b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346

ht t p: / /www.bjmicrobio l .com.br /

Veterinary Microbiology

Evolution of equine influenza viruses (H3N8)during a Brazilian outbreak, 2015

Patricia Filippsen Favaroa,∗, Wilson Roberto Fernandesb, Dilmara Reischakc,Paulo Eduardo Brandãoa, Sheila Oliveira de Souza Silvaa, Leonardo José Richtzenhaina

a Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Medicina Veterinária Preventiva e SaúdeAnimal, São Paulo, SP, Brazilb Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Médica, São Paulo, SP, Brazilc Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Nacional Agropecuário, Campinas, SP, Brazil

a r t i c l e i n f o

Article history:

Received 17 January 2017

Accepted 7 July 2017

Available online 13 October 2017

Associate Editor: João Araujo

Keywords:

Equine influenza

H3N8

Hemagglutinin

Neuraminidase

Evolutionary analysis

a b s t r a c t

Equine influenza is one of the major respiratory infectious diseases in horses. An equine

influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veteri-

nary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza

viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino

acid sequences were compared with the recent isolates from North and South America and

the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The

hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine

strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K)

and another not in antigenic site (K304E). The four substitutions changed the hydrophobic-

ity of hemagglutinin. Three distinct genetic variants were identified during the outbreak.

Eleven variants were found in four quasispecies, which suggests the equine influenza virus

evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines

without the production of protective antibody titers might be the major contributing factors

on virus dissemination during this outbreak.

© 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is

an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

pneumonia.

Introduction

Equine influenza (EI) leads to epithelial destruction in theupper respiratory tract of horses, resulting in fever, coughing,

∗ Corresponding author at: Depto. Medicina Veterinária Preventiva e

Universidade de São Paulo, R. Dr. Orlando Marques de Paiva, 87, 05508-E-mail: patriciafilippsen@gmail.com (P.F. Favaro).

https://doi.org/10.1016/j.bjm.2017.07.0031517-8382/© 2017 Sociedade Brasileira de Microbiologia. Published by

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

nasal discharge, performance impairment and occasionally1

Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia,270, Cidade Universitária, São Paulo, SP, Brazil.

The classification of influenza A viruses is based ontwo major surface antigens, hemagglutinin (HA) and neu-raminidase (NA) and are related to virus infection and

Elsevier Editora Ltda. This is an open access article under the CC.

r o b i

pr

aerKs11Caat

btO2wbStwA

ibtcHgo

M

S

NtpU(lpapfsc

I

Twsvt

b r a z i l i a n j o u r n a l o f m i c

ropagation.1 Minor changes in amino acid constitution canesult in different antigenicity and lead to immune scape.2

Equine influenza viruses (EIVs) (H3N8) were initiallyssigned to a single cluster3 and evolved into two sublin-ages, American and Eurasian, according to the geographicegion.4 The American lineage diverged into South American,entucky and Florida sublineages.5 The strains of the Floridaublineage had amino acid mutations in the hemagglutinin

(HA1) subunit and this lineage diverged into Florida Clade (FC1), with the substitutions A78V and S159N, and Floridalade 2 (FC2),6,7 represented by A/equine/South Africa/4/2003nd A/equine/Richmond/1/07, respectively.7 The lineages FC1nd FC2 have been identified in outbreaks worldwide and arehe predominant circulating EIVs.8–16

EIV is considered endemic in Brazil, where the first out-reak caused by a H3N8 virus was reported in 1963,16–22 whilehe most recent H7N7 EIV outbreak happened in 1976.23,24

utbreaks of H3N8 occurred in this country in 200825 and010,26 but data on the lineages to which the viruses belongedere not published.25,26 H3N8 EIVs isolated from a further out-reak in 2012 in the states of São Paulo and Rio Grande doul belonged to FC116,22 and, regardless of several substitu-ions in the HA1 subunit, no major differences in antigenicityere found in comparison with the Florida 1 vaccine strain/equine/South Africa/4/2003.16

The aims of this study were (a) to describe an EIV outbreakn a veterinary school hospital in Brazil in 2015 that affectedoth vaccinated and unvaccinated horses; (b) to characterizehe HA1 and neuraminidase (NA) amino acid sequences inomparison with the vaccine strain and the Brazilian 20123N8 EIV; and (c) to access the inter and intra-host EIVenetic variation that might have caused the impact of thisutbreak.

aterials and methods

amples

asal swabs were collected in triplicates from all the 32 horseshat were in the hospital barns. The nasal swabs were trans-orted in minimum essential medium (Gibco, Carlsbad, CA,SA), with penicillin (200 U/mL) and streptomycin (200 �g/mL)

Gibco, Carlsbad, CA, USA). Transtracheal washes were col-ected along with the nasal swabs, from eight horses thatresented nasal discharge, cough and fever. The nasal swabsnd transtracheal washes were kept at −80 ◦C until use. Aanel of 64 serum samples was collected from the horses: 32rom the first day on which they showed respiratory clinicaligns; and 32 on the 15th day thereafter. The serum samplesollected were stored at −20 ◦C until use.

nfluenza A virus screening

he DirectigenTM EZ FluA & B test kit (BD, Sparks, MD, USA)as used on nasal swabs from the first 13 horses that were

uspected of having EI. This assay targets the influenza Airus nucleoprotein and was performed in accordance withhe manufacturer’s instructions.

o l o g y 4 9 (2 0 1 8) 336–346 337

Virus isolation

Virus isolation was undertaken in 10-day-old specificpathogen-free (SPF) embryonated chicken eggs by means ofthe allantoic fluid route. The SPF eggs were kindly donatedby Laboratório Biovet, located in Brazil. All horses that werepositive for EIV by means of DirectigenTM EZ FluA & B testkit (BD, Sparks, MD, USA) or amplification of M gene27,28 hadnasal swabs (or transtracheal wash, in case it was done) usedin virus isolation. Virus isolation (maximum of two passages)was confirmed by polymerase chain reaction (RT-PCR), target-ing the M gene of influenza A viruses,27 under the conditionspreviously described.28

Amplification of M, HA and NA genes and DNAsequencing

Total RNA was extracted from 300 �L of allantoic fluid of eachsample using the Purelink

®RNA mini kit (Ambion, Carlsbad,

CA, USA) and cDNA was synthesized using 2.5 �M of Uni12primer (5′-NNAGCRAAAGCAGGNNN-3′, modified from Hoff-mann et al., 2001) and SuperScriptTM III (Life Technologies,Carlsbad, CA, USA). The RNA extraction and cDNA synthesiswere done as per the manufacturer’s instructions.

