Determinants of Enteric Calicivirus Infection.

项目摘要 人类诺如病毒（HuNoV）胃肠炎是全球范围内的重大公共卫生负担。缺乏一个强大的 可重复的HuNoV细胞培养系统仍然限制了我们研究HuNoV基本方面的能力 感染虽然最近的几个突破增加了我们在体外增殖HuNoV（例如B细胞）的能力 和人肠类培养物），这些系统不够稳健，可能是耗时和昂贵的，或者 很难复制。可用于HuNoV研究的替代模型不一定反映基本的 HuNoV及其天然宿主的生物学特征。在这项提案中，我们将使用我们的新恒河猴肠 杯状病毒（ReCV）模型，反映了HuNoV的生物学特征和多样性，以识别宿主 感染的决定因素。ReCV和HuNoV在人与非人之间的动物源性/种间传播 人类灵长类宿主表明，在进化过程中， 两个属。在这里，我们试图使用CRISPR-Cas9鉴定肠道杯状病毒感染的宿主决定因素。 全基因组筛选我们最近发现了一种功能性ReCV进入受体， 在细胞培养中的宽容。我们假设ReCV进入受体也参与了HuNoV 感染.这一假设将在以下具体目标中得到检验。目标1。表征受体介导 ReCV感染。目的2：肠样细胞和B细胞中HuNoV和ReCV感染的比较表征 cultures.在第一个目标中，我们将通过使用ReCV来剖析HBGAs在ReCV受体介导的进入中的作用。 具有不同HBGA结合的分离株，表达不同HBGA和/或受体的细胞系，肠杆菌属 SENG-6 EPS和不同的合成HBGA。我们还将评估不同跨膜蛋白的作用。 研究感染中受体的亚型，并绘制ReCV相互作用位点和糖基化的重要性。在 第二个目的是研究ReCV进入受体在HuNoV感染肠道和B细胞中的作用 培养和鉴定在HuNoV感染中起作用的其他细胞表面组分。我们还将评估 胆汁或胆汁盐促进HuNoV感染的机制以及M细胞在感染中的作用。 极化细胞我们的长期目标是鉴定和表征ReCV的病毒和宿主决定因素， HuNoV感染，并开发新的干预/预防策略。我们的建议使用一种新型的肠溶 杯状病毒模型，以了解病毒进入以及胆汁和HBGAs在肠道病毒感染中的作用。我们 ReCV的发现可能直接转移到HuNoV感染。本项目的主要意义在于 确定肠道杯状病毒感染易感性的决定因素， HuNoV细胞培养系统和新的干预/预防策略。

PROJECT SUMMARY Human norovirus (HuNoV) gastroenteritis is a significant public health burden worldwide. The lack of a robust and reproducible HuNoV cell culture system still limits our ability to study fundamental aspects of HuNoV infection. While several recent breakthroughs increased our ability to propagate HuNoVs in vitro (e.g. B cell and human enteroid cultures), these systems are not robust enough, can be time consuming and expensive or hard to replicate. The surrogate models available for HuNoV research do not necessarily reflect essential biological features of HuNoVs and their natural host. In this proposal we will use our novel rhesus enteric caliciviruses (ReCV) model that reflects the biological features and diversity of HuNoVs to identify host determinants of infection. Zoonotic/interspecies transmission of ReCVs and HuNoVs between human and non- human primate hosts suggests evolutionary conservation of shared factors of host susceptibility between the two genera. Here we seek to identify host determinants of enteric calicivirus infection using CRISPR-Cas9 genome wide screening. We recently identified a functional ReCV entry receptor that is necessary for ReCV permissiveness in cell culture. We hypothesize that the ReCV entry receptor is also involved in HuNoV infections. This hypothesis will be tested in the following specific aims. Aim 1. Characterize receptor mediated ReCV infection. Aim 2: Comparative characterization of HuNoV and ReCV infections in enteroid and B cell cultures. In the first aim we will dissect the role of HBGAs in ReCV receptor mediated entry by using ReCV isolates with disctinct HBGA binding, cell lines expressing different HBGAs and/or the receptor, Enterobacter SENG-6 EPS and different synthetic HBGAs. We will also evaluate the role of the different transmembrane isoforms of the receptor in infection and map the ReCV interaction site and importance of glycosylation. In the second aim, we will evaluate the role of ReCV entry receptor in HuNoV infections in enteroid and B cell cultures and identify other cell surface components playing a role in HuNoV infections. We will also evaluate the mechanism of bile or bile salts in promoting HuNoV infections and the role of M cells in infections of polarized cells. Our long-term goal is to identify and characterize viral and host determinants of ReCV and HuNoV infections and to develop novel intervention/prevention strategies. Our proposal uses a novel enteric calicivirus model to understand viral entry and the role of bile and HBGAs in enteric viral infections. Our findings with ReCVs may be directly transferable to HuNoV infection. The major significance of this project is the identification of determinants of susceptibility to enteric calicivirus infection that can lead to improved HuNoV cell culture systems and new intervention/prevention strategies.