Norovirus Expression and Replication Systems to Enhance Viral Detection and Devel

本计划项目2的重点是继续探索培养人类杯状病毒的方法，并了解限制病毒基因表达、复制和细胞系统生长的分子基础。虽然人类诺如病毒作为全球食源性和水媒疾病的最重要原因的临床和经济意义现在已被认识到，但对影响环境中活病毒的持久性和传播的因素以及致病和免疫的潜在机制知之甚少。这种缺乏了解主要是由于缺乏可用的体外培养或感染的动物模型。我们的长期目标是利用上一个项目期间的基本发现，可以测量体外感染性和新的体外细胞培养系统的胃肠上皮。在具体目标1中，我们将改进和开发有效的体外培养诺瓦克病毒和其他诺如病毒的方法。这方面的研究将确定RNA转染细胞中病毒传播的限制是否是由于缺乏共受体。这些研究还将开拓新的思路，以获得和维持原代人肠上皮细胞的培养。在具体目标2中，我们建议了解诺如病毒蛋白表达调节细胞先天反应的分子机制，以及这些细胞反应是否调节病毒复制和传播。在具体目标3中，我们建议使用基因表达和复制系统来剖析VP1，VP2和VPg相互作用以及与基因组RNA相互作用以进行RNA转录的机制。这些研究的结果将产生关于诺如病毒复制的新知识，并将补充和允许对项目1和3中开发的病毒检测方法以及抗病毒方法进行功能评估。

The focus of Project 2 of this Program Project is to continue to pursue approaches to cultivated human caliciviruses and to understand the molecular basis of restriction of virus gene expression, replication and growth in cell systems. Although the clinical and economic significance of the human noroviruses as the most important cause of global foodborne and waterborne illness is now recognized, factors affecting the persistence and transmission of viable virus in the environment and the underlying mechanisms of pathogenesis and immunity are poorly understood. This lack of understanding is largely due to the lack of available in vitro cultivation or animal models of infection. Our long-term goal is to exploit fundamental discoveries made in the previous project period that can measure in vitro infectivity and new in vitro cell culture systems for gastrointestinal epithelium. In Specific Aim 1, we will improve and develop efficient in vitro methods for cultivation of Norwalk virus and other noroviruses. Studies in this aim will determine if the restriction of viral spread in RNA-transfected cells is due to the lack of a co-receptor. These studies will also exploit new ideas to obtain and maintain primary human intestinal epithelial cells in culture. In Specific Aim 2, we propose to understand the molecular mechanisms by which norovirus protein expression regulates cellular innate responses as well as whether these cellular responses regulate viral replication and spread. In Specific Aim 3, we propose to use gene expression and replication systems to dissect the mechanisms by which VP1, VP2, and VPg interact with each other and with genomic RNA for RNA encapsidation. The results from these studies will generate new knowledge about the replication of noroviruses and will complement and allow functional assessment of methods of virus detection as well as of antiviral that are developed in Projects 1 and 3.