Calcivirus or calicivirus
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This document provides information about Caliciviruses/Calciviruses including their classification, morphology, genome organization, replication cycle, pathogenesis, clinical manifestations, epidemiology, and methods of diagnosis, prevention and control. It discusses that Caliciviruses have a non-enveloped structure, a positive-sense RNA genome, and cause acute gastroenteritis in humans. Noroviruses are the most common cause of outbreaks worldwide and spread primarily through fecal-oral transmission. Diagnosis can be done through electron microscopy, ELISA/RIA, or RT-PCR methods. Prevention focuses on hand hygiene, disinfection, and isolation of infected individuals.
Calcivirus or calicivirus
- 1. Calicivirus/Calcivirus By- Sanju Sah St. Xavier’s College, Maitighar, Kathmandu Department of Microbiology
- 3. Genera (five) within the family Caliciviridae Norovirus Nebovirus Lagovirus Sapovirus Vesivirus 1. Feline calicivirus (species) 2. Vesicular Exanthema of Swine (species)* • Swine isolates 1932-1956 (13 Serotypes) • San Miguel Sea-lion virus 1972-present (17 serotypes) • Cetacean, bovine, reptile, primate, rabbit, walrus, human, skunk etc. 1978-present (>10) * Colloquially grouped as Marine Caliciviruses/Vesiviruses 3 Laboratory for Calicivirus Studies
- 5. 5 • The prototypic Norwalk virus described as non-enveloped, round, 27-nm particles with a ‘ragged’ outer edge but lacking a definite surface structure. • Many other Noroviruses have - in common a buoyant density of 1.33–1.41 g/cm3, an inability to propagate in vitro. • Noroviruses possess a single capsid protein. • The genome is a non-segmented, single-stranded, positive polarity RNA genome. • The RNA genome is present in a 27-nm naked (non- enveloped) capsid consisting of 60,000-Da capsid protein. • The genome does not contain any virion polymerase. • Ten prominent spikes and 32 cup-shaped depressions can be seen on the virion by microscopy.
- 6. • The structure of the capsid protein is organized into two domains joined by a flexible hinge. • The inner shell (S) domain is composed of the N-terminal 225 residues and is involved in the formation of the icosahedral capsid shell. • The shell domain also shows the classic eight-stranded barrel structure typical of many viral capsid proteins. 6
- 7. 7 • The protruding (P) domain forms prominent structures extending from the surface of the shell, and is formed from the C-terminal half of the protein. • The P domain is further organized into two subdomains (P1 and P2), and it has been suggested that these structures may be involved in binding to cellular receptors and may also be the determinants of strain specificity.
- 8. 8 Genome organization • Phylogenetic analyses of the RNA polymerase and capsid regions of the genome indicate that human Noroviruses are divided into two genetic groups. • The genome of group I viruses is slightly larger (7.7 kb) than the group II genome (7.5 kb), although the reading frame usage of these two genetic groups is very similar. • The genomes of both group I and group II Noroviruses are characterized by a large 50 open reading frame (ORF) encoding a nonstructural polyprotein of approximately 1700 amino acids, followed by a smaller ORF at 3’ end encoding the capsid structural protein and small basic protein.
- 9. 9 • This genome organization clearly distinguishes Noroviruses from other positive-strand RNA viruses, such as the picornaviruses. • The smaller size of the group II Norovirus genome is attributable to its smaller ORF1. • A characteristic feature of the animal calicivirus genomic and subgenomic RNAs (smaller sections of the original transcribed template strand) is that the 5’ termini are highly conserved.
- 10. 10 • The 5’ terminal sequence of ORF1 is repeated around the 5’ terminal region of ORF2, suggesting that human enteric caliciviruses, like their animal counterparts, also produce a subgenomic RNA. • The 5’ genomic and subgenomic termini of both groups I and II Noroviruses begin with the sequence GU. • The conserved GU residues at the 5’ genomic termini appears to be a common feature of the Caliciviridae. • The number of serotypes are not known.
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- 12. 12 Polyprotein processing • In common with picornaviruses, caliciviral synthesized as a large polyprotein precursor which proteins are is subsequently processed in a proteolytic cascade by the viral 3C-like protease. • In vitro transcription and translation of genomic clones does not appear to generate the intact polyprotein, suggesting that proteolytic processing probably occurs cotranslationally.
- 13. 13 Viral replication • The Norwalk virus replicates in the cytoplasm with release of viral particles on cell destruction. • The virus is presumed to replicate in a manner similar to that of picornaviruses.