All nasal swabs were tested by means of RT-PCR target-ing the M gene of influenza A viruses,27 under conditionsdescribed previously.28 Amplification of the complete hemag-glutinin (HA) (1762 nt) and neuraminidase (NA) (1413 nt) genesfrom the isolates was undertaken using modified primersoriginally designed by Gildea et al.29 and Hoffmann et al.30

(Table S1) and PlatinumTM Taq DNA Polymerase High Fidelity(Life Technologies, Carlsbad, CA, USA), as per the manufac-turer’s instructions. HA and NA amplicons were purified fromagarose gels using IllustraTM GFX PCR DNA and gel band purifi-cation kit (GE Healthcare, Little Chalfont, Buckinghamshire,UK), as per the manufacturer’s instructions. The sequencing

reaction was carried out using the BigDye®

Terminator v.3.1Cycle Sequencing kit (Applied Biosystems, Carlsbad, CA, USA),in accordance with the manufacturer’s instructions, in theApplied BiosystemsTM 3500 Genetic Analyzer (Applied Biosys-tems, Foster City, CA, USA). All fragments were amplified andsequenced three times, in order to increase confidence in theanalysis.

Molecular cloning

In order to evaluate intra-host EIV genetic variation (quasis-pecies), a segment of HA gene was cloned and sequencedfrom four isolates. The HA RT-PCR products from theprimers HA1F and HA466R29 were purified using ExoSAP-It

®(USB, Cleveland, OH, USA) and were inserted into

the pTZ57R/T vector of the Thermo ScientificTM InsTA-clone PCR cloning kit (Thermo Fisher Scientific Inc.,Vilnius, Lithuania) in Escherichia coli (JM109). Plasmids

were extracted using Nucleospin®

Plasmid (Macherey-Nagel,

Düren, Germany) and were sequenced with M13/pUC primers(Thermo Fisher Scientific Inc., Vilnius, Lithuania) as describedabove.

i c r o

338 b r a z i l i a n j o u r n a l o f m

Phylogenetic analysis

The confidence levels of all sequences were evaluated usingthe FinchTVTM software. Nucleotide sequences were appliedusing the Basic Local Alignment Search Tool (BLASTn),available in GenBank, National Center for BiotechnologyInformation (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/), tosearch for homologous sequences. EIV sequences from theEpiFluTM database of the Global Initiative on Sharing AllInfluenza Data (GISAID) were also used in the analysis. Theaccession numbers in the GenBank and GISAID databasesare provided as Supplementary data, in Tables S2 and S3.Sequences were aligned using Muscle,31 and maximum-likelihood amino acid trees were built using 1000 bootstrapreplicates,32 through MEGA 7.33 The mean evolutionary diver-sity and distances for complete HA sequences were estimatedusing MEGA 7.33 HA nt sequences of eight isolates were com-pared in order to evaluate inter-host EIV genetic variation.

Evolutionary analysis

A molecular clock test on 77 EIV HA1 nucleotide sequencesrepresenting the five clades was performed using the max-imum likelihood method, with the Hasegawa–Kishino–Yano(HKY) model34 with discrete gamma distribution, in MEGA 7.33

The nucleotide substitution rates and phylogenetic rela-tionships were assessed through Bayesian Markov ChainMonte Carlo (MCMC) analysis,35 using BEAST v.1.7.536 withthe HKY model with four categories of gamma-distributed ratevariation among sites.34 The strict and relaxed (lognormal andexponential) molecular clocks were calibrated using the straindates. The effective sample size (ESS) was accessed by meansof Tracer v.1.6.037 and the final MCC clock-tree was viewedusing FigTree 1.4.2.38

Anti-influenza A virus antibodies

A commercial enzyme-linked immunosorbent assay (ELISA;

FlockChek®

Avian Multiscreen Antibody Test Kit, IDEXX Lab-oratories Inc., Westbrook, ME, USA) that had been developedto detect antibodies against the highly conserved viral nucle-oprotein (NP) of the influenza A virus was used with the horseserum samples. The test consists in a competitive ELISA thus ahigher concentration of specific antibodies will result in lowertest result. Samples showing a ratio of the serum sample opti-cal density (OD) to the negative control OD provided by theassay (S/N) ≤ 0.60 were considered positive; while S/N > 0.60was considered negative for the presence of anti-influenzaantibodies.

Results

EI outbreak description

The Veterinary School Hospital of the School of Veterinary

Medicine and Animal Science, University of São Paulo, SãoPaulo, SP, Brazil, experienced an EIV outbreak in vaccinatedand unvaccinated horses from September 16 to September 25,2015. Twenty-six of the 32 horses housed at the hospital at

b i o l o g y 4 9 (2 0 1 8) 336–346

the time of the outbreak (29 adult horses, two foals and onepony) displayed at least one clinical sign of EI within an eight-day period, including nasal discharge, dry cough and sneezing,along with fever, inappetence and mucosal congestion.

Among the 32 horses, ten horses had been immunized witha vaccine containing an inactivated A/equine/Kentucky/97strain, and two had been immunized with the inactivatedA/equine/South Africa/4/2003 and A/equine/Kentucky/94strains. Three horses were immunized but the name of thevaccine and/or strain was not available. The two foals werefrom vaccinated mares but there was no full vaccinationhistory available. For the remaining horses, the anti-EIVvaccination history was unknown.

Influenza A virus screening and isolation

The initial screening on the 13 horses that first showed res-piratory clinical signs was carried out using the DirectigenTM

EZ FluA & B test kit (BD, Sparks, MD, USA), on nasal swabs.There were six positive results and one inconclusive result.The matrix (M) gene of the influenza A virus was identifiedby means of RT-PCR on 23 nasal swabs out of 32 that weretested. For the eight horses from which transtracheal washeswere collected along with the nasal swabs, both samples wereanalyzed by means of RT-PCR for the M gene. Among these,seven swabs and three transtracheal washes showed positiveresults. The transtracheal washes (positive or negative) frompositive horses (positive at nasal swab sampling) were used atvirus isolation. Twelve EIVs were isolated from the 23 horsesthat were found to be positive through RT-PCR. Six of the iso-lates were from transtracheal washes and five were from nasalswabs. Twelve of the 15 vaccinated horses were infected withEIV, ten with clinical signs. One of the horses that showedclinical signs had no virus detected but had seroconversion atELISA. Among the vaccinated horses that had EIV, two receivedthe OIE recommended strain, eight the A/equine/Kentucky/97and two with no description of which EIV strain received.

