Recently, we reported the high prevalence of NoV-, SaV-, and TV-specific antibodies in captive rhesus macaques (10
). Although the existence of cross-reactive binding antibodies between human NoVs and the prototype TV suggested that anti-NoV antibodies detected in NHPs (10
) are a consequence of TV infections, there also is evidence suggesting that NHPs are susceptible to at least experimental NoV infection (38
). In this study, we targeted the molecular detection of CVs in TNPRC rhesus macaques with a generic primer set (P289/P290) that has been used previously for the detection of a variety of CVs (11
). A high prevalence of recoviruses was detected, with 57 (11.6%) of the 500 stool samples containing viruses closely related (62 to 90% nucleotide homology) to the prototype TV. According to phylogenetic analysis, the recovirus strains detected in this study can be grouped into four genetic types within two genogroups. Of the 57 isolates, 15 (26%), 11 (19%), 25 (44%), and 6 (11%) grouped to GI.1, GI.2, GI.3, and GII.1, respectively (Fig. ). Such genetic diversity of recoviruses and circulation of genetically distinct strains in the TNPRC colony resembles the features of human NoVs. All the recoviruses detected in this study exhibited at least a 10% difference in nucleotides compared to those of the prototype TV, which was isolated from stool samples collected in 2004 from the same colony (11
). The existence of homologous antibody protection and the existence of distinct serotypes that likely are associated with genotypes were suggested by the high prevalence (69%) of TV (GI.1)-neutralizing antibodies in the colony (herd immunity) and by the higher TV-neutralizing antibody titers in animals shedding viruses of heterologous (other than GI.1) genotypes (Fig. ). Consequently, tissue culture adaptation of the different recovirus strains, identification of serotypes, assessment of protective level of VN antibodies, T- and B-cell epitope mapping, and relation of antigenic epitopes to carbohydrate binding sites will provide valuable information for NoV vaccine design and development.
Interestingly, in one rhesus macaque stool sample, a NoV was detected. The susceptibility of NHPs to experimental NoV infection was demonstrated previously (38
). Nevertheless, this report is the first to describe the natural NoV infection of a NHP. Laboratory contamination was excluded by the repeated detection of the NoV from different aliquots of the original sample in two collaborating laboratories. Since stool samples were collected as individual rectal loops, contamination during sample collection could be excluded. The rhesus NoV isolate exhibited a 89 to 93% nucleotide homology with GII.6, GII.7, and GII.9 human NoVs (Fig. ). Since the separation of these genotypes in the RdRp region analyzed is poor, capsid-based (ORF2) sequence analysis of strain FT244 will be necessary to determine its exact genotype. Nevertheless, data generated in this study clearly placed the rhesus NoV isolate in a close proximity with human NoV isolates, indicating the possibility of interspecies transmission. Despite the high prevalence of anti-NoV and anti-SaV antibodies in the TNPRC colony (10
), only one NoV-infected animal was detected in this study, and no SaV was found. Whether the low detection rate of NoVs and SaVs compared to the high seroprevalence is due to the seasonal occurrence of these viruses in colony macaques, the unfitness of the primers, or other reasons remains to be elucidated. The possible zoonotic nature of enteric CV infections also is suggested by the high prevalence of TV-neutralizing antibodies in serum samples of animal caretakers and the involvement of HBGAs in TV infection. The end titers of human samples were lower than the end titers of NHP samples. However, these human samples were collected nearly 20 years ago, and antibody titers could be reduced during long-term storage. Parallel investigation of the epidemiology of enteric CVs in human and NHP populations therefore would be of interest.
Based on results obtained from in vitro
binding assays and volunteer challenge studies, HBGAs have been identified as the putative cellular receptors for human NoVs (16
). The association between the HBGA binding abilities of NoVs and susceptibility to infection was established for some strains (27
); however, for others, such a link was not found (26
), indicating that different NoVs use different strategies to establish infection, and that factors other than HBGAs also might be involved. Since there is no effective tissue culture or animal model available to study this relationship, the potential involvement of HBGAs in recovirus infection was examined in this study. Despite the fact that NHPs and humans have four phenotypes of ABO blood groups (A, B, O, and AB), only humans and the anthropoid apes express the ABH antigens on the surface of red blood cells (1
). Both New and Old World monkeys secrete ABH antigens in their saliva and possess anti-A and/or anti-B antibodies in their sera. Thus, ABO blood groups of rhesus macaques can be determined by the detection of A/B antigens in saliva or antibodies in serum. In this study, based on the reported involvement of ABO, Lewis, and host secretor status in NoV infection (16
), saliva-based assays were used for the HBGA phenotyping of rhesus macaques. All commercially available MAbs used were confirmed to recognize the corresponding HBGA antigens except the anti-H type I (BG-4) antibody, which showed no reaction with any of the human saliva samples, even at a 1:10 dilution.
Saliva-based ABO phenotyping of the 500 animals used in this study revealed an almost-homotypic population, with 97% of the animals being type B. These results created difficulty in linking any particular ABO phenotype to susceptibility to recovirus infection or in excluding that possibility. Similarly, no association could be established for a particular Lewis phenotype. Among the 57 recovirus-positive macaques, 55 (96%) were type B and 2 (4%) were type O. The recovirus infection of type O animals also was indicated by the presence of VN serum antibodies. Thus, at least both type B and type O macaques are susceptible to recovirus infection. To assess the role of type A antigen and the possible differences among the different strains in population-based studies, macaques with a more-polymorphic distribution of the ABO types need to be tested.
