Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

Karol Sestak,Stephanie Feely,Jan Vinjé,Bapi Pahar,Nicole Gregoricus,Tibor Farkas,Xavier Alvarez,Jason Dufour,Brittney Fey,Edwin Hargitt,D William Cameron

doi:10.1371/journal.pone.0037973

Abstract

BackgroundTissue 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 & FindingsTwo 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.Conclusions/SignificanceTaken together, our results show that ReCVs represent an alternative cell culture and animal model to study enteric calicivirus replication, pathogenesis and immunity.

Highlights

Caliciviruses (CV) are small, non-enveloped, icosahedral viruses with a,7.5–8.5 kb positive sense, single stranded, polyadenylated RNA genome
Human NoVs are a common cause of epidemic and sporadic acute gastroenteritis worldwide [4,32]
The prototype Norwalk virus was discovered in 1972 [33], NoV research and the development of prevention strategies have since been hampered by the lack of robust cell culture system and animal model that closely mimics the clinical symptoms of NoV gastroenteritis in humans

Summary

Introduction

Caliciviruses (CV) are small, non-enveloped, icosahedral viruses with a ,7.5–8.5 kb positive sense, single stranded, polyadenylated RNA genome. The Caliciviridae family consists of five established genera, Norovirus, Sapovirus, Lagovirus, Vesivirus and Nebovirus. Rhesus enteric CVs (Recovirus), St. Valerian-like viruses (Valovirus) and chicken CVs represent three additional, yet unassigned CV genera [1,2,3]. Noroviruses (NoV) are important etiologic agents of acute gastroenteritis in humans and account for the majority of nonbacterial gastroenteritis outbreaks as well as .50% of all food-related gastroenteritis outbreaks [4,5]. NoVs can be subdivided into five genogroups (GI-V) and at least 33 genotypes [6,7]. No robust cell culture or in vivo model exists to study human NoVs. Tissue culture-adapted Tulane virus (TV), a GI. rhesus enteric calicivirus (ReCV), and a mixture of GII. and GII. 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

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