VIRAL AGENTS CAUSINGVIRAL AGENTS CAUSINGGASTROENTERITISGASTROENTERITIS

Helmut Albrecht, MDHelmut Albrecht, MDID Division Chief, USC DOMID Division Chief, USC DOM

(based on lecture by (based on lecture by Nandini Narayan, MD)Nandini Narayan, MD)

Viruses found in the gut (1)

A. Associated with gastroenteritis• Rotaviruses• Adenoviruses 40/41• Caliciviruses• Norwalk like viruses or SRSV (Small Round

Structured Viruses)• Astroviruses• SRV (Small Round Viruses)• Coronaviruses• Toroviruses

Viruses found in the gut (2)

B. Found in the gut, not normally associated with gastroenteritis• Polio• Coxsackie A• Coxsackie B• Echo• Enteroviruses 68-71• Hepatitis A• Hepatitis E• Adenoviruses 1-39• Reoviruses

C. Found in the gut as opportunistic infection • CMV• HSV• VZV• HIV

VIRAL AGENTS CAUSINGVIRAL AGENTS CAUSINGGASTROENTERITISGASTROENTERITIS

Pathogens discussed in today’s lecture

1. Rotavirus2. Enteric adenoviruses3. Caliciviruses4. Astroviruses5. Toroviruses

ROTAVIRUSROTAVIRUS

Family Reoviridae

Genus Rotavirus

Other genera Orthreovirus,

Coltivirus, orbivirus (sheep)

ROTAVIRUS- discoveryROTAVIRUS- discovery• First isolated in 1973 in Australia by Ruth Bishop at

the Royal Children's Hospital in Melbourne. • EM identification from duodenal biopsies from

children with diarrhea.• "Virus particles in epithelial cells of duodenal mucosa

from children with acute non-bacterial gastroenteritis," Lancet, 1:1281-3, 1973.

• Described in stool samples from children by Albert Z. Kapikian, in the US

• Human and animal strains are recognized

Rotavirus

ROTAVIRUS STRUCTUREROTAVIRUS STRUCTURE

STRUCTURAL FEATURES OF STRUCTURAL FEATURES OF ROTAVIRUSROTAVIRUS

• 60-80nm in size

• Non-enveloped virus

• EM appearance of a wheel with radiating spokes

• Icosahedral symmetry

• Double capsid• Double stranded (ds) RNA in 11 segments

STRUCTURAL FEATURES OF STRUCTURAL FEATURES OF ROTAVIRUSROTAVIRUS (contd.)(contd.)

• Double capsid (outer and inner capsid)

• Core with genome

• Capsid is cleaved by trypsin to form ISVP

[intermediate/infective sub-viral particle]

Rotavirus structure

ROTAVIRUS- ultrastructureROTAVIRUS- ultrastructure

ROTAVIRUSROTAVIRUSEM STRUCTUREEM STRUCTURE

VIRAL STRUCTURAL PROTEINS VIRAL STRUCTURAL PROTEINS (VP)

• Outer structural proteins - VP7 and VP4

VP7 -VP7 - Glycoprotein

VP4 -VP4 - protease-cleaved, P protein, viral

hemagglutinin; forms spikes from the surface

• Inner core structural proteins VP 1, 2, 3, 6

• VP6 is an important antigenic determinant

CLASSIFICATION- GroupsCLASSIFICATION- Groups

•7 Groups (A through G) and 2 subgroups (Iand II)

based on VP6 differences

•Group A is the most common

•Group B (outbreaks in China)

•Group C (worldwide)

CLASSIFICATION - SerotypesCLASSIFICATION - Serotypes• Serotypes based on viral capsid proteins inducing neutralizing Ab

• 14 G serotypes based on G protein (VP 7) differences

– 5 predominant strains in U.S. (G1-G4, G9) account for 90% of isolates

– Strain G1 accounts for 73% of infections

• 20 P serotypes based on P protein (VP4) with P4/P8 predominance

• Common PG combinations are: P8G1, P8G2, P4G2, P8G4

CLASSIFICATIONCLASSIFICATION (contd.)

