Norovirus

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A Microbial Biorealm page on the genus Norovirus

Classification

Higher order taxa

Full lineage: root; Viruses; ssRNA viruses; ssRNA positive-strand viruses, no DNA stage; Caliciviridae; Norovirus

Species

NCBI: Taxonomy

Genus species

Description and significance

Non-cultivable human norovirus belongs to the Calciviridae family of viruses and is classified as a Group B biodefense pathogen. Examination of negatively stained Norovirus by electron microscopy showed that the virus has an amorphous appearance with a feathery outer edge and a diameter of 27nm(Kapikian et al., 1972). The Norwalk virus, as it was formerly known, before the genus Norovirus was commonly accepted, is a non-enveloped virus that contains a ~7.7kb postitive-sense single-stranded RNA genome. Its genome is polyadenylated at its 3’ end and encodes three primary open reading frames (ORFs) (Jiang et al., 1993). Human noroviruses are responsible for almost all outbreaks (<95%) of nonbacterial gastroenteritis in the United States and Europe (Billgren et al., 2002; Fankhauser et al., 1998).

Norovirus infection may be spread through person-to-person contact or be waterborne or foodborne. Foodborne infection has been particularly prominent, and the CDC estimates that 40-50% of all U.S. foodborne gastroenteritis outbreaks are caused by norovirus. A wide variety of foods have been implicated, including salads, salad dressings, baked goods, deli meats, fruits and vegetables, water, and ice. Secondary transmission has occurred in most outbreaks, which usually terminate spontaneously after one to two weeks (Dolin, 2007).

Transmission appears to occur through fecal-oral spread; respiratory spread has been suspected but never proven. Virus shedding (as assayed by RT-PCR) can last from a few days to several weeks and can begin before symptoms occur and continue after they have resolved, complicating the management of outbreaks. These infections are worldwide in distribution and affect all age groups, although mostly school-aged children and adults (Dolin, 2007).

Norovirus is an important virus to consider because of its biologic, physicochemical, and epidemiologic features, which present serious challenges for infection control. Noroviruses extremely infectious, and as few as 10 to 100 particles may be needed to cause infection. They are highly resistant to inactivation by freezing, heating to 60°C, exposure to chlorine in concentrations of 0.5 to 1.0mg per liter, pH levels of 2.7, and treatment with ether, ethanol, or detergent-based cleaners (Dolin, 2007). It is clear that Norovirus is an environmental threat for humans, with the manifestation of the virus causing mild-severe gastroenteritis. Currently there is no specific therapy available, although most cases are self-limited (Dolin, 2007).

Genome structure

Norovirus has a positive-sense, single stranded RNA genome, which contains three open reading frames (ORFs)(Zheng et al., 2006). The genome is linear and is 7,654 nucleotides in length (Hardy and Estes, 1996). The ORF1 encodes the nonstructural polyprotein that is cleaved by viral 3C-like protease into probably 6 proteins, including the deduced RNA-dependent RNA polymerase (Belliot et al., 2003). ORF2 and ORF3 encode the major (VP1) and minor (VP2) capsid proteins, respectively (Green et al., 2001; Jiang et al., 1990, 1993).

Virion Structure

A study done by Prasad and colleagues in 1994 revealed that the Norovirus capsid has a diameter of 38.0nm and exhibits T=3 icosahedral symmetry, with a defined surface structure that resembles typical animal and human caliciviruses, in which cup-like depressions or hollows are evident at the three- and fivefold axes of symmetry. Each virus particle is composed of 180 molecules of the capsid protein, which form 90-arch-like capsomers at all the local and strict twofold axes surrounding the hollows(Bertolotti-Ciarlet et al., 2002). The capsid protein folds into two principal domains, a shell (S) domain and a protruding (P) domain, which contains two subdomains, P1 and P2 (Bertolotti-Ciarlet et al., 2002). Studies done by Bertolotti-Ciarlet and colleagues indicate that the shell domain of the Norovirus capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between dimeric subunits—which may control the size of the capsid. In the modular structure of the Norovirus capsid protein, the S domain is typically involved in the icosahedral contacts, and the P domains are involved in the dimeric contacts (Prasad et al., 1999).

Noroviruses and other caliciviruses are unique among the animal viruses because they posses a capsid composed of a single major structural protein. Because of this, all the functional entities required for calicivirus structural integrity, immunogenicity, and infectivity are encoded in one structural protein. It is believed that the capsid protein not only provides shell structure for the virus but also contains cellular receptor binding site(s) and viral phenotype or serotype determinants. The function of VP2 associates with upregulation of VP1 expression in cis and stabilization of VP1 in the virus structure (Bertolotti-Ciarlet et al., 2002). Understanding the structure and functions of this viral capsid protein should facilitate the development of antiviral strategies for caliciviruses (Bertolotti-Ciarlet et al., 2002).

