Antiviral responses in iPSC-derived human primary cells to Ebola virus infection

高致病性新出现的病毒是全球公共卫生的一个主要问题，目前西非爆发的埃博拉病毒突出表明了这一点。埃博拉病毒家族的不同成员在人类中的致病性方面表现出显著差异，从埃博拉病毒（EBOV）的40-90%的病死率到莱斯顿病毒（RESTV）的无症状感染。确定导致RESTV在人类中减毒表型的因素受到缺乏概括人类中RESTV和EBOV感染的观察到的差异的感染模型的阻碍。在本申请的目的1中，我们提出建立基于人原代细胞的新型模型系统，以剖析宿主对EBOV和RESTV感染的应答，并鉴定可能的保护相关性。使用人诱导多能干细胞（iPSC），我们将产生被认为在EBOV致病性中起主要作用的EBOV感染的两种靶细胞类型，骨髓树突状细胞（mDC）和原代肝细胞。虽然有证据表明EBOV感染损害或失调骨髓细胞中的抗病毒反应（见意义和初步数据），但认为肝细胞参与EBOV感染中观察到的大量不受控制的病毒产生。我们将使用基于iPSC的平台来定义宿主对高致病性和低致病性埃博拉病毒反应的差异，以确定可能的保护相关性。在目的2中，我们提出建立EBOV和RESTV的复制子-VLP（病毒样颗粒）系统，其可用于原代细胞中以剖析病毒生命周期中的不同步骤，并将它们与观察到的宿主对感染的反应的差异联系起来。通过检查原代细胞中高致病性和低致病性成员之间的差异来更好地理解埃博拉病毒的发病机制，将为人类致病性的决定因素提供急需的见解，并将为未来治疗方法的发展提供更有针对性的方法。

Highly pathogenic emerging viruses are a major concern for global public health as highlighted by the current Ebola virus outbreak in West Africa. Different members of the ebolavirus family demonstrate significant differences in pathogenicity in humans ranging from case fatality rates of 40-90% for Ebola virus (EBOV) to asymptomatic infections with Reston virus (RESTV). Determining the factors that lead to the attenuated phenotype of RESTV in humans is hampered by the lack of infection models that recapitulate the observed differences of RESTV and EBOV infection in humans. In Aim 1 of this application, we propose to establish a novel model system based on human primary cells to dissect the host responses to EBOV and RESTV infection and identify possible correlates of protection. Using human induced pluripotent stem cells (iPSCs), we will generate two target cell types of EBOV infection which are thought to play a major role in EBOV pathogenicity, myeloid dendritic cells (mDCs) and primary hepatocytes. While there is evidence that EBOV infection impairs or dysregulates the antiviral response in myeloid cells (see significance and preliminary data), hepatocytes are thought to be involved in the massive, uncontrolled virus production observed in EBOV infection. We will use the iPSC-based platforms to define the differences in the host response to high and low pathogenic ebolaviruses in order to identify possible correlates of protection. In Aim 2, we propose to establish a replicon-VLP (viral like particle) system for EBOV and RESTV which can be used in primary cells to dissect the different steps in the virus life cycle and connect them to observed differences in the host response to infection. A better understanding of ebolavirus pathogenesis by examination of differences between high and low pathogenic members in primary cells will provide much needed insights in determinants of pathogenicity in humans and will inform more targeted approaches for the future development of therapeutics.