Host and Viral Determinants of Orthobunyavirus Vertical Transmission: Novel Model Systems to Understand the Mechanisms of Congenital Disease in Humans and Ruminants

项目摘要/摘要 新出现的传染病对人类和农业健康构成重大威胁；因此，必须 我们采取积极主动的方法来了解与感染相关的病毒和宿主因素 和发病机制。许多布尼亚病毒感染，包括裂谷热(RVFV)、缓存谷(CVV)和 Schmallenberg(SBV)病毒会在家畜中引起大规模的流产事件，从而导致重大的经济 菌株，增加了人类感染的易感性。拉克罗斯病毒(LCV)是一种相关的布尼亚病毒，目前尚不清楚 在家畜中引起垂直传播，然而，垂直传播在实验中被牵连 感染。两例RVFV垂直传播病例发生在孕妇和感染者中 RVFV有较高的晚期流产风险。垂直传播是否发生在反刍动物和 人类是由于LACV感染，还是在人类中由SBV和CVV感染，目前尚不清楚。给定LACV和 CVV感染会导致人类患上危及生命的疾病，先天性感染可能只是简单地 由于大多数本亚病毒感染的性质温和，因此被忽视。我们假设SBV、CVV和LACV 可以感染人类和反刍动物胎盘，以及毒力因子，如NSM，和可变诱导 跨病毒的抗病毒反应决定了致病的严重性，从而决定了宿主物种的致畸性。 这项建议将利用两个模型系统来研究胎盘中的布尼亚病毒感染。首先，我们将 检测LACV、CVV、SBV和RVFV是否在体外感染人、羊和大鼠的胎盘外植体。 利用野生型和NSM敲除病毒，我们将确定NSM是否有助于宿主或细胞趋向性， 感染的免疫反应、程序性细胞死亡途径和先天性发病机制。第二，我们 将利用遗传上易处理的2D人类滋养层细胞和滋养层类器官(TO)系统来比较细胞- 布尼亚病毒感染时免疫反应和细胞死亡途径的特异性差异。这将是 首次利用人胎盘有机体研究先天性本雅氏病毒感染。要成功完成 在提议的项目中，我在初级导师的指导下制定了一项特殊的职业发展计划 艾米·哈特曼博士(匹兹堡大学(U Pitt))和共同导师卡罗琳·科因博士(杜克大学) 和伦纳德·达乌托(U Pitt)。我的训练将包括人体器官的实际操作和教学训练。 除了在病理学、跨物种胎盘生物学和免疫学方面的教学培训外，还发展。U·皮特 为支持拟议的研究和培训提供必要的环境 杰出的科学家，高密封性的实验室和动物设施，实验室培训机会 管理，负责任的研究行为和勇气，以及展示研究的机会。 成功的培训和完成拟议的研究将支持我的最终职业目标，即建立 研究先天性本雅氏病毒跨病毒和宿主物种机制的独立研究计划 感染在人类和反刍动物胎盘器官样研究和先天性病理学中具有特殊的生态位。

PROJECT SUMMARY/ABSTRACT Emerging infectious diseases pose a significant threat to human and agricultural health; therefore, it is imperative that we take a proactive approach toward understanding virus and host factors that are associated with infection and pathogenesis. Many bunyavirus infections, including Rift Valley fever (RVFV), Cache Valley (CVV), and Schmallenberg (SBV) viruses, cause massive abortogenic events in livestock that lead to significant economic strain and increased susceptibility of human infection. La Crosse virus (LCV), a related bunyavirus, is not known to cause vertical transmission in livestock, however vertical transmission has been implicated upon experimental infection. Two cases of vertical transmission of RVFV have occurred in pregnant women and those infected with RVFV have a higher risk for late-term miscarriages. Whether vertical transmission occurs in ruminants and humans due to LACV infection or in humans from SBV and CVV infection is unknown. Given LACV and CVV infections can cause life-threatening diseases in humans, it is plausible that congenital infection may simply be overlooked due to the mild nature of most bunyavirus infections. We hypothesize that SBV, CVV, and LACV can infect human and ruminant placentas and that virulence factors, such as NSm, and variable induction of antiviral responses across viruses dictate pathogenesis severity, and thus teratogenicity, across host species. This proposal will utilize two model systems to study bunyavirus infection of the placenta. First, we will examine whether LACV, CVV, SBV, and RVFV infect placenta explants from humans, sheep, and rats in vitro. Using wild type and NSm knockout viruses, we will identify whether NSm contributes to host or cellular tropism, immune responses to infection, programmed cell death pathways, and congenital pathogenesis. Second, we will utilize genetically tractable 2D human trophoblast and trophoblast organoid (TO) systems to compare cell- specific differences in immune responses and cell death pathways upon bunyavirus infection. This will be the first study to utilize human placenta organoids to study congenital bunyavirus infection. To successfully complete the proposed project, I have developed an exceptional career development plan under the primary mentorship of Dr. Amy Hartman (University of Pittsburgh (U Pitt)) and co-mentors, Drs. Carolyn Coyne (Duke University) and Leonard D’Aiuto (U Pitt). My training will consist of hands-on and didactic training in human organoid development in addition to didactic training in pathology, cross-species placenta biology, and immunology. U Pitt provides the necessary environment to support the proposed research and training through accessibility to outstanding scientists, high containment laboratory and animal facilities, training opportunities in laboratory management, responsible conduct of research and grantsmanship, and opportunities to present research. Successful training and completion of the proposed research will support my ultimate career goal to establish an independent research program studying the cross virus- and host-species mechanisms of bunyavirus congenital infections with a special niche in human and ruminant placenta organoid research and congenital pathology.