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 ...