L.N. 173Morgado, M. 175Morgado, M.G. 14, 39, 154, 168, 182, 189,

251Mori, C.M.C. 246Mori, E. 246Morillo, S.G. 142Moriones, E. 212Mósena, A.C.S. 233, 235, 236, 251Mota, B.E.F. 74Mota, G.C. 86Mota, M.T. de O. 73Mota, T.Q. 152Moura, F. de B.P. 93Moura, F.E.A. 147, 148Moura, M. 11, 46Moura, M.E.G. 157, 158Muller, E.C.A. 191, 196Müller, U.B. 230Muller, V. 78Muller, V.D.M. 93, 94, 186Munin, F.S. 14, 58, 258Myiazaki, R.D. 132

NNagasse-Sugahara, T.K. 112Nagata, T. 76, 133, 172, 210, 216Nagel, N.E. 11, 51, 269Nali, L.H. da S. 11, 32, 33Nara, J.M. 255, 256Nardi, M.S. 14, 60Nascimento, B.L.S. 213Nascimento, I. 215Nascimento, M.A. 104, 114Nascimento, V.A. 14, 27, 40Nascimento, Y.M. 92Nassar, A.F.C. 225Nava, A. 14, 60Navarro, I.T. 166Navarro, J.A.S. 10, 13, 17, 49Navarro, J. de O. 14, 58, 252, 257Navarro, J.S. 13Naveca, F.G. 14, 27, 40, 180, 184Naves, J.H.F. de F. 260Negri Filho, L.C. 229Nelson, M. 13, 53Nepomuceno, A. 108

Nepomuceno, A.M. 110, 111Neto, A.C. 270Neto, D.B. 229Neto, D.F.L. 245Neto, E.A. 91Neto, E.D. 11, 32Neto, F.C. 151, 155Neto, J.P.N. 213Neto, L.L.S. 146Neto, M.M. 157Neurauter, A.L. de A. 90Neves, A.P. 64Neves, M.A.O. 138Nguyen, J.B. 14, 25Nicolini, C. 216Nogueira, A.H.C. 225Nogueira, G.B. 157Nogueira, J.S. 11, 31Nogueira, L.M. 123Nogueira, M.F.T. de L. 259Nogueira, M.L. 73, 120, 131, 135, 137,

155, 167, 196Nogueira, R. 172Nonogaki, S. 194Nozawa, C. 66, 67, 118Nunes, C.F. 14, 37, 243Nunes, E.M. 154Nunes, M.R.T. 12, 21Nunes, M.S. 13, 37

OOcadaque, C.J. 147, 148Oda, J.M.M. 188Ogata, R.A. 255, 256Ogawa, J. 12, 24Okano, W. 229Okino, C.H. 12, 52, 231, 238Okuda, L.H. 225, 264, 265, 268Olival, G.S. do 11, 33Oliveira, A. 12, 24Oliveira, A.C. 85Oliveira, A.C.R. 125, 126Oliveira, A. do S. 191, 196Oliveira, A.L.R. 11, 31, 239Oliveira, A.M. 266Oliveira, A.S. 78, 88, 91Oliveira, C.P. 157Oliveira, D. 122, 123Oliveira, D.B. 10, 17, 191, 198Oliveira, D.C.A. 203Oliveira, F.M.S. 147Oliveira, G.P. 68, 69Oliveira, I.B. 95Oliveira, I.B.R. 210


Index286


Oliveira, J.G. 138Oliveira, J.M. 13, 37Oliveira, L. do H. dos S. 11, 32Oliveira, L.G. 239Oliveira, L.M. 133Oliveira, M.D. 117Oliveira, M.M. 65, 135Oliveira, R. 219Oliveira, R.A. 70Oliveira, R. das N. 203Oliveira, R.G. de O. 90Oliveira, R.N. 247Oliveira, R.N. 13, 55Oliveira, R.S. 139Oliveira, S. 210Oliveira, T.F. de M.S. 62, 160Oliveira, T.F.M.S. 65Oliveira, T.F.P. 266Oliveira, T.S. 131Oliveira, V.M. 12, 30Ometto, T. 11, 14, 50, 60, 224, 236, 271Ometto,T. 224Ordonez, F.J.P. 226Ordoñez, F.J.P. 227Orellana, M.D. 10, 13, 19, 36Orílio, A.F. 213, 215Orsi, M.A. 13, 54Ortamnn, C.F. 149Osaki, S.C. 267Otaguiri, K.K. 13, 36Otonel, R.A.A. 247, 262, 267Oyafuso, M.K. 248

