Genetic diversity and histo-blood group antigen interactions of rhesus enteric caliciviruses

Abstract

Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol. 82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus within Caliciviridae, with the proposed name of Recovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involving in vitro and in vivo models of human NoV gastroenteritis.

FIG. 1.

Rhesus enteric caliciviruses are genetically diverse and can be classified into four genetic types within the two genogroups. The dendrogram was constructed by the neighbor-joining clustering method of the MEGA (version 3.1) software with Jukes-Cantor distance calculations. The confidence values of the internal nodes were obtained by performing 1,025 bootstrap analyses. The prototype Tulane virus (M33) isolated in 2004 from the TNPRC rhesus colony is circled.

FIG. 2.

Rhesus NoV isolate is closely related to human NoV isolates. The dendrogram, based on the alignment of 274-nucleotide RdRp sequences, was constructed by the neighbor-joining clustering method of the MEGA (version 3.1) software with Jukes-Cantor distance calculations. The rhesus NoV (FT244) isolate is circled.

FIG. 3.

Mean VN titers against the prototype TV (GI.1) in macaques with virus shedding. Animals were grouped by the genetic types of the recovirus isolates. VN titers were higher in animals infected with GI.2, GI.3, or GII.1 strains than in animals infected with GI.1 strains. Statistical significance was calculated between GI.1 (test virus) and the other groups using two-tailed t test with unequal variances. P < 0.05 was considered significant. Error bars represent SD. n, number of samples available for VN; *, P < 0.05.

FIG. 4.

Saliva and synthetic oligosaccharide binding of the prototype TV. Eight type A, type B, and type O saliva samples (A) and BSA- or PAA-conjugated type A and type B trisaccharides (B) were tested, respectively. Mean OD values of separate experiments were calculated. Error bars represent SD. **, P < 0.01; ***, P < 0.0005.

FIG. 5.

Saliva blocking of TV replication. Type A and type B, but not type O, saliva samples blocked TV replication. Eight type A, type B, and type O saliva samples were tested individually. C, virus control. ABO and Lewis types are indicated. Plaque reduction was calculated by 1 − (PFU of sample/PFU of virus control) and is expressed as a percentage. The means were calculated from the results of two independent tests with two different TV concentrations (∼50 and ∼100 PFU). Error bars represent SD. *, P < 0.05; ***, P < 0.0005.

FIG. 6.

BSA-conjugated type A and type B trisaccharides increase TV plaque numbers in a concentration-dependent manner. C, virus control. Fold increase/decrease was calculated by PFU of sample/PFU of control. The mean was calculated from the results of two independent tests with two TV concentrations (∼50 and ∼100 PFU). Error bars represent SD. P values indicate statistical significance. *, P < 0.05.