Anti-influenza A virus antibodies

The ELISA results among the horses that were positive for EIVswere S/N 1.0918 (95% CI: 0.8612; 1.3225) at the first samplingand S/N 0.2620 (95% CI: 0.1607; 0.3633) on the 15th day there-after (Table S4). Increased levels of influenza A antibodies werealso seen with paired serum samples from the horses thatvirus was not detected or isolated from samples: S/N 0.6626(0.3437; 0.9815) on day 1 and S/N 0.3615 (0.0995; 0.6235) on day15. Nineteen horses seroconverted, nine were positive at bothsampling and four remained negative. Twenty seven horseswere considered infected due to virus detection in nasal swabor transtracheal wash, or to seroconversion at ELISA (negative-positive). The description of the samples, EIV screening, virusisolation, ELISA and vaccination status are presented in TableS4.

HA and NA gene analyses

HA sequences were obtained from ten of the isolates. Sixnucleotide (nt) sequences comprised full codons of the hemag-glutinin gene (1698 nt), which included the signal peptide (nt

r o b i

1f1t(atgHd

HwrFi(

(s(99

lpEiftC

1wAhnA(aaAatG

H

Tsw2c(

ramat

b r a z i l i a n j o u r n a l o f m i c

–45) (SP/8, SP/10, SP/11, SP/12, SP/15 and SP/32), and four wererom partial segments of the uncleaved protein: nucleotides

to 1016 (SP/4), 27 to 1698 (SP/25), 1 to 1165 (SP/30) and 1o 437 (SP/18). Five neuraminidase sequences were obtainedSP/8, SP/10, SP/12, SP/15 and SP/25) comprising nt 53–1366,nd no nucleotide differences were shown. For one represen-ative strain (SP/8), NA was resequenced to achieve better NAene coverage, from which nt 28–1395 were obtained. TheA and NA sequences of the EIV São Paulo/2015 strains wereeposited in GenBank (KX954135–KX954164).

Nine amino acid substitutions were found in theA1 sequences of São Paulo/2015 isolates, in comparisonith the World Organisation for Animal Health (OIE)-

ecommended vaccine strain A/equine/South Africa/4/2003.our of these substitutions were not present in the Brazil-an 2012 outbreak strain (N3S, T121S, G142R and K304E)Table 1).

Three genetic variants were identified from the outbreakvariant 1, V1; variant 2, V2; and variant 3, V3) with single sub-titutions in residues 121 and 304 (T-K/T-E and S-K) at HA1Table 2). The sequence similarities between variants were9.8% (V2/V3) and 99.9% (V1/V2 and V3) for nucleotides and9.6% (V2/V3) and 99.8% (V1/V2 and V3) for amino acids.

The HA and NA amino acid maximum composite like-ihood phylogenetic trees showed similar topologies andresented five groups: Florida Clade 1, Florida Clade 2,urasian, Kentucky and Pre-divergence (Figs. 1 and 2), show-ng eight HA1 sequences and one representative NA sequencerom samples from this study. The viruses isolated inhe present study belonged to the H3N8 subtype Floridalade 1.

The hydrophobicity of HA1 changed in residues 62, 138,42 of V1, V2 and V3 and 304 of V2, in comparisonith A/equine/Rio Grande do Sul/1/2012 and A/equine/Southfrica/4/2003 (Fig. 3). NA substitutions also changed theydrophobicity profile. São Paulo/15 NA sequences had 39t changes and there were 11 aa changes in relation to/equine/Ohio1/03 (FC1): one in the transmembrane helix

V35A) and ten in the head domain. The numbering ofctive sites followed the N2 numbering pattern.9 Five aminocid substitutions were found in SP-2015 NA but not in/equine/Rio Grande do Sul/1/2012: L36P, R40G, R171K, E411Gnd H450Y. The 2012 and 2015 Brazilian strains sharedhe substitutions V35A, N205S, R260K, E271G, S337N and416E.

A evolutionary analyses

he mean evolutionary diversity for the 157 complete HAequences representing the five clades from 1963 to 2015as 0.034 (SE 0.004) and 0.0019 (SE 0.0012) for the Brazilian

015 lineages. In addition, the amino acid distance was cal-ulated using the Poisson correction model40 in MEGA 733

Table 3).The molecular clock tests on 77 EIV HA1 sequences, rep-

esenting the five clades, showed that the evolutionary rates

mong sequences were not equal. In both of the relaxedolecular clocks (lognormal and exponential), the ESS was

bove the minimum threshold of 200, which means thathe sequences had good posterior representation and were

o l o g y 4 9 (2 0 1 8) 336–346 339

uncorrelated. The lognormal clock better represented the evo-lutionary analysis than did the exponential clock, since ithad lower standard deviation (SD): lognormal SD: 12.1418;exponential SD: 13.0462. Thus, the lognormal molecular clockwas used for the MCMC analysis, with the HKY model andcalibration using strain dates from BEAST v.1.7.536 (Fig. 4).The lineages grouped into two different clades: Clade 1for V1 and V2; and Clade 2 for V3. The estimation forthe time that has elapsed since the most recent commonancestor (TMRCA) indicated that the Brazilian-2015 strainsare significantly younger than the root height and FloridaClade 1.

HA sequences (1–437 nt of the gene) comprising thesignal peptide and the first 130 amino acids of HA1 (nt1–392) were cloned from three isolates (A/equine/SãoPaulo/8.FMVZ/2015, A/equine/São Paulo/10.FMVZ/2015,A/equine/São Paulo/15.FMVZ/2015) and one nasal swab(A/equine/São Paulo/18.FMVZ/2015). Four clones from eachstrain were sequenced and the amino acid and nucleotidemean diversities were estimated. The number of nucleotideand amino acid substitutions per site, relating to the meandiversity40 among the clonal populations of the four horses,was 0.0055 and 0.015, with standard error estimations of0.001 and 0.004, respectively. The amino acid clone diversitywas obtained using the Poisson model,41 and the nucleotideclone diversity was assessed using the maximum compositelikelihood, both with gamma distributed rates and 1000bootstrap replicates in MEGA 7.33

The descriptions of amino acid substitutions in partial HA1clones from the strains A/equine/São Paulo/8.FMVZ/2015,A/equine/São Paulo/10.FMVZ/2015, A/equine/SãoPaulo/15.FMVZ/2015, A/equine/São Paulo/18.FMVZ/2015,A/equine/São Paulo/6/1963, A/equine/São Paulo/1/1969 andA/equine/South Africa/4/2003 are presented in Table S5.

Discussion

Twelve EIVs (H3N8) were isolated from horses during theoutbreak, which ten were sequenced and three genetic vari-ants (V1-3) were identified. The difference between V1/V2and V3 consists of substitution in the residues 121 and304, been V1 T121/K304, V2 T121/E304 and V3 S121/K304.Up to the time of this study, the residue S121 was uniqueto V3 and T121/E304 to V2 Brazilian 2015 strains, exceptby A/equine/Romania/1980 that have the V2 pattern. Dur-ing the outbreak, this substitution first appeared in samplescollected between September 19 and September 22, but wasnot seen in samples collected between September 14 andSeptember 16, thus suggesting that it appeared during theoutbreak.

