Genetic basis of virus induced Biliary Atresia

项目概要/摘要 胆道闭锁是儿童终末期肝病的最常见原因，其数量 小儿肝移植的适应症之一。因为在体内发现了致病病毒 对于患病儿童的肝脏，胆道闭锁的拟议病因是围产期病毒感染 触发免疫介导的胆管上皮破坏。小鼠胆道模型 闭锁支持病毒发病机制，因为新生小鼠感染了恒河猴轮状病毒（RRV） 发生门管内炎症和肝外胆管阻塞。 RRV 目标 胆管细胞受到感染，除了直接损伤胆管细胞外，还诱发自然 杀伤细胞介导胆管上皮损伤。 我们之前已经证明序列“SRL”（氨基）内的氨基酸精氨酸（R） RRV VP4 蛋白上的酸 445-447) 是病毒结合和进入胆管上皮所必需的 细胞。我们开发了反向遗传学系统来创建 RRV 突变体 (RRVVP4-R446G)，该突变体 与野生型RRV相比，VP4蛋白有一个氨基酸变化。在体外，突变体 病毒在胆管细胞中的结合和感染性降低。在体内，它产生的症状较少 和新生小鼠的死亡率，导致胆道闭锁的减弱形式。我们将使用这个 突变株以及其他 VP4 突变体，以确定 RRV 如何结合、进入、运输 通过细胞，最终激活胆管细胞的先天免疫反应。我们会 还确定了这些 VP4 突变体感染和激活浆细胞样树突状细胞的能力 自然杀伤细胞激活。这些互补的方法将产生病毒式传播的新见解 诱发胆道闭锁。

Project Summary/Abstract 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 extrahepatic bile duct obstruction. RRV targets the cholangiocyte for infection and in addition to direct cholangiocyte injury also induces Natural Killer cell mediated injury to the biliary epithelium. We have previously shown that the amino acid, arginine (R) within the sequence “SRL” (amino acids 445-447) on the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G), which had a single amino acid change in VP4 protein compared to wild type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced less symptoms and mortality in neonatal mice, resulting in an attenuated form of biliary atresia. We will use this mutant strain along with additional VP4 mutants to determine how RRV binds to, enters, traffics through the cell, and ultimately activates the cholangiocyte's innate immune response. We will also ascertain these VP4 mutants' ability to infect and activate plasmacytoid dendritic cells leading to Natural Killer cell activation. These complimentary approaches will generate new insight in viral induced biliary atresia.