Calicivirius Norovirus

Noroviruses are RNA viruses that tend to cause gastroenteritis in a range of species, including humans. They have been labeled the “perfect pathogen” and “the Ferrari of viruses” because of their ability to evolve rapidly and spread at high rates. The physical symptoms of noroviral illness are commonly vomiting and diarrhea, which is the principle mode of viral shedding. Other symptoms include myalgia, stomach cramps, and headaches.

Caliciviridae Norovirus

Classification

Noroviruses were first identified in 1972, following the initial 1968 outbreak in Norwalk, Ohio, where the name originates. Originally called “Norwalk-like viruses,” norovirus strains are grouped into the Caliciviridae family. The virus itself has been classified into 40 different strains, which has been further divided into genomic groups, I-V. I, II, and IV are known to be pathogenic in humans, with GII being the most common in massive breakouts. Specific strains are usually named after the location of their outbreak such as GII.4 Sydney or Snow Mountain virus.

Genome Structure

The norovirus genome is an approximately 7.5 kb, plus-sense, single stranded RNA that contains 3 Open Read Frames (ORFs). ORF1 constitutes nonstructural protein synthesis, and is cleaved by virus protease 3C into 6 proteins that are used in RNA polymerase and other essential cell functions. ORF2 encodes capsid proteins, along with protruding domains (P1 and P2 and shell S). It is believed that the protruding domain plays a significant role in lethality.

A basic norovirus genome structure. The ORF2 capsid region has been enlarged to illustrate protruding domains

These arch-like protruding structural proteins are grouped into what is known as VP1 (viral protein 1), which is the main binding site to histo-blood group antigens HBGA. P1 and P2 sites are crucial for cell interactions and immune recognition, because it protrudes from the surface of the capsid. The protruding groups are also considered extremely variable, especially the P2 region. Variation rates between members of the same genogroup are between 45-61% and within genotypes at a rate of 14-44% (Zheng). This is alarming because of the rate of evolution occurring in areas most common for immune response will make permanent immunization like polio unlikely. ORF3 contains a basic protein sequence that codes for a small capsid protein, and is important for VP1 stability and interaction with genome RNA during virion formation. It is contested whether ORF3 or ORF2 is more variable, but there is definite consensus that this segment of the genome displays greater change over time.

Evolution and New Strains

Since it is understood that the protruding regions of the Norovirus are extremely variable, research on the P2 region has accelerated our understanding of the evolution of human strains. Evolution in Noroviruses are centralized on this sequence because it controls whether the virus can infect a given host. It has been found that the carbohydrate binding sites used in HBGA attacks are evolving rapidly, and is creating a strong immune-driven selective force (Rocha-Pereira). It is through the binding of HBGA sites that norovrius manages to enter the epithelial cells of the GI tract, specifically the small intestine. A common term in the explanation of this selectivity is herd immunity, which is the result of a significant portion of a population developing immunity to a specific pathogen and passing it on to offspring. In the case of the norovirus, this exertion of a strong selective pressure on a small and highly variable section of RNA code can cause a new outbreak every 2-3 years. This leads to what is known as “epochal shifts” which track the antigenic shift that occurs regularly. This is identical to the pathogenicity patterns of seasonal influenza, which also undergo immune-driven selection. It is suggested that evolution in noroviruses occur in spurts, usually in response to the threat of human immunity. A study on frozen fecal samples from the 1970’s showed a nucleotide substitution rate of approximately 4.3 x 10^-3 per site per year (Bok et al.). This relatively high rate of evolution can definitely be linked to the diversity of protruding sites, especially P2. Because the genome is positive sense, the rate of translation into viral proteins is expedited greatly and is likely a culprit in the virus’ rapid proliferation. This simplicity means anti-noroviral drugs will have to target the RNA directly after it has been injected into the host cell.

Spread

Because the main shedding mechanism is vomiting and diarrhea, most cases arise from exposure to vomitus. Studies have examined the viral content of potential norovirus victims and have found fecal samples to contain 10₅ - 10₁₁ viral cells per gram. What’s more, viral shedding continues asymptomatically, as long as three weeks after physical illness has subsided. This leads to higher spread rates in populations due to ignorance of prolonged contagiousness. Incubation periods last between 10-48 hours, and has an extremely low infectious dose of approximately 17 cells. Further alarming is the hardiness of the pathogen itself. Norovirus has been known to persist on dry surfaces for several weeks. It can withstand numerous strong cleaning agents such as alcohol-based hand sanitizer and chlorine-based washes because it lacks a protein envelope. Although boiling can kill Norovirus, moderate heating and freezing are ineffective (Glass et al). Coupled with the high shedding rate is the ease at which Norovirus is transmitted. Normal fecal-oral transmission is common, along with contaminated food ingestion and contact with surfaces carrying the virus. The quickest form of transmission is that of aerosolized particles being taken up by nearby hosts, which is unique to intestinal viruses

