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（WNV）是一种对中枢神经系统（CNS）和人体产生高毒力的人类病原体。 在美国，这是流行性脑炎的最常见原因。没有疫苗或特定的 西尼罗河病毒感染者可获得抗病毒治疗，而开发这样的武器库需要 对其发病机制有了更全面的了解。 WNV 主要通过位传输 被感染的蚊子。一旦沉积到皮肤中，病毒就会在局部引流淋巴结中复制，然后再进入皮肤。 病毒血症，使病毒能够传播到各个器官，包括中枢神经系统，其中神经元是 主要感染目标。目前，尚不清楚哪些细胞在中枢神经系统之外被感染，但有许多细胞被感染。 体外研究表明，骨髓细胞，例如单核细胞/巨噬细胞，可能是主要目标。 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，西尼罗河病毒在其中复制强劲。我们对感染 WNV 的细胞进行了免疫表型分析 并发现其中很大一部分是CD4 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) 靶标 (miR-Ts) 的 WNV 突变株可限制 WNV 复制 以细胞特异性的方式。我们将淋巴或骨髓特异性 miR-T 工程化到 WNV 基因组中 并发现无法在骨髓细胞中复制的西尼罗河病毒株能够以几乎相同的方式复制 野生型 WNV 毒株，而无法在淋巴细胞中复制的 WNV 毒株则无法 在同一模型中复制。因此，我们假设淋巴细胞是必不可少的初始 外周 WNV 复制的细胞靶标。在此应用中，我们试图识别细胞 对于 WNV 在离体人体淋巴组织（目标 1）以及小鼠体内的复制至关重要 （目标 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).