Development of a human norovirus cell culture model

 描述(申请人提供)：人类诺如病毒(HuNov)是全球非细菌性胃肠炎的主要原因，导致显著的死亡率、发病率和经济损失。 成本。然而，目前还没有针对HuNoV的有效疫苗或抗病毒药物。这在很大程度上是由于无法在实验室培养HuNov。由于缺乏HuNoV细胞培养模型，科学家无法研究HuNoV生物学的基本方面，无法确定治疗靶点，也扼杀了快速识别抗病毒候选药物的能力。因此，迫切需要开发一种可重复使用的组织培养体系。长期目标是阐明HuNoV与宿主相互作用的机制，并开发有效的HuNoV疾病预防和控制策略。该项目的目标是确定一种在培养中可重复支持人新城疫病毒感染的细胞类型。中心假设是HuNov单独或与肠道上皮细胞共同培养感染肠道免疫细胞。这 假设是基于我们在一种新开发的HuNoV小鼠模型中的数据，该模型表明HuNoV感染巨噬细胞。此外，其他人的工作表明，HuNoV感染黑猩猩肠道固有层中的B细胞和树突状细胞，HuNoV病毒样颗粒结合 人十二指肠活检标本的肠道固有层细胞和组织切片的HuNoV抗原染色呈阳性。长期以来，人们一直认为HuNoV感染肠上皮细胞，但肠道免疫细胞的感染将为在肠上皮细胞培养HuNoV失败提供解释。这一假说将通过追求两个特定目标来验证：1)确定HuNoV是否在人类肠道免疫细胞中复制；2)确定HuNoV与3D原代人类肠道微型器官共培养后是否在免疫细胞中复制。在第一个目标中，我们将直接感染来自人类小肠组织的肠道免疫细胞，来自人类多能干细胞的巨噬细胞或树突状细胞，以及建立的人类免疫细胞系。在第二个目标中，原代和干细胞来源的免疫细胞将在感染前首先与人类肠道微型器官共培养。这种方法 在概念上是创新的，因为这项研究测试了一个范式转换假说，即HuNov在肠道的免疫细胞而不是肠细胞中复制。此外，人类原代免疫细胞/上皮共培养的发展代表了人类肠道微型器官系统的技术创新，将为研究肠道感染提供一个新的模型。这项拟议的研究具有进一步的重要意义，因为它有望导致建立一种可重复使用的HuNov组织培养模型。这样的发展将克服该领域的一大障碍，过去40年来，这一障碍一直阻碍着胡诺夫病毒的研究。最终，这样的技术突破将为未来在基础和翻译层面上的HuNoV研究提供基础。

 DESCRIPTION (provided by applicant): Human noroviruses (HuNoVs) are the major cause of non-bacterial gastroenteritis worldwide, resulting in significant mortality, morbidity and economic cost. However, no effective vaccines or antivirals exist for HuNoV. This is in large part due to the inability to culture HuNoV in the laboratory. This lack of a HuNoV cell culture model has prevented scientists from studying fundamental aspects of HuNoV biology, identifying therapeutic targets, and it has stifled the ability to rapidly identify antiviral drug candidates. hus, there is a critical need to develop a reproducible tissue culture system for HuNoV. The long-term goal is to elucidate mechanisms of HuNoV-host interactions and develop effective HuNoV disease prevention and control strategies. The objective of this project is to determine a cell type that reproducibly supports HuNoV infection in culture. The central hypothesis is that HuNoVs infect intestinal immune cells alone or in co-culture with intestinal epithelial cells. This hypothesis is based on our data in a newly developed HuNoV mouse model demonstrating that HuNoV infects macrophages. In addition, work by others shows that HuNoV infects B cells and dendritic cells in the intestinal lamina propria of chimpanzees, HuNoV virus-like particles bind to intestinal lamina propria cells, and tissue sections of human duodenal biopsy specimens stain positive for HuNoV antigen. It has long been presumed that HuNoV infects intestinal epithelial cells, but infection of intestinal immune cells would provide an explanation for the failed attempt to culture HuNoV in epithelial cells. The hypothesis will be tested by pursuing two specific aims: 1) Determine whether HuNoV replicates in human intestinal immune cells, and 2) Determine whether HuNoV replicates in immune cells following co-culture with 3D primary human intestinal mini-organs. In the first aim, we will directly infect intestinal immune cells from human small intestinal tissue, macrophages or dendritic cells derived from human pluripotent stem cells and established human immune cell lines. In the second aim, primary and stem cell-derived immune cells will first be co-cultured with human intestinal mini-organs prior to infection. This approach is innovative conceptually because the research tests a paradigm-shifting hypothesis, i.e., that HuNoVs replicate in immune cells instead of enterocytes in the intestine. In addition, the development of primary human immune cell/epithelial co-cultures represents a technical innovation of the human intestinal mini-organ system and will provide a new model for studying enteric infections. The proposed research is further significant because it is expected to result i the development of a reproducible tissue culture model for HuNoVs. Such a development would overcome a major barrier in the field, which has hampered HuNoV research for the past 40 years. Ultimately, such technical break-through will provide the basis for future advances in HuNoV research both at the basic and translational level.