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- 15. 15 • Norwalk virus enter the body predominantly via the oral route. • Virions are acid stable, consistent with an ability to survive passage through the stomach. • The virion causes infection first by binding to the cell receptor on the cell membrane and enter the cell. • Receptor binding triggers conformational change which results in release of viral RNA into cell cytoplasm. • VPg is removed from the viral RNA. – VPg (viral protein genome-linked) is a protein that is covalently attached to the 5′ end of positive strand viral RNA and acts as a primer during RNA synthesis in a variety of virus families
- 16. 16 • Positive stranded RNA serves both as genomic and mRNA for these viruses and is translated into a large polypeptide known as non capsid viral protein. • Subsequently the viral protein is utilized by the viral enzymes protease to form capsid protein of the progeny as well as several noncapsid protein including the RNA polymerase. • RNA polymerase initiates the synthesis of progeny RNA genomes.
- 17. 17 • The infecting viral RNA is copied and the complementary strand serves as template for the synthesis of new plus strands. • The subgenomic RNA serves as a template for translation of both the capsid and terminal ORF proteins. • Replication is followed by packaging of plus strands into virions and maturation involves the several cleavage events. • The progeny virion assembly occurs by coating of genomic RNA with the capsid protein in the cell cytoplasm called as encapsidation. • The release of progeny virions occurs by the lysis of the cell.
- 18. 18 Incubation Period • Based on volunteer studies with the Norwalk virus, the incubation period ranges from 10 to 51 hours, with a mean of 24 hours. • Norwalk viruses are highly contagious. • 10–100 infectious particles may be needed to initiate infection. • Human caliciviruses enter the body predominantly via the oral route. Pathogenesis and pathology
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- 20. 20 • Virions are acid stable, consistent with an ability to survive passage through the stomach. • Indirect evidence from epidemiologic studies suggests that viruses may enter also via aerosols, such as in those generated from the explosive vomiting that often occurs during illness . • The site of primary replication for the human caliciviruses has not been established, but it is assumed that they replicate in the upper intestinal tract.
- 21. 21 • Biopsies of the jejunum of volunteers who develop gastrointestinal illness following oral administration of the Norwalk or Hawaii virus exhibit histopathologic lesions . • Partial flattening and broadening of villi with disorganization of the mucosal epithelium. • Lamina propria infiltrated with mononuclear cells and vacuolization of mucosal epithelium. • Crypt cell hyperplasia is common. • Dilatation of the rough and smooth ER with an increase in multivesiculate bodies in mucosal epithelial cells.
- 22. 22 • In general, Norovirus infections seem to cause mild atrophy (waste away, especially as a result of the degeneration of cells) of the villi of the small intestine, assumed to arise from limited virus replication that damages the mucosal cells. • The appearance of mucosal lesions was paralleled by a decrease in brush border (the microvilli-covered surface of simple cuboidal epithelium and simple columnar epithelium cells) enzymes, which returned to normal values during convalescence.
- 23. 23 Host immunity • Norwalk virus infection confers a brief and short immunity. • Recurrent infection occurs throughout life, because of the absence of long-term immunity, lack of cross-strain immunity, and because of the diversity of Norwalk virus strains.
- 24. 24 Clinical manifestations • Norwalk viruses cause gastroenteritis in adults. • The illness in symptomatic cases typically begins after an incubation period of 24–48 hours. • The illness is characterized by sudden onset of nausea, vomiting, which can be projectile and severe. • Low grade fever and diarrhea usually occur, the latter being relatively mild.
- 25. 25 • In contrast to bacterial gastroenteritis, diarrheal stools do not contain blood, mucus, or white cells. • Fecal leukocytes are absent. • Other symptoms- mild abdominal pain , malaise and headache. • Vomiting may arise from a decrease in gastric motility, giving rise to a reflux action into the stomach. • Norwalk virus gastroenteritis is short-lived and typically lasts for 24–48 hours.
- 26. 26 • In general, Norovirus infections are self-limiting and affected patients rarely need to be hospitalized. • There have, however, been occasional reports of severe dehydration that required the administration of intravenous fluids. • Deaths associated with Norovirus infections are exceptionally rare and have not been directly attributed to this group of viruses. • Gastric emptying is delayed, and malabsorption of fat, D-xylose, and lactose has been observed.