•Electropherotypes are based on the mobility

of

RNA segments by PAGE

• Useful in epidemiologic studies

ROTAVIRUS- PROPERTIESROTAVIRUS- PROPERTIES

• Virus is stable in the environment (months)

• Relatively resistant to handwashing agents

• Susceptible to disinfection with 95% ethanol, ‘Lysol’, formalin

PATHOGENESISPATHOGENESIS

• Targeted host cells - mature enterocytes lining the

tips of intestinal villi

• Intermediate/infective sub-viral particle (ISVP)

produced through proteolysis

• Enter host cell by endocytosis

• Virus replicates in the host cell cytoplasm

REPLICATIONREPLICATION

• mRNA transcription with viral RNA

polymerase

• Capsid proteins formed, assembled into

immature capsid

• RNA replicated to form double stranded

RNA genome

HISTOPATHOLOGYHISTOPATHOLOGY

• Mature enterocytes lining the tips of

intestinal villi are affected

• Villous atrophy and blunting

• Death of the mature enterocytes

HISTOPATHOLOGYHISTOPATHOLOGY

• Infiltration of lamina propria with

mononuclear cells

• Repopulation of the villous tips with

immature secretory cells [crypt

hyperplasia]

HISTOPATHOLOGYHISTOPATHOLOGY

EPIDEMIOLOGYEPIDEMIOLOGY

• A major cause of diarrhea-associated

hospitalizations and deaths

• Sero-prevalence studies show that

antibody is present in most by age 3y

ROTAVIRAL DISEASE BURDENROTAVIRAL DISEASE BURDENWorldwide

WORLDWIDE DISTRIBUTION OF WORLDWIDE DISTRIBUTION OF ROTAVIRUSROTAVIRUS

(Source- Centers for Disease Control and Prevention)

DISEASE BURDENDISEASE BURDENUSAUSA

• 20-40 deaths/year

• Cases with dehydration ~ 1-2.5%

• 50,000-70,000 hospitalizations/year

• Physician visits ~ 500,000/year

• 2.7- 3.5 million affected each year

• Economic impact

ROTAVIRAL DISEASE BURDEN- U.S.A.ROTAVIRAL DISEASE BURDEN- U.S.A.Source- MMWR August 11, 2006 / 55(RR12);1-13

EPIDEMIOLOGYEPIDEMIOLOGY• Age- children 4mo - 2 years are most affected

Protection of younger infants through through transplacental antibody

transfer • Asymptomatic infections are common,

especially in adults

• Nosocomial infections • Outbreaks

• Severe Disease young, immunocompromised

EpidemiologyEpidemiology (contd.)

• Seasonality Winter months (Nov. through May in US)

Gradual spread W to E

Year-round in the tropics

• Incubation period - thought to be <4 days

EPIDEMIOLOGYEPIDEMIOLOGY(Source- Centers for Disease Control and Prevention)

SEASONAL SPREAD - U.S.SEASONAL SPREAD - U.S.(Source- CDC)

TRANSMISSIONTRANSMISSION

• Mainly person to person via fecal-oral route

• Food and water-borne spread is possible

• Fomites

• Spread via respiratory route is speculated

EPIDEMIOLOGYEPIDEMIOLOGY - spread

• Contagious from before onset of diarrhea to a few days after end of diarrhea

• Large amounts of viral particles are shed in diarrheal stools

• Infective dose 10-100 pfu

EPIDEMIOLOGYEPIDEMIOLOGYDifferences in Groups

• Group A infections are most common

• Group B has been associated with outbreaks in

adults in China

• Group C is responsible for sporadic cases of

diarrhea in infants around the world

Rotavirus Immunity

• Antibody against VP7 and VP4 partially protective

• First infection usually does not lead to permanent immunity

• Reinfection can occur at any age

• Subsequent infections generally less severe

CLINICAL CASECLINICAL CASE

• A 22 month old female is admitted to the pediatric ward for cough and fever up to 103°F.