Several equine flu outbreaks occurred around the worldin 2010–2012 and were related to the FC1 and FC2sublineages.11–16 The Brazilian-2015 isolates were similar tothe Dubai, Uruguay, Argentina, Chile and Kentucky strainsfrom 2012. The Brazilian-2015 strains were placed into two

clusters in a major cluster that contains the FC1 EIV strainsfrom 2011 and 2012. The short distances between the vari-ants of the present study shown in each branch wouldbe expected in strains from same outbreaks. The MCMC

340 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346

Table 1 – Amino acid substitutions in complete HA sequences among Brazilian-2015 isolates from the present study (*),North and South American-2012 strains and the OIE-recommended Florida Clade 1 A/equine/South Africa/4/2003vaccine strain. Numbers below sites E and A are the amino acid positions.

Strain Site E Site A

3 7 62 104 121 138 142 223 304

A/equine/South Africa/4/2003 N G R D T A G V KA/equine/Sao Paulo/4.FMVZ/2015* S D K N T S R I KA/equine/Sao Paulo/8.FMVZ/2015* S D K N T S R I KA/equine/Sao Paulo/10.FMVZ/2015* S D K N T S R I EA/equine/Sao Paulo/11.FMVZ/2015* S D K N T S R I EA/equine/Sao Paulo/12.FMVZ/2015* S D K N S S R I KA/equine/Sao Paulo/15.FMVZ/2015* S D K N S S R I KA/equine/Sao Paulo/18.FMVZ/2015* S G/D K N S – – – –A/equine/Sao Paulo/25.FMVZ/2015* S D K N S S R I KA/equine/Sao Paulo/30.FMVZ/2015* S D K N T S R I KA/equine/Sao Paulo/32.FMVZ/2015* S D K N S S R I KA/equine/Rio Grande do Sul/2012 N D K N T S G I KA/equine/Dubai/1/2012 N D K N T S G I KA/equine/Sao Paulo/6/1963 N D R D M A G V K

A/equine/Dubai/1/2012 representing KF026412.1, KF026413.1, KF026407.1, KF026408.1, KF026409.1 and KJ372713.1.A/equine/Sao Paulo/6/1963 representing CY032397.1(–): sequence not available.

Table 2 – Amino acid and nucleotide substitutions in the hemagglutinin gene (HA1 subunit) of eight equine influenzaviruses isolated from an outbreak in São Paulo, SP, Brazil, 2015.

Genetic variant EIV Residue 121 Codon Residue 304 Codon

V1 8/2015 T ACA K AAAV2 10/2015 T ACA E GAAV2 11/2015 T ACA E GAAV3 12/2015 S TCA K AAAV3 15/2015 S TCA K AAAV3 25/2015 S TCA K AAAV1 30/2015 T ACA K AAA

V3 32/2015 S

analysis indicated that the Brazilian 2015 strains and the 2012strains from Dubai, USA and Brazil have the same ancestor.These variants clustered into two clades in the MCC tree:Clades 1 and 2.

Substitutions at HA antigenic sites can interfere with theantibody binding response.42 The EIV isolates in the presentstudy had two amino acid substitutions at site A (residues138 and 142) and one at site E (residue 62) when comparedto A/equine/South Africa/4/2003. Site A showed decreasedhydrophobicity and is situated in an external portion at theHA head.16,42 The two substitutions at site A (aa 138 and142), which were seen in all the Brazilian EIV strains from2015, showed that the combination of these two resultedin greater change when compared to the single substitu-tion (A138S) found in A/equine/Rio Grande do Sul/2012 andA/equine/Dubai/1/2012 (Fig. 3), thus could affect antibodybinding. Part of the lower hydrophobicity was due to the 138substitution, which occurred in FC1-2012 strains but did notresult in great antigenic changes.16 The substitution G142Rwas unique to V1, V2 and V3 Brazilian-2015 strains and was

not found in other EIVs from public databases, thus suggestingthat this substitution might have happened before or duringthe outbreak.

TCA K AAA

The substitutions R62K (site E), D104N and A138S (site A)6

and V223I were previously described in FC1 strains.14 Thesubstitutions V78A and N159S that differentiate FC1 fromFC2 remained.8,14 As noticed by Legrand et al.,14 the sevensubstitutions in residues G7D, R62K, V78A, D104N, A138S,N159S and V223I have apparently remained unchanged since2008.

The nucleotide and amino acid diversity among the clonesshow that there were occurrences of quasispecies duringthe outbreak. This supports the hypothesis of variant evolu-tion during outbreaks. A transmission network and evidenceof genetic variants were previously studied in a single EIVoutbreak.43

The catalytic function of the NA enzyme is related tothe residues R-116, D-149, R-150, R-223, E-275, R-291, R-368 and Y-402,44 which were conserved in São Paulo-2015strains. Epitopes at the NA head involving the residues 150,199, 344–346, 367, 399 and 40045 were conserved among theEIVs analyzed, with three exceptions: the changes Q199Hon A/equine/Kentucky/2/12 and A/equine/Kentucky/3/12 and

R150Q on A/equine/Spain/1/07.

The ELISA used in this study has an epitope-blocking for-mat and this format allows to be used for anti-influenza

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346 341

A/equ ine /FL/146609 /201 1 A/equ ine /Rio Grande do Sul/1/201 2 A/equ ine /Yokoha ma/aq5 /201 1 A/equ ine /Yokoha ma/aq29 /201 1 A/equ ine /Ken tucky/1/201 1 A/equ ine /Ken tucky/4/201 1 A/equ ine /New York/1/201 1

A/equ ine /Penn sylvan ia/1-2/201 1 A/equine/Argentina/E-2345-1/2012 A/equine/Kentucky/1/2012 A/equine/Dubai/3/2012 A/equine/Dubai/2/2012 A/equine/Dubai/1/2012

A/equine/Kentucky/2/2012 A/equ ine /Ken tucky/3/201 2

A/equine/Tennessee/28A/2014 A/equ ine /Tenne ssee /4A/201 4 A/equ ine /Tenne ssee /30 A/201 4 A/equ ine /Tenne ssee /29 A/201 4 A/equ ine /Tenne ssee /28 B/201 4 A/equ ine /Tenne ssee /27 A/201 4

A/equ ine /Sao Pau lo/10 .FMVZ/201 5 A/equ ine /Sao Pau lo/11 .FMVZ/201 5

A/equine/Sao Paulo/8.FMVZ/2015 A/equ ine /Sao Pau lo/30 .FMVZ/201 5

A/equ ine /Sao Pau lo/12 .FMVZ/201 5 A/equ ine /Sao Pau lo/15 .FMVZ/201 5 A/equ ine /Sao Pau lo/25 .FMVZ/201 5 A/equ ine /Sao Pau lo/32 .FMVZ/201 5