PPacca, C.C. 73Pacheco, S.M. 52Pacheco, T. dos 166Pacheco, T.J.A. 218Padilla, M.A. 98, 245Pádua, R.M. 76Paglia, A. 248Paglia, A.P. 64, 227Paixão, C.G.S da 141Paixão, I. 71Paixão, I.C.N. de P 87, 90Paixão, I.C.N. de P. 90Paixão, I.C.N.de P. 98Paixão, I.C.N.P. 12, 28, 77, 95, 107, 108,

268Paixão, IC.N.P. 14, 59Paixão, I.C.P. 110, 111Paixão, I.C.P.N. 64Paixão, L.C.F. 150Paixão, S. 133

Pallás, V. 10, 13, 17, 49Pandolfi, J.R. 228Pannuti, C.S. 11, 32Pantoja, M.B. 222Pardo-Vargas, A. 95Parente, A.F. 120Parente, A.M.B. 146Parente, J. 65Parra, M.C.P. 155Pascoal, J. de O. 160Pascoli, G.V.T. 160Passetti, F. 119Passos, A.M. 165Paterson, A. 11, 46Paula, D.F. 96Paula, F.L. 146Paula, J.E.F.B. 12, 28, 107, 108, 110Paulini, I.J. 204Paulo, W.M. 229Pedrosa, C.F. 176Pedrosa, F.C. 179, 197Pedrosa, L.G. 12, 28Pedrosa, L.G.M. 111Pedrosa-Macena, L.G. 107, 108, 110Peiró, A. 13, 49Peña, B.P. 205Penalva de Oliveira, A.C. 11, 33Penido, B.A.F. 235Perecin, D. 208Pereira, A.B. 14, 59Pereira, A.F. 225Pereira, A.V.C. 194, 197Pereira, F.L. 258, 262Pereira, H. de S.P 90Pereira, H. de S.P. 90Pereira, L.E. 239Pereira, M.G. 127, 152Pereira, O.M.D. 11, 32Pereira, P.L. 263Pereira, P.M.C. 13, 55, 247Pereira, P.S. 110Pereira, S.A.R. 147, 148Pereira, U.P. 15, 41Pereira, W.V.E.G. 126, 153Pérez, R. 264Périco, J.M.B. 196Perissini, L.H. 83Perosa, A.H. 14, 38Pessoa, C.R. 78Pestana, N.F. 14, 38Petersen, E. 224Petry, M.V. 224, 271Pianowski, L.F. 15, 43Pierrotti, L.C. 11, 32

Pilotto, J.H. 168Pilotto, M.R. 109Pimenta, S.T.S. 191Pimentel, F.B. 232, 244Pinhatti, A.K. 73Pinheiro, M. da S. 95, 140Pinheiro, T.M. 137, 196Pinheiro, T.M.L. 95, 140Pinho, J.B. 224Pinto, A.M. 71Pinto, A.M.V. 14, 59, 268Pinto, A.R. 77Pinto, L.B. 224Pinto, L.R. 208Pinto, M.A. 13, 37, 173, 178, 245Pinto, M.T. 10, 19Pinto, V.B. 209Pinto, V. da S.C. 264, 265, 268Pires, L.M. 196Pires, M.A. 74Pituco, E.M. 225, 264, 265, 268Piva, M.R. 190Polaro, A.A. 150, 177Polloni, L.C. 62Ponce, E.L. 154Pontes, C.M.L. 146Portal, T.M. 160Portari, E.A. 193Possati, F. 248, 249, 262Possatti, F. 247, 258Prado, A.A.O. 69, 130Prates, M. 178, 181Prates, M.C. 14, 38Price, S.L. 15, 43Primo, F.L. 75Provazzi, P.J.S. 167, 169Pujol, S. 103Pusterla, N. 273

QQueiroz, A. 93Queiroz, A.T.L. 210Queiroz, L.H. 270Queiroz, S.R. de A. 14, 58Quintana, S.M. 169Quintana, V.H.A. 127, 145, 152Quirino, M.S. 213