• Chest X ray shows left lower lobe pneumonia.• She is being treated with intravenous Ceftriaxone and

her fever is gradually improving.• On hospital day #5, she develops diarrhea with 4

watery stools and a fever of 102°F. • Stool studies showed no traces of blood and no fecal

leukocytes. • Further studies are pending.

CLINICAL FEATURESCLINICAL FEATURES

• Incubation period - thought to be <4 days

• Fever- can be high grade (>102F in 30%)

• Vomiting, nausea precede diarrhea • Diarrhea

• usually watery (no blood or leukocytes)

• lasts 3-9 days

• longer in malnourished and immune deficient individuals.

• NEC and hemorrhagic GE seen in neonates

MECHANISM OF DIARRHEAMECHANISM OF DIARRHEA

• Watery diarrhea due to net secretion of intestinal fluid and loss of absorptive surface

• Activation of the enteric nervous system• Role of NSP4 peptide regions as an

enterotoxin

CLINICAL FEATURESCLINICAL FEATURES (contd.)(contd.)

• Dehydration is the main contributor to mortality

• Secondary malabsorption of lactose and fat, and chronic diarrhea are possible

Clinical appearance of dehydration

Photo Credit: Dr. D. Mahalanabis, World Health Organization

DIAGNOSISDIAGNOSIS

• Antigen detection in stool

ELISA, LA (Group A rotavirus), immunochromatographic assay

• EM- non-Group A viruses also

• Culture- Group A rotaviruses can be cultured in monkey kidney cells

• Serology for epidemiologic studies

TREATMENT AND TREATMENT AND PREVENTIONPREVENTION

• Treatment Treatment Supportive- oral, IV rehydration

• PreventionPreventionHand hygiene and disinfection of surfaces

• VaccineVaccine

Rotavirus Vaccine (Rota)Human

Animal

TissueCulture

Reassortant

•Created by genetic reassortment•Causes nonhuman rotavirus strains to express human rotavirus antigens on their surface•Nonhuman rotaviruses have low pathogenicity for humans•Replicate but do not cause disease

ROTAVIRAL VACCINE: Historical lessonsROTAVIRAL VACCINE: Historical lessonsPhoto source- PATH vaccine resource library

• Live tetravalent rhesus-human reassortant vaccine (Rotashield)

• Licensed for use in August 1998

• Removed from the market in October 1999 due to risk of intussusception

• Cases were seen 3-20 days after vaccination, especially the first dose

• Approx. 15 cases/1.5 million doses given

• Attributable risk 1:10,000 (1st dose)

RotaTeq® (Merck)

• Live oral vaccine licensed 2006 in US• Contains 5 reassortants (WC3 bovine strain

with viral surface proteins of human serotypes G1-4 and P1A)

• Contains no preservatives or thimerosal• 3-dose schedule – age 2,4,6 month • Minimum age of first doses is 6 weeks• First dose should be administered between 6

and 12 weeks of age (until age 13 weeks)• Do not initiate series after 12 weeks of age

MMWRMMWR 2006;55:(RR-12):1-13. 2006;55:(RR-12):1-13.

Rotavirus Efficacy and Safety Trial (REST)

• ~70,000 enrollees in 11 countries• 34,000 enrolled in the US• Conducted from 2001 to 2004 • Double-blind (with sponsor blinding), placebo-

controlled, randomized• Data published in Jan. 2006

Vesikari T, Matson DO, Dennehy P, et al. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006;354:23--33.

Efficacy of RotaTeq®

• 98%98% reduction in severe RV GE within first year of vaccination

• 96%96% reduction in hospitalization• 74%74% reduction of RV GE (any grade of

severity) within first year of vaccination• 71% reduction of RV GE in first 2 years

of vaccination

Incidence of intussuception-RotaTeq®

New Eng J Med 2006; 354:23-33

02468

10121416

42 days 1 yr.

vaccinePlacebo

Rotarix• Live attenuated monovalent vaccine• Manufactured by Avant Immunotherapeutics• Contains the G1P[8] human rotavirus strain • Studied in Latin America in large trials• 2 dose schedule • Safety established- intussusception not

increased, efficacious• Licensed in other countries

GASTROENTERITIS DUE GASTROENTERITIS DUE TO ENTERIC ADENOVIRUSTO ENTERIC ADENOVIRUS

Adenovirus GastroenteritisAdenovirus Gastroenteritis• Isolated 1953 by Rowe from adenoidal tissue