A/equine/California/1/2010 A/equ ine /OR/78356 /201 2

A/Equ ine /Sweden /SVA111206 SZ0085 /VIR165837 /201 1 A/equ ine /Lincolnshire/1/200 7 A/equ ine /Tott ori/1/200 7

A/equ ine /Mon tana /9233 /200 7 A/equ ine /Sou th Africa/4/200 3 A/equ ine /Wiscon sin/1/200 3 A/equ ine /Ohio/113461 -1/200 5 A/equ ine /Virginia/131054 -3/200 5 A/equ ine /Texas/117793 /200 5

A/equ ine /Newmarket/5/200 3 A/equ ine /California/8560 /200 2 A/equ ine /Ken tucky/5/200 2

A/equ ine /Huabe i/1/200 7 A/equ ine /Richmond /1/200 7

A/equ ine /Baizak/09 /201 2 A/equ ine /LKZ/09 /201 2

A/equ ine /Heilong jiang /SS 1/201 3 A/equ ine /Xuzhou /01 /201 3

A/equ ine /Heilong jiang /1/201 0 A/equ ine /North Rhine Westpha lia/1/201 4 A/equ ine /Ayrshire/1/201 3 KR534268 .1 A/equ ine /Rome/1/201 4

A/equ ine /Worcestershire/2/201 2 A/equ ine /Coun ty Durha m/2/201 2

A/equ ine /Shrop shire/8/201 3 A/equ ine /Scott ish Borde rs/3/201 5

A/equ ine /Hertfordshire/1/201 3 A/equ ine /Northa mpton shire/1/201 3 A/equ ine /Worcestershire/1/201 3 A/equ ine /Warwickshire/1/201 3 A/equ ine /East Yorks hire/1/201 3 A/equ ine /Lana rks hire/1/201 3 A/equ ine /Lana rks hire/2/201 3 A/equ ine /Northa mpton shire/7/201 3 A/eq ine /Shrop shire/5/201 3 A/equ ine /Norfolk/1/201 5 A/equ ine /North Yorkshire/1/201 5 A/equ ine /Le icestershire/1/201 5 A/equ ine /North Yorkshire/2/201 5

A/equ ine /Ken tucky/8/199 4 A/equ ine /Ken tucky/1/199 2

A/equ ine /Berlin/1/198 9 A/equ ine /Miami/1/196 3

A/equ ine /Urugua y/1/196 3 A/equ ine /Sao Pau lo/1/196 9

A/equ ine /Sao Pau lo/6/196 399

99

60

60

99

89

75

69

61

87

65

62

71

63

94

84

63

97

61

63

82

HA

Florida

subli neage

Cla de 1

Florida

subli neage

Cla de 2

Eurasia n lineage

Pre divergent lineage

American

Lineage

Kentucky lineage

Fig. 1 – Maximum composite likelihood amino acid tree, using Poisson model with 1000 bootstrap replicates of 77 HA1subunit sequences (nt 1–987) of H3N8 EIVs available in GenBank and GISAID EpiFluTM for the Florida Clade 1 (FC1), FloridaClade 2 (FC2), Kentucky, Eurasian and Pre-divergent lineages. The viruses characterized in the present study are indicatedby diamonds (�).

awtis

ntibody detection in several species.46–48 Anti-NP antibodiesere detected in nine horses in the first collection while in

he second collection 28 samples were positive demonstrat-ng serum-conversion after virus infection. Two horses thathowed no clinical signs and were negative in EIV screening,

seroconverted, confirming that they were infected during theoutbreak.

The EIV variants isolated from this single outbreak sug-gest that a quasispecies evolutionary pattern was present.The substitutions (residues 138 and 142) found in HA1

342 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346

497571 | NA | EPI ISL 151846 | A/equine/Warwickshire/1/2013 497575 | NA | EPI ISL 151847 | A/equine/East Yorkshire/1/2013 497570 | NA | EPI ISL 151845 | A/equine/Worcestershire/1/2013 493623 | NA | EPI ISL 151986 | A/equine/Shropshire/8/2013 493620 | NA | EPI ISL 151985 | A/eqine/Shropshire/5/2013 493617 | NA | EPI ISL 151984 | A/equine/Northamptonshire/7/2013 493613 | NA | EPI ISL 151981 | A/equine/Lanarkshire/1/2013

KF049192.1 A/equine/County Durham/2/2012 KF049190.1 A/equine/Roxburghshire/1/2012 KF049174.1 A/equine/Worcestershire/1/2012 493625 | NA | EPI ISL 151841 | A/equine/Northamptonshire/1/2013

710986 | NA | EPI ISL 212044 | A/equine/Scottish Borders/3/2015 686739 | NA | EPI ISL 205764 | A/equine/North Yorkshire/1/2015 694842 | NA | EPI ISL 205957 | A/equine/Leicestershire/1/2015

686737 | NA | EPI ISL 205763 | A/equine/Norfolk/1/2015 699681 | NA | EPI ISL 207225 | A/equine/North Yorkshire/2/2015

KF049194.1 A/equine/Devon/1/2011 AB618505.1 A/equine/Yokohama/aq13/2010

KF049172.1 A/equine/East Renfrewshire/2/2011 KF049191.1 A/equine/Lichtenfeld/1/2012

KF559336.1 A/equine/Richmond/1/2007 GU571145.1 A/equine/Huabei/1/2007 KF309035.1 A/equine/Heilongjiang/1/2010

KF806987.1 A/equine/Xuzhou/01/2013 KC986392.2 A/equine/Heilongjiang/SS1/2013 KP202376.1 A/equine/Kostanay/09/2012 KP202374.1 A/equine/LKZ/09/2012 KP202373.1 A/equine/Baizak/09/2012

CY075852.1 A/equine/Spain/1/2007 497568 | NA | EPI ISL 151796 | A/equine/Ayrshire/1/2013

FJ375224.1 A/equine/Newmarket/5/2003 CY030761.1 A/equine/California/8560/2002 98508 | NA | EPI ISL 9534 | A/equine/Ohio/1/2003

DQ222914.1 A/equine/Wisconsin/1/03 CY067320.1 A/equine/Ohio/113461-1/2005 CY067304.1 A/equine/Texas/117793/2005

CY067560.1 A/equine/Virginia/131054-3/2005 CY067512.1 A/equine/Montana/9233/2007

JX844148.2 A/equine/Kyonggi/SA1/2011 AB591843.1 A/equine/Tottori/1/07

584272 | NA | EPI ISL 177498 | A/equine/California/1/10 KF049187.1 A/equine/Kentucky/2/2012 KF049186.1 A/equine/Kentucky/3/2012

