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LECTURE 69, 2007 Helmut Albrecht, MD ID Division Chief Department of Medicine USC SOM halbrecht@gw.mp.sc.edu based on a lecture by Nandini Narayan, MD

VIRAL GASTROENTERITIS AGENTS The following are well-established causal agents of viral gastroenteritis in humans: a. Rotavirus b. Enteric adenoviruses c. Human Caliciviruses d. Astrovirus. e. Torovirus f. HSV, CMV in immunocompromised patients (not discussed here)

ROTAVIRUS Rotavirus was first identified by electron microscopy in 1973 from duodenal biopsies of children with diarrhea and was later detected in stool samples by negative EM. Human and animal rotaviruses are known. CLASSIFICATION Family Reoviride includes genus Rotavirus, Reovirus and Colorado Tick Fever virus STRUCTURE ♦ Non-enveloped, icosahedral, 60-80nm in size ♦

Electron microscopic appearance of a wheel with radiating spokes (Latin, rota=wheel)

Double capsid (outer capsid, inner capsid) and a core.

Major structural proteins1. Outer capsid structural proteins- VP7 (G protein) and VP4 (the viral hemagglutinin or P protein). Both elicit neutralizing antibodies. 2. Inner capsid structural proteins- Mainly VP6, also VP1, 2, 3.

Inner core contains the genome that has double stranded (ds) RNA in 11 segments. Genome codes for 6 virus proteins (VP1-6) and 6 non-structural proteins (NSP1-6). RNA segments can be separated by gel electrophoresis; segmented genome can reassort with co-infection of cells with different strains.

CLASSIFICATION ♦ Groups: Seven different groups (A to G) and 2 subgroups based on antigenic specificity of VP6. Most human pathogens belong to groups A, B, and C; group A viruses with either subgroup I or II, are the most common. ♦

Serotypes: 14 human G serotypes (specified by VP7) and 20 P serotypes (specified by VP4)

E-types: based on differences in relative migration rates of genome segments in PAGE

PROPERTIES Virus is acid-labile but rather stable in the environment. It is susceptible to disinfection with 95% ethanol, ‘Lysol’ and formalin. 1


PATHOGENESIS Affected host cells are mature enterocytes lining the middle and upper end of the intestinal villi in the small intestine. In laboratory animals, hepatocytes are also infected. The infectious particle is thought to be an “intermediate sub-viral particle” (ISVP) that is generated after removal of some of the outer capsid. The viral attachment protein is probably exposed after protease digestion in the GI tract removes some or all of the outer capsid (VP4). Virions enter the host cell by endocytosis. Virus replicates in the host cell cytoplasm- viral mRNA is transcribed using the viral RNA polymerase that is already present in the virion to form structural protein units of the capsid. The mRNA segments are assembled into the immature capsid and then replicated to form double stranded RNA genome. Large amounts of viral particles are shed in diarrheal stools. Histopathology of infected intestines shows villous atrophy and blunting, due to death of the mature enterocytes and infiltration of lamina propria with mononuclear cells. Subsequently there is repopulation of the villous tips with immature secretory cells [crypt hyperplasia]. Mechanism of watery diarrhea has been postulated to be due to cell dysfunction/death, activation of the enteric nervous system, and via NSP4, a non-structural protein, that may play a role as a viral enterotoxin and secretory agonist. During convalescence, repopulation with immature secretory cells may contribute to the secondary lactose intolerance that is sometimes seen. EPIDEMIOLOGY ♦ Distribution Worldwide: 600,000-850,000 deaths per year (20% of diarrhea-related deaths under age 5 years and 40% of severe diarrhea cases) and billions of cases of diarrhea that account for ~22% of hospitalizations due to diarrheal illness. ♦

In the U.S.: 20-40 deaths per year and >50,000 hospitalizations per year. Seasonality Winter months (November through May) in the US, starting from Mexico and SW USA and gradually progressing N/NE to reach East Coast and Canada in spring. Seen year round in the tropics.

Age Infects children at a young age. Older infants and young children (4mo-2 years) tend to be more symptomatic with diarrhea. Young infants may be protected due to transplacental transfer of maternal antibody.

Spread-mainly person to person via fecal-oral route and through fomites. Spread by food and water is also possible. Spread via respiratory route is speculated.

ContagiousnessContagious period starts before onset of diarrhea to a few days after end of diarrhea. Highly contagious among susceptible individuals due to the following: [a] Small infective dose (only 10 pfu) [b] High numbers of viral particles being shed in diarrheal stools (1010/gm) [c] Shedding of virus from asymptomatic hosts

Nosocomial cases and outbreaks are known to occur.

Asymptomatic infections are common, especially in adults.

♦ Severe infections are seen in young, elderly, immune compromised hosts. ♦

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.

