DEVELOPMENT OF Q PCR ASSAY FOR DETECTION OF ENTERIC CALICIVIRUSES

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 表示子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 最近，我们报道了一种可培养杯状病毒（杜兰病毒; TV）分离自恒河猴的发现。基于初步结果显示TV和诺如病毒（NV）在其遗传多样性、流行病学和组织血型抗原（HBGA）结合方面具有密切的相似性，加上内部反向遗传学系统的可用性，我们正在寻求开发基于TV的非人灵长类动物替代模型用于人NV胃肠炎。为了支持本研究中的这一努力，我们开发了qRT-PCR，目的是通过恒河猴的受控感染过程来计数生物样品中的TV RNA负载。用TV对未成年恒河猴进行血清学预筛选，诱导实验感染。用恒河猴和人来源的NoV接种另外的猕猴。虽然TV接种的动物发生了以病毒脱落、发热和腹泻为特征的感染，但在NoV接种的猕猴中仅观察到无症状感染。在TV接种动物中观察到病毒中和血清抗体的显着增加。在接种后第三天获得的十二指肠活检中观察到TV抗原的存在。这些结果表明，TV杯状病毒可用于肠道杯状病毒的致病性和免疫性研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Recently we reported the discovery of a cultivable calicivirus (Tulane virus; TV) isolated from a rhesus macaque. Based on preliminary results that show close similarities between TVs and noroviruses (NVs) in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding, plus the availability of in-house reverse genetics system, we are pursuing the development of a TV-based non-human primate surrogate model for human NV gastroenteritis. To support this effort in this study, we developed a qRT-PCR with the objective to enumerate TV RNA load in biological samples through the course of controlled infections of rhesus macaques. A TV was used to inoculate serologically pre-screened juvenile rhesus macaques to induce experimental infection. Additional macaques were inoculated with rhesus- and human-derived NoVs. While TV-inoculated animals developed infection characterized by virus shedding, fever and diarrhea, only asymptomatic infection was noted in NoV-inoculated macaques. A remarkable increase in virus-neutralizing serum antibodies was observed in TV-inoculated animals. Presence of TV antigens was visualized in duodenum biopsies obtained at post-inoculation day three. These results suggest that TV calicivirus might be used to study the pathogenesis and immunity of enteric caliciviruses.