Genetic basis of virus induced Biliary Atresia

项目总结/摘要 胆道闭锁是小儿终末期肝病最常见的原因， 小儿肝移植的一个适应症。因为致病病毒已经在 胆道闭锁的病因是围产期病毒感染 引发免疫介导的胆管上皮破坏。胆管炎小鼠模型 新生小鼠感染恒河猴轮状病毒（RRV）后， 门脉道内出现炎症和肝外胆管阻塞。RRV目标 胆管细胞的感染，除了直接的胆管细胞损伤也诱导自然 杀伤细胞介导的胆管上皮损伤。 我们先前已经表明，序列“SRL”（氨基）内的氨基酸精氨酸（R） 氨基酸445-447）是病毒结合和进入胆管上皮细胞所必需的 细胞我们开发了反向遗传学系统来创建RRV的突变体（RRVVP 4-R446 G）， 与野生型RRV相比，在VP 4蛋白中具有单个氨基酸变化。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， 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 突变株沿着额外的VP 4突变体，以确定RRV如何结合、进入、运输 并最终激活胆管细胞的先天免疫反应。我们将 还确定了这些VP 4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新的见解， 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.