Using apathogenic and mammalian-ubiquitous reoviruses to safely discover genetic and phenotypic changes in viruses that promote adaptation to new host species and distinct niches within a host.

病毒不断进入新的宿主，它们是否茁壮成长取决于它们在抑制抗病毒宿主反应的同时完成所有复制步骤的能力。了解遗传和表型变化如何促进病毒适应新的生态位是很重要的。哺乳动物正呼肠孤病毒（MRV）是研究适应的完美病毒，因为它感染各种各样的哺乳动物而不引起疾病。MRV自然感染肠上皮；然而，我们对MRV的大部分理解来自于70年来适应非肠细胞的病毒，如L929致瘤小鼠成纤维细胞。使用来自初级废水的4种“野生”mrv，我们发现实验室适应mrv的知识不一致。例如，据说MRV在细胞内经历由溶酶体蛋白酶介导的必要的蛋白水解衣壳剥膜步骤。我们发现野生mrv在细胞内很难脱壳，并且在非肠细胞培养中其他尚未确定的复制步骤中表现出缺陷。呼肠孤病毒在L929细胞上传代5次并分离出单个克隆亚分离株后，呼肠孤病毒外衣壳蛋白s3的多态性使细胞内脱壳成为可能。本研究旨在进一步应用MRV作为模型系统，以更好地了解病毒对来自不同组织和哺乳动物来源的细胞的适应性。目的1将建立MRV的真正分子特征。我们将评估MRV复制的所有主要步骤，分别是实验室适应MRV与野生MRV，以及L929细胞与来自猪、牛和大鼠的正常极化肠上皮细胞。鉴于MRV在肠道感染过程中自然遇到消化蛋白酶，我们还将建立细胞外MRV脱膜的真实动力学和对不同哺乳动物蛋白酶的敏感性。考虑到即使是实验室适应的MRV菌株在诱导宿主IFN和NfkB信号通路的能力上也存在很大差异，我们还将在不同宿主细胞培养的背景下建立宿主对野生MRV的真正反应。最后，我们将推导出MRV在肠细胞中复制的数学模型，该模型可用于表征宿主和病毒变体之间的MRV适应性。AIM 2将发现mrv适应的途径。利用适应细胞内脱壳的野生mrv的克隆性亚分离株，我们现在将确定s3中的哪些多态性能够实现细胞内脱壳，不同的野生mrv对细胞培养的适应途径是否相同或不同，s3多态性是否存在于野生准种中或在传代中产生，s3多态性与其他s3功能（如细胞信号传导）之间的关系。以及其他MRV基因中出现的多态性促进了适应的非剥膜步骤。我们还将在不同的肠道细胞培养物上传递野生mrv，并应用我们的克隆分离方法来发现在适应不同哺乳动物物种的肠道培养物过程中产生的多态性。

Viruses are continuously entering new hosts, and whether they thrive depends on their ability to complete all steps of replication while subduing anti-viral host responses. It is important to understand how genetic and phenotypic changes promote virus adaptation to new niches. Mammalian orthoreovirus (MRV) is a perfect virus to study adaptation since it infects a wide assortment of mammals without causing disease. MRV naturally infects intestinal epithelia; yet most of our understanding of MRV comes from viruses adapted for 70 years to non-enteric cells such as the L929 tumorigenic mouse fibroblasts. Using 4 "wild" MRVs from primary effluent, we discovered inconsistencies in knowledge from lab-adapted MRVs. For example, MRV are said to undergo the essential proteolytic capsid uncoating step within cells, mediated by lysosomal proteases. We discovered that wild MRVs poorly uncoat intracellularly and exhibit deficiencies in other yet-to-be identified replication steps in non-enteric cell cultures. After 5 passages on L929 cells and isolation of single clonogenic sub-isolates, polymorphisms in the reovirus outercapsid protein s3 enable intracellular uncoating. This proposal seeks to further apply MRV as a model system to better understand virus adaptation to cells from distinct tissues and mammalian sources. Aim 1 will establish the true molecular features of MRV. We will evaluate all major steps of MRV replication for lab-adapted versus wild MRVs, and in L929 cells versus normal polarized intestinal epithelial cells from porcine, bovine and rat origins. Given MRV naturally encounters digestive proteases during enteric infection, we will also establish the true kinetics of extracellular MRV uncoating and sensitivity to proteases from distinct mammals. Given that even lab-adapted MRV strains differ substantially in their ability to induce host IFN and NfkB signaling pathways, we will also establish the true host response to wild MRVs in the context of different host cell cultures. Finally, we will derive a mathematical model for replication of MRVs in intestinal cells that can be applied to characterize MRV fitness between hosts and virus variants. AIM 2 will discover the path(s) to adaptation of MRVs. Using the clonogenic sub-isolates of wild MRVs that adapted to uncoat intracellularly, we will now determine which polymorphisms in s3 enable intracellular uncoating, whether different wild MRVs take the same or different paths of adaptation to cell culture, whether the s3 polymorphisms were present in the wild quasispecies or arose in passage, what relationship s3 polymorphisms have on other s3 functions such as cell signaling, and what polymorphisms in other MRV genes arise to promote non-uncoating steps of adaptation. We will also passage wild MRVs on distinct intestinal cell cultures and apply our clonogenic isolation approach to discover what polymorphisms arise during adaptation to intestinal cultures from distinct mammalian species.