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2000万人，导致330万临床病例 肝炎病例和因肝胆疾病死亡44,000人。作为一种无包膜病毒，HEV的存在令人惊讶 作为循环血液中抵抗中和作用的准包膜外体样病毒粒子。1型HEV(HEV-1)感染 与重型肝炎有关，孕妇死亡率高(&gt；25%)。HEV-1病毒在胎盘组织中复制， HEV-1垂直传播与新生儿死亡率高相关。由于缺乏有效的细胞培养或 对于HEV-1的动物模型，妊娠期HEV-1相关严重疾病的发病机制尚不清楚。显著 在患有重型肝炎的感染戊型肝炎的孕妇和感染戊型肝炎的猪中发现较高的肿瘤坏死因子-α水平。 在中枢神经系统组织中可检测到的HEVRNA水平显著高于 在中枢神经系统组织中未检测到HEV RNA的猪。长期目标是勾勒出有助于 妊娠期间HEV相关的高死亡率。不幸的是，我们目前还没有一个合适的系统，无论是在体外还是在 活体，研究母胎界面HEV-1感染情况。在目标1中，我们将在体外建立一个胎盘屏障 Transwell插入以研究母胎界面的HEV-1感染。我们推测血液中的HEV-1病毒 在高峰期，病毒血症穿过胎盘屏障，导致胎儿感染。我们将发展胎盘屏障文化 模仿在体内构成人类胎盘屏障的关键的母体血液和胎儿血层，通过 BeWo胎盘滋养层细胞与人脐静脉内皮细胞在基底外侧和根尖侧共培养 一种细胞外基质包裹的Transwell植入物。屏障的完整性将通过测量TEER和 对小分子的屏障渗透性。我们将确定HEV-1是否可以通过感染屏障培养物穿越屏障 分别在携带HEV-1和HEV-3的母体腔内检测胎儿腔内HEV的含量。 障碍。在目标2中，我们将确定HEV-1在母胎界面感染导致胎儿 感染。我们推测，在病毒血症高峰期，母血循环中的准包膜外切体样HEV 当胎盘屏障被促炎细胞因子如肿瘤坏死因子-α和白介素IL-2炎症时，更容易通过胎盘屏障。 18在HEV感染期间持续产生，并且HEV-1在屏障中感染产生III型IFN 限制病毒感染。我们将用肿瘤坏死因子-α和白介素18单独或联合作用于屏障培养物，然后 将未包膜的HEV-1、HEV-3、准包膜的HEV-1、HEV-3分别感染它们，以确定其数量 已经越过障碍的混合动力汽车。我们还将检测干扰素-α、干扰素-β和干扰素-λ在S感染后的表达水平 细胞，并确定在屏障诱导的抗病毒耐受环境是否可以转移到对HEV敏感的肝脏 细胞。我们期望建立一种胎盘屏障，模拟关键的母体血液和胎儿血液面向层 体内，并表明准包膜HEV-1更容易通过屏障，特别是当屏障被炎症时 促炎细胞因子肿瘤坏死因子-α和白介素18。我们还预计，HEV-1诱导干扰素-λ1/λ2/λ3具有限制病毒的屏障作用 在屏障处诱导的抗病毒耐药环境可转移到对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.