Leishmania RNA virus (LRV) infectivity and host responses

描述(申请人提供)：感染原生动物寄生虫利什曼原虫表现出各种疾病病理，从轻微的皮肤到致命的内脏疾病到毁容的粘膜皮肤疾病。这种多样性的基础尚不清楚；利什曼原虫菌株或物种之间的差异以及人类宿主遗传背景的差异已被牵连。粘膜皮肤利什曼病(MCL)尤其令人衰弱，主要与万年虫亚属内物种的感染有关。在实验动物模型中，感染某些利什曼原虫株的dsRNA病毒的存在，通过依赖TLR3的炎症反应，增加了病理和转移。LRV是唯一已证实的与疾病严重程度增加和转移有关的毒力因子，这两个特征与人类MCL有关，主要由巴西乳杆菌(LBR)引起。最近，我们利用基因组序列数据鉴定了一株同时表现LRV和MCL的巴西乳杆菌分支，并与利什曼原虫LRV病假说一致，LRV分离株显示出极高的毒力。因此，这一分支将成为详细研究巴西钩端螺旋体中LRV的作用和反应的重点(S)。利用一种新的利什曼原虫转移小鼠模型，将产生克隆的LRV和LRV-株，并确定它们的感染性和转移潜力。我们将利用RNAi靶向LRV dsRNA基因组的方法，开发一种令人兴奋的新方法来产生等基因LRV系，以剖析LRV的作用和对LRV的响应。在目标2中，我们重点研究哺乳动物对LRV感染的利什曼原虫的反应，包括对LRV与LRV-L.braziliensis和L.guyanensis的免疫反应的详细表征。最近的数据表明，RNA干扰途径与巴西钩端螺旋体的感染性有关，这将在古扬氏钩端螺旋体中使用RNAi缺失的ArgAerte(AGO1)敲除进行测试。我们的研究表明，LRV和RNAi之间可能存在一个或两个物种的机械性联系。这将通过使用携带相关dsRNA信号通路变化的小鼠，用不同LRV或RNAi状态的寄生虫挑战来从机械上证实和探索这一点。最后，我们将重点介绍利什曼原虫对LRV的反应，包括基因表达的变化、LRV对RNAi活性的调控或RNAi途径对病毒siRNA形成的调控，以及siRNA生物学。我们将探索产生具有感染性的LRV克隆的可能性，这将从根本上提高我们剖析LRV在利什曼原虫生物学中的作用的能力。

DESCRIPTION (provided by applicant): Infections with the protozoan parasite Leishmania exhibit a variety of disease pathologies, ranging from mild cutaneous to fatal visceral to disfiguring mucocutaneous disease. The basis for this diversity is not understood; differences amongst strains or species of Leishmania have been implicated as well as differences in the human host genetic background. Mucocutaneous leishmaniasis (MCL) is especially debilitating, associated primarily with infections by species within the Viannia subgenus. In experimental animal models the presence of a dsRNA virus infecting some strains of Leishmania confers elevated pathology and metastasis, mediated by a TLR3-dependent inflammatory response. LRV is the only proven virulence factor associated with increased disease severity and metastasis, two hallmarks relevant to human MCL, caused primarily by L. braziliensis (Lbr). Recently we used genome sequence data to identify a L. braziliensis clade manifesting both LRV and MCL, and consistent with the Leishmania LRV-disease hypothesis, an LRV+ isolate exhibited greatly elevated virulence. This clade will thus be the focus of detailed studies of the role(s) and responses to LRV in L. braziliensis. Clonal LRV+ and LRV- lines will be generated and their infectivity and metastatic potential defined, making use of a new mouse model for Leishmania metastasis. We will exploit an exciting new approach for generating isogenic LRV- lines to dissect the roles of and response to LRVs, utilizing RNAi targeting of the LRV dsRNA genome . In Aim 2 we focus on the mammalian host response to LRV-infected Leishmania, including a detailed characterization of the immune response to LRV+ vs. LRV- L. braziliensis and L. guyanensis. Recent data implicate the RNA interference pathway in L. braziliensis infectivity, which will be tested in L. guyanensis using RNAi-null Argonaute (AGO1) knockouts. Our studies suggest the possibility of a mechanistic link in either or both species between LRV and RNAi. This will be confirmed and explored mechanistically through the use of mice bearing alterations in relevant dsRNA signaling pathways, challenged with parasites of varying LRV or RNAi status. Lastly we will focus on the Leishmania response to LRV, including changes in gene expression, modulation of RNAi activity by LRV or of viral siRNA formation by the RNAi pathway, and siRNA biology. We will explore the possibility of generating an infectious LRV clone, which would radically improve our ability to dissect the role of LRV in Leishmania biology.