Norovirus Expression and Replication Systems to Enhance Viral Detection and Devel

本计划项目2的重点是限制寻求培养人的途径 目的：了解限制病毒基因表达、复制和转录的分子基础 细胞系统中的生长。尽管作为人类诺如病毒的临床和经济意义 全球食源性和水源性疾病的最重要原因现已被认识到，影响 活病毒在环境中的持续和传播及其潜在机制 发病机制和免疫机制知之甚少。这种理解的缺乏在很大程度上是由于缺乏 可用于体外培养或动物感染模型。我们的长期目标是利用根本 前一个项目期的发现可以测量体外感染性和新的体外细胞 胃肠末梢上皮的培养系统。在具体目标1中，我们将改进和发展高效的 诺瓦克病毒和其他诺沃克病毒的体外培养方法。这一目标的研究将确定是否 限制病毒在RNA转基因细胞中的传播是由于缺乏辅助受体。这些研究还将 开发新的想法来获得和维持原代培养的人类最终上皮细胞。以特定的目标 2、我们提出了解诺如病毒蛋白表达调控的分子机制。 细胞的先天反应以及这些细胞反应是否调节病毒的复制和传播。 在特定的目标3中，我们建议使用基因表达和复制系统来剖析这些机制 其中Vp1、VP2和Vpg相互作用并与基因组RNA相互作用以进行RNA封装。这个 这些研究的结果将产生关于诺如病毒复制的新知识，并将 补充并允许对病毒检测方法以及下列抗病毒药物进行功能性评估 在项目1和3中开发。

The focus of Project 2 of this Program Project is to confinue to pursue approaches to culfivate human caliciviruses and to understand the molecular basis of restricfion of virus gene expression, replicafion 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 culfivafion or animal models of infecfion. 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 gastrointesfinal epithelium. In Specific Aim 1, we will improve and develop efficient in vitro methods for culfivafion 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 intesfinal 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 replicafion and spread. In Specific Aim 3, we propose to use gene expression and replicafion systems to dissect the mechanisms by which VPl, 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 replicafion of noroviruses and will complement and allow funcfional assessment of methods of virus detection as well as of antivirals that are developed in Projects 1 and 3.