Presence of Antibodies against Genogroup VI Norovirus in Humans

Noroviruses (NoVs) are the leading cause of epidemic and sporadic acute gastroenteritis in humans with worldwide an estimated 1 million hospitalizations and up to 200,000 deaths in children < 5 years of age each year [1, 2]. Outbreaks occur in various settings including long-term care facilities, hospitals, schools, restaurants and cruise ships. The main modes of transmission of NoV are person-to-person and through the consumption of contaminated food or water. During outbreaks, however, multiple transmission routes may play a role [3]. In recent years NoVs have been detected in a number of mammalian species and several studies have suggested that zoonotic transmission from animal to humans may occur [4‐6] and that an animal reservoir might be the source of the introduction of new strains in the human population. Although no zoonotic events have been reported, there are several indications that NoVs may be able to cross the species barrier. Gnotobiotic pigs have been experimentally infected with a human NoV strain [4], and viruses closely related to human NoVs have been detected in swine [7]. Moreover, NoV sequences have been detected in livestock and in retail meat samples highlighting a possible route for indirect zoonotic transmission of NoVs through the food chain and the risk for emergence of animal/human recombinants [8].

Noroviruses are a group of non-enveloped, single-stranded, RNA viruses with an icosahedral capsid symmetry classified into the genus Norovirus of the family Caliciviridae. They can be grouped in at least 5 different genogroups (designated GI-GV) [1, 9]. Strains infecting humans are found in GI, GII and GIV. Porcine NoVs are classified in distinct genotypes within GII, bovine and ovine viruses belong uniquely to GIII, and murine NoVs are grouped in GV. Recently, several research groups have reported NoVs in domestic carnivores with diarrhea [10, 11]. Canine NoVs (CaNoVs) genetically related to GIV have been reported in Italy, Greece and Japan [10‐13], whereas viruses belonging to a proposed new genogroup (GVI) were found in fecal samples from dogs with diarrhea in Portugal and Italy [9, 14‐16].

The zoonotic potential of an infectious disease agent has been inferred by comparing pathogen-specific antibody levels between individuals that are in close contact with a particular animal and a matched control population with no professional exposure to animals [17, 18]. For example, a higher serum antibody level against bovine NoV was detected in large animal veterinarians compared to the general population, indicating that bovine NoV strains could infect the human population [18]. Additionally, antibodies to human NoVs have been detected in pigs highlighting the possibility of human-to-animal transmission of NoV [5].

In most industrialized countries, pets are an integral part of the household leading to well-documented health risks associated with owning a pet. Bites, scratches and allergies are more frequent; however, infections including parasitic, bacterial, fungal and viral diseases can be transmitted to humans [19]. In a recent report, human NoV sequences were detected in fecal samples from pet dogs which had been in direct contact with humans with NoV gastroenteritis, suggesting that human NoVs can at least survive in the gastrointestinal tract of dogs [20].

To investigate if CaNoV may infect humans, sera from pet veterinarians and age-matched population controls were tested for IgG antibodies to recombinant virus-like particles of CaNoV. This Study Protocol was published elsewhere [21].

Of the 373 veterinarians, 83 (22.3%) had IgG antibodies against CaNoV compared to 7 (5.8%) of the 120 matched population controls (p < 0.001). Moreover, the mean corrected OD₄₅₀ values for CaNoV antibodies was significantly higher in veterinarians than in controls (p < 0.001) (Figure 1). CaNoV antibodies were detected in veterinarians from all four countries.

To evaluate possible cross-reactivity between these antibodies and human NoV, two serum samples from veterinarians with high (HAT) and low antibody titers (LAT) to CaNoV were pre-incubated with 2-fold serial dilutions of CaNoV VLPs. The corrected OD₄₅₀ values for CaNoV antibodies in both serum samples decreased significantly with increasing concentration of CaNoV VLPs (β = −0.150 ± 0.043, 95%CI −0.287 to −0.013, p < 0.05 and β = −0.073 ± 0.018, 95%CI −0.132 to −0.014, p <0.05 for serum HAT and LAT, respectively) (Figure 2A). By contrast, no significant change in the corrected OD₄₅₀ values was observed when the pre-incubated sera were tested for the presence of GII.4 New Orleans antibodies (β = −0.011 ± 0.031; 95%CI −0.11 to 0.089 and β = 0.0219 ±0.056; 95%CI 0.158 to 0.202 for samples 68 and 25, respectively) (Figure 2B).

We detected IgG antibodies against CaNoV in 22.3% of the small animal veterinarians and in 5.8% of the age-matched population controls. These findings suggest that CaNoV may infect humans and that small animal veterinarians are at an increased risk for exposure and possibly infection with this virus. The presence of antibodies in the population control samples may be explained by household contact as dogs are popular pet animals.

An increased exposure risk to bovine NoV has been reported for large animal veterinarians in the Netherlands who had a higher seroprevalence of bovine NoV antibodies than the general population [18]. Conversely, a high prevalence of antibodies against human NoV (Norwalk strain) was detected in pigs and captive juvenile macaques [5, 22]. These data suggest that NoV may be able to cross the species barrier. Exposure to zoonotic agents is a widely recognized risk in veterinary medicine. In an Australian survey, 4% of veterinarians reported having acquired at least one zoonotic disease from animal-related exposure [23]. In another study, IgG antibodies against Brucella spp and Coxiella burnetii were higher among veterinarians working in an endemic region [24]. The zoonotic risk of veterinarians and pig handlers for hepatitis E virus (HEV) infections has been demonstrated in studies in Taiwan and the US [25, 26]. In the Taiwan study, 27% of pig handlers tested positive for anti-HEV antibodies compared to 8% of the control subjects [25], while in the US study 26% of veterinarians working with swine and 17% of blood donors were seropositive for HEV, suggesting that veterinarians may be at a higher risk of HEV infection through animal contact, compared to normal blood donors [26].

A limitation of our study was that the antibodies detected by the CaNoV VLPs may be cross-reactive against human NoVs. However, the blocking assay data showed that binding of CaNoV antibodies but not human NoV antibodies, could be blocked by CaNoV VLPs, demonstrating the VLP-based ELISA used in this study measured CaNoV-specific antibodies.

In conclusion, our data suggest that CaNoV may infect humans and that small animal veterinarians are at an increased risk. Studies that test human stool samples in households with dogs with CaNoV diarrhea are needed to confirm that this virus is able to cause diarrheal disease in humans.