Canine Noroviruses

      Keywords

      Noroviruses (NoVs) were first identified in humans in 1972 on immune electron microscopy observation of the stools of volunteers infected with filtrates of faecal samples collected from a nonbacterial gastroenteritis outbreak occurred in 1968 in Norwalk, Ohio, USA.
      • Kapikian A.Z.
      • Wyatt R.G.
      • Dolin R.
      • et al.
      Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis.
      Nonenveloped, small, rounded viruses (SRVs), 27 nm in size, were observed in the fecal filtrates and specific antibodies were detected in both experimentally and naturally infected individuals, suggesting that the particles were the etiologic agent of Norwalk gastroenteritis.
      On genetic characterization, NoVs have been classified as a distinct genus of the Caliciviridae family.
      • Xi J.N.
      • Graham D.Y.
      • Wang K.N.
      • et al.
      Norwalk virus genome cloning and characterization.
      NoVs have been now recognized as the major etiologic agent of nonbacterial acute gastroenteritis worldwide and they are estimated to cause more than 1 million hospitalizations and up to 200,000 deaths in children younger than 5 years on an annual basis.
      • Patel M.M.
      • Hall A.J.
      • Vinjé J.
      • et al.
      Noroviruses: a comprehensive review.
      NoVs have been also identified in cows, pigs, mice, and carnivores, and the role of some animal species as potential source of novel human NoVs via interspecies transmission and eventually recombination has been hypothesized.
      • Koopmans M.
      Progress in understanding norovirus epidemiology.