Our findings are consistent with previous studies that reported group B as the major blood type (~97%) found in rhesus macaques (25
). On the other hand, Malaivijitnond et al. described the polymorphism of the ABO blood group in rhesus macaques in Thailand, although variation among troops from different geographic locations was significant (30
). Since ABO typing is not included in the routine assessment of research colony macaques, the identification of a colony with polymorphic HBGA distribution may pose a challenge.
In saliva binding assays, the prototype TV bound to both type A and B saliva but not to type O saliva (Fig. ). The type A and type B binding was confirmed by the binding of the prototype TV to BSA-conjugated type A and B trisaccharides (Fig. ). Interestingly, no binding to the PAA-conjugated type A or B trisaccharides was observed, even though the anti-A (ABO1) and anti-B (ABO2) MAbs recognized both of the corresponding BSA- and PAA-conjugated trisaccharides. Similar discrepancies have been noted previously for NoV VLP binding (17
). Huang et al. demonstrated that while specific MAbs recognize the HBGAs present in human milk or saliva regardless of the carrier molecule, the binding of NoV VLPs depends on both the specific carbohydrate structure and the carrier molecule (18
). Marionneau et al. suggested that the optimal binding of NoVs to the carbohydrate ligands requires an optimal density of the ligand, and binding may disappear at low densities (31
). Since PAA-conjugated trisaccharides with various carbohydrate-to-PAA ratios were not available for our study, this possibility remains to be elucidated.
Both previous observations with NoV VLPs and our experiments with TV indicate that the nature of carrier molecules plays a critical role in the binding of enteric CVs to carbohydrate structures. HBGA binding interfaces of different NoV strains have been characterized by cocrystallizing NoV P domain dimers with tri- and pentasaccharides (2
). In our study, there was no demonstrable interaction between the prototype TV and unconjugated type A and B pentasaccharide structures or PAA-conjugated type A and B trisaccharides. This indicates that interaction between enteric CVs and HBGAs is more complex than is described in these structural studies, and future studies with more-complex HBGA structures are necessary.
Results of plaque reduction assays confirmed the involvement of the type A and B HBGAs in recovirus infection. The preincubation of the prototype TV with type A or B saliva samples resulted in a significant reduction of plaque numbers (Fig. ). Type O saliva samples, the PAA-conjugated type A and B trisaccharides, and ALeb and BLeb pentasaccharides had no effect in plaque assays (data not shown). Contrary to our expectation, both BSA-conjugated type A and B trisaccharides increased the plaque numbers by 2- to 12-fold in a dose-dependent manner. This effect started at a lower concentration with the BSA-conjugated type B (2.5 μg/ml) than the type A (7.5 μg/ml) trisaccharide, indicating a higher-affinity interaction with the prototype TV.
These observations create several controversies. According to the ability of saliva samples to block TV infectivity in vitro
, saliva should act as an antiviral during natural infection. It also is difficult to explain how, contrary to saliva samples, BSA-conjugated synthetic oligosaccharides increased TV infectivity. One explanation is the involvement of coreceptor-receptor interactions in TV infection. We hypothesize that attachment to the HBGA (coreceptor) is required to prime TV, possibly through conformational changes, to enable interaction with a yet-unknown cell surface molecule (receptor). In saliva, HBGA structures are attached to a wide variety of molecules, including high-molecular-weight mucins. It was shown that NoV VLPs bind to HBGA structures attached to high-molecular-weight carriers but not to HBGAs attached to low-molecular-weight carriers present in human milk and saliva (18
). These large carriers of HBGAs, possibly due to steric hindrance, interfered with receptor binding when saliva was used in our TV plaque assays. On the other hand, the smaller BSA molecules had no interference with the receptor binding site. Assuming that TV receptor and coreceptor molecules need to be in physical proximity to support optimal attachment and entry, isolated receptor molecules far from the coreceptor will not be able to support virus attachment and entry. In the case of HBGA-primed TV virions, these isolated receptor molecules alone could be sufficient, which will lead to the increased efficiency of infection and plaque numbers.
The involvement of coreceptor/receptor interactions in enteric CV infections also is supported by previous studies showing that the cell surface expression of HBGAs alone does not confer susceptibility to NoV infection, even in cell types that are able to support the intracellular steps of NoV replication (14
Finally, the discrepancy with saliva acting as a natural antiviral in vitro could be explained by a yet-unknown mechanism (pH, digestive enzymes, etc.) that in the stomach or small intestine dissociates the HBGA-mucin complexes or otherwise digests the large carrier structures, thus exposing the receptor interaction sites.
In summary, this study describes the high prevalence of genetically diverse rhesus enteric CVs, in conjunction with evidence that suggests the zoonotic potential of enteric CV infections and the involvement of HBGAs in recovirus infection. Although more-detailed studies of recovirus HBGA interactions are necessary, including the characterization of the different isolates described in this study and in vivo
studies to link HBGAs to susceptibility to recovirus infection, the data presented here, together with our previous reports (11
), collectively indicate the potential that recoviruses can be used to address research questions applicable to NoV gastroenteritis.