RRabelo, A. 207Rabelo, D.C.C. 143, 180Racaniello, V.R. 14, 25Raeva, L.M.G. 13, 36


Index287


Rafael, E. 120Rahal, P. 12, 13, 23, 36, 72, 73, 75, 79, 82,

83, 120, 167, 169, 243Raissa, L. 177Rajao, D. 15, 41Rajão, D.S. 270Ramalho, T.O. 13, 47Ramirez, G. 253Ramírez, G. 253Ramos, A. da C. 162Ramos, C.J.B. 14, 59Ratti, D. 262Real-Hohn, A. 85Rebouças, A.S.J. 70Rebouças, M.S. 273Rechenchoski, D.Z. 66, 67, 118Reginatto, F.H. 81, 147, 149Rehfeld, I.S. 14, 58Reinaldo, M.R. 165Reis, C.F. 14, 26Reischak, D. 13, 54Reis, J. 263Reis, M.B.A. 217Rendon, D.S.C. 13, 36Resende, R. 216Resende, R. de O. 13, 215Resende, R.O. 10, 13, 17, 49, 96Resque, H.R. 160, 161Reys, C. 158Rezende, B. 70Rezende, I.M. 121, 233Rezende, I.M.de 64Ribeiro, A. de S. 267Ribeiro, A.L.M. 132, 166Ribeiro, A.S. 232, 237Ribeiro, B.M. 13, 48, 207, 213, 218Ribeiro, C. 64Ribeiro, C.P. 98, 225Ribeiro, G.P. 222Ribeiro, G.S. 13, 48Ribeiro, J. 258Ribeiro, L.F.C. 210, 211Ribeiro, M. 71, 77Ribeiro, M. de S.R 90Ribeiro, M. de S.R. 90Ribeiro, M.G. 273Ribeiro, M.G.M. 189, 190Ribeiro, M.R. 73Ribeiro, M.S. 143Ribeiro, S.G. 217, 218, 220Ribeiro, Z.M.A. 217Ricardo, N.M.P.S. 66, 67, 118Ricci, E. 157Richtzenhain, L.J. 226, 227, 256

Rigotto, C. 12, 29, 96, 103, 105, 106, 111, 113

Rios, W.M. 78Ritterbusch, G.A. 12, 52Rivetti Jr, A.V. 266Rocco, I.M. 11, 31Rocha,B.A. 203Rocha, D.R. 80Rocha, E.S. de O. 15, 40Rocha Junior, M.C. 10, 19Rocha, L.B. 204Rocha, M.A.L. 93, 94Rocha, N. 170Rocha, N. de S.P.D. 150Rocha, R.P. 15, 40, 131Rocha, S. 111Rodrigues, A.P. de S. 260, 266Rodrigues, C.M. 80Rodrigues, E.A.M. 161Rodrigues, E.S. 10, 13, 19, 36, 172Rodrigues, F.C. das C. 193Rodrigues, I.C. 138Rodrigues, J.S.V. 132, 166Rodrigues, M.C. 141Rodrigues, M.V. 261Rodrigues, N.F. 15, 40, 69, 130Rodrigues, N.F.S. 74Rodrigues, R.A. 12, 21Rodrigues, R.A.L. 66, 102, 103Rodrigues, S.V. 171Roehe P.M. 55Roehe, P.M. 11, 12, 31, 50, 75, 99, 113,

247, 272Rohrmann, G.A. 11, 46Rollie, J.C. 11, 46Roma, E.H. 170Romanel, E. 11, 44, 46, 221Romano, C.M. 11, 14, 32, 33, 37, 137, 243Romano, E. 217Romeiro, F. 134Romeiro, M.F. 135, 162Rosa, J.C.A. 13, 52, 55Rosales, C.A.R. 13, 54Rosa, L.H. 86Rosa, M.P. 252Rosa, P.C.R. 167Rostal, M. 14, 60Roxo, T. 85Rribeiro, J. 14, 57Russomano, F.B. 170, 193Russo, R. 243Russo, R.R. 78

