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Can noroviruses be zoonotic? Sarah Caddy, Imperial College London Introduction Human noroviruses are responsible for an estimated 3 million cases of vomiting and diarrhoea in the UK every year 1. Outbreaks
caused by this formidable virus are common in
hospitals, care homes, schools and cruise ships.
Noroviruses are typically spread via the faecooral route, most often by direct person-to-person
contact 2. In recent years, novel noroviruses have
been characterized from a number of animal
species. In addition, reports of animals being
infected with human noroviruses have emerged.
These findings have raised questions over the
role of animals in the spread of noroviruses.
Many humans are in direct contact with animals
on a daily basis, be they pets or livestock, hence
exposure to noroviruses carried by animals is a
concern. This commentary aims to weigh up the
evidence available to assess the likely
significance of norovirus transmission between
animals and humans. Similarities between human and animal norovirus strains Noroviruses are not a single type of virus, but instead a large group of viruses with significant diversity. Noroviruses are divided into six genogroups based on their capsid sequence 3,4. Table 1 summarises the noroviruses that have been identified to date. Genogroup Species I Humans II Humans, pigs 5 III Cattle 6, sheep 7 IV Humans, dogs 8, cats 9 V Mice 10 VI Dogs 4 Table 1. Noroviruses of different species The animal noroviruses within the same genogroups as the human strains theoretically have the greatest potential ability to infect humans, for example the GII human and swine strains. GII human strains account for 96% of all human norovirus infections worldwide, hence are a very important genogroup 11. The swine GII strains are distinct from the human strains with less than 86% amino acid identity in their capsid sequences. However, noroviruses have been shown to be undergo genetic recombination 12. This ‘antigenic shift’ is believed to occur when two strains infect the same cell, and can result to antigenically novel viruses being generated. Despite this concern, sequence data for several thousand norovirus strains obtained from infected humans have found no swine norovirus sequences, hence these have been classified as non-­‐zoonotic agents 13. Similarly, there are no reports identifying any of the other animal noroviruses in humans, regardless of their genetic relatedness. Given that noroviruses typically cause acute, self limiting infections however, it is estimated that only 15% gastrointestinal infections in humans are brought to the attention of a medical practitioner, diagnostic testing is only performed in 13% of such cases 14. Discovery of animal norovirus specific antibodies in humans Indirect evidence for previous infection with viruses can be obtained by identifying virus specific antibodies. All noroviruses (with the exception of murine norovirus) cannot be grown in cell culture 15, hence obtaining sufficient antigen for antibody screening assays have proven challenging. A solution to this problem was provided by Jiang et al, who showed that if the norovirus capsid protein is expressed synthetically, it will spontaneously fold into the correct capsid shape 16. These virus-­‐like particles (VLPs) can be used to screen sera for norovirus specific antibodies. Production of bovine norovirus VLPs 17 has led to the testing of human serum samples across the world for possible infection with these cattle viruses. As with the pig noroviruses, no bovine noroviruses have been detected in human stools 13, but serological surveys have revealed unexpected results. Humans in India had a 10.7% prevalence to bovine norovirus 18 and in the Netherlands a 20% seroprevalence to bovine noroviruses was detected. This proportion rose to 28% amongst dutch veterinarians 19. Antibody production against bovine norovirus strongly suggests that humans can become infected with the virus, but whether bovine norovirus actually causes disease in humans has not yet been proven. Swine norovirus VLP production has recently been achieved 20, but as of yet no serological studies in humans have been reported. Given the close genetic relatedness between GII swine and norovirus strains, differentiating antibody responses to each virus is likely to be complicated. Dog strains of noroviruses were first identified in 2007 in Italy, Portugal and Greece 4,8,21. Some of these strains are remarkably similar to the rare human strains in genogroup IV. Canine norovirus VLPs have been generated to determine the presence and prevalence of anti-­‐canine norovirus antibodies in humans, and as with the bovine VLPs, the results are interesting. A total of 493 humans were screened for the presence of anti-­‐canine antibodies in their blood 22, and of the 120 participants who did not have regular contact with dogs, 5.8% were seropositive to canine norovirus. For the 373 study participants with daily contact with dogs (all were veterinarians), 22.3% humans were seropositive. This result does suggest zoonotic transmission of canine norovirus, but the clinical significance is unknown. Crossing the carbohydrate species barrier Noroviruses are known to bind to specific carbohydrates on the surface of cells 23, a process predicted to play a key role in cell entry. The types of carbohydrates expressed by cells of different animals can vary significantly, and it has been hypothesized that this could determine the species specificity of certain noroviruses. An innovative study has recently determined