KF049185.1 A/equine/Kentucky/4/2012 KF049184.1 A/equine/Kentucky/5/2012 753427 | NA | EPI ISL 220480 | A/equine/Tennessee/28A/2014

KF049183.1 A/equine/Pennsylvania/1-2/2011 594018 | NA | EPI ISL 180684 | A/Equine/Sweden/SVA111206SZ0085/VIR165837/2011

584297 | NA | EPI ISL 177502 | A/equine/Rio Grande do Sul/1/12 KF049173.1 A/equine/Dubai/1/2012

A/equine/Sao Paulo/8.FMVZ/2015 584280 | NA | EPI ISL 177500 | A/equine/Kentucky/1/11

AB727556.2 A/equine/Yokohama/aq5/2011 AB727558.2 A/equine/Yokohama/aq29/2011

EU926630.1 A/equine/Lonquen/1/2006 CY030151.1 A/equine/Kentucky/1/1992

CY030183.1 A/equine/Kentucky/8/1994 KF049176.1 A/equine/Lincolnshire/2006 KF049177.1 A/equine/Aboyne/2005

CY032415.1 A/equine/Berlin/1/1989 CY032311.1 A/equine/Santa Fe/1/1985

CY028838.1 A/equine/Miami/1/1963 CY032423.1 A/equine/Uruguay/1/1963

CY032399.1 A/equine/Sao Paulo/1/1969 CY032295.1 A/equine/Sao Paulo/6/1963100

79

100

100

94

74

97

75

74

73

77

87

93

94

80

92

77

NA

Americanlineage

Pre divergent lineage

Eurasian lineage

Floridasublineage

Clade 2

Floridasublineage

Clade 1

Kentucky lineage

Fig. 2 – Maximum composite likelihood amino acid tree, using Poisson model with 1000 bootstrap replicates of 65 NAsequences (nt 28–1395) of H3N8 EIVs available in GenBank and GISAID EpiFluTM for the Florida Clade 1 (FC1), Florida Clade 2(FC2), Kentucky, Eurasian and Pre-divergent lineages. The virus characterized in the present study is indicated by a

diamond (�).

might have affected antibody binding, especially to thehorses that had been vaccinated with the OIE-recommendedstrain, A/equine/South Africa/4/2003. Also, the use of an

out of date vaccine strain or updated vaccines withoutthe production of protective antibody titers might be themajor contributing factors on virus dissemination during thisoutbreak.

Ethical approval

This study was approved by the Ethics Committee

on Animal Use of the School of Veterinary Medicineand Animal Science of the University of São Paulo(no. 4480120314).

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346 343

South Africa/4/2003Sao Paulo/8.FMVZ/2015Sao Paulo/10.FMVZ/2015Sao Paulo/11.FMVZ/2015Sao Paulo/12.FMVZ/2015Sao Paulo/15.FMVZ/2015Sao Paulo/25.FMVZ/2015Sao Paulo/30.FMVZ/2015Sao Paulo/32.FMVZ/2015Rio Grande do Sul/1/2012

Rio Grande do Sul/1/2012

Ohio/1/2003Sao Paulo/8.FMVZ/2015

HA

NA

0

-0.6

-1.2

Mea

n hy

drop

hobi

city

Mea

n h y

drop

hobi

city

-1.8

-2.4

R62K

V35A

L36P

R40G

R171K

N205S

R260K/E271G

S337N

E411G H450Y

G416E

A138SG142R

K304E

-3

-3.6

-4.2

-3.6

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450

-3

-2.4

-1.0

-1.2

-0.0

0

0.6

1.2

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160Position

Positio n

Eisenberg scale mean hydrophobicity profilescan-window size = 13

Eisenberg scale mean hydrophobicity profilescan-window size = 13

170 180 190 200 210 220 230 250 260 270 280 290 300 310 320240

Fig. 3 – Eisenberg scale mean hydrophobicity profile for the equine influenza virus HA1 and partial NA amino acidsequences (465 aa) of the OIE-recommended strains, São Paulo/2015 isolates and A/equine/Rio Grande do Sul/1/2012 isolate.This profile was built using BioEdit v.7.2.5.39

Table 3 – Amino acid distances of 157 EIV HA sequences between the sublineages of EIVs, including the seven completeHA sequences from the present study. Distances were calculated using the Poisson correction model40 in MEGA 7.33

EIV groups Distance (%) Standard error

Pre-divergence/Brazil 2015 6.29 0.009Brazil 2015/Florida I 1.40 0.003Brazil 2015/Florida II 3.07 0.006Brazil 2015/American 3.85 0.007

4.42.3

C

T

A

TBCL

Brazil 2015/Eurasian

Florida II/Florida I

onflicts of interest

he authors declare no conflicts of interest.

cknowledgements

his work was financially supported by FAPESP-razil [2011/03234-7]; CNPq-Brazil [141677/2014-7] andAPES/PROEX-Brazil [2327]. We would like to thank theaboratório Biovet, located in Brazil, for the donation of

0 0.0087 0.004

the SPF embryonated chicken eggs used in this study. Wegratefully acknowledge the contribution of the authors of theoriginating and submitting laboratories of the sequences inthe EpiFluTM database of GISAID on which this research isbased. The list is detailed in Supplementary data shown inTables S2 and S3.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.bjm.2017.07.003.

344 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 336–346

A/equine/Sao_Paulo/12.FMVZ/20150.280.38

0.99

0.090.5

0.630.02

0.120.05

0.10.11

0.71

0.17

0.130.950.99

0.150.050.04

0.13

0.91

0.93

0.76

0.94

0.21

0.1

0.31

0.93

0.37

0.220.160.65

10.1

0.460.220.17

0.180.120.170.96

0.11

0.9

0.96

0.970.810.22

0.99

0.98

0.99

0.99

0.97

0.81

1950 1960 1970 19901980 2000 2010 2020

1

0.98

1

1

11

1

0.49

1

1

1

1

1

0.91

11

1

1

1

1

11

A/equine/Sao_Paulo/15.FMVZ/2015A/equine/Sao_Paulo/25.FMVZ/2015A/equine/Sao_Paulo/32.FMVZ/2015A/equine/Sao_Paulo/11.FMVZ/2015A/equine/Sao_Paulo/10.FMVZ/2015A/equine/Sao_Paulo/30.FMVZ/2015A/equine/Sao_Paulo/8.FMVZ/2015

A/equine/Dubai/3/2012

A/equine/Dubai/1/2012A/equine/Kentucky/1/2012

A/equine/Kentucky/4/2011A/equine/New_York/1/2011A/equine/FL/146609/2011A/equine/kentucky/1/2011