      Etiology

      Caliciviruses are nonenveloped SRVs with a single-stranded, positive-sense, polyadenylated RNA genome of 7 to 8.5 kb.
      • Green K.Y.
      Caliciviridae: the noroviruses.
      Based on their genetic relationships and genome organization, caliciviruses have been classified into 4 genera: namely Vesivirus, Lagovirus, Sapovirus, and Norovirus.
      • Green K.Y.
      Caliciviridae: the noroviruses.
      More recently, other caliciviruses have been discovered and proposed as members of distinct genera: Nebraska-like viruses
      • Smiley J.R.
      • Chang K.O.
      • Hayes J.
      • et al.
      Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus.
      (Nebovirus) in cows, rhesus caliciviruses,
      • Farkas T.
      • Sestak K.
      • Wei C.
      • et al.
      Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae.
      Saint Valerienè–like viruses in swine,
      • L'Homme Y.
      • Sansregret R.
      • Plante-Fortier E.
      • et al.
      Genomic characterization of swine caliciviruses representing a new genus of Caliciviridae.
      and avian caliciviruses.
      • Wolf S.
      • Reetz J.
      • Otto P.
      Genetic characterization of a novel calicivirus from a chicken.
      Caliciviruses have been associated to a variety of clinical signs, ranging from gastroenteric disease to exanthematic lesions, to severe systemic diseases and hemorrhagic forms, and they are recognized as important pathogens in both humans and animals.
      NoVs are important human enteric pathogens
      • Patel M.M.
      • Hall A.J.
      • Vinjé J.
      • et al.
      Noroviruses: a comprehensive review.
      and they have also been detected in the stools of livestock animals, although their role as pathogens in these animals remains controversial.
      • Souza M.
      • Azevedo M.S.P.
      • Jung K.
      • et al.
      Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.
      • Cheetham S.
      • Souza M.
      • Meulia T.
      • et al.
      Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs.
      In mice, NoV is able to invade the central nervous system (CNS) in STAT1-deficient animals, causing fatal disease.
      • Karst S.M.
      • Wobus C.E.
      • Lay M.
      • et al.
      STAT1-dependent innate immunity to a Norwalk-like virus.
      Mouse NoV has also been adapted to in vitro growth, thus providing an excellent model/surrogate for the study of human NoVs, which are noncultivatable.
      • Wobus C.E.
      • Karst S.M.
      • Thackray L.B.
      • et al.
      Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.
      • Duizer E.
      • Schwab K.J.
      • Neill F.H.
      • et al.
      Laboratory efforts to cultivate noroviruses.
      NoV genome is 7.5 to 7.7 kb in length and contains 3 distinct open reading frames (ORFs).
      • Green K.Y.
      Caliciviridae: the noroviruses.
      ORF1 encodes a large polyprotein that is post-translationally cleaved into 6 nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp). ORF2 encodes the capsid protein VP1, while ORF3 encodes a small basic protein, VP2 (Fig. 1) .
      • Green K.Y.
      Caliciviridae: the noroviruses.
      The viral capsid contains 180 copies of VP1 protein and a few copies of VP2. The VP1 contains 2 main domains, S and P. The S (shell) domain is highly conserved and connected through the P1 subdomain to the highly variable P2 (protruding) subdomain.
      • Prasad B.V.
      • Hardy M.E.
      • Dokland T.
      • et al.
      X-ray crystallographic structure of the Norwalk virus capsid.
      • Chen R.
      • Neill J.D.
      • Estes M.K.
      • et al.
      X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity.
      The P2 region possesses several motifs that control binding to the host cell and virus antigenicity.
      • Siebenga J.J.
      • Vennema H.
      • Renckens B.
      • et al.
      Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006.
      • Tan M.
      • Jiang X.
      Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle.
      Figure thumbnail gr1
      Fig. 1Norovirus (strain Norwalk, accession M87661) genome organization. Proteolytic clivage map of the non-structural polyprotein encoded by ORF1. The NH2-terminal portion (N) of the highly conserved shell (S) domain and the protruding region (P) subdomains (P1 and P2) are also indicated.
      • Prasad B.V.
      • Hardy M.E.
      • Dokland T.
      • et al.
      X-ray crystallographic structure of the Norwalk virus capsid.
      NoVs are genetically and antigenically highly heterogeneous. Accumulation of punctate mutations and recombination drive their evolution, generating an impressive diversity. The highly conserved ORF1/ORF2 junction region is a preferential site for NoV recombination.
      • Bull R.A.
      • White P.A.
      Mechanisms of GII.4 norovirus evolution.
      Recombination may create chimeric viruses with intermediate genetic features between the parental viruses, generating inconsistencies in the classification/nomenclature. A consistent and reliable classification of NoVs is based on the analysis of the complete capsid gene.
      • Zheng D.-P.
      • Ando T.
      • Fankhauser R.L.
      • et al.
      Norovirus classification and proposed strain nomenclature.
      Strains within the same genotype (or cluster) share greater than 85% amino acid identity, while strains of different genotypes within the same genogroup share 55% to 85% amino acid identity.
      • Zheng D.-P.
      • Ando T.
      • Fankhauser R.L.
      • et al.
      Norovirus classification and proposed strain nomenclature.
      Humans NoVs belong to genogroups (G) I, II, and IV.
      • Koopmans M.
      Progress in understanding norovirus epidemiology.
      In addition, NoVs classified as GII have been detected in pigs,
      • van Der Poel W.H.
      • Vinjé J.
      • van Der Heide R.
      • et al.
      Norwalk-like calicivirus genes in farm animals.
      • Wang Q.-H.
      • Han M.G.
      • Cheetham S.
      • et al.
      Porcine noroviruses related to human noroviruses.
      and GIII NoVs in large and small ruminants.
      • Oliver S.L.
      • Dastjerdi A.M.
      • Wong S.
      • et al.
      Molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (Norwalk-like viruses) unlikely to be of risk to humans.
      • Wolf S.
      • Williamson W.
      • Hewitt J.
      • et al.
      Molecular detection of norovirus in sheep and pigs in New Zealand farms.
      NoVs proposed as GV have been detected in mice.
      • Karst S.M.
      • Wobus C.E.
      • Lay M.
      • et al.
      STAT1-dependent innate immunity to a Norwalk-like virus.

