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

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

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

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

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

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

Antibodies used for immunofluorescent staining of duodenum biopsy tissues.

a<p>Tulane National Primate Research Center,^bnot applicable,^cFluorescein-Iso-Thio-Cyanate, ^dDigestive enzyme.</p

TV-antigen containing lymphocyte populations in cultured, TV-inoculated PBMCs.

<p>A & B– One day-old cultures of TV-inoculated PBMCs show the trend (p>0.05) for TV antigen expression by CD20+HLA-DR+ B cells. C & D– Six days-old cultures show significant presence of TV antigens predominantly inside the CD20+HLA-DR+ cells (p<0.05). Several other T and B cell subsets also contain TV while no virus was detected in negative control, mock-inoculated PBMCs. Asterisks indicate significant differences between the TV-inoculated and control cells.</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