Rotavirus: Studies of Intestinal Tropism, Viral Entry, and Rescue

描述（由申请人提供）： 轮状病毒（RV）是全球婴幼儿严重腹泻的唯一最重要原因。这些病毒也是健康成人、老年人和免疫功能低下者的常见病因。事实上，所有哺乳动物物种都能被其自身的同源宿主物种RV有效感染，但不能被异源RV感染。最近，有效的减毒活RV疫苗已上市，但其作用机制和减毒的分子基础尚不清楚。RV感染主要局限于小肠的绒毛上皮。值得注意的是，肠细胞中异源RV复制的宿主范围限制的基础仍然未知。由于其相对较小的基因组大小、宿主范围和组织嗜性，轮状病毒是研究宿主上皮和肠道病原体之间相互作用的高度易处理的模型系统。RV也是靶向人小肠上皮的潜在载体和评估针对肠道病原体的疫苗接种策略的理想系统。我们的目标是继续研究RV与宿主的相互作用，以更好地了解肠道微生物的发病机制。该提案的具体目的是：1）在鼠模型中使用病毒抑制剂表征选定的同源和异源RV的宿主限制性肠细胞嗜性的遗传基础。这是我们的假设，RV表面蛋白（VP4），它介导的细胞进入和RV IRF3拮抗剂蛋白，NSP1，它阻断宿主干扰素反应，是负责确定宿主限制性肠道复制的表型。2)在极化上皮的体外模型中表征RV细胞进入的早期事件，并确定介导细胞进入的VP 4和VP 7的构象。目前尚不清楚大的，无包膜的二十面体RV颗粒如何进入极化肠上皮细胞的细胞质。我们将使用高度特异性的单克隆抗体，共聚焦显微镜和细胞生物学技术的组合来阐明这一过程。细胞进入事件导致不同同源和异源RV株的不同肠细胞嗜性。我们推测，进入过程的特点是两个RV表面蛋白的结构和细胞定位的关键变化。3)开发一个易于操作的反向遗传学系统来操纵具有复制能力的轮状病毒的基因组。为了将RV发病机制和细胞进入研究提升到下一个水平，需要直接修饰RV双链、分段RNA基因组的方法。这样的系统也将使我们和其他研究人员能够利用RV的精密肠向性来设计用于其他传染病的独特靶向载体。我们的假设是，我们可以通过利用最近验证的呼肠孤病毒和环状病毒的方法来实现这一目标。 公共卫生相关性： 7.一般的急性肠道感染，特别是轮状病毒感染，是儿童和成人，尤其是老年人和免疫功能低下者发病率相当高的原因。它们也是我们的武装部队成员和退伍军人患病的重要原因。此外，了解感染胃肠道的病原体与宿主之间相互作用的性质，对我们促进更好的生物防御策略的努力具有重要意义。这里提出的研究将产生更好的理解和增加的知识的微生物致病原因在人类肠道疾病。

DESCRIPTION (provided by applicant): Rotaviruses (RV) are the single most important cause of severe diarrhea in infants and young children worldwide. These viruses are also common causes of disease in healthy adults, the elderly and the immunocompromised. Virtually all mammalian species are efficiently infected with their own homologous host- species RV but not by heterologous RV. Recently, effective, live attenuated RV vaccines became commercially available, but their mechanisms of action and molecular basis for attenuation are not understood. Infection with RV is predominantly restricted to the villous epithelium of the small intestine. Of note, the basis for host-range restriction of heterologous RV replication in intestinal cells remains unknown. Due to their relatively small genomic size, host-range and tissue tropism, rotaviruses are a highly tractable model system for study of the interaction between host epithelium and enteric pathogens. RVs are also potential vectors for targeting the human small bowel epithelium and an ideal system for assessing vaccination strategies against enteric pathogens. It is our objective to continue studies of RV-host interactions with the goal of better understanding enteric microbial pathogenesis. The specific aims of the proposal are to: 1) Characterize the genetic basis for host-restricted intestinal cell tropism of selected homologous and heterologous RVs using viral reassortants in a murine model. It is our hypothesis that one of the RV surface proteins (VP4), which mediates cell entry and the RV IRF3 antagonist protein, NSP1, which blocks the host interferon response, are responsible for determining the phenotype of host restricted intestinal replication. 2) Characterize early events in RV cell entry in an in vitro model of polarized epithelium and determine the conformations of VP4 and VP7 that mediate cell entry. It is not clear how the large, non-enveloped icosahedral RV particle enters the cytoplasm of a polarized enterocyte. We will use a combination of highly specific monoclonal antibodies, confocal microscopy and cell biologic techniques to illuminate this process. Cell entry events contribute to the varying intestinal cell tropisms of different homologous and heterologous RV strains. We hypothesize that the entry process is characterized by critical changes in the structure and cellular localization of the two RV surface proteins. 3) Develop a tractable reverse-genetics system to manipulate the genome of a replication competent rotavirus. In order to take RV pathogenesis and cell entry studies to the next level, methodologies to directly modify the RV double-stranded, segmented RNA genome are needed. Such a system will also potentially enable us and other investigators to utilize the exquisite entero-tropism of RVs to design unique targeting vectors for other infectious diseases. It is our hypothesis that we can accomplish this aim by utilizing approaches recently validated for reovirus and orbiviruses. PUBLIC HEALTH RELEVANCE: 7. Project Narrative Acute enteric infections in general and rotavirus infections in particular are a cause of considerable morbidity both in children and in adults, especially the elderly and immunocompromised. They are also important causes of morbidly for members of our armed services and our veteran population. In addition, understanding the nature of the interaction between pathogens that infect the gastrointestinal tract and the host has substantial relevance to our efforts to promote better biodefense strategies. The studies proposed here will yield better understanding and increased knowledge of the microbial pathogeneses that cause enteric diseases in man.