Mechanisms of antiviral immunity and tolerance in the intestinal epithelium of Jamaican Fruit Bats

总结 胃肠道（GI）是蝙蝠病毒感染的主要靶器官，但感染很少引起 病毒性肠炎的典型症状，如腹泻。我们项目的长期目标是了解 肠上皮细胞的特异性先天免疫反应机制，使蝙蝠能够维持病毒 感染而不经历细胞病变效应和肠屏障功能障碍。我们假设 牙买加果蝠（Artibeus jamaicensis，JFBs）的I型和III型干扰素（IFN）保护性应答 使胃肠道上皮持续无症状的病毒感染。为了验证我们的假设，我们将 利用一种新的JFB肠体外模型，3-D肠，这是一种复杂的长期培养物， 由成体组织来源的干细胞产生的原代肠上皮细胞。肠道反应 将JFB的上皮与在人类肠中诱导的上皮进行比较。在目标1中，我们将定义I/III型IFN JFB类肠体对病毒感染的反应。在目标2中，我们将确定I/III型IFN影响的程度 病毒复制和JFB胃肠上皮屏障功能。我们将分析肠道 上皮细胞对可感染JFB细胞的三种单链RNA病毒的反应：H18 N11流感病毒 病毒，严重急性呼吸道综合征冠状病毒2（SARS-CoV-2），和雪松病毒，一种非致病性 亨尼帕病毒具体来说，我们将分析病毒复制和I/III型干扰素激活的动力学， 肠样组织，并将对病毒感染的肠上皮细胞进行全面的蛋白质组学分析。 为了评估病毒诱导的I/III型IFN对胃肠道上皮病理学的作用，将研究IFN信号传导。 诱导或抑制，并分析对病毒感染的肠道易感性的影响。我们也将 测量病毒性肠炎期间通常被破坏的上皮细胞活力、屏障和修复功能。 我们的项目在技术上是创新的，因为我们将首次分析肠道病毒感染， 因为我们也将对JFB进行完整的蛋白质组分析， 肠上皮拟议的工作是概念上的创新，因为它将定义如何病毒诱导 I/III型IFN影响肠上皮功能和完整性。我们提出的工作是重要的，因为它将 提供了新的见解上皮细胞内在先天免疫机制参与无症状 用病毒病原体感染蝙蝠肠道，这对潜在的病原体具有重要意义 溢出事件

Summary The gastrointestinal (GI) tract is a major target organ for viral infection in bats, but infections rarely cause typical symptoms of viral enteritis such as diarrhea. The long-term goal of our project is to understand the specific innate immune response mechanisms of the intestinal epithelium that enable bats to sustain viral infection without experiencing cytopathic effects and intestinal barrier dysfunction. We hypothesize that protective type I and type III interferon (IFN) responses in Jamaican Fruit Bats (Artibeus jamaicensis, JFBs) enable persistent, asymptomatic viral infection of the gastrointestinal epithelium. To test our hypothesis, we will utilize a novel in vitro model of the JFB intestine, 3-D enteroids, which are complex long-term cultures of primary intestinal epithelial cells generated from adult tissue-derived stem cells. Responses in the gut epithelium of JFBs will be compared to those induced in human enteroids. In Aim 1, we will define type I/III IFN responses of JFB enteroids to viral infection. In Aim 2, we will determine to what extent type I/III IFNs impact viral replication and barrier function in the gastrointestinal epithelium of JFBs. We will analyze intestinal epithelial cell responses to three single-stranded RNA viruses that can infect JFB cells: H18N11 influenza virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Cedar virus, a non-pathogenic henipavirus. Specifically, we will analyze the kinetics of viral replication and type I/III IFN activation in the enteroids and will also perform a comprehensive proteomics analysis of virus-infected intestinal epithelial cells. To assess the role of virus-induced type I/III IFNs for gastrointestinal epithelial pathology, IFN signaling will be induced or inhibited, and the impact on enteroid susceptibility to viral infection will be analyzed. We also will measure epithelial cell viability, barrier and repair functions that are commonly disrupted during viral enteritis. Our project is technologically innovative, because we will, for the first time, analyze viral infection in enteroid cultures derived from JFBs and because we also will perform a complete proteome analysis of the JFB intestinal epithelium. The proposed work is conceptually innovative, because it will define how virus-induced type I/III IFNs impact intestinal epithelial function and integrity. Our proposed work is significant, because it will provide novel insights in the epithelial cell-intrinsic innate immune mechanisms involved in asymptomatic infection of the bat intestine with viral pathogens, which has important implications for potential pathogen spillover events.