VIRAL AGENTS CAUSING GASTROENTERITIS

Helmut Albrecht, MD
ID Division Chief, USC DOM
(based on lecture by 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 CAUSING GASTROENTERITIS

Pathogens discussed in todays lecture 1. Rotavirus 2. Enteric adenoviruses 3. Caliciviruses

4. Astroviruses
5. Toroviruses

ROTAVIRUS
Family Reoviridae

Genus Rotavirus
Other genera Orthreovirus, Coltivirus, orbivirus (sheep)

ROTAVIRUS- 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 STRUCTURE

STRUCTURAL FEATURES OF ROTAVIRUS

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 ROTAVIRUS (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- ultrastructure

ROTAVIRUS
EM STRUCTURE

VIRAL STRUCTURAL PROTEINS (VP)

Outer structural proteins - VP7 and VP4
VP7 - Glycoprotein

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- 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 - 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

CLASSIFICATION (contd.)
Electropherotypes are based on the mobility of RNA segments by PAGE Useful in epidemiologic studies

ROTAVIRUS- PROPERTIES
Virus is stable in the environment (months) Relatively resistant to handwashing agents

Susceptible to disinfection with 95% ethanol,

Lysol, formalin

PATHOGENESIS
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

REPLICATION
mRNA transcription with viral RNA
polymerase Capsid proteins formed, assembled into immature capsid RNA replicated to form double stranded RNA genome

HISTOPATHOLOGY
Mature enterocytes lining the tips of
intestinal villi are affected

Villous atrophy and blunting

Death of the mature enterocytes

HISTOPATHOLOGY
Infiltration of lamina propria with
mononuclear cells

Repopulation of the villous tips with

immature secretory cells [crypt

hyperplasia]

HISTOPATHOLOGY

EPIDEMIOLOGY
A major cause of diarrhea-associated
hospitalizations and deaths

Sero-prevalence studies show that

antibody is present in most by age 3y

ROTAVIRAL DISEASE BURDEN

Worldwide

WORLDWIDE DISTRIBUTION OF ROTAVIRUS

(Source- Centers for Disease Control and Prevention)

DISEASE BURDEN
USA

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.

Source- MMWR August 11, 2006 / 55(RR12);1-13

EPIDEMIOLOGY
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

Epidemiology (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

EPIDEMIOLOGY
(Source- Centers for Disease Control and Prevention)

SEASONAL SPREAD - U.S.

(Source- CDC)

TRANSMISSION
Mainly person to person via fecal-oral route Food and water-borne spread is possible Fomites Spread via respiratory route is speculated

EPIDEMIOLOGY - 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

EPIDEMIOLOGY
Differences 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 CASE
A 22 month old female is admitted to the pediatric ward for cough and fever up to 103F. 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 102F. Stool studies showed no traces of blood and no fecal leukocytes. Further studies are pending.

CLINICAL 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 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 FEATURES
(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

DIAGNOSIS
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 PREVENTION

Treatment
Supportive- oral, IV rehydration

Prevention
Hand hygiene and disinfection of surfaces

Vaccine

Rotavirus Vaccine (Rota)

Human

Animal

Reassortant Tissue Culture

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 lessons

Photo 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
MMWR 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), placebocontrolled, 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% reduction in severe RV GE within first year of vaccination

96% reduction in hospitalization 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
16 14 12 10 8 6 4 2 0 42 days 1 yr. vaccine Placebo

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

TO ENTERIC ADENOVIRUS

Adenovirus 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 Gastroenteritis
Types 40, 41 Belong to serogroup F Isolation requires special media-Graham 29 ELISA for rapid detection is available

Diarrhea due to Enteric Adenovirus

Age <4 years Ubiquitous Year round Spread via fecal-oral route

CLINICAL FEATURESAdenovirus 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 CALICIVIRUSES

HUMAN CALICIVIRUSES (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 HuCV
NLV (Norovirus)
Norwalk virus Hawaii virus Snow Mountain virus Montgomery county virus Taunton (England)

SLV (Sapovirus)
Sapporo virus Manchester virus Houston/86 London/92

MORPHOLOGY- typical
32 cup-like depressions
EM appearance of Star of David

31-35 nm size
E.g.- Sapporo-like viruses

HUMAN CALICIVIRUSES - SLV

Morphology of HuCV- atypical

Smaller size- approx. 27 nm Rough, feathery surface but no

internal pattern
Small Round Structured viruses E.g.- Norwalk-like viruses

NOROVIRUS
Photo 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 - 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

SPREAD

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

Fecal contamination of food or water Fomites (stool/vomitus) Ingestion of aerosolized particles

SPREAD 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 60C Evidently survive in steamed shellfish

EPIDEMIOLOGY
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 : Outbreaks
Cruise ships, schools, nursing homes,
Can involve infants and school-age children
Photo source- http://www.seattlest.com

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

DIAGNOSIS
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 ASTROVIRUS

ASTROVIRUS
Described in relation to an outbreak of gastroenteritis in 1975 Detected by EM Immunologically distinct from Hu CV Animal strains are known

Classification of Astrovirus
Genus Astrovirus Family Astroviridae Human serotypes: HuAstV 1-8

ASTROVIRUS- 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- STRUCTURE
Photo source- http://www.ncbi.nlm.nih.gov/ICTVdb/Images/Cornelia/astro3.htm

ASTROVIRUS - 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

ASTROVIRUS - 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 Astrovirus
Source- http://www.ozestuaries.org/indicators/shellfish_closures.jsp

ASTROVIRUS - 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:
Hospital 2: Hospital 3: Hospital 4: Hospital 5:

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