Ecology

Progress on understanding the basic mechanisms of Norovirus replication has been far slower due to the inability to cultivate the virus in the laboratory. What is known, however, is the low infectivity and the unusually high stability outside of the host. Because the life cycle of the Norovirus is not known, the only information that can be reported on are studies done by specific groups of researchers in attempts to uncover parts of the replication process. In one study, it was found that the expressed genomic RNA was found to replicate and Norovirus sub-genomic RNA was transcribed from genomic RNA by use of Norovirus nonstructural proteins expressed from genomic RNA and was subsequently translated into Noroviral capsid protein VP1. Viral genomic RNA is packaged into virus particles generated in mammalian cells. These observations indicate that mammalian cells have the ability to replicate Norovirus genomic RNA (Asanaka et al., 2005).

The host cell range of Noroviruses has expanded quite a bit: the virus is now found in mice, cows, pigs, and humans (Widdowson et al., 2005). Human Norovirus does have a binding specificity within the human species, however. ABH and Lewis histo-blood group antigens are carbohydrate epitopes present throughout many tissue of the human body. The type 1 and 3 chain ABH histo-blood group antigens are present on mucosal epithelial cell surfaces and in salivary secretions, with variations in the carbohydrates in different individuals based on their secretor status and blood type. Recent observations suggest that Norovirus likely attaches to either H types 1 or 3 present on gastroduodenal epithelial cells (Harrington et al., 2002). Harrington and colleagues determined that Norovirus virus-like particles bound poorly to saliva containing ABH antigens in blood group B individuals, and may also account for the observation that blood group B individuals are more resistant to NV challenge. This data also suggests that blood group secretor status may be susceptibility alleles for some, but not all, Norovirus infections in humans (Harrington et al., 2002). These are just some recently proposed mechanisms for the ways in which the Human Norovirus may select for its human host.

Recent reports have surfaced that norovirus strains can periodically emerge either globally or nationally, displace other strains, and increase disease incidence. Norovirus spreads best in a common source such as water or food. It is incredibly infectious—as it takes as few as 10-100 particles to spread. Its environmental impacts are almost exclusively pathogenic. It causes gastroenteritis epidemics all over the world (Dolin, 2007).

Pathology

Human Norovirus infects humans. The virus is specific to certain humans—this has to do with binding specificity. Binding specificity is based upon the histo-blood group antigens. Virulence factors include the binding to several histo-blood group antigens (complex carbohydrate structures expressed on many cell types, including gastrointestinal epithelial cells; three distinct antigens exist: A, B, and O). Different viral genotypes have different affinity for ABO antigens (Tan et al., 2003). GI noroviruses preferentially recognize blood group antigens A and O. GII noroviruses preferentially recognize blood group antigens A and B. The binding domain for these antigens centers on the P2 domain of the viral capsid (Harrington et al., 2002). Blood group antigens likely serve as receptors for noroviruses or some other function critical for infection, because human blood group type is closely linked to susceptibility to norovirus gastroenteritis (Rockx et al., 2005).

Upon infection with Norovirus, lesions in the jejunum but not the stomach or rectum and manifests with vomiting and diarrhea. Changes appear within 24 hours of viral infection and remain through the height of the illness, persisting for a variable time after the illness. Intestinal villi appear blunted, but the mucosa remains intact. Symptoms that usually accompany Norovirus-induced illness usually entails both vomiting and diarrhea, and is often accompanied by nausea, abdominal cramps, and systemic symptoms such as malaise, myalgia, chills, and headaches. Fever is present in approximately half the cases and is usually low-grade. The illness typically has an incubation period of 24-51 hours. The actual viral mechanisms, which cause vomiting and diarrhea are unknown (Dolin, 2007).

Current Research

Previously, the development of an effective therapy for noroviral gastroenteritis had been hampered by the lack of a cell culture system. The Chang and George study in 2007 reported the generation of Norwalk virus replicon-bearing cells in BHK21 and Huh-7 cells and furthermore demonstrated that alpha interferon (IFN-α) effectively inhibited the replication of Norovirus in these cells. IFN-γ also inhibited the replication of Norovirus in the replicon-bearing cells. They discovered that the combination of IFN-α and ribavirin showed additive effects in the inhibition of Norovirus replication. Their findings indicated that IFNs and ribivirin may be good therapeutic options for noroviral gastroenteritis (Chang and George, 2007).

