Identification of the initial cells infected by West Nile virus ex vivo and in vivo

项目概要： 西尼罗河病毒（West Nile virus，WNV）是一种高度致命的人类中枢神经系统（CNS）病原体， 在美国流行性脑炎的最常见原因。没有疫苗或特定的 抗病毒治疗可用于WNV感染的个体，而开发这样的武器库需要 对其发病机制有更完整的认识。WNV主要是通过一个 被感染的蚊子一旦沉积到皮肤中，病毒在局部引流淋巴结中复制， 病毒血症，使病毒扩散到各种器官，包括中枢神经系统，其中神经元是 感染的主要目标。目前，尚不清楚哪些细胞在CNS外被感染，但许多 体外研究表明，骨髓细胞，如单核细胞/巨噬细胞，可能是主要靶。 为了开始在更生理相关的模型中理解这一点，我们利用了离体人类 淋巴组织模型，其中WNV复制稳健。我们对WNV感染的细胞进行了免疫表型分析 发现其中有很大一部分是CD 4 + T细胞。为了进一步验证这些发现，我们生成了 含有细胞特异性microRNA（miR）靶点（miR-Ts）以限制WNV复制的WNV突变株 以细胞特异性的方式。我们将淋巴或骨髓特异性miR-Ts工程化到WNV的基因组中， 并发现不能在骨髓细胞中复制的WNV菌株能够与 野生型WNV毒株，而不能在淋巴细胞中复制的WNV毒株不能在淋巴细胞中复制。 在同一个模型中复制。因此，我们假设淋巴样细胞是重要的初始细胞， WNV在外周复制的细胞靶点。在本申请中，我们试图识别细胞 对WNV在离体人淋巴组织（Aim 1）以及小鼠中的复制是必需的 (Aim 2）。

PROJECT SUMMARY: West Nile virus (WNV) is a highly virulent human pathogen of the central nervous system (CNS) and the most common cause of epidemic encephalitis in the United States. There are no vaccines or specific antiviral treatments available for WNV-infected individuals, and development of such an arsenal requires a more complete understanding of its pathogenesis. WNV is primarily transmitted through the bit of an infected mosquito. Once deposited into the skin, the virus replicates in a local draining lymph node prior to viremia, which allows the virus to spread to various organs, including the CNS, where neurons are the major targets of infection. Currently, it is unclear which cells are infected outside of the CNS, but numerous in vitro studies suggest that myeloid cells, such as monocytes/macrophages, are likely the primary targets. To begin understanding this in a more physiologically relevant model, we utilized an ex vivo human lymphoid tissue model, where WNV replicates robustly. We immunophenotyped the WNV-infected cells and found that a significant proportion were CD4+ T cells. To further validate these findings, we generated WNV mutant strains that contain cell-specific microRNA (miR) targets (miR-Ts) to restrict WNV replication in a cell-specific manner. We engineered lymphoid- or myeloid-specific miR-Ts into the genome of WNV and found that WNV strains unable to replicate in myeloid cells were able to replicate nearly identically to wild-type WNV strains, while WNV strains incapable of replicating in lymphoid cells were unable to replicate in this same model. Therefore, we hypothesize that lymphoid cells are essential initial cellular targets for WNV replication in the periphery. In this application, we seek to identify the cells that are essential for WNV replication in the human lymphoid tissue ex vivo (Aim 1) as well as in mice (Aim 2).