The Molecular Determinants of Virus Induced Biliary Atresia

描述（申请人提供）：胆道闭锁是小儿终末期肝病最常见的病因，也是小儿肝移植的第一适应症。由于在患病儿童的肝脏中发现了致病病毒，因此提出的胆道闭锁的病因是围产期病毒感染引发免疫介导的胆道上皮破坏。小鼠胆道闭锁模型支持病毒发病机制，因为感染了恒河轮状病毒（RRV）的新生小鼠在门道内发生炎症和肝外胆管阻塞。RRV以胆管细胞为感染目标，除了直接损伤胆管细胞外，还诱导t细胞介导的胆管上皮损伤。轮状病毒是一种由11个基因片段组成的dsRNA病毒。我们假设特定的轮状病毒基因控制着感染胆管细胞和诱导免疫介导损伤的能力。为了验证这一假设，我们从亲本菌株RRV和TUCH中产生了一套完整的单基因重组。这些重组为我们提供了一套独特的工具，以确定特定轮状病毒基因如何促进发病机制。在初步研究中，我们发现RRV第3、4、6、8、9和11段是感兴趣的基因。考虑到对基因片段4的观察结果的可靠性，我们把重点放在了这个基因上。我们将使用RRV -胆管细胞感染和t细胞活化的体外模型来确定基因片段4在疾病发病中的作用机制。我们还将利用反向遗传学产生突变基因片段4的感染性病毒，以确定其在更复杂环境下完整宿主的基础。这些互补的方法将对病毒性胆道闭锁产生新的认识。

DESCRIPTION (provided by applicant): Biliary atresia is the most common cause of pediatric end stage liver disease and the number one indication for pediatric liver transplantation. Because pathogenic viruses have been found in the liver of afflicted children, a proposed etiology for biliary atresia is a perinatal viral infection triggering immune mediated destruction of the biliary epithelium. The murine model of biliary atresia supports a viral pathogenesis as newborn mice infected with rhesus rotavirus (RRV) develop inflammation within the portal tract and extra- hepatic bile duct obstruction. RRV targets the cholangiocyte for infection and in addition to direct cholangiocyte injury also induces T-cell mediated injury to the biliary epithelium. Rotavirus is a dsRNA virus comprised of 11 gene segments. We hypothesized that specific rotavirus genes govern the ability to infect the cholangiocyte and induce immune mediated injury. To test this hypothesis, we generated a complete set of single gene reassortants derived from the parental strains RRV and TUCH. These reassortants give us a unique set of tools to determine how specific rotavirus genes contribute to the pathogenesis. In preliminary studies, we found that RRV segments 3, 4, 6, 8, 9 and 11 are genes of interest. Given the robust nature of the observations made with gene segment 4, we focused on this gene. We will use in vitro models of RRV - cholangiocyte infection and T-cell activation to determine the mechanisms by which gene segment 4 contributes to disease pathogenesis. We will also use reverse genetics to generate mutate gene segment 4 infectious virus to determine the basis in the more complex environment of the intact host. These complimentary approaches will generate new insight in viral induced biliary atresia. PUBLIC HEALTH RELEVANCE: Biliary atresia is the most common cause of pediatric end stage liver disease and the number one indication for pediatric liver transplantation. Because pathogenic viruses have been found in the liver of afflicted children, a proposed etiology for biliary atresia is a perinatal viral infection triggering immune mediated destruction of the biliary epithelium resulting in biliary obstruction. Our goal, using a unique set of rotavirus gene knockouts, is to determine the molecular basis for this process. In so doing develop we hope to develop new treatment strategies to alter the course of this challenging disease. This project is in complete accord with the NIH mission to reduce illness and disability.