• Linear double-stranded DNA

• 70-75nm in diameter

• Genus: Mammalian AV (mastadenoviruses) – 6 human subgenera (A-F)

– 51 human serotypes (1-51)

• Known oncogenic potential of some serotypes

• Commonly used as vectors in

gene therapy and vaccine delivery

Adenoviral core/capsid proteins

Adenovirus GastroenteritisAdenovirus Gastroenteritis

• Types 40, 41

• Belong to serogroup F

• Isolation requires special media-Graham 29

• ELISA for rapid detection is available

Diarrhea due to Diarrhea due to Enteric AdenovirusEnteric Adenovirus

• Age <4 years

• Ubiquitous

• Year round

• Spread via fecal-oral route

CLINICAL FEATURES- CLINICAL FEATURES- Adenovirus gastroenteritisAdenovirus gastroenteritis

• Incubation period 3 -10 days

• Diarrhea lasts for 10 -14 days

• Can also cause intussusception, mesenteric adenitis, appendicitis

• Treatment with cidofovir promising for severe disease in immunocompromised

HUMAN CALICIVIRUSESHUMAN CALICIVIRUSES

HUMAN CALICIVIRUSESHUMAN CALICIVIRUSES(HuCV)(HuCV)

•Family Caliciviridae

•Non-enveloped RNA viruses

with ss [+] sense RNA

•27-35 nm in size

•Contains single capsid protein

Calicivirus

• Norwalk virus and “Norwalk-like”• “Sapporo-like” viruses• Vesivirus• Lagovirus

CLASSIFICATION OF HuCVCLASSIFICATION OF HuCV

NLV (Norovirus)Norwalk virusHawaii virusSnow Mountain virusMontgomery county virusTaunton (England)

SLV (Sapovirus)Sapporo virusManchester virusHouston/86London/92

MORPHOLOGY- typicalMORPHOLOGY- typical

• 32 cup-like depressions

• EM appearance of “Star of David”

• 31-35 nm size

• E.g.- Sapporo-like viruses

HUMAN CALICIVIRUSES - SLVHUMAN CALICIVIRUSES - SLV

Morphology of HuCV- atypicalMorphology of HuCV- atypical

• Smaller size- approx. 27 nm

• Rough, feathery surface but no internal pattern

• Small Round Structured viruses

• E.g.- Norwalk-like viruses

NOROVIRUSNOROVIRUSPhoto source- http://www.kyobiken.or.jp/norovirus.JPG

Norwalk virus• “winter vomiting disease” 1968, Norwalk OH• Cause 40% of nonbacterial epidemics

– 45% foodborne, 52% shell fish associated outbreaks• Explosive epidemics

– camps, cruise ships, nursing homes• Food borne illness

– raw shellfish

EPIDEMIOLOGY - NorovirusesEPIDEMIOLOGY - Noroviruses• Worldwide distribution

• >23 million cases/year in the U.S.

• Major cause of food-borne outbreaks of GE (>50%)

• Most people have had infections by age 4 years (by sero-prevalence studies)

Norwalk virus: Clinical Features

• 24 hour (range 12-96) incubation period• Vomiting prominent • Headache, myalgia, fever• Diarrhea 1-3 days, less severe than rotavirus• Affects all ages• Treatment symptomatic

– rehydration, antidiarrheals• Complications rare

– immunocompromised

SPREADSPREAD

• Person-to-person fecal-oral spread (stool/vomitus)

• Fecal contamination of food or water

• Fomites (stool/vomitus)

• Ingestion of aerosolized particles

SPREAD OF NOROVIRUSSPREAD OF NOROVIRUS“A study has shown that where fingers come into

contact with virus-contaminated material, Norovirus can be consistently transferred via the fingers to melamine surfaces and from there to other typical hand-contact surfaces, such as taps, door handles and telephone receivers…..……It was found that contaminated fingers could, sequentially transfer virus to up to seven clean surfaces. Norovirus can survive for up to several days in the environment and still cause infection. ”