S

Sabino, U. 224Sacchetto, L. 64, 227, 248Sacchetto, L.P. 121Sacramento, P.R. 158Sakata, S.T. 13, 54Salcedo, J.M.V. 173, 194, 197Sales, C. de B.P.M. 93Salla, P. de F. 228Salles, S.T. 73Salles, T.S. 84Salustiano, S.G. 172Salvador, F.S. 137Sá, M. 103Samara, S.I. 239, 250, 269Sampaio, M.L. da S. 146, 202Sampaio, S.V. 243Sanches, M.M. 217Sanchez, E.F. 72Sangrillo, F.S.S. 90Santana-Pereira, P. 12, 28, 107, 108, 111Santiago, J.C.L. 222Santos, B.A. 214Santos, C. 12, 31Santos, C.J. 89Santos, D.A. 10, 18, 102Santos, D.F. 172Santos, D.I. 220Santos, D.M. da S. 13, 35Santos, E.S. 88, 245Santos, F.M. 117, 185Santos, G.R. 261Santos, H.C.P. 134, 136Santos, H.F. 113Santos, I.G. de A. 189, 190Santos, I.M. 77Santos, J. das V. 194Santos, J.R. 203Santos Jr, C.D. 14, 26Santos Jr, J.A. dos 185Santos, L.F.P. 150Santos, L.L. 123Santos, L.L.R. 143Santos, L.S. 11, 32Santos, M. 195Santos, M.C. 171, 174, 274Santos, M.M.A.B. 245Santos, R. 85Santos, R.F. 191, 261Santos, S.P. 265Santos, T.C. 149Santos, T.F.Q. 98Santos, T.V. 178Santos, W.K. da S. 127, 150, 152Saporiti, V. 255


Index288


Saraiva, H.A.C. 213Sarda, F.N. 147Sardi, S.I. 146, 202Sato, M.I.Z. 12, 29Saturno, T. 14, 38Saturno, T.H. 181Sbegue, A. 193Scagion, G.P. 275Schaefer, R. 13, 53, 234Schenkel, E.P. 147Schissi, C.D. 12, 28, 105Schissi; C.D. 104Schrago, C.G. 11, 44Sciani, J.M. 203Sebastiani, M.C. 275Segura, M. de N.O. 213Seixas, M. 224, 236, 271Seixas, M.M.M. 11, 50Semaan, L.M. 197Sena, A. 157, 165Senem, J.V. 211Sepulveda, L. 239Serra, O.P. 130, 132, 166Serrão, V.H.B. 234Shimizu, J.F. 243Shirata, N.K. 194Sichero, L. 79, 154, 163Sihler, W. 11, 45Silva, A. 64Silva, A.A.B. 273Silva, A.C.M. 125Silva, A.E.B. 86, 122Silva, A.K. 133, 141Silva, A.K.F. 219Silva, A.L. de S. 157Silva, A.P. 264Silva, B.F.G. 164Silva, B.M. 69Silva, C.C. 12, 30Silva, C.E.C. 137Silva, C.L. 78Silva, C.O. 11, 32Silva, D.C.P.S. 219, 221Silva, D.F. 11, 33Silva, D.M. 143, 156Silva, E. 188Silva, F. de O. 72, 84Silva, G.A.V. 184Silva, G.C.D. 196Silva, G.C.P. 261Silva, G.F. 167Silva, I.T. 76, 81, 149Silva, Í.T.S.S. 70Silva, J. de M. 186

Silva, J.L. 85, 92Silva, J.L.A. 156Silva, J.P. 13, 37, 209, 212Silva Jr, J.V.J. 15, 42, 192Silva, J.S. 204Silva Júniro, J.V.J. 228Silva, K.N. 216Silva, L.A. 13, 49Silva, L.C. 229Silva, L.C.F. 10, 12, 17, 21, 30, 101, 102,