      Caliciviruses in Dogs

      Unlike calicivirus infections in cats,
      • Radford A.D.
      • Coyne K.P.
      • Dawson S.
      • et al.
      Feline calicivirus.
      canine caliciviruses are not regarded as important pathogens and they are not usually included in diagnostic algorithms for canine infectious diseases. Calicivirus-like particles have been occasionally identified by electron microscopy in specimens from dogs with fluid diarrhea and, in some instances, glossitis, balanitis, or vesicular vaginitis. Most isolates were feline caliciviruses (FCVs) and were likely acquired from cats.
      • Crandell R.A.
      Isolation and characterization of caliciviruses from dogs with vesicular genital disease.
      • Evermann J.F.
      • McKeirnan A.J.
      • Smith A.W.
      • et al.
      Isolation and identification of caliciviruses from dogs with enteric infections.
      • Martella V.
      • Pratelli A.
      • Gentile M.
      • et al.
      Analysis of the capsid protein gene of a feline-like calicivirus isolated from a dog.
      • San Gabriel M.C.
      • Tohya Y.
      • Sugimura T.
      • et al.
      Identification of canine calicivirus capsid protein and its immunoreactivity in western blotting.
      • Evermann J.F.
      • Bryan G.M.
      • McKiernan A.J.
      Isolation of a calicivirus from a case of canine glossitis.
      Thus far, there are only 2 documented reports on the identification of authentic canine caliciviruses in dogs. In 1985 a calicivirus was isolated from the feces of a 4-year-old dog with bloody diarrhea and central nervous system disturbance in Tennessee, USA. The virus was found to replicate in experimentally infected dogs and to elicit seroconversion, although disease was not reproduced. Also, the virus was antigenically unrelated to FCV and antibodies against the virus were identified in 76% of the canine sera collected.
      • Schaffer F.L.
      • Soergel M.E.
      • Black J.W.
      • et al.
      Characterization of a new calicivirus isolated from feces of a dog.
      However, it was not characterized molecularly and its taxonomic status remains uncertain. In 1990, another calicivirus was identified in Japan in a 2-month-old pup with intermittent watery diarrhea.
      • Mochizuki M.
      • Kawanishi A.
      • Sakamoto H.
      • et al.
      A calicivirus isolated from a dog with fatal diarrhoea.
      The virus, strain 48, was found to be antigenically and genetically unrelated to FCV and was tentatively proposed as a “true” canine calicivirus (CaCV) and included in the Vesivirus genus.
      • Matsuura Y.
      • Tohya Y.
      • Nakamura K.
      • et al.
      Complete nucleotide sequence, genome organization and phylogenic analysis of the canine calicivirus.
      • Roerink F.
      • Hashimoto M.
      • Tohya Y.
      • et al.
      Organization of the canine calicivirus genome from the RNA polymerase gene to the poly(A) tail.
      Antibodies to CaCV 48 have been detected in 57% of dogs in Japan
      • Mochizuki M.
      • Hashimoto M.
      • Roerink F.
      • et al.
      Molecular and seroepidemiological evidence of canine calicivirus infections in Japan.
      and in 36.5% of dogs in Korea.
      • Jang H.K.
      • Tohya Y.
      • Han K.Y.
      • et al.
      Seroprevalence of canine calicivirus and canine minute virus in the Republic of Korea.

      Noroviruses in Dogs

      The first evidence of NoV in carnivores was documented in 2006 in a captive lion cub that died of severe hemorrhagic enteritis at 4 weeks of age in Pistoia, Italy.
      • Martella V.
      • Campolo M.
      • Lorusso E.
      • et al.
      Norovirus in captive lion cub (Panthera leo).
      The animal tested negative to all potential lion viral pathogens, and on bacteriologic investigations it was found to be infected by toxigenic Clostridia. Unexpectedly, NoV RNA was detected in the intestinal tract and, on genomic characterization, the virus was found to resemble human GIV NoVs (Alphatron-like), with 69.3% to 70.1% amino acid identity in the full-length capsid protein, and it was proposed as a distinct NoV genotype, GIV.2, while human Alphatron-like NoVs are GIV.1. Human GIV.1 NoVs are usually identified only sporadically in the human population, although they may be commonly detected in sewage samples from treatment plants,
      • La Rosa G.
      • Iaconelli M.
      • Pourshaban M.
      • et al.
      Molecular detection and genetic diversity of norovirus genogroup IV: a yearlong monitoring of sewage throughout Italy.
      • La Rosa G.
      • Pourshaban M.
      • Iaconelli M.
      • et al.
      Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends.
      indicating that there are open gaps in the understanding of their ecology and in the diagnosis.
      As lions are susceptible to the majority of canine and feline pathogens, the detection of NoV in lions raised the question of whether domestic carnivores represented the source of infection for the captive lion cub. By expressing in baculovirus the capsid protein of the lion NoV, virus-like particles (VLPs) were produced and used to set up an ELISA, revealing specific antibodies in 16.1% of feline and 4.8% of canine sera.
      • Di Martino B.
      • Marsilio F.
      • Di Profio F.
      • et al.
      Detection of antibodies against norovirus genogroup GIV in carnivores.
      Also, by screening a collection of stools from dogs with gastroenteritis in Italy in 2007, NoV was detected in 2.2% (4 of 183) of the pups.
      • Martella V.
      • Lorusso E.
      • Decaro N.
      • et al.
      Detection and molecular characterization of a canine norovirus.
      • Martella V.
      • Decaro N.
      • Lorusso E.
      • et al.
      Genetic heterogeneity and recombination in canine noroviruses.
      The age of the pups ranged between 60 and 70 days and 3 of 4 pups were also co-infected by canine parvovirus. These direct and indirect pieces of evidence confirmed that domestic carnivores might harbor NoVs.
      Shortly after the first identification, additional evidence about the circulation of NoVs in dogs has been documented. During an epidemiologic study in 2008 in Greece, a cluster of NoV infection was identified in a kennel in Thessaloniki in 6 pups, 2.5 to 3 months old, that were housed together,
      • Ntafis V.
      • Xylouri E.
      • Radogna A.
      • et al.
      Outbreak of canine norovirus infection in young dogs.
      suggesting the highly infectious nature of canine NoVs for young pups. All the NoV-infected animals were also co-infected by canine coronavirus.
      In a 1-year survey in Portugal in 2008 of dogs from municipal shelters, veterinary clinics, and pet shops, NoV was detected in the stools of 25 of 63 (40%) of dogs with diarrhea and 4 (9%) of 42 asymptomatic animals. In most cases, the NoV-infected dogs displayed mixed infections by either canine parvovirus or coronavirus or both.
      • Mesquita J.R.
      • Barclay L.
      • Nascimento M.S.J.
      • et al.
      Novel norovirus in dogs with diarrhea.
      Also, NoV RNA was detected in 3 of 106 stools collected from pups with parvovirus gastroenteritis in 2007 in the United Kingdom (Martella and colleagues, unpublished information, 2011). These findings indicate the canine NoVs circulate in several European countries.

