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

项目总结： 西尼罗河病毒(West Nile Virus，WNV)是一种高度毒力的人类中枢神经系统(CNS)病原体。 流行性脑炎在美国最常见的原因。没有疫苗或特效药 为西尼罗河病毒感染者提供抗病毒治疗，而开发这样的武器库需要 对其发病机制有了更全面的认识。WNV主要通过一个比特传输 被感染的蚊子。一旦病毒沉积到皮肤，病毒就会在局部引流的淋巴结内复制，然后 病毒血症，允许病毒扩散到各种器官，包括中枢神经系统，在那里神经元是 感染的主要目标。目前，尚不清楚哪些细胞是在中枢神经系统之外感染的，但有许多 体外研究表明，髓系细胞，如单核/巨噬细胞，可能是主要的目标。 为了开始在更具生理学相关性的模型中理解这一点，我们利用了一名体外人类 淋巴组织模型，其中西尼罗河病毒复制旺盛。我们对感染西尼罗河病毒的细胞进行了免疫表型鉴定 并发现其中很大一部分是CD4T细胞。为了进一步验证这些发现，我们生成了 包含细胞特异性microRNA(MiR)靶标(miR-ts)以限制西尼罗河病毒复制的西尼罗河病毒突变株 以特定于细胞的方式。我们将淋巴或髓系特异的miR-ts基因工程到西尼罗河病毒的基因组中 并发现无法在髓系细胞中复制的西尼罗河病毒株能够复制到几乎相同的 野生型WNV毒株，而不能在淋巴细胞中复制的WNV毒株 以同样的模式复制。因此，我们假设淋巴样细胞在最初阶段是必不可少的 在外围地区复制西尼罗河病毒的蜂窝目标。在本应用程序中，我们试图识别细胞 对西尼罗河病毒在体外人淋巴组织(AIM 1)和小鼠体内复制是必不可少的 (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).