IMMUNITY AND PATHOGENESIS OF A NOVEL NOROVIRUS

描述（由申请人提供）：这是一项A1（-06 149年=13.5%）竞争性更新，旨在获得题为“新型诺如病毒的免疫力和发病机制”的R 01 AI 054883资助。诺如病毒（Noroviruses，NoV，B类生物防御因子）是世界范围内非细菌性流行性胃肠炎的主要病原体，造成严重的疾病、死亡和经济损失。在美国，80%至96%的非细菌性流行性胃肠炎病例归因于NoV。人NoV不易培养，不会感染小动物。目前还没有NoV疫苗，尽管对当NoV衣壳蛋白过度表达时产生的病毒样颗粒的免疫应答的研究已经有了重要的见解。我们在2003年鉴定了鼠NoV（MNV）1，并且在第一个资助期内，在将MNV感染发展成用于理解NoV复制、免疫和发病机制的方面的稳健的仅感染实验模型方面取得了重大进展。我们于2004年开发了第一个NoV培养系统2，并于2007年开发了第一个与培养系统耦合的NoV反向遗传学系统3。发现MNV感染了相当大比例的实验室小鼠，需要大量投资开发封闭的小鼠繁殖群以产生非免疫小鼠，用其进行免疫学和致病性实验。进一步的工作导致了21个新菌株的分离和全测序，以及T=3病毒粒子和由MNV衣壳蛋白组装的迷人的较小T=1颗粒的冷冻EM结构的解决方案。最重要的是，我们在这里表明，它是可能的，有效地接种疫苗抗MNV，MNV从肠道的清除涉及B细胞，抗体，CD 4 T细胞，CD 8 T细胞，和穿孔素。有趣的是，某些MNV毒株在WT小鼠的肠道中持续复制，并且尽管有显著的免疫应答，但仍在粪便中脱落数周。反向遗传学、疫苗接种模型、小鼠遗传学以及持久性和非持久性MNV毒株的组合将使我们能够通过以下特定目的来定义MNV免疫的分子和免疫学机制：目的1。确定MNV疫苗接种的机制。目标2.确定粪便中MNV持续脱落的机制。目标3.检验T=1型MNV的产生是一种免疫逃避策略的假设。1. Karst等人，STAT1-dependent innate immunity to a Norwalk-like virus. 2003.科学299：1575。2. Wobus等人，诺如病毒在细胞培养物中的复制揭示了对树突状细胞和巨噬细胞的向性。2004. PLOS Biology. E432 3. Ward等人，使用polII驱动的全长cDNA表达回收感染性鼠诺如病毒。2007. PNAS. 6月20日（Epub ahead of print）项目简介：诺如病毒是世界上90%以上流行性胃肠炎的罪魁祸首，造成了难以估量的危害。我们已经开发了一种诺如病毒感染的小鼠模型，并首次证明我们可以接种疫苗对抗口服诺如病毒感染。此外，我们已经建立了分子方法，使我们能够绘制负责重要生物表型的病毒基因。在这项资助中，我们建议定义负责诺如病毒免疫和疫苗接种的机制，确定尽管有积极的免疫反应，但病毒仍能持续存在的机制，并定义诺如病毒感染细胞中产生的新型病毒颗粒在感染中的作用，我们假设该病毒颗粒可能是逃避免疫的病毒工具。

DESCRIPTION (provided by applicant): This is an A1 (year -06 149=13.5%) competing renewal for grant R01 AI054883 entitled "Immunity and Pathogenesis of a Novel Norovirus". Noroviruses (NoV, Category B Biodefense agents) are the major etiologic agent of nonbacterial, epidemic gastroenteritis worldwide, causing significant suffering, morbidity, and economic loss. Eighty to 96% of all cases of non-bacterial epidemic gastroenteritis in the US are attributed to NoV. Human NoV are not readily cultured and do not infect small animals. There is no NoV vaccine, although significant insight has come from studies of the immune response to virus-like-particles generated when NoV capsid proteins are over expressed. We identified murine NoV (MNV) in 20031, and during the first grant period made significant strides towards developing MNV infection into a robust infection-only experimental model for understanding aspects of NoV replication, immunity, and pathogenesis. We developed the first NoV culture system in 20042, and the first NoV reverse genetics system coupled to a culture system in 20073. MNV was found to infect a significant proportion of laboratory mice, requiring a significant investment in developing a closed mouse breeding colony to generate non-immune mice with which to perform immunology and pathogenesis experiments. Further work has led to isolation and full sequencing of 21 new strains, and the solution of the cryo-EM structure of both the T=3 virion and a fascinating smaller T=1 particle assembled from the MNV capsid protein. Of fundamental importance, we show here that it is possible to effectively vaccinate against MNV, and that clearance of MNV from intestine involves B cells, antibody, CD4 T cells, CD8 T cells, and perforin. Interestingly, certain MNV strains persistently replicate in intestine of WT mice and are shed in feces for weeks despite a significant immune response. The combination of reverse genetics, a vaccination model, mouse genetics, and persistent and non-persistent MNV strains will allow us to define molecular and immunologic mechanisms of MNV immunity through the following Specific Aims: Aim 1. Determine the mechanism(s) responsible for vaccination against MNV. Aim 2. Determine the mechanism(s) responsible for persistent shedding of MNV in feces. Aim 3. Test the hypothesis that production of the T=1 form of MNV is an immune evasion strategy. 1. Karst, et al. STAT1-dependent innate immunity to a Norwalk-like virus. 2003. Science. 299:1575. 2. Wobus et al., Replication of a Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. 2004. PLOS Biology. E432. 3. Ward, et al., Recovery of infectious murine norovirus using polII-driven expression of full-length cDNA. 2007. PNAS. June 20 [Epub ahead of print]. PROJECT NARRATIVE: Noroviruses are responsible for more than 90% of the epidemic gastroenteritis in the world, causing untold harm. We have developed a mouse model for norovirus infection, and have shown for the first time that we can vaccinate against oral norovirus infection. In addition we have set up molecular methods that will allow us to map viral genes responsible for important biological phenotypes. In this grant we propose to define the mechanisms responsible for norovirus immunity and vaccination, determine the mechanisms that allow the virus to persist despite an active immune response, and define the role in infection of a novel viral particle made in norovirus infected cells that we hypothesize may be a viral tool to evade immunity.