A chicken model to study hepatitis E virus pathogenesis

项目摘要：戊型肝炎病毒（HEV）每年感染全球超过2000万人，导致超过44,000人死亡， 与HEV相关的肝胆疾病。戊型肝炎病毒感染的一个独特特征是暴发性肝炎，死亡率高，>25% 在孕妇中。由于缺乏动物模型，HEV相关的发病机制尚不清楚。 妊娠期间暴发性肝炎尚不清楚。性激素水平的升高，如黄体酮和雌激素 在怀孕期间被称为促进某些病毒复制，包括戊型肝炎病毒。越来越多的证据表明， HEV的复制是性激素依赖的，因为在性激素存在的情况下，HEV的复制显著增强 或妊娠血清中的感染细胞。最近从兔子身上发现的戊型肝炎病毒导致了妊娠模型的发展 对于戊型肝炎，作为具有高死亡率的暴发性肝炎，令人信服地和独立地在戊型肝炎感染者中复制。 怀孕的兔子长期目标是确定宿主（激素和免疫）和病毒因素 与HEV相关的暴发性肝衰竭在目的1中，我们将确定妊娠相关的影响和机制， 性激素对人肝细胞中HEV复制的影响。我们假设HEV感染抑制了 雌激素受体ER-α和ER-β在怀孕期间，这导致抑制宿主先天免疫， 增强HEV复制。E1、E2和P4单独或组合对肝细胞中HEV复制的影响将被描述。 确定，和性激素在调节戊型肝炎病毒复制的潜在机制，通过表达 雌激素受体ER-α/ER-β和孕激素受体PGRMC 1/PGRMC 2及其对细胞因子表达的影响将 被描绘出来。目的2：阐明妊娠相关性激素在妊娠期子宫内膜异位症发生发展中的作用及机制 暴发性肝炎的实验。我们推测，妊娠相关性激素的变化抑制了 宿主先天性和细胞介导的免疫应答导致增强的HEV复制和暴发性肝炎。怀孕 兔将感染戊型肝炎病毒，以确定性激素和免疫学相关的发展， 妊娠期肝炎。P4和E1/E2介导的暴发性肝炎的机制将被阐明 通过定量ER-α/ER-β、PGRMC 1/PGRMC 2和孕酮诱导的卵巢癌细胞中ER-α/ER-β、PGRMC 1/PGRMC 2的mRNA和蛋白表达水平， 阻断因子及其对感染兔细胞因子表达的影响。在目标3中，我们 定义与暴发性肝衰竭相关的病毒遗传因子。从案例研究中，8个独特的氨基酸残基 与暴发性肝功能衰竭有关。我们假设这些突变显著增强了 HEV复制导致更高的病毒载量和暴发性肝衰竭。我们将首先利用HEV荧光素酶复制子 以确定8个氨基酸突变对肝细胞中HEV复制的影响。最大的前2个突变 对HEV复制的增强作用将单独或组合引入基因型3 HEV感染性克隆， 拯救突变病毒，将用于感染怀孕的兔子，以确定所确定的突变是否有助于 暴发性肝炎我们期望确定性激素、潜在机制、免疫学相关因素， 以及在HEV感染期间促成暴发性肝衰竭发展的病毒遗传元件。

Project Summary: Hepatitis E virus (HEV) infects >20 million people worldwide annually leading to >44,000 deaths due to HEV-related hepatobiliary diseases. A unique feature of HEV infection is fulminant hepatitis with high mortality of >25% in pregnant women. Due to the lack of an animal model, the underlying mechanism in the pathogenesis of HEV-associated fulminant hepatitis during pregnancy is unknown. The elevated levels of sex hormones such as progesterone and estrogen during pregnancy are known to promote certain virus replications including HEV. Accumulating evidence indicate that HEV replication is sex hormone-dependent, as HEV replication was significantly enhanced in the presence of sex hormones or pregnancy sera in infected cells. The recent discovery of HEV from rabbits led to the development of a pregnancy model for HEV, as fulminant hepatitis with high mortality was convincingly and independently reproduced in HEV-infected pregnant rabbits. The long-term goal is to identify the host (hormonal and immunological) and viral factors contributing to HEV-associated fulminant hepatic failure. In aim 1, we will determine the effect and mechanism of pregnancy-associated sex hormones on HEV replication in human liver cells. We hypothesize that HEV infection inhibits the expression of estrogen receptor ER-α and ER-β during pregnancy, which results in suppression of host innate immunity leading to enhanced HEV replication. The effect of E1, E2 and P4, singly or in combination, on HEV replication in liver cells will be determined, and the underlying mechanisms of sex hormones in regulating HEV replication through the expression of estrogen receptor ER-α/ER-β, and progesterone receptor PGRMC1/PGRMC2 and their effect on cytokine expressions will be delineated. In aim 2, we will elucidate the role and mechanism of pregnancy-associated sex hormones in the development of fulminant hepatitis using a rabbit model. We hypothesize that changes of pregnancy-associated sex hormones suppress host innate and cell-mediated immune responses leading to enhanced HEV replication and fulminant hepatitis. Pregnant rabbits will be infected with HEV to identify the sex hormones and immunological correlates in the development of fulminant hepatitis during pregnancy. The mechanisms of P4- and E1/E2-mediated fulminant hepatitis will be delineated by quantifying mRNA and protein expression levels of ER-α/ER-β, PGRMC1/PGRMC2, and progesterone-induced blocking factor in liver and placenta tissues, and their effect on cytokine expressions in infected rabbits. In aim 3, we will define the viral genetic element(s) associated with fulminant hepatic failure. From case studies, 8 unique amino acid residues within HEV ORF1 are associated with fulminant hepatic failure. We hypothesize that these mutations significantly enhance HEV replication leading to higher viral loads and fulminant hepatic failure. We will first utilize HEV luciferase replicons to determine the effect of the 8 amino acid mutations on HEV replication in liver cells. The top 2 mutations with greatest enhancement on HEV replication will be introduced, singly and in combination, into genotype 3 HEV infectious clones to rescue mutant viruses, which will be used to infect pregnant rabbits to determine whether the identified mutations contribute to fulminant hepatitis. We anticipate to identify the sex hormone(s), underlying mechanisms, immunological correlates, and viral genetic element(s) contributing to the development of fulminant hepatic failure during HEV infection.