Leishmania RNA viruses and pathogenesis

项目摘要。 以前我们在实验动物模型中发现，感染LRV 1的dsRNA病毒的存在， Guyanensis利什曼原虫菌株赋予升高的病理学和转移，由TLR 3依赖性 炎症反应。这一发现与人类的相关性是由最近的发现确立的， 感染携带LRV 1的利什曼原虫的患者也显示出药物治疗失败的频率升高 病理学和细胞因子反应增加。在这里，我们将这些研究扩展到L。巴西，第一个 开发新的动物模型和工具。新发现的类似布尼亚病毒的病毒“TOP”将是一个高 优先级; TOP发生在>95%的所有L。巴西菌株和大多数相关的Viannia 物种大量的初步数据表明，在毒力方面的作用很强，可能超过LRV 1。在 目的1研究LRV 1和TOP依赖的毒力机制。精心挑选的一系列等基因系 显示LRV 1和/或TOP（病毒型）的不同组合， 表征了然后，这些将用于探测LRV 1依赖性毒力的机制， 与类似于I型干扰素诱导的强巨噬细胞反应相关。TOP依赖 毒力似乎通过一种完全不同的机制起作用，不依赖于干扰素， 包括树突状细胞。在目标2中，我们关注分子病毒学，特别是TOP，因为这些研究可能会 为直接抑制这些病毒的努力提供信息。我们确定，TOP是一个高度分化的血统， 布尼亚病毒目的一个新科，称为“利什布尼亚病毒科”。由于这些新的特点正好出现在 利什曼原虫从单种寄生转变为异种/脊椎动物寄生时， 这些结构差异可能有助于TOP致病机制。我们将探索他们的 编码潜力和预测的蛋白质的存在和作用，并使用遗传学方法来功能 测试它们在病毒复制和毒力中的作用。

Project summary. Previously we showed that in experimental animal models, the presence of the dsRNA virus LRV1 infecting strains of Leishmania guyanensis confers elevated pathology and metastasis, mediated by a TLR3-dependent inflammatory response. The relevancy of this finding to humans was established by recent findings that patients infected with Leishmania bearing LRV1 show an elevated frequency of drug treatment failures, as well increased pathology and cytokine responses. Here we extend these studies to L. braziliensis, first by developing new animal models and tools. The newly discovered bunyavirus- like virus “TOP” will be a high priority; TOP occurs in >95% of all L. braziliensis strains examined and the majority of related Viannia species. Extensive preliminary data suggest a strong role in virulence, potentially exceeding that of LRV1. In Aim 1 we will study mechanisms of LRV1- and TOP-dependent virulence. A well-chosen series of isogenic lines showing different combinations of LRV1 and/or TOP (virotypes) will be established and their virulence characterized. These will then be used to probe the mechanism of LRV1-dependent virulence, which is associated with a strong macrophage response similar to that induced by type I interferons. TOP-dependent virulence seems to act through a completely different mechanism, independent of interferon and likely involving dendritic cells. In Aim 2 we focus on molecular virology, especially of TOP, as these studies will likely inform efforts to inhibit these viruses directly. We established that TOP is a highly divergent lineage within a new family of the Bunyavirales termed “Leishbunyaviridae”. Since these novel features arose precisely at the time deep in evolution when Leishmania transitioned from monxenous to dixenous/vertebrate parasitism, these structural differences are likely to contribute to the TOP pathogenic mechanism. We will explore their coding potential and the existence and role(s) of predicted proteins, and use genetic approaches to functional test their role in viral replication and virulence.