the carbohydrate binding preferences of bovine norovirus, and extrapolated from this the likelihood of viral transmission to humans 24. Bovine norovirus binds a Galα1,3 carbohydrate motif, which is commonly expressed on bovine gut cells. The enzyme that synthesizes Galα1,3 motif is present in all mammalian species, with the noteable exception of humans. This suggests that bovine norovirus cannot bind to human gut cells, and thus cannot cause infection. Animal infection with human noroviruses Multiple studies have shown that pigs are regularly exposed to human norovirus. Over half of the pigs in a US report were seropositive to both GI and GII human noroviruses. Similarly, a third of Japanese pig sera analysed were seropositive 25. This finding was supported by a study that demonstrated human strains can replicate and induce an immune response in gnotobiotic pigs 26. The hypothesis that pigs can be infected by human norovirus has been further supported by the detection of human norovirus RNA (GII.4) in pigs in Canada and Taiwan 27,28 Alarmingly a Canadian pork retail product was positive for human norovirus, posing a speculative risk to human consumers. A GII.4 human norovirus has also bee detected in stool samples from cattle 27. On a daily basis most humans in the western world do not have close contact with cattle and pigs. However, there are approximately 10 million dogs in the UK 29, divided amongst 26% of the households. Pet dogs are often considered members of the family, and as such it is expected they would come into contact with human norovirus particles if any family members are infected. An unusual Finnish study sought to investigate this by collecting stool samples from dogs if their owners were experiencing acute gastroenteritis lasting 1-­‐3 days 30. A proportion of these human gastroenteritis cases were predicted to be human norovirus Canine stool samples were tested for the presence of GI, GII and GIV human noroviruses, and 4 dogs were found to be positive for GII human noroviruses. The quantity of human norovirus detected from the stools in 3 dogs was low and could be attributed to human norovirus merely passing through the canine gastrointestinal tract and not replicating. Dogs could theoretically act as fomites (surfaces that carry virus but no replication occurs) if personal hygiene is not optimal. However, the fourth positive dog in this study had surprisingly high levels of human norovirus in their stools, and the strain identified was identical to that isolated from stools of the owner. This strongly implies that human norovirus had replicated within the gastrointestinal tract of this dog. Mild signs of gastroenteritis were reported in this dog, but these were very non-­‐specific and the overall health status of the dog is not reported. From this single case it cannot be determined if human norovirus has the ability to replicate in all dogs, a subset of dogs, or only immunosuppressed dogs. Importantly, this report also cannot determine who became infected with the virus first. Was it the human or the dog? Concluding remarks Many studies have sought to explore the roles of animals in transmission of noroviruses to humans. It has been shown that humans can mount immune responses to bovine and canine noroviruses, but no animal noroviruses have been detected in human stools. In addition, carbohydrate-­‐binding preferences of different norovirus strains and differing carbohydrate expression between species makes zoonotic infections seem unlikely. As for animal infection with human noroviruses, viral RNA has been detected in stools from pigs and dogs. This provides a theoretical zoonotic risk to humans, but the overall role of animals in human norovirus infections is believed to be low. However, the immunosuppressed and people working closely with animals are at greater risk, and the fast evolution rates of these RNA viruses means that if zoonotic strains are not present now, they may well emerge in the future. Key words Norovirus, zoonosis, virus-­‐like particle, gastroenteritis References 1. Tam, C. et al. Longitudinal study of infectious intestinal disease in the UK (IID2 study): incidence in the community and presenting to general practice. Gut 61, 69–77 (2012). 2. Mathijs, E. et al. A review of known and hypothetical transmission routes for noroviruses. Food and environmental virology 4, 131–52 (2012). 3. Zheng, D.-­‐P. et al. Norovirus classification and proposed strain nomenclature. Virology 346, 312–23 (2006). 4. Mesquita, J. R., Barclay, L., Nascimento, M. S. J. & Vinjé, J. Novel Norovirus in Dogs with Diarrhea. Emerging Infectious Diseases 16, 980–982 (2010). 5. Sugieda, M., Nagaoka, H., Kakishima, Y., Ohshita, T. & Nakamura, S. Detection of Norwalk-­‐like virus genes in the caecum contents of pigs. Public Health 1215–1221 (1998). 6. Woode, G. N. & Bridger, J. C. Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves. 1, (1978). 7. Wolf, S. et al. Molecular detection of norovirus in sheep and pigs in New Zealand farms. Veterinary microbiology 133, 184–9 (2009). 8. Martella, V. et al. Detection and Molecular Characterization of a Canine Norovirus. Emerging Infectious Diseases 14, 1306–1308 (2008). 9. Pinto, P. et al. Discovery and Genomic Characterization of Noroviruses from a Gastroenteritis Outbreak in Domestic Cats in the US. PloS one 7, e32739 (2012). 10. Karst, S. M., Wobus, C. E., Lay, M., Davidson, J. & Virgin, H. W. STAT1-­‐dependent innate immunity to a Norwalk-­‐like virus. Science 299, 1575–1578 (2003). 11. Tran, T. N. 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