A/equine/Tennessee/27A/2014A/equine/Tennessee/28B/2014A/equine/Tennessee/29A/2014A/equine/Tennessee/28A/2014A/equine/Tennessee/30A/2014A/equine/Tennessee/4A/2014

A/equine/Kentucky/3/2012A/equine/Kentucky/2/2012

A/Equine/Sweden/SVA111206SZ0085/VIR165837/2011A/equine/Pennsylvania/1-2/2011A/equine/yokohama/aq5/2011A/equine/yokohama/aq29/2011

A/equine/California/1/2010A/equine/Lincolnshire/1/2007A/equine/Montana/9233/2007A/equine/Tottori/1/2007

A/equine/Texas/117793/2005A/equine/Ohio/113461-1/2005A/equine/Virginia/131054-3/2005

A/equine/Wisconsin/1`/2003A/equine/South_Africa/4/2003

A/equine/California/8560/2002A/equine/Norfolk/1/2015A/equine/Leicestershire/1/2015A/equine/North_Yorkshire/2/2015A/equine/North_Yorkshire/2/2015A/equine/Scottish_Broders/3/2015

A/equine/Hertfordshire/1/2013A/equine/Worcestershire/1/2013A/equine/Northamptonshire/1/2013A/equine/Warwickshire/1/2013A/equine/Lanarkshire/1/2013A/equine/Shropshire/5/2013A/equine/Lanarkshire/2/2013A/equine/East_Yorkshire/1/2013A/equine/Northamptonshire/7/2013A/equine/Shropshire/8/2013

A/equine/worcestershire/2/2012A/equine/County_Durham/2/2012

A/equine/North_Rhine_Westphalia/1/2014

A/equine/Baizak/09/2012A/equine/Ayrshire/1/2013

A/equine/LKZ/09/2012A/equine/Heilongjiang/SS1/2013A/equine/Xuzhou/01/2013

A/equine/Heilongjiang/01/2010A/equine/Huabei/01/2007A/equine/Richmond/01/2007

A/equine/Newmarket/5/2003A/equine/Kentucky/5/2002

A/equine/Kentucky/8/1994A/equine/Kentucky/1/1992

A/equine/Berlin/1/1989A/equine/Miami/1/1963A/equine/Uruguay/1/1963A/equine/Sao_Paulo/6/1963

A/equine/Sao_Paulo/6/1969

KR534268.1_A/equine/Rome/1/2014

A/equine/OR/78356/2012

A/equine/Dubai/2/2012A/equine/Rio_Grande_do_SuI/1/2012

A/equine/Argentina/E-2345-1/2012

Clade 1

Clade 2

V1 and 2

V3

Fig. 4 – MCMC evolutionary analysis on the HA gene of the EIV H3N8, using a lognormal molecular clock, HKY model andcalibration with 77 HA1 sequences (nt 1–987) and strain dates from BEAST v.1.7.5.36 The viruses characterized in the

r

present study are indicated by diamonds (�).

e f e r e n c e s

1. Wright P, Neumann G, Kawaoka Y. Orthomyxoviridae: TheViruses and Their Replication. vol. 5. Philadelphia: LippincottWilliams and Wilkins; 2007.

2. Park AW, Daly JM, Lewis NS, Smith DJ, Wood JLN, Grenfell BT.Quantifying the impact of immune escape on transmissiondynamics of influenza. Science. 2009;326:726–728.

3. Kawaoka Y, Bean WJ, Webster RG. Evolution of thehemagglutinin of equine H3 influenza viruses. Virology.

1989;169(2):283–292.

4. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M,Mumford JA. Antigenic and genetic evolution of equine H3N8influenza A viruses. J Gen Virol. 1996;77(Pt 4):661–671.

5. Lai AC, Chambers TM, Holland RE Jr, et al. Diverged evolutionof recent equine-2 influenza (H3N8) viruses in the WesternHemisphere. Arch Virol. 2001;146(6):1063–1074.

6. Bryant NA, Rash AS, Russell CA, et al. Antigenic and geneticvariations in European and North American equineinfluenza virus strains (H3N8) isolated from 2006 to 2007. VetMicrobiol. 2009;138(1–2):41–52.

7. World Organisation for Animal Health. Equine Influenza.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals.Paris: OIE; 2016. www.oie.int/fileadmin/Home/fr/Health standards/tahm/2.05.07 EQ INF.pdf Accessed08.11.16.

8. Bryant NA, Rash AS, Woodward AL, et al. Isolation andcharacterisation of equine influenza viruses (H3N8) fromEurope and North America from 2008 to 2009. Vet Microbiol.2011;147(1–2):19–27.

r o b i

b r a z i l i a n j o u r n a l o f m i c

9. Virmani N, Bera BC, Shanumugasundaram K, et al. Geneticanalysis of the matrix and non-structural genes of equineinfluenza virus (H3N8) from epizootic of 2008–2009 in India.Vet Microbiol. 2011;152(1–2):169–175.

10. Bera BC, Virmani N, Shanmugasundaram K, et al. GeneticAnalysis of the Neuraminidase (NA) Gene of EquineInfluenza Virus (H3N8) from Epizootic of 2008-2009 in India.Indian J Virol. 2013;24(2):256–264.

11. Yondon M, Heil GL, Burks JP, et al. Isolation andcharacterization of H3N8 equine influenza A virusassociated with the 2011 epizootic in Mongolia. InfluenzaOther Respir Viruses. 2013;7(5):659–665.

12. Gildea S, Fitzpatrick DA, Cullinane A. Epidemiological andvirological investigations of equine influenza outbreaks inIreland (2010–2012). Influenza Other Respir Viruses.2013;7(suppl 4):61–72.

13. Karamendin K, Kydyrmanov A, Kasymbekov Y, et al.Continuing evolution of equine influenza virus in CentralAsia, 2007–2012. Arch Virol. 2014;159(9):2321–2327.

14. Legrand LJ, Pitel PH, Cullinane AA, Fortier GD, Pronost SL.Genetic evolution of equine influenza strains isolated inFrance from 2005 to 2010. Equine Vet J. 2015;47(2):207–211.

15. Perglione CO, Gildea S, Rimondi A, et al. Epidemiological andvirological findings during multiple outbreaks of equineinfluenza in South America in 2012. Influenza Other RespirViruses. 2016;10(1):37–46.

16. Beuttemmüller EA, Woodward A, Rash A, et al.Characterisation of the epidemic strain of H3N8 equineinfluenza virus responsible for outbreaks in South Americain 2012. Virol J. 2016;13:45.

17. Andrews C, Pereira HG, Wildy P. Viruses of Vertebrates. 4th ed;1978. London.

18. Cunha RG. Isolamento do vírus de influenza A/equi 2 noEstado de Guanabara. Rev Bras Biol. 1970;30(4):491–498[Portuguese].