Due to the lack of suitable tissue culture or animal models, the true nature of the norovirus pathogenesis remains unknown. Straub et al., demonstrates that noroviruses can infect and replicate in a physiologically relevant 3-dimentional organoid model of human small intestine epithelium. This group of researchers achieved this by growing the cells on porous collagen-I coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in situ hybridization provided evidence of norovirus infection. Their results demonstrate overall that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts that used differentiated monolayer cultures failed (Straub et al., 2007).

To understand the extent of heterotypic norovirus antibody specificity to inter- and intra-genogroup strains and its applicability to vaccine design, LoBue and colleagues collected sera from humans infected with different norovirus strains and from mice inoculated with alphavirus vectors expressing strain-specific recombinant norovirus-like particles (VLPs). They used VLPs that were assembled from Norwalk virus (NV), Hawaii virus (HV), Snow Mountain virus (SM), and Lordsdale virus (LV) as antigens to define and compare heterotypic antibody responses in humans and mice. Furthermore, they examined whether or not heterotypic antibodies could block specific binding of the ABH histo-blood group antigens (receptors for norovirus binding and entry) to norovirus VLPs. Their studies suggest that infection with one of several different genogroup I strains in humans induces heterotypic antibodies which block NV binding to ABH antigens. They also found that inoculating mice with vaccine cocktails encoding multiple norovirus VLPs enhances heterotypic and ligand attachment—better protection from a broader range of noroviruses than monovalent vaccination (LoBue et al.,2006). Vaccination studies in regards to norovirus are very important due to the fact that there currently is no vaccination against norovirus.

References

Asanaka, M., Atmar, R.L., Ruvolo, V., Crawford, S.E., Frederick, H.N., and Estes, M.K. "Replication and packaging of Norwalk virus RNA in cultured mammalian cells". Proceedings of the National Academy of Sciences. 2005. Volume 10(29). p. 10327-10332.

Belliot, G., Sosnovtsev, S.V., Mitra, T., Hammer, C., Garfield, M., Green, K.Y. "In vitro proteolytic processing of the MD145 Norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in Calcivirus-infected cells." Journal of Virology. 2003. Volume 77. p. 10957-10974. In:Zheng, D., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I., and Monroe, S.S. “Norovirus classification and proposed strain nomenclature”. Virology. 2006. Volume 346. p. 312-323.

Bertolotti-Ciarlet, A., White, L.J., Chen, R., Venkataram Prasad, B.V., and Estes, M.K. “Structural Requirements for the assembly of Norwalk virus-like particles”. Journal of Virology. 2002. Volume 76. p. 4044-4055.

Billgren, M., Chistenson, B., Hedlund, K.O., and Vinje, J. "Epidemiology of Norwalk-like Human Caliciviruses in Hospital Outbreaks of Acute Gastroenteritis in the Stockholm Area in 1996". Journal of Infection. 2002. Volume 44. p. 26-32. In: Asanaka, M., Atmar, R.L., Ruvolo, V., Crawford, S.E., Frederick, H.N., and Estes, M.K. "Replication and packaging of Norwalk virus RNA in cultured mammalian cells". Proceedings of the National Academy of Sciences. 2005. Volume 10(29). p. 10327-10332.

Chang, K., and George, D.W. “Interferons and ribavirin effectively inhibit Norwalk virus replication in replicon-bearing cells”. Journal of Virology. 2007. Volume 81(22). p. 12111-12118.

Clarke, I.N., and Lambden, P.R. “Organization and Expression of Calicivirus Genes”. The Journal of Infectious Diseases. 2000. Volume 181. p. S309-16.

Dolan, R. “Norovirus—challenges to control”. New England Journal of Medicine. 2007. Volume 357(11). p. 1072-1073.

Fankhauser, R.L., Noel, J.S., Monroe, S.S., Ando, T., and Glass, R.I. "Molecular Epidemiology of “Norwalk-like Viruses” in Outbreaks of Gastroenteritis in the United States". Journal of Infectious Diseases. 1998. Volume 178. p. 1571-1578. In: Asanaka, M., Atmar, R.L., Ruvolo, V., Crawford, S.E., Frederick, H.N., and Estes, M.K. "Replication and packaging of Norwalk virus RNA in cultured mammalian cells". Proceedings of the National Academy of Sciences. 2005. Volume 10(29). p. 10327-10332.