ENVIRON. HEALTH JOURNAL March 2005, pages 22-24

SPREAD- Viability of Caliciviruses

• Survive on surfaces for several days• Survive in water chlorinated at routine levels

(up to 10 ppm)• Survive freezing, heating up to 60°C• Evidently survive in steamed shellfish

EPIDEMIOLOGYEPIDEMIOLOGY

•Asymptomatic infections- seroconversion but

asymptomatic shedding of virus•Low infective dose (~10 pfu)•Viral excretion during convalesence (~ 2

wk.)•Protective immunity short-lived•NLV cross protection?•Blood group antigens predilection?

EPIDEMIOLOGY : OutbreaksEPIDEMIOLOGY : Outbreaks

• Cruise ships, schools, nursing homes,

• Can involve infants and school-age children

• Source usually is contaminated food and water (E.g. seafood-oyster and shellfish, salads, cake icing, raw fruit etc.)

• Rapid secondary spread

Photo source- http://www.seattlest.com

DIAGNOSISDIAGNOSIS

• Specimen- stool, vomitus, food, environmental swabs (during outbreak investigations)

• RT-PCR in state public health labs.

• Serology for epidemiologic purposes

• Immune EM is less used

Control of spread of CV GE

• Unclear as to which is the best method of disinfection of surfaces

• CDC recommends environmental disinfection of non-porous surfaces with 1000 ppm bleach solution (1 part bleach: 50 parts water)

HUMAN HUMAN ASTROVIRUSASTROVIRUS

ASTROVIRUSASTROVIRUS

• Described in relation to an outbreak of

gastroenteritis in 1975

• Detected by EM

• Immunologically distinct from Hu CV

• Animal strains are known

Classification of AstrovirusClassification of Astrovirus

• Genus Astrovirus

• Family Astroviridae

• Human serotypes: HuAstV 1-8

ASTROVIRUS- structureASTROVIRUS- structure• Small ss RNA virus • Non-enveloped • 27-32nm in size• Round with an unbroken, smooth surface• EM appearance of a 5 or 6 pointed star within

smooth edge • Contain 3 structural proteins• Genome has been sequenced

ASTROVIRUS- STRUCTUREASTROVIRUS- STRUCTUREPhoto source- http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Cornelia/astro3.htmPhoto source- http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Cornelia/astro3.htm

ASTROVIRUSASTROVIRUS - Clinical Features

• Infants and children are most often affected• Elderly and immune compromised persons also• Short incubation period 1-4 days• Nausea, vomiting, abdominal cramping and

watery diarrhea• Constitutional symptoms-fever, malaise,

headache

ASTROVIRUSASTROVIRUS - Epidemiology

• Endemic worldwide

• True disease burden is not yet clear

• Mainly in children <7 years of age

• Transmission- person-to-person [fecal-oral]

• Outbreaks due to fecal contamination of

sea-food or water

Outbreaks of AstrovirusOutbreaks of AstrovirusSource- http://www.ozestuaries.org/indicators/shellfish_closures.jsp

ASTROVIRUSASTROVIRUS - Diagnosis

• EM (virus shed in stool in great numbers)

• EIA

• RT-PCR

Toroviruses

• Family Coronaviridae

• Genus Torovirus

• Human and animal pathogen

• Pleomorphic, coated ss (+) RNA virus

• Core “doughnut-shaped” (torus)

• Watery diarrhea in 2 – 12 months old

• Diagnosis: EM

Cytomegalovirus

• Herpesvirus• Immune competent

– Mononucleosis-type syndrome• Immunocompromised

– retinitis– hepatitis– gastrointestinal involvement (colon)

Cytomegalovirus colitis

Request: Stool for viruses

• Hospital 1: Enterovirus, adenovirus, herpesviruses (CMV, HSV)

• Hospital 2: CMV• Hospital 3: Enterovirus• Hospital 4: Stop sending us this!• Hospital 5: Please resubmit in viral cx

medium! => HSV, CMV