19. Cunha RG, Passos WS, Pagano MCC, Souza DM. Surto degripe eqüina produzido por vírus de influenza A/Equi 2, noEstado do Rio de Janeiro, Brasil. Rev Bras Med Vet.1986;8(3):88–91 [Portuguese].

20. Mancini DAP, Geraldes EA, Pinto JR, Soares MA. An outbreakof influenza in equines of São Joaquim farm, São Roque, SP,Instituto Butantan. Rev Fac Med Vet Zootec Univ São Paulo.1988;25(1):93–100.

21. Loureiro BO [M.Sc. Dissertation] Estudo de um surto deinfluenza eqüina ocorrido em 2001 no Estado do Rio de Janeiromediante a caracterizacão do vírus e avaliacão do esquemavacinal. Rio de Janeiro, Brazil: Instituto Oswaldo Cruz; 2004[Portuguese].

22. Villalobos EMC, Mori E, Lara MCCSH, et al. Isolation,sequencing and phylogenetic analysis of equine influenzavirus causing the 2012 outbreak in São Paulo, Brazil. In: XXIVCongresso Brasileiro de Virologia, VIII Encontro de Virologia doMercosul., vol. 18. 2013:246.

23. Piegas NS, Takimoto S, Barbosa HC, Lacerda JP, Guarnieri R,Ishimaru T. Isolamento do virus influenza de surto de gripeequina em São Paulo. In: XV Congresso Brasileiro de MedicinaVeterinária. 1976.

24. Cunha RG, Passos WS, Valle MDC. Surto de gripe eqüinaproduzido por vírus influenza A/Eq1 no Estado do Rio deJaneiro, Brasil. Rev Bras Biol. 1978;38(3):549–554 [Portuguese].

25. World Organisation for Animal Health. Expert SurveillancePanel on Equine Influenza Vaccines – Conclusions andRecommendations; 2009. www.oie.int/doc/ged/D6325.PDFAccessed 23.12.16.

26. World Organisation for Animal Health. Expert Surveillance

Panel on Equine Influenza Vaccines – Conclusions andRecommendations; 2011. www.oie.int/doc/ged/D10847.PDFAccessed 23.12.16.

o l o g y 4 9 (2 0 1 8) 336–346 345

27. Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L,Rimmelzwaan GF, Osterhaus AD. Detection of influenza Aviruses from different species by PCR amplification ofconserved sequences in the matrix gene. J Clin Microbiol.2000;38(11):4096–4101.

28. Filippsen P [M.Sc. Dissertation] Pesquisa de anticorpos inibidoresda hemaglutinacão contra o vírus da influenza equina (subtipos:H7N7 e H3N8) em equídeos provenientes do Estado de São Paulo.São Paulo, SP, Brazil: Faculdade de Medicina Veterinária eZootecnia, Universidade de São Paulo; 2014 [Portuguese].

29. Gildea S, Quinlivan M, Arkins S, Cullinane A. The molecularepidemiology of equine influenza in Ireland from 2007–2010and its international significance. Equine Vet J.2012;44(4):387–392.

30. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR.Universal primer set for the full-length amplification of allinfluenza A viruses. Arch Virol. 2001;146(12):2275–2289.

31. Edgar RC. MUSCLE: multiple sequence alignment with highaccuracy and high throughput. Nucleic Acids Res.2004;32(5):1792–1797.

32. Felsenstein J. Phylogenies and the comparative method. AmNat. 1985;125(1):1–15.

33. Kumar S, Stecher G, Tamura K. MEGA7: MolecularEvolutionary Genetics Analysis Version 7.0 for BiggerDatasets. Mol Biol Evol. 2016;33(7):1870–1874.

34. Hasegawa M, Kishino H, Yano T. Dating of the human-apesplitting by a molecular clock of mitochondrial DNA. J MolEvol. 1985;22(2):160–174.

35. Drummond AJ, Nicholls GK, Rodrigo AG, Solomon W.Estimating mutation parameters, population history andgenealogy simultaneously from temporally spaced sequencedata. Genetics. 2002;161(3):1307–1320.

36. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesianphylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol.2012;29(8):1969–1973.

37. Rambaut A, Suchard MA, Xie W, Drummond AJ. TreeAnnotatorv1.7.5; MCMC Output Analysis; 2003–2013.http://beast.bio.ed.ac.uk/ Accessed 30.09.16.

38. Rambaut A. FigTree, Tree Figure Drawing Tool, v 1.4.2; 2014.http://tree.bio.ed.ac.uk/software/figtree/ Accessed 30.09.16.

39. Hall TA. BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows95/98/NT. Nucleic Acids Symp Ser. 1999;41:95–98.

40. Nei M, Kumar S. Molecular Evolution and Phylogenetics. NewYork: Oxford University Press; 2000.

41. Zuckerkandl E, Pauling L. Molecules as documents ofevolutionary history. J Theoret Biol. 1965;8(2):357–366.

42. Woodward A, Rash AS, Medcalf E, Bryant NA, Elton DM.Using epidemics to map H3 equine influenza virusdeterminants of antigenicity. Virology. 2015;481:187–198.

43. Hughes J, Allen RC, Baguelin M, et al. Transmission of equineinfluenza virus during an outbreak is characterized byfrequent mixed infections and loose transmissionbottlenecks. PLoS Pathogens. 2012;8(12):e1003081.

44. Shtyrya YA, Mochalova LV, Bovin NV. Influenza virusneuraminidase: structure and function. Acta Nat.2009;1(2):26–32.

45. Saito T, Taylor G, Laver WG, Kawaoka Y, Webster RG.Antigenicity of the N8 influenza A virus neuraminidase:existence of an epitope at the subunit interface of theneuraminidase. J Virol. 1994;68(3):1790–1796.

46. de Boer GF, Back W, Osterhaus AD. An ELISA for detection ofantibodies against influenza A nucleoprotein in humans andvarious animal species. Arch Virol. 1990;115(1–2):47–61.

47. Starick E, Werner O, Schirrmeier H, Kollner B, Riebe R, MundtE. Establishment of a competitive ELISA (cELISA) system forthe detection of influenza A virus nucleoprotein antibodies

i c r o

346 b r a z i l i a n j o u r n a l o f m

and its application to field sera from different species. J VetMed. 2006;53(8):370–375.

48. Ciacci-Zanella JR, Vincent AL, Prickett JR, Zimmerman SM,Zimmerman JJ. Detection of anti-influenza A nucleoprotein

b i o l o g y 4 9 (2 0 1 8) 336–346

antibodies in pigs using a commercial influenzaepitope-blocking enzyme-linked immunosorbent assaydeveloped for avian species. J Vet Diagn Invest. 2010;22(1):3–9.