Relationship with Nervous System

Because of the degree of violence that the virus evokes for the spread has led some to speculate it influences the nervous system in some way. One relationship that has been determined is the slowing of the digestion of food during the incubation period. Food at this stage is likely being used a transport for later expulsion from the host, and is thus pumped with viruses. Another connection norovirus makes to the nervous system is controlling muscle contrctions in and around the stomach. This provokes and sustains projectile vomiting that is characteristic of the virus. Unfortunately, not much is known as to whether these NS interactions are actually carried out by norovirus, so further research will probably be directed in this area.

The Perfect Pathogen?

A major source of frustration in the research of norovirus is the fact that we have been unable to culture it within human cells in the laboratory setting. Because of this, most research done on norovirus is through surrogate viruses such as porcine enteric Sapovirus and feline Vesivirus ^{4<\sup>. While research on the virus has been extensive, the underlying problem of antigenic shift and constant evolution within the virus genome means pattern-finding will ultimately prove more useful than isolated breakthroughs in specific GII.4 strains. Except in individuals with compromised immune systems, the very old and the very young, Norovirus is not considerably lethal; fatalities average around 800 per 21 million cases in the US, or about 3.81 x 10 -5 <\sup> % . However, developing countries have higher death rates from gastroenteritic illnesses, especially in children. The brilliance of the norovirus lies in the fact that it doesn’t kill the host, but makes it extremely efficient at shedding, long after physical symptoms subside. Furthermore, its rate of evolution leaves vaccine researchers less than optimistic. Since it is not as great of a threat in terms of mortality, it is possible that vaccine progress has less priority than annual influenza outbreak preventative measures. Studies highlighting the limited immunity expressed by volunteers repeatedly expsed to norovirus note that even rechallenges present failure completely resist the virus. This limited immunity on conjunction with the epochal evolution suggests control efforts would be best if concentrated on prevention and education rather than regular immunizations}

Notable Cases

Norwalk, Ohio, 1968: In a 2-day period, half (116/232) the students and teachers fell ill. This was the first outbreak, and gave the virus it's name.

Cruiseships: because of the cramped living conditions aboard cruiseships, these are ideal spreading environments for norovirus. Especially harrowing is the virus ability to survive on surfaces, long after a cruise ship has travelled from one port to another. This transports viruses across oceans, and makes the world much smaller.

Airline case: an individual contracted it before boarding, and vomited into the aisle of the packed airplane. It was cleaned and a new crew was sent on, and the virus managed to persist in the rug after heavy cleaning. 5 of the passengers on this flight were infected, and 27 cases occurred at least 51 hours after exposure to the contaminated plane^9<\sup>.

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3. Zheng, D.P., "Norovirus classification and proposed strain nomenclature". Virology March 15 2006 (New York, N.Y.) (0042-6822), 346 (2), p. 312.
4. Rocha Pereira, J., Nascimento, M. S. J., "Targeting Norovirus: Strategies for the Discovery of New Drugs" 2012 Aspects of Clinical Use and Recent Advances, Dr. Patrick Arbuthnot (Ed.), ISBN: 978-953-51-0256-4, InTech, DOI: 10.5772/32677
5. Bok, K., Abente, E.J., Realpe-Quintero, M., Mitra, T., Sosnovtsev, S. V., Kapikian, A. Z., Green, K. Y.. “Evolutionary Dynamics of GII.4 Noroviruses over a Thirty-four Year Period” Journal of Virology, 2009; DOI: 10.1128/JVI.00864-09
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7. Parrino, T. A., Schreiber, D. S., Trier, J. S.,. Kapikian, A. Z., Blacklow, N. R., “Clinical Immunity in Acute Gastroenteritis Caused by Norwalk Agent” New England Journal of Medicine 1977; 297:86-89July 14, 1977DOI: 10.1056/NEJM197707142970204
8. Hall, A., "Noroviruses: The Perfect Human Pathogen?" Journal of Infectious Diseases. (2012) 205(11): 1622-1624 doi:10.1093/infdis/jis251
9. Thornley, C. N., Emslie, N. A., Sprott, T. W., Greening, G. E., Rapana, J. P., Clin “Recurring Norovirus Transmission on an Airplane” Infectious Diseases. (2011) 53 (6):515-520.doi: 10.1093/cid/cir465
10. Strong, D. W., Thackray, L. B., Smith, T. J., Virgin, H. W., "Protutruding Domain of Capsid Protein Is Necessary and Sufficient to Determine Murine Norovirus Replication and Pathogenesis In Vivo" Journal of Virology 2012 86(6) 295-2958