Leishmaniasis is the spectrum of diseases that are caused by single cell protozoan parasites of Leishmania
genus. Second only to malaria, leishmaniasis is one of the most important human parasitic diseases in the world. Affecting over 12 million people across the globe with 1.6 million new cases annually, it takes lives of 30 000 people each year while 350 million are living at risk. Endemic to tropical and subtropical regions, the disease is spread by phlebotomine sandflies that transmit the parasites during their blood meal. The clinical manifestations may differ depending on a variety of factors such as parasite species, environmental conditions and the competence of the host immune system. The broad spectrum of pathologies spans from a relatively mild cutaneous leishmaniasis (CL) with skin lesions localized at the site of the sandfly bite, to a very severe and potentially lethal visceral leishmaniasis (VL) when parasites invade internai organs. Most of the CL cases are self-healing due to strength of the immune system that is the natural guardian and protector of human health. One of the main weapons of antimicrobial immune ammunition is inducible nitric oxide synthase (iNOS) which is essential for production of nitric oxide (NO). This molecule is critical to control many pathogenic infections including cutaneous forms of leishmaniasis. Being an extremely successful pathogen, Leishmania has evolved to evade and even to capitalize on complex and stratified host immune system, and thus persevering and parasitizing on humans for thousands of years. Among the plethora of dodging and subversion techniques, one relies on a symbiotic relationship between some species of Leishmania and dsRNA viruses, such as Leishmania RNA virus 1 (LRVl), that are harbored inside the parasites. Presence of LRVl serves as an aggravating factor contributing to development of CL complications such as mucocutaneous leishmaniasis (MCL) and disseminated cutaneous leishmaniasis (DCL). These pathologies are characterized by metastatic highly inflamed lesions coming from primary sites of infection to the mucosal nasopharyngeal tissues in the case of MCL, and other skin areas in the case of DCL. The last decade's research has unraveled that recognition ofLRVl by innate immune sensor Toll­ like receptor 3 (TLR-3) triggers proinflammatory signaling cascades leading to disease exacerbation in mice. In particular it results in production type I interferons (type I IFNs) that govern the deterioration phenotype. Additionally, LRVl-TLR-3 interaction leads to a prolonged survival of a host cell, therefore allowing parasites to persist within it. Moreover, the innate recognition of LRVl contributes to disease metastasis in immunocompromised murine mode! and inefficient treatment and relapse in human patients. While these studies underline mutualistic nature between a parasite and its viral tenant, potential host­ protective and parasite-detrimental outcomes of innate recognition of LRVl have not been investigated. In this study we describe for the first time that LRVl-TLR-3-type I IFN signaling can trigger parasite­ detrimental iNOS. We show that Leishmania is able to mitigate this process by minimizing iNOS expression and even to capitalize on produced NO in order to facilitate its dissemination and metastasis. Our data provide new insights on the complex tripartite interaction between a host cell, a parasite and its virus and support the existing evidence on how this relationship benefits Leishmania. Detailing these mechanisms is crucial to combat leishmaniasis in the future especially considering limitations of the current drugs in general and in LRVl-associated infections in particular. With this in mind we develop a method for automated image-based high-throughput drug screening (HTDS) against LRVl-containg parasites that will accelerate search for new therapeutics against this devastating disease.