Green, K.Y., Chanock, R.M., Kapikian, A.Z., 2001. In: Knipe, D.M., Howley, P.M. et al. (Eds.), "Human Caliciviruses: Fields Virology, 4th ed., vol. 1. Lippincott Williams and Wilkins, Philadelphia, p. 841-874. In: Zheng, D., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I., and Monroe, S.S. “Norovirus classification and proposed strain nomenclature”. Virology. 2006. Volume 346. p. 312-323.

Hardy, M.E., and Estes, M.K. "Completion of the Norwalk virus genome sequence". Virus Genes. 1996. Volume 12(3). p. 287-290.

Harrington, P.R., Lindesmith, L., Yount, B., Moe, C.L., and Baric, R.S. “Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice”. Journal of Virology. 2002. Volume 76(23). p. 12335-12343.

Jiang, X., Graham, D.Y., Wang, K., Estes, M.K. "Norwalk virus genome cloning and characterization." Science. 1990. Volume 250. p. 1580-1583. In: Zheng, D., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I., and Monroe, S.S. “Norovirus classification and proposed strain nomenclature”. Virology. 2006. Volume 346. p. 312-323.

Jiang, X., Wang, M., Wang, K., Estes, M.K. "Sequence and genomic organization of Norwalk virus". Virology. 1993. Volume 195. p. 51-61. In: Zheng, D., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I., and Monroe, S.S. “Norovirus classification and proposed strain nomenclature”. Virology. 2006. Volume 346. p. 312-323.

Kapikian, A.Z., Wyatt, R.G., Dolin, R., Thornhill, T.S., Kalica, A.R., Chanock, R.M. "Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis". Journal of Virology. 1972. Volume 10. p. 1075-81. In:Clarke, I.N., and Lambden, P.R. “Organization and Expression of Calicivirus Genes”. The Journal of Infectious Diseases. 2000. Volume 181. p. S309-16.

Lindesmith, L., Moe, C., Marionneau, S., Ruvoen, N., Jiang, X., Lindblad, L., Stewart, P., LePendu, J., and Baric, R. “Human susceptibility and resistance to Norwalk virus infection”. Nature Medicine. 2003. Volume 9(5). p. 548-553.

LoBue, A.D., Lindesmith, L., Yount, B., Harrington, P.R., Thompson, J.M., Johnston, R.E., Moe, C.L., and Baric, R.S. “Multivalent Norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains”. Vaccine. 2006. Volume 24. p. 5220-5234.

Prasad, B.V., Rothnagel, V.R., Jiang, X., and Estes, M.K. “Three-dimensional structure of baculovirus-expressed Norwalk virus capsids”. Journal of Virology. 1994. Volume 68. p. 5117-5125. In: Bertolotti-Ciarlet, A., White, L.J., Chen, R., Venkataram Prasad, B.V., and Estes, M.K. “Structural Requirements for the assembly of Norwalk virus-like particles”. Journal of Virology. 2002. Volume 76. p. 4044-4055.

Rockx, B.H., Vennema, H., and Hoebe, C.J. "Association of histo-blood group antigensand susceptibility to norovirus infection". Journal of Infectious Disease. 2005. Volume 191(5). p. 749-54.

Straub, T.M., Honer zu Bentrup, K., Orosz-Coghlan, P., Dohnalkova, A., Mayer, B.K., Bartholomew, R.A., Valdez, C.O., Bruckner-Lea, C.J., Gerba, C.P., Abbaszadegan, M., and Nickerson, C.A. “In vitro cell culture infectivity assay for human Noroviruses”. Emerging Infectious Diseases. 2007. Volume 13(3). p. 396-403.

Tan, M., Huang, P., and Meller, J. "Mutations within the P2 domain of Norovirus capsid affect binding to human histo-blood group antigens evidence for a binding pocket". Journal of Virology. 2003. Volume 23. p. 12562-71.

Waters, A., Coughlan, S., and Hall, W.W. “Characterization of a novel recombination event in the Norovirus polymerase gene”. Virology. 2007. Volume 363. p. 11-14.

Widdowson, M.A., Monroe, S.S., and Glass, R.I. “Are Noroviruses emerging?”. Emerging Infectious Diseases. 2005. Volume 11(5). p. 735-737.

Zheng, D., Ando, T., Fankhauser, R.L., Beard, R.S., Glass, R.I., and Monroe, S.S. “Norovirus classification and proposed strain nomenclature”. Virology. 2006. Volume 346. p. 312-323.

Edited by Rabia Bajwa, student of Emily Lilly at University of Massachusetts Dartmouth.