103Silva, L.D. 138Silva, L.K. dos S. 66, 102, 103Silva, L.M. 170Silva, L.P. 134Silva, M. 175Silva, M.A.N. 170Silva, M.C. 234Silva, M.C.C. 89, 164, 194, 245Silva, M.C. de M. 138Silva, M.F. 208Silva, M.G. 163Silva, M.H.R. 188Silva, M.J.M. 12, 53Silva, M.L. 14, 38, 88, 91Silva, M.S. 216, 233Silva, M.X. 117Silva, P.A. 87, 172Silva, R. 107Silva, R.J.C. 154Silva, R.K. 275Silva, R.R. 12, 53Silva, R.U. 138Silva, S. 242Silva, S.O.S. 256Silva, T.C. 87Silva, T. da F. 11, 44Silva,T.F. 221Silva, T.G. 264, 265, 268Silva, V.S. 228Silveira, A.C. 163Silveira, F.A. 159Silveira, R. 242Silveira, S. 233, 235Silveira, V.R. 11, 31Silvestre, R.V.D. 119, 133, 141Silvia, A.P. 272Simabuco, F. 12, 24Simas, P.V.M. 11, 51, 269Simões, C.M.O. 76, 81, 147, 149Simões, M. 124Simões, R.S. de Q. 193Simoes, R.S.Q.S. 170Siqueira, A.B. 191

Siqueira, C.E.H. 162, 167Siqueira, J.A.M. 144, 160Siqueira, T.R. 119, 121Skums, P. 13, 36Slavov, S.N. 13, 36, 172Slhessarenko, R.D. 130, 132, 166Snak, A. 267Soares, A. 242Soares, A.C. 14, 37, 243Soares, A.M. 78Soares, C. 65, 120Soares, L. da S. 112, 138, 144, 161, 182Soares, L.S. 12, 53Soares, M.S. 236Sobrinho, J.S. 79Soliman ,M. 113Soliman, M.C. 12, 29, 103, 105, 106, 111Solorio, M.R. 14, 60Sotero, A. de J. 13, 47Sotero, A.J. 13, 47Sousa, C.A. 142Sousa, D.D. 184Sousa, J.G.A. 217Sousa Jr, E.C. 119Sousa, M.P. 12, 30Sousa, M.S. de 112Souza, A.C.S. 89Souza, C. 203Souza, C.K. 252Souza, E. 195Souza, F.G. de 12, 29, 105, 106, 111Souza, J.G. 63, 81Souza, K. 120Souza, K.C. 15, 41Souza, K.F.C.S. 87Souza, K.M. 136Souza, K.M.C. 11, 34Souza, K.S. 71Souza, L.B.F.C. 150, 201Souza, L.F.B. 173, 194, 197Souza, L.M.S. 127, 150, 152Souza, L.R.G. 235, 237Souza, M. 11, 34, 134, 136Souza, M.B. de L.D. 125, 126, 127, 163Souza, M.C.P. 14, 60Souza, M.L. 11, 45Souza, M.M. 91Souza, N.V. 146Souza, R.P. 239Souza, S.F. 225, 268Souza, V.C. 14, 27, 40Souza, W.M. 82, 156, 162Spada, P.K. de P. 112Spano, L.C. 139, 141, 153


Index289


Spera, C.G. 264, 272Sperança, M.A. 88Spiegel, M. 12, 21Spilki, F.R. 12, 29, 96, 103, 105, 106, 111,

113, 228Staggemeier, R. 12, 29, 96, 103, 105, 106,

111, 113Stancioli, E.F.B. 13, 35, 63, 81, 241, 273Stangherlin L.M. 245Stangherlin, L.M. 164, 194Stefanello, F. 260Steffens, R. 248Steindel, M. 147Stephens, P.R.S. 90Stoppa, G.F.Z. 241Streck, A.F. 13, 52, 56, 235, 236, 250, 251,

252Suhette, R. 12, 30Suzuki, A. 11, 31, 239Szabo, M.P.J. 160

TTaffarel, L.E. 232Takayanagui, O.M. 10, 13, 19, 36Takiuchi, E. 166, 232, 237, 248, 249, 267Tamashiro, E. 11, 14, 33, 38, 175, 181Tanaka, T. 187Tanaka, Y. 10, 19Tanuri, A. 15, 43Tarboda, R.L.M. 173Tavares, D.C. 231Tedesco, A.C. 75Teixeira, B. 263Teixeira, D.M. 112Teixeira, G. 64, 77Teixeira, M.M. 120Teixeira, S. 14, 39Teixeira, T.F. 113Teixeira, V. 71Teixeira, V.L. 14, 59, 77, 87, 90, 95Tenório, E.C.N. 203Terzian, A.C.B. 120Tesh, R.B. 213Tessari, E.N.C. 241Thiry, E. 63Thomazelli, L. 236, 271Thomazelli, L.M. 11, 50, 224Thomaz, L. 204Thomaz, M.M. 266Tigre, D.M. 146Timenetsky, M.C.S.T. 239Timenetsky, M. do C.S.T. 142Tinôco, R.S. 13, 49Toffoli, B. 63