      Genetic Heterogeneity in Canine NoVs

      Thus far, 6 canine NoV strains have been analyzed molecularly. Sequence information has been gathered on the RdRp region, at the 3′ end of ORF1, the full-length capsid protein (ORF2), and the minor basic protein (ORF3). The prototype canine NoV strain, Bari/170/07/ITA,
      • Martella V.
      • Lorusso E.
      • Decaro N.
      • et al.
      Detection and molecular characterization of a canine norovirus.
      resembles the virus lion NoV Pistoia/387/06/ITA, as the 2 viruses share 96.7% amino acid identity in the RdRp and 90.1% amino acid identity in the capsid protein. Likewise, the Greek strain Thessaloniki/30/2008/GRC resembles the canine virus Bari/170/07/ITA, both in the RdRp (100% amino acid identity) and the capsid gene (99.4% amino acid identity).
      • Ntafis V.
      • Xylouri E.
      • Radogna A.
      • et al.
      Outbreak of canine norovirus infection in young dogs.
      A large insertion of 20 residues can be observed in the P2 hypervariable domain of GIV.2 animal NoVs with respect to GIV.1 human viruses. By homology modeling and 3-dimensional alignment, the P insertion appears to form a loop protruding from the compact barrel-like structure of the P2 subdomain and exposed on the outer surface of the capsid.
      Interestingly, another canine NoV strain, Bari/91/07/ITA, although sharing the same pol (RdRp) type as strains Dog/Bari/170/07/ITA and Lion/Pistoia/387/06/ITA, possesses a novel ORF2 gene, with the highest identity (57.8% amino acid) to the unclassified human strain Chiba/040502/04/JAP. This canine virus is distantly related (36.0%–54.5% amino acid identity) to all other NoVs,
      • Martella V.
      • Decaro N.
      • Lorusso E.
      • et al.
      Genetic heterogeneity and recombination in canine noroviruses.
      suggesting the existence in dogs of NoVs with a novel capsid genotype. The UK strain FD210/07/GBR resembles both in the RdRp (98.5% amino acid identity) and the capsid (95.0% amino acid identity) canine virus Bari/91/07/ITA.
      The Portuguese NoV strain Viseu/C33/08/PRT and the UK strain FD53/07/GBR display a third capsid genotype. These viruses are related to each other (99.5% amino acid identity in the RdRp and 98.6% amino acid in the VP1), while they have only 63.1% to 63.9% amino acid identity in the full-length VP1 to the strain Bari/91/07/ITA and FD210/07/GBR (Fig. 2, Table 1).
      Figure thumbnail gr2
      Fig. 2Phylogenetic tree constructed on the full-length amino acid sequence of the capsid protein. The tree was constructed using a selection of NoV strains representative of the genogroups I to V. bo, bovine; po, porcine; mu, murine; hu, human.
      Table 1Classification of canine NoVs based on the full-length capsid protein VP1
      GenogroupGIGIIGIIIGIVGVGVIClassification
      GenotypeGIV.1GIV.2GVI.1GVI.2GVI.3ORF1ORF2
      Lion/Pistoia/387/06/ITA41,6-37,849,7-45,836,6-36,569,2-68,990,136,950,054,4-54,554,1-53,8GIV.2GIV.2
      Dog/Bari/170/07/ITA41,1-36,950,2-45,935,9-35,168,0-67,790,136,650,054,3-54,053,8-53,4GIV.2GIV.2
      Dog/Bari/91/07/ITA40,8-38,054,4-50,237,7-37,054,4-54,254,5-54,436,057,89563,8-63,2GIV.2GVI.2
      Dog/FD/210/07/GBR40,7-38,154,7-50,437,6-37,153,4-53,354,6-54,136,457,59563,9-63,1GIV.2GVI.2
      Dog/FD/53/07-2/GBR41,5-38,453,4-48,839,6-38,153,7-53,554,1-53,836,755,263,9-63,898,6GIV.2GVI.3
      Dog/C33-Viseu/07/PRT41,2-38,153,9-48,639,3-37,853,4-53,253,8-53,436,454,963,2-63,198,6GIV.2GVI.3

