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

项目摘要Lauretta A.Lacko，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% 异常现象。寨卡病毒(ZIKV)感染导致自然流产风险增加 胎儿宫内生长，尽管胎儿丢失的机制仍不确定。人们对此知之甚少 ZIKV感染在植入前和围产期的影响，因为目前大多数研究都是在妊娠期 模型的重点是植入后阶段。最近的ZIKV疫情是一个日益令人担忧的公共卫生问题，因为 孕妇感染不仅会导致流产和发育不良，还会导致严重的神经系统疾病 小头畸形等后果。值得注意的是，最严重的胎儿异常与 在怀孕的第一和第二个三个月期间的感染。鉴于这一毁灭性事件的迅速出现 感染性疾病，迫切需要在早期确定ZIKV的宫内致病机理 怀孕了。这项研究的总体目标是确定寨卡病毒感染在早期阶段的影响。 怀孕，或植入前和植入前后，对胎儿和胎盘发育的影响，并揭示可能的 早期感染增加疾病风险的机制。最近的研究表明，ZIKV 可感染早孕永生化滋养层细胞系和胎盘外植体，但不能感染成熟人 从妊娠晚期分离出的滋养细胞或外植体。我们假设滋养外胚层细胞(TEC) 是怀孕早期有效的母亲向胎儿传播病毒的途径，并可以传播病毒 随着时间的推移，进一步感染发育中的神经前体细胞。事实上，我们的初步研究(以下手稿 Peer Review)证明了小鼠胚泡和人类植入前胚胎的体外感染 结果可有效感染TECs。此外，植入前和植入围术期的ZIKV感染可以诱导神经 妊娠中期祖细胞死亡。在本提案中，我们将定义TECs对ZIKV的细胞响应 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的全基因组基因敲除筛查中确定的候选基因(目标2)。我们会 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 研究将提供对寨卡病毒在早期阶段垂直传播的基本见解 怀孕了。更重要的是，它将揭示黄病毒和/或其他病毒的调控机制。 怀孕期间的感染。

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.