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CLINICAL FEATURES ♦ Incubation period is < 4 days ♦ Fever- can be high grade (>102°F in 30%) ♦

Nausea and vomiting precede diarrhea.

Diarrhea is usually watery (no blood or leukocytes), lasting 3-9 days, but longer in malnourished and immune deficient individuals. Necrotizing enterocolitis and hemorrhagic gastroenteritis is seen in neonates. Dehydration is the main contributor to mortality. Secondary malabsorption of lactose and fat, and chronic diarrhea are possible

DIAGNOSIS ♦ ♦ ♦ ♦ ♦

Antigen-detection in stool-by ELISA (uses a monoclonal antibody) and LA is used for rapid diagnosis. Several kits are commercially available. Detects only Group A rotavirus. Electron microscopy- non-group A viruses also Culture- group A rotaviruses can now be cultured in monkey kidney cells. Epidemiological studies use patterns of viral RNA migration by polyacrylamide gel electrophoresis (electropherotyping). Different genetic strains may circulate in a given community. RT-PCR

TREATMENT Supportive-rehydration

PREVENTION OF SPREAD ♦ ♦ ♦ ♦

Hand washing- partly effective Alcohol-based hand gels Disinfection of surfaces, toilets, toys etc. Vaccines Rotashield, a live tetravalent rhesus-human reassortant vaccine was first licensed for use in infants in August 1998. Post-licensure surveillance indicated a possible relationship between the occurrence of intussusception 3-14 days after the vaccine especially the first dose. (15 cases/1.5 million doses were reported). The vaccine was eventually removed from the market in October 1999, when studies confirmed the temporal link between vaccination and intussusception. RotaTeq® (Merck) is a new live pentavalent human-bovine reassortant vaccine that was licensed in 2006 in the US. Contains WC3 bovine strain with viral surface proteins of human serotypes G1-4 and P[8]. Vaccine appears to confer protection that lasts for at least 2 years. Other vaccine candidates are also being studied; Rotarix® (Avant Immunotherapeutics) is a live attenuated monovalent G1P[8] human rotavirus vaccine that has been studied in Latin America and is licensed in several countries.

Further Reading

1. Textbook- Medical Microbiology, 5th ed. (2005) Elsevier Mosby (Murray, Rosenthal, Pfaller), Chapter 632, p 627. 2. The discovery of Rotavirus- Bishop RF et al. Lancet 1974;1(7849):149-151. 3. Worldwide disease burden- Parashar U et al. “Rotavirus and severe childhood diarrhea”. Emerging Infectious Diseases. February 2006; 12(2):304-306. http://www.cdc.gov/eid 4. Details of Rotavirus serotypes- PM Howley (ed.) Fields Virology 4th edition, 2001. Lippincott Williams & Wilkins. Vol2 p.1787-1833. 5. Topic review- Pediatric Annals Vol.35, No.1, Jan. 2006. 6. RotaTeq® web site- http://www.rotateq.com 7. REST- “Safety and Efficacy of a pentavalent Human-Bovine reassortant Rotavirus vaccine” N Engl J Med 2006;354:23-33, Jan 5, 2006. 8. Viral Gastroenteritis. New England Journal of Medicine; July 1991, p252-264

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HUMAN CALICIVIRUSES CLASSIFICATION ♦

Members of the family Caliciviridae

Now classified into genera Norovirus and Sapovirus, based on antigenic differences. Previously named Norwalk-like virus (NLV) and Sapporo-like virus (SLV)

Norwalk virus is the prototypic member of the Genus Norovirus. It was first detected in stools of patients with gastroenteritis in Norwalk, Ohio in 1972. There are 3 genogroups within the Genus Norovirus, containing about >25 genetic clusters.

Examples of Sapoviruses include Manchester virus, London/92 virus, etc.

Many agents have not yet been cultured. Norwalk virus has been cloned.

STRUCTURE ♦

Non-enveloped, single stranded RNA viruses with a plus-sense genome.

27-35 nm in size.

Two morphologic groups are based on electron microscopic appearance:-

Atypical, Small round structured viruses (SRSVs) –smaller, approx. 27 nm, they have a rough, feathery edge, but no internal pattern. (These are the members of Norovirus group.) Typical, spiked appearing viruses- are 31-35nm and have 32 cup-shaped depressions on surface (‘calici’= chalice or calyx i.e. cup-like). The description of EM appearance is named after the 6-pointed “Star of David”. (These are members of the Sapovirus group.)

