Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes.Taken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity

Genetic Diversity and Histo-Blood Group Antigen Interactions of Rhesus Enteric Caliciviruses▿

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

Flow cytometry detection of TV antigens-containing cells <i>in vitro</i>.

<p>A– PBMCs isolated from healthy rhesus macaques were used for <i>in vitro</i> inoculation with TV. After being cultured <i>in vitro</i> for 6 days, cells were sorted out into CD3+ T cell and CD20+ B cell populations. Presence of TV antigens was detected in CD20+ B cells as shown by histograms (black peak). B– The CD20+ B cells were further subdivided into populations expressing the HLA-DR, CD11c or CD123 antigens. Presence of TV was revealed predominantly in CD20+HLA-DR+ cells while some of the other lymphocyte populations including the CD20+CD11c+ and CD20+CD123+ B cells also contained TV antigens.</p

Virus shedding in stools following experimental inoculation.

<p>Virus shedding for the TV-inoculated animals started on day 1 (HC55 and HI77) and on day 2 (HB61). For the NoV-inoculated animals virus shedding started on day 1 (HB11) and day 2 (GI96) as determined by nested RT-PCR. No samples from GI96 were however positive by qRT-PCR and only samples corresponding to HB11 days 1, 2 and 9 were positive and quantitatively evaluated. Considering that juvenile rhesus macaques of 2.5–4.0 years of age produce daily 80±20 grams of stool, the three TV-inoculated and HB11 macaques produced more virus in stools than what they received in inoculum.^anot applicable.</p

Antibodies used for flow cytometry of <i>in vitro</i> cultured PBMCs.

a<p>Tulane National Primate Research Center,^bnot applicable.</p

Virus shedding in stools and virus-neutralizing (VN) serum antibody response following TV inoculation.

<p> All of the three TV-inoculated macaques showed presence of virus-specific RNA in stools (upper panel) and developed VN antibody responses (≥4-fold increase) by PID 7 (lower panel). Duration of shedding ranged in TV-inoculated macaques between 8–10 days while reaching the peak of ∼10⁵ RNA copies per gram of stools in HB61 and HC55 and ∼10⁴ in HI77. The detection limit of the assay was 10–100 copies per reaction. Antibody serum levels reached their peak in all three macaques by PID 7–9 (1∶160–1∶640) while returned to pre-inoculation levels by PID 80.</p

Immunofluorescent confocal microscopy of duodenum biopsies from TV-inoculated macaque HC55.

<p> A– The cytokeratin+ epithelial cells (red) did not co-localize with TV (green). The TV+ cells appear in subepithelium i.e. lamina propria (arrows). B– The subepithelial CD3+ T lymphocytes (red) did not co-localize with TV (green). C– Co-localization of some of the CD20+ B cells (red) with TV (green) is indicated by arrows. D– The IBA-1+ macrophages (red) did not co-localize with TV (green). Nuclear DNA is in blue. E– The CD11c+ myeloid dendritic cells (red) did not co-localize with TV (green) while co-localization between CD11c+ and calnexin (blue) markers resulted in purple cell coloration. The co-localization of TV+ cells with calnexin is indicated by arrows. F– Absence of co-localization between TV (green) and TUNEL (red) markers indicates that TV-infected cells did not undergo apoptosis. Differential interference contrast (gray) is highlighted for the better visualization of tissue architecture.</p

Colocalization of TV and calnexin antigens inside the HC55 macaque's duodenum lamina propria.

<p>A– While most of the cells are calnexin-single-positive, simultaneous presence of both TV (green) and calnexin (red) antigens is seen in some of the (yellowish) cells. DIC– differential interference contrast. B– Spectral overlap of TV and calnexin antigens is shown by arrows.</p

Duodenum biopsies from healthy control and TV-inoculated macaques.

<p>A– Duodenum tissue from healthy control macaque with normal tissue architecture of intestinal villi. Magnification 10x. B– Duodenum tissue obtained at PID 3 from TV-inoculated HC55 macaque: Superficial mucosa contains diffuse mononuclear cell infiltrate with formation of an intra-epithelial lymphoid follicle. Villi are slightly blunted. Magnification 10x. C– Superficial mucosa of TV-inoculated macaque contains macrophages, plasma cells and scattered eosinophils. Magnification 40x. D– The brush border (green lining = villi) was preserved in TV-inoculated macaques. Purple blue cells indicate underlying subepithelium (TG2 is a digestive enzyme).</p

Rhesus macaques and enteric caliciviruses used for experimental inoculation.

a<p>Tulane Virus i.e. rhesus enteric CV; ^bHuman NoVs GenBank #s: JQ320072 and JQ320073; ^cBacteria-free filtrate of 20% stool suspension in PBS. TV and NoV-inoculated animals were kept separated in devoted BSL2 rooms. The total dosage of TV was 10⁶ viral RNA copies per animal.</p