Placental barrier culture to delineate the mechanism of hepatitis E virus infection at the maternal and fetal interface

项目摘要：戊型肝炎病毒（HEV）每年感染全球超过2000万人，导致330万临床病例 肝炎病例和超过44 000例肝胆疾病死亡。作为一种无包膜病毒，HEV令人惊讶地存在于 作为循环血液中抵抗中和的类包膜外泌体样病毒体。基因1型戊型肝炎病毒（HEV-1）感染 与妊娠妇女的高死亡率（>25%）的暴发性肝炎有关。HEV-1在胎盘组织中复制， HEV-1垂直传播与高新生儿死亡率相关。由于缺乏有效的细胞培养或 HEV-1的动物模型，HEV-1相关的妊娠期严重疾病的机制尚不清楚。显著 在患有暴发性肝炎的HEV感染孕妇中发现了较高水平的TNF-α， CNS组织中可检测到的HEV RNA的促炎细胞因子（TNF-α和IL-18）水平显著高于 在CNS组织中检测不到HEV RNA的猪中。长期目标是阐明有助于 妊娠期间HEV相关的高死亡率。不幸的是，我们目前还没有合适的系统，无论是体外还是体内。 体内，以研究母胎界面的HEV-1感染。在目标1中，我们将在体外建立胎盘屏障， Transwell插入研究母胎界面中的HEV-1感染。我们假设循环血液中的HEV-1 在病毒血症高峰期穿过胎盘屏障导致胎儿感染。我们将培养胎盘屏障细胞 模拟体内构成人胎盘屏障的关键的母体血液和胎儿血液面对层， 在基底侧和顶侧共培养BeWo胎盘滋养层细胞和人脐静脉内皮细胞 细胞外基质包被的Transwell插入物屏障的完整性将通过测量TEER和 屏障对小分子的渗透性。我们将通过感染屏障培养物来确定HEV-1是否可以穿过屏障 分别用HEV-1和HEV-3在母体腔室中，并测量胎儿腔室中HEV的量， 屏障。在目的2中，我们将确定母亲-胎儿界面中HEV-1感染导致胎儿死亡的机制。 感染我们假设，在病毒血症高峰期，母血循环中的类包膜外泌体样HEV 当胎盘屏障被促炎细胞因子如TNF-α和IL-12炎症时， 18，在HEV感染期间持续产生，并且屏障中的HEV-1感染产生III型IFN， 限制病毒感染。我们将单独或联合使用TNF-α和IL-18刺激屏障培养物，然后 分别用无包膜HEV-1、HEV-3、准包膜HEV-1、HEV-3感染，测定其含量 已经越过屏障的混合动力汽车。我们还将测定感染的大肠杆菌中IFN-α、IFN-β和IFN-λs的表达水平。 细胞，并确定在屏障处诱导的抗病毒耐药环境是否可以转移到HEV敏感的肝脏 细胞我们期望建立一个胎盘屏障，模仿关键的母体血液和胎儿血液面对层， 体内，并显示准包膜的HEV-1将更容易穿过屏障，特别是当屏障被感染时， 促炎细胞因子TNF-α和IL-18。我们还预期HEV-1在屏障中诱导IFN-λ1/λ2/λ3以限制病毒 复制，并且在屏障处诱导的抗病毒抗性环境可转移到HEV敏感的肝细胞。

Project Summary: Hepatitis E virus (HEV) infects >20 million people worldwide annually leading to 3.3 million clinical cases of hepatitis and >44,000 deaths due to hepatobiliary diseases. As a non-enveloped virus, HEV is surprisingly present as quasi-enveloped exosome-like virions in circulating blood that resist neutralization. Genotype 1 HEV (HEV-1) infection is associated with fulminant hepatitis with high mortality (>25%) in pregnant women. HEV-1 replicates in placental tissues, and HEV-1 vertical transmission is associated with a high neonatal mortality. Due to the lack of an efficient cell culture or animal model for HEV-1, the mechanism of HEV-1-associated severe diseases during pregnancy is unknown. Significantly higher levels of TNF-α were found in HEV-infected pregnant women with fulminant hepatitis, and HEV-infected pigs with detectable HEV RNA in CNS tissues had significantly higher levels of proinflammatory cytokines (TNF-α and IL-18) than in pigs without detectable HEV RNA in CNS tissues. The long-term goal is to delineate the mechanisms contributing to HEV-associated high mortality during pregnancy. Unfortunately, we currently do not have a suitable system, in vitro or in vivo, to study HEV-1 infection at the maternal-fetal interface. In aim 1, we will establish an in vitro placental barrier in Transwell insert to study HEV-1 infection in the maternal-fetal interface. We hypothesize that HEV-1 in circulating blood during peak viremia crosses the placental barrier leading to fetal infection. We will develop a placental barrier culture mimicking the critical maternal blood- and fetal blood-facing layers that constitute the human placental barrier in vivo, by co-culturing BeWo placental trophoblastic cells and human umbilical vein endothelial cells on basolateral and apical sides of an extracellular matrix-coated Transwell insert. The integrity of the barrier will be confirmed by measuring TEER and barrier permeability to small molecules. We will determine whether HEV-1 can cross the barrier by infecting barrier cultures in the maternal chamber with HEV-1 and HEV-3, respectively, and measuring the amount of HEV in the fetal chamber of the barrier. In aim 2, we will determine the mechanisms of HEV-1 infection in the maternal-fetal interface leading to fetal infection. We hypothesize that quasi-enveloped exosome-like HEVs in circulating maternal blood during peak viremia more easily cross the placental barrier when the barrier is inflamed by pro-inflammatory cytokines such as TNF-α and IL- 18 that are consistently produced during HEV infection, and that HEV-1 infection in the barrier produces type III IFNs to limit viral infection. We will inflame the barrier cultures with TNF-α and IL-18, separately or in combination, and then infect them with non-enveloped HEV-1, HEV-3, quasi-enveloped HEV-1, HEV-3, respectively, to determine the amounts of HEV that have crossed the barrier. We will also determine the expression levels of IFN-α, IFN-β and IFN-λs in infected cells, and determine if the antiviral resistance environment induced at the barrier can be transferred to HEV-susceptible liver cells. We anticipate to establish a placental barrier mimicking the critical maternal blood- and fetal blood-facing layers in vivo, and show that quasi-enveloped HEV-1 will more easily cross the barrier especially when the barrier is inflamed with proinflammatory cytokines TNF-α and IL-18. We also expect that HEV-1 induces IFN-λ1/λ2/λ3 in the barrier to limit virus replication, and that the antiviral resistance environment induced at the barrier is transferable to HEV-susceptible liver cells.