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.