Detection of Leishmania RNA Virus in Leishmania Parasites

Abstract

Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis. The endosymbiosis of viruses in microbes is a well-described and prevalent environmental partnership, where viruses offer their cellular host incentives of fitness in exchange for the use of their metabolic machinery. We have recently exposed this as an important factor in certain metastatic leishmaniases of South America, where the nucleic acid of a virus residing within some Leishmania parasites acts as a potent innate immunogen causing a destructive inflammatory response, which worsens disease. Leishmania RNA Virus (LRV) exists within many species of Leishmania as a stable infection; these LRV positive strains have been found throughout South America in cutaneous leishmaniases that are often complicated by the occurrence of infectious metastasis with an underlying hyperinflammatory response. In this report, we describe the use of an anti-dsRNA monoclonal antibody (J2), which specifically recognizes dsRNA in a quantitative and sequence-independent fashion. Refined versions of these methods could be transferred to the field as diagnostic tools for detecting the presence of LRV (or other dsRNA viruses), and potentially assessing the LRV-related risk of complicated cutaneous leishmaniasis.