Effect of Pre- and Peri-Implantation Zika Virus Infection on Fetal Development

项目概要Lauretta A.拉科，F32重新提交，2018年8月8日 围产期感染是孕产妇和胎儿疾病的主要原因，占所有先天性疾病的2- 3 异常寨卡病毒（ZIKV）感染导致自然流产和贫困的风险增加 胎儿宫内生长，但胎儿丢失的机制尚未确定。知之甚少 ZIKV感染在着床前和着床期的影响，因为目前大多数妊娠研究 模型集中在植入后阶段。最近的ZIKV流行是一个日益增长的公共卫生问题， 感染孕妇不仅会导致流产和发育不良，而且会导致严重的神经系统疾病， 如小头畸形等。值得注意的是，最严重的胎儿异常与 在怀孕的第一个和第二个三个月感染。鉴于这场毁灭性的 由于ZIKV感染性疾病，迫切需要在早期阶段确定ZIKV的子宫内发病机制。 怀孕本研究的总体目标是确定ZIKV感染在肿瘤的最早阶段的影响。 妊娠，或植入前和植入期，对胎儿和胎盘发育的影响，并深入了解可能的 早期感染增加疾病风险的机制。最近的研究表明ZIKV 能感染孕早期永生化滋养层细胞系和胎盘外植体，但不感染成熟人 滋养层或从晚期妊娠分离的外植体。我们假设滋养外胚层细胞（TEC） 是怀孕早期母亲向胎儿传播病毒的有效途径， 随着时间的推移进一步感染发育中的神经祖细胞。事实上，我们的初步研究（手稿根据 同行评审）证明鼠胚泡和人植入前胚胎的离体感染 导致TEC的有效感染。此外，植入前和植入期ZIKV感染可诱导神经细胞凋亡。 妊娠中期的祖细胞死亡。在本提案中，我们将定义TEC对ZIKV的细胞应答 体内感染（目的1），并通过研究ZIKV感染的分子机制来剖析控制ZIKV感染的分子机制。 从基于CRISPR的全基因组范围基因敲除筛选中鉴定的候选基因（Aim 2）。我们将 联合收割机结合免疫染色、基因表达和基因编辑技术来解决这些问题。这些 这些研究将为ZIKV在早期阶段的垂直传播提供基本的见解。 怀孕更重要的是，它将揭示黄病毒和/或其他病毒的调控机制， 怀孕期间感染。

PROJECT SUMMARY Lauretta A. Lacko, F32 Resubmission, 8/8/2018 Perinatal infections are a major cause of maternal and fetal illness, accounting for 2-3% of all congenital abnormalities. Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth, although the mechanisms underlying fetal loss remain undetermined. Little is known about the impact of ZIKV infection during pre- and peri-implantation because most current studies in pregnancy models focus on post-implantation stages. The recent ZIKV epidemic is a growing public health concern as infection in pregnant woman not only causes abortion and poor growth, but also severe neurological consequences such as microcephaly. Notably, the most severe fetal abnormalities have been associated with infection during the first and second trimester of pregnancy. Given the rapid emergence of this devastating infectious disease, there is an urgent need to determine the in utero pathogenesis of ZIKV during early gestation. The overall objective of this study is to determine the effect of ZIKV infection at the earliest stages of pregnancy, or pre- and peri-implantation, on fetal and placental development, and to shed insight into possible mechanisms by which early infection increases risk of the disease. Recent studies have demonstrated ZIKV can infect first trimester immortalized trophoblast cell line and placental explants, but not mature human trophoblasts or explants isolated from late stage pregnancies. We hypothesize that trophectoderm cells (TECs) are a route for efficient mother to fetus viral transmission during early pregnancy, and can propagate the virus to further infect developing neural progenitor cells over time. Indeed, our preliminary studies (manuscript under peer review) demonstrate that ex vivo infection of murine blastocysts and human pre-implantation embryos results in efficient infection of TECs. Further, pre- and peri-implantation ZIKV infection can induce neural progenitor cell death at mid-gestation. In this proposal, we will define the cellular response of TECs to ZIKV infection in vivo (Aim 1) and dissect the molecular mechanism controlling ZIKV infection by studying the candidate genes identified from a CRISPR-based whole genome wide gene knockout screen (Aim 2). We will combine immunostaining, gene expression, and gene editing technologies to address these questions. These studies will provide fundamental insight into vertical transmission of ZIKV during the earliest stages of gestation. More importantly, it will shed a light into the mechanism regulating flavivirus and/or other viral infections during pregnancy.