Togawa, R.C. 217Tolardo, A.L. 135, 162Tommasino, M. 154Tonietti, P. de O. 246Toniollo, G.H. 231Toppa, N.H. 116Torikachvili, M. 235, 252Torquato, E.B 211Torrelio, C.M.Z. 14, 60Torres, A.P.R. 12, 30Torres, F.D. 272Torres, S. 186Torres, S.V.S. 193Tort, L.F.L. 101Tozato, C.C. 14, 57Trabuco, A.C. 78, 145Trento, C.L. 189, 190Trevisol, I.M. 12, 52, 231, 238Trindade, A.C.G. 157Trindade, G. de S. 13, 54, 64, 74, 149, 227,

230, 233, 248Trindade, G.S. 12, 21Trindade, G.T. 225Tunin, A. 225

UUeira,C.U.V. 14, 26Ullmann, L.S. 13, 56, 120, 137, 196, 259,

273Ulmann, L.S. 261Urbano, P.R. 11, 14, 32, 37, 243Urbano, P.R.P. 11, 33

VVago, A.R. 116, 117Valera, F. 175, 181Valera, F.C.P. 11, 14, 33, 38Valério, O.C. 273Vancini, R. 84Varella, R. 242Vargas, M.D. 64Vasconcelos, K. de O. 210Vasconcelos, P.F.C. 145Vasconcelos, P.F. da C. 213Vasli, M.F.S. 11, 44Vaslin, M.F.S. 11, 44, 219, 221Vaughan, G. 13, 36Vaz, E.R. 14, 26Vecchi, L. 14, 26Vedovello, D. 120, 135, 137, 196Vedovello,D. 73Veiga, A.B.G. 99Veiga, R. 119

Veloso, V. 14, 39Veloso, V.G. 182, 189Venditti, L.L.R. 268Ventura, A. 12, 24Veras, R.M. de A. 93, 94Vera, V. 253Vergueiro, M. 195Vessaro Silva, S.A. 210Vicentini, F. 141Victoria, M. 101Vieira, C.J. da S.P. 167Vieira, D.S. 173, 194, 197Vieira, E.M. 14, 57Vieira, F.N. 64, 227Vieira, H.R. 142, 159Vieira, R. de S. 160Viillalobos, E.M.C. 246Vilela, A.P.P. 266Villa, L.L. 75, 79, 154, 163Villamil, V. 253Villani, F.N.A. 138Villeth, G.R.C. 218Vincent, L.A. 15, 41Vitral, C.L. 13, 37, 173, 178Vogel, V.A.R. 110, 111Voguel, V.R. 108Volpini, L.P.B. 139, 153Vorsatz, C. 182, 189Vubil, A. 175

WWalker, D. 11, 50Wang, R. 13, 47Watanabe, A.S.A. 94, 179, 188Webby, R.J. 11, 50Weber, M.N. 52, 233, 235, 236, 251, 252Webster, R.G. 11, 50Weidmann, D.M.D.S. 12, 21Wood,C. 260

XXavier, A.A. 76Xavier, C.A.D. 11, 43, 207Xavier, M.A. 208

YYamamoto, A.Y. 169Yamamoto, K.A. 84Yamamoto, K.I. 73Yamasaki, L.H.T. 13, 36Yamatogi, R.S. 254Yokosawa, J. 14, 26, 62, 65, 68, 135, 160


Index290


ZZacalusni, S. 187Zaguini, J. 12, 28, 105Zanatto, D.A. 246Zanella, E.L. 260Zanella, J.R.C. 13, 53, 234, 260, 270Zanluca, C. 202Zapana, E.M. 205Zerbini, F.M. 11, 43, 207, 209, 212Zeredo, A.C.B. 12, 28Ziber, G. 103Zirbes, G. 12, 29Zonta, W. 63Zubiaur, Y.M. 212Zuchi, N. 130, 132, 166

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