      Pathogenic Potential of Canine NoVs

      The pathogenicity of canine NoVs in experimental infections in gnotobiotic or specific-pathogen-free (SPF) animals has not been assessed. Viral shedding could be monitored in a naturally infected pup with mixed infection by NoV and canine parvovirus type-2. The pup recovered from the disease 4 days after hospitalization but NoV was shed at detectable levels for 3 weeks.
      • Martella V.
      • Lorusso E.
      • Decaro N.
      • et al.
      Detection and molecular characterization of a canine norovirus.
      Prolonged NoV shedding after infection/disease has been documented for weeks or even months in human patients.
      • Atmar R.L.
      • Opekun A.R.
      • Gilger M.A.
      • et al.
      Norwalk virus shedding after experimental human infection.
      • Siebenga J.J.
      • Beersma M.F.C.
      • Vennema H.
      • et al.
      High prevalence of prolonged norovirus shedding and illness among hospitalized patients: a model for in vivo molecular evolution.
      Likewise, murine NoV shedding can last for several weeks in immune-competent mice,
      • Karst S.M.
      • Wobus C.E.
      • Lay M.
      • et al.
      STAT1-dependent innate immunity to a Norwalk-like virus.
      • Hsu C.C.
      • Riley L.K.
      • Wills H.M.
      • et al.
      Persistent infection with and serologic cross-reactivity of three novel murine noroviruses.
      and this has been interpreted as a mechanism of virus persistence in the host population.
      In most cases, NoV-infected dogs were also co-infected by other pathogens. That mixed infections can elicit mechanisms of synergism, as observed between coronaviruses and parvoviruses,
      • Appel M.J.
      Does canine coronavirus augment the effects of susbequent parvovirus?.
      • Pratelli A.
      • Tempesta M.
      • Roperto F.P.
      • et al.
      Fatal coronavirus infection in puppies following canine parvovirus 2b infection.
      cannot be ruled out. Interestingly, the frequency of detection of NoV has been found to differ significantly between symptomatic and asymptomatic dogs in a 1-year survey in Portugal.
      • Mesquita J.R.
      • Barclay L.
      • Nascimento M.S.J.
      • et al.
      Novel norovirus in dogs with diarrhea.
      Interpretation of these findings is not clear, as several factors can influence the course of NoV infection. As canine NoVs appear to display a number of capsid genotypes, there could be differences in the biological properties (eg, virulence, ability to bind to canine cellular receptors, and so on) among the various NoV strains. In addition, mechanisms of genetic resistance could alter the outcome of NoV infection in some canine breeds, thus confounding the picture. Experimental human infection studies with the prototype Norwalk virus (GI.1) showed that the study participants were repeatedly susceptible or resistant to symptomatic infection following repeated virus challenge.
      • Parrino T.A.
      • Schreiber D.S.
      • Trier J.S.
      • et al.
      Clinical immunity in acute gastroenteritis caused by Norwalk agent.
      Subsequent studies have revealed that human NoVs recognize histoblood group antigens (HBGAs) as receptors or co-receptors. HGBAs are complex carbohydrates present on the surface of red blood cells and mucosal epithelia, or free in biological fluids such as milk and saliva. HGBAs are synthesized under the control of highly polymorphic ABO, Lewis, and secretor gene families. Different NoV genotypes variously recognize these antigens, and the recognition patterns have been found to correlate with susceptibility to infection and illness.
      • Tan M.
      • Xia M.
      • Chen Y.
      • et al.
      Conservation of carbohydrate binding interfaces: evidence of human HBGA selection in norovirus evolution.
      • Hutson A.M.
      • Atmar R.L.
      • Graham D.Y.
      • et al.
      Norwalk virus infection and disease is associated with ABO histo-blood group type.
      • Hutson A.M.
      • Atmar R.L.
      • Marcus D.M.
      • et al.
      Norwalk virus-like particle hemagglutination by binding to histo-blood group antigens.
      • Chakravarty S.
      • Hutson A.M.
      • Estes M.K.
      • et al.
      Evolutionary trace residues in noroviruses: importance in receptor binding, antigenicity, virion assembly, and strain diversity.
      The global spread and predominance of pandemic GII.4 NoV variants have been related to the broad range of recognized HBGA types.
      • Tan M.
      • Xia M.
      • Chen Y.
      • et al.
      Conservation of carbohydrate binding interfaces: evidence of human HBGA selection in norovirus evolution.
      Similar mechanisms appear to influence genetic resistance of pigs to NoV infection under experimental conditions.
      • Cheetham S.
      • Souza M.
      • McGregor R.
      • et al.
      Binding patterns of human norovirus-like particles to buccal and intestinal tissues of gnotobiotic pigs in relation to A/H histo-blood group antigen expression.