- 27. 27 Epidemiology • Noroviruses are now established as the most important cause of epidemic nonbacterial outbreaks of gastroenteritis worldwide. • Norwalk virus gastroenteritis is found worldwide in adults. • National surveillance and diagnosis by EM of outbreaks of nonbacterial gastroenteritis in the UK have shown that Noroviruses are a more common cause of infective gastroenteritis than Salmonella or Campylobacter. • The infection typically occurs in group settings, such as schools, hospitals, nursing homes, etc.
- 28. 28 • It is a strict human infection, and humans are the major source of infection. • The virus is excreted in the vomitus and feces for several weeks after recovery; hence vomitus and feces are important sources of infection. • Infection is transmitted from person to person by ingestion of food or water contaminated with the virus. • Subsequent surveillance and investigations of outbreaks in many parts of the world identified the potential of Noroviruses for causing epidemic gastroenteritis in semiclosed or community wide populations, for example families, healthcare institutions, holiday locations including cruise ships, educational establishments, and the catering industry.
- 29. 29 • Outbreaks occur among children and adults, but rarely among neonates or very young children. • Although considerable data are available on the causal role of Noroviruses in outbreaks, less is known about their role in endemic disease, perhaps because of the relative mildness of the illness. • Hospitalization of individual cases is thus unusual, and opportunities to investigate sporadic cases are scarce.
- 30. 30 Laboratory Diagnosis VIRUS ISOLATION • There are no reports of the isolation of Noroviruses in either cell or human intestinal organ cultures. • Tests with a wide range of animals have also failed to identify a suitable model, although recently it was reported that macaques could be experimentally infected with human Noroviruses. • Some success has been achieved with the transmission of Norwalk virus in chimpanzees, in which serological responses and excretion of Norwalk virus antigen in stools were described. • It is, however, unlikely that isolation in cell cultures will ever be used for diagnosis, as in vitro methods are too slow and labor-intensive.
- 31. 31 ELECTRON MICROSCOPY • direct detection of Noroviruses in stools. • Virions possess an amorphous surface structure, lacking a defined symmetry, with a ragged outline that probably explains the wide range of particle diameters (32– 38 nm) reported.
- 32. 32 • It is essential that preparations of Noroviruses are examined in the absence of antibody, which can mask the surface structure and lead to incorrect identification. • Noroviruses are commonly excreted in feces as small aggregates and in very low numbers at 106 viruses/g ofstool. • These concentrations are close to the limit of sensitivity of the EM, so careful examination of preparations is necessary.
- 33. 33 SEROLOGICAL METHODS • Radioimmunoassay (RIA) or ELISA are also used to detect the virus and viral antigen in the stool. • Both ELISA and RIA are the serodiagnostic tests frequently used to detect specific antibodies to Norwalk virus in the serum. • The EIA format for detection of Noroviruses in clinical specimens offers considerable advantages over RT-PCR. • EIA is cheap, rapid, and simple, and thus easy to deploy in the routine diagnostic setting.
- 34. 34 RT-PCR • The characterization of the Norovirus genome has allowed the application of molecular techniques to the diagnosis of Norovirus infection. • Based on RNA polymerase and capsid antigen of Norovirus. • These techniques are not used for routine diagnosis because of cost considerations and the need for specialized laboratory equipment (e.g. thermal cyclers), and are thus restricted to the research or reference laboratory.
- 36. 36 Prevention and control • No specific treatment is available for Norwalk virus. • No vaccine is available against the virus. • The high infectivity of Noroviruses and the explosive outbreaks of gastroenteritis that often occur in semiclosed communities present a major challenge to control of infection and require aggressive intervention. • Measures for interrupting the various modes of transmission must include ‘enteric’ precautions, but these alone are ineffective and should be supplemented by measures to deal with patients who vomit.
- 37. 37 • Symptomatic healthcare workers should be excluded from contact with patients for at least 2 days after resolution of symptoms. • Strict personal hygiene, such as effective hand washing should be routine • People caring for patients should wear disposable gloves, gowns, and masks when cleaning vomit, fecal material, or contaminated clothing • All potentially contaminated surfaces in toilets, bathrooms, and rooms occupied by patients should be disinfected with a chlorine-based product and cleaned with a hot detergent solution.
- 38. 38 • Contact with other patients should be avoided • Common-source outbreaks are well documented. • Symptomatic food handlers have often been incriminated and must be excluded from the workplace for at least 2 days after resolution of symptoms. • Decontamination of all potentially infected surfaces in the kitchens and associated rest and toilet facilities is essential .
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- 42. 42 • Thank you…