CLINICAL FEATURES

♦ ♦ ♦ ♦ ♦ ♦

Causal agent referred to for illnesses commonly known as- “stomach flu” or viral gastroenteritis. Relatively short incubation period (Range 12hrs. to 4 days) Illness is brief (3-5 days) and usually self-limited GI symptoms -nausea, vomiting, abdominal cramping and watery diarrhea (fecal leukocytes are absent). Other symptoms-headache, fever, malaise, myalgia, ? respiratory tract symptoms. Outbreaks in institutions, cruise ships, etc. through contaminated food or water (feco-oral)

EPIDEMIOLOGY ♦ ♦ ♦ ♦

♦ ♦ ♦ ♦ ♦ ♦

Worldwide distribution. Higher seroprevalence in developing countries. Established as the most frequent etiology of infectious GE in some countries. Infections occur year-round with an apparent predominance in winter season. Transmission-direct fecal-oral (person-to-person) spread or indirect fecal-oral spread via contamination of food/water, and through fomites contaminated by vomitus or stool. Possible spread through aerosolization of vomitus and ingestion (rather than inhalation) of infective particles. Low infective dose (~10 virions). Viral excretion in stool can continue beyond the cessation of symptoms. Asymptomatic infections (>30% cases) can result in transmission of infection and also in seroconversion. Outbreaks can involve infants and school-age children Outbreaks related to consumption of contaminated oyster and shellfish have been described. It is thought that the seafood or water or ice becomes contaminated with fecal material from sewage or food handlers. Estimated to cause >50% of food-borne gastroenteritis outbreaks in the US. Clinical and microbiologic features as well as epidemiologic criteria (Kaplan’s criteria) suggest viral etiology of gastroenteritis outbreaks. 4


DIAGNOSIS ♦ ♦ ♦ ♦

Analysis of stool or suspected food material; also environmental samples (outbreak investigations) State public health laboratories mainly use realtime RT-PCR of stool and emesis samples, as well as environmental samples during outbreak investigations. Serology is used for epidemiological purposes, with paired sera. Commercial ELISA is thought to have poor sensitivity

CONTROL Unclear as to which is the best method of disinfection of surfaces CDC recommends environmental disinfection of non-porous surfaces with 1000ppm bleach solution (1 part bleach: 50 parts water)

ASTROVIRUS First described in relation to an outbreak of gastroenteritis in 1975, by direct electron microscopy. Genus Astrovirus, family Astroviridae, contains 8 human species (antigenic types) that are known as HuAst 1 to 8.

STRUCTURE ♦

Small ss RNA, non-enveloped virus 27-32nm in size.

EM appearance of a smooth, round structure with an unbroken surface (unlike indented surface of calicivirus) and a 5 or 6 pointed star within.

Immunologically distinct from Human Caliciviruses.

CLINICAL FEATURES Incubation period 1-4 days Diarrhea, headache, nausea, low-grade fever, vomiting (Similar to other viruses)

EPIDEMIOLOGY ♦ ♦

♦ ♦ ♦ ♦ ♦

Worldwide Mainly affects children infants and young children <7 years of (clinical illness is less common in adults) More common in winter season Transmission by direct person-to-person, via fecal-oral route or ingestion of aerosolized vomitus Transmission indirectly through fecal contamination of sea-food/water (outbreaks described) and contaminated fomites Low infective dose (<10 pfu) Outbreaks have been seen in child-care centers.

DIAGNOSIS ♦ ♦ ♦

EM especially useful since the virus is often shed in large amounts in stool Enzyme Immunoassay (EIA) test is available commercially in Europe RT-PCR in used by research laboratories.

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ENTERIC ADENOVIRUSES ♦

Serotypes implicated are 40 and 41 (enteric serotypes). These are difficult to culture and require special cell lines

Cause diarrheal disease in infants and children<4 years of age

Feco-oral spread

Incubation period is 8-10 days

Diarrhea lasts 5-12 days, longer than other viral gastroenteritis

Diagnosis- Latex agglutination, ELISA, EM

TOROVIRUS ♦

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

FURTHER READING 9. Textbook- Medical Microbiology, 5th ed. (2005) Elsevier Mosby (Murray, Rosenthal, Pfaller). 10. Manual Of Clinical Microbiology, 8th Ed.-2003; ASM Press (Murray et al eds.). 11. Red Book 2006; 27th ed. American Academy of Pediatrics. 12. Fact sheets on Noroviruses- Centers for Disease Control and Prevention. 13. http://www.cdc.gov/ncidod/dvrd/revb/gastrp/norovirus.htm 14. Astrovirus gastroenteritis. Pediatr Infect Dis J. 2002 Nov; 21(11):1067-9. 15. Update on Caliciviruses and human acute gastroenteritis. Pediatr Infect Dis J. 2002 Nov; 21(11):1069-1071. 16. Human Caliciviruses and pediatric gastroenteritis. Semin Pediatr Infec Dis. 2004 Oct;15(4):23745.

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viral gastero intestinal tractGI-2007