      Diagnosis

      Several sets of primers have been designed for molecular diagnosis of human NoVs in different diagnostic regions (A–C) spanning the ORF1 and ORF2.
      • Vinjé J.
      • Hamidjaja R.A.
      • Sobsey M.D.
      Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses.
      Diagnostic tools can be greatly affected by NoV genetic diversity.
      • Mattison K.
      • Grudeski E.
      • Auk B.
      • et al.
      Multicenter comparison of two norovirus ORF2-based genotyping protocols.
      In most cases, diagnosis of canine NoV was accomplished using broadly reactive primers sets targeting diagnostic region A within the RdRp, such as p289-p290 or JV12Y/YV13I.
      • Jiang X.
      • Huang P.W.
      • Zhong W.M.
      • et al.
      Design and evaluation of a primer pair that detects both Norwalk- and Sapporo-like caliciviruses by RT-PCR.
      • Vennema H.
      • de Bruin E.
      • Koopmans M.
      Rational optimization of generic primers used for Norwalk-like virus detection by reverse transcriptase polymerase chain reaction.
      However, it has been shown that designing more specific primers can allow increasing significantly the detection rates of canine NoVs (from 1.9% to 27.6%).
      • Mesquita J.R.
      • Barclay L.
      • Nascimento M.S.J.
      • et al.
      Novel norovirus in dogs with diarrhea.
      Several unsuccessful attempts have been made to adapt to in vitro cultivation the prototype canine NoV strain Bari/170/07/ITA, using both canine and feline cell lines and primary cells. With the exception of murine NoVs,
      • Wobus C.E.
      • Karst S.M.
      • Thackray L.B.
      • et al.
      Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.
      NoVs appear to be noncultivatable in vitro.
      • Duizer E.
      • Schwab K.J.
      • Neill F.H.
      • et al.
      Laboratory efforts to cultivate noroviruses.
      • Lay M.K.
      • Atmar R.L.
      • Guix S.
      • et al.
      Norwalk virus does not replicate in human macrophages or dendritic cells derived from the peripheral blood of susceptible humans.
      Replication of human NoVs in vitro has been demonstrated in a 3-dimensional organoid model of human small intestinal epithelium, displaying a high level of cellular differentiation.
      • Straub T.M.
      • Höner zu Bentrup K.
      • Orosz-Coghlan P.
      • et al.
      In vitro cell culture infectivity assay for human noroviruses.
      However, these results have not been reproduced in other laboratories.
      An ELISA has been set up using the baculovirus-expressed capsid protein of the GIV.2 lion NoV.
      • Di Martino B.
      • Marsilio F.
      • Di Profio F.
      • et al.
      Detection of antibodies against norovirus genogroup GIV in carnivores.
      This assay was successfully used to assess exposure of domestic carnivores to NoVs. However, considering the extent of the genetic heterogeneity of canine NoVs, generating synthetic antigens based on other capsid genotypes (GVI.2 and GVI.3) would be necessary to portray a more precise picture.

