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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 最近，我们报道了从恒河猴身上分离到的一种可培养杯状病毒(杜兰病毒；TV)。基于初步结果显示TV和诺如病毒(NVS)在遗传多样性、流行病学和组织血型抗原(HBGA)结合方面非常相似，加上内部反向遗传学系统的可用性，我们正在开发一种基于TV的人类NV胃肠炎的非人类灵长类代理模型。为了在这项研究中支持这一努力，我们建立了一种定量RT-PCR方法，目的是通过恒河猴的受控感染过程来计数生物样本中的TV RNA载量。用电视对经过血清学筛选的幼年恒河猴进行接种，诱导其实验性感染。更多的猕猴被接种了恒河猴和人类来源的Nov。接种TV的动物出现以脱毒、发热和腹泻为特征的感染，而接种NOV的猕猴仅出现无症状感染。病毒中和血清抗体在接种TV的动物中显著升高。在接种后第三天的十二指肠活检组织中可见TV抗原的存在。提示TV杯状病毒可用于研究肠道杯状病毒的致病机制和免疫功能。

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.