      Zoonotic Potential of Canine NoVs

      Dogs are regarded as vectors of viral, bacterial, or parasitic zoonosis,
      • Heyworth J.S.
      • Cutt H.
      • Glonek G.
      Does dog or cat ownership lead to increased gastroenteritis in young children in South Australia?.
      but the risks linked to transmission of enteric viruses are almost ignored. However, several pieces of evidences indicate that enteric viruses may have a zoonotic potential: (1) infection of young children by rotavirus strains of canine and feline origin has been documented repeatedly
      • Martella V.
      • Bányai K.
      • Matthijnssens J.
      • et al.
      Zoonotic aspects of rotaviruses.
      ; (2) having dogs in or near a home has been recognized as a risk factor for acquisition of IgA antibodies specific for NoV in infants in a seroepidemiologic study conducted in rural Mexico
      • Peasey A.E.
      • Ruiz-Palacios G.M.
      • Quigley M.
      • et al.
      Seroepidemiology and risk factors for sporadic norovirus/Mexico strain.
      ; and (3) a calicivirus gastroenteritis outbreak occurred in a nursing home in Exeter, UK, in 1983 and was found to be epidemiologically linked to the household dog. Acute gastroenteric disease in the dog occurred 24 hours before the human index case and antibodies specific for the human caliciviruses were identified in the dog, thus suggesting a possible association.
      • Humphrey T.J.
      • Cruickshank J.G.
      • Cubitt W.D.
      An outbreak of calicivirus associated gastroenteritis in an elderly persons home A possible zoonosis?.
      (4) Also, under experimental conditions, NoVs have been found to be able to cross the host species barriers. A GII.4 human NoV was able to infect and induce diarrhea in gnotobiotic piglets and calves,
      • Cheetham S.
      • Souza M.
      • Meulia T.
      • et al.
      Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs.
      • Souza M.
      • Azevedo M.S.P.
      • Jung K.
      • et al.
      Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.
      thus indicating that heterologous infections can occur. (5) In addition, NoV strains genetically similar to the canine virus Bari/91/07/ITA (88.9% nucleotides and 98.9% amino acid identities in a short fragment spanning the 5′ end of ORF2) have been detected in oysters destined for raw consumption in Japan (strains Yamaguchi/C34/03/JAP, Yamaguchi/24B/02/JAP, and Yamaguchi/24C/02/JAP
      • Nishida T.
      • Nishio O.
      • Kato M.
      • et al.
      Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan.
      ). This could indicate that canine-like GVI NoVs are common in some geographic settings and that they can contaminate the coastal areas and accumulate at detectable levels in bivalve molluscs destined for raw consumption. Contamination of shellfish by animal (porcine and bovine) enteric caliciviruses, alone or in conjunction with human viruses, has been demonstrated in 22% of oysters in United States.
      • Costantini V.
      • Loisy F.
      • Joens L.
      • et al.
      Human and animal enteric caliciviruses in oysters from different coastal regions of the United States.
      However, while the impact of sewage pollution on the water environment by livestock may be relevant, especially in the areas of high livestock production, it is difficult to explain the presence of canine-like NoVs in oysters. A possible explanation for this is that similar viruses are harbored in other animal species or in settled human populations. (6) Finally, human GIV (Alphatron-like) NoVs are genetically much more related to animal GIV NoVs (GIV.2) than to GI and GII human NoVs, suggesting points of intersection during their evolution. The modalities of this intersection are uncertain but likely they were favored by the strict social interactions between humans and pets.

      Summary

      NoV are regarded as emerging pathogens in humans, and the creation of worldwide surveillance networks has allowed the researchers to gather important epidemiologic information and to gain unforeseen insights into the mechanisms of NoV evolution. The discovery of NoVs in carnivores and the genetic relationship between them and some human viruses raise interesting questions inherent in the ecology of these viruses and the possibilities of interspecies transmission. Also, it will be interesting to assess whether and to which extent NoVs impact on pet health.

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