Development of an RT-PCR assay for American Phlebotomine-transmitted Bunyaviricetes and isolation of Pacuvirus (Peribunyaviridae) in dogs with canine visceral Leishmaniasis in Rio de Janeiro, Brazil

Sand fly vectors harbor and transmit Leishmania parasites and other pathogens, including arboviruses, to vertebrate hosts. The geographical distribution of phleboviruses transmitted by sand flies in areas affected by leishmaniasis has prompted research into coinfection in vectors and in Leishmania reservoirs in Europe and Africa. Despite the widespread occurrence of canine visceral Leishmaniasis in Brazil and the identification of several viruses carried by sand flies, the issue of coinfection remains unclear. We developed an RT-PCR-based assay to detect segmented (-) RNA arboviruses from the families Phenuiviridae and Peribunyaviridae in the Americas. This assay was based on a phylogenetic analysis of the genomes of 29 American species within these families. We processed serum and bone marrow samples from 25 dogs with confirmed cases of visceral leishmaniasis in Rio de Janeiro City, Brazil. Samples from two of the dogs were amplified using a set of primers tailored to the predicted amplicon size. DNA sequencing analysis revealed a high identity to members of the Peribunyaviridae family, specifically within the genus Pacuvirus. We successfully cultivated the virus from one of the samples and sequenced its genome, confirming the identification of a virus belonging to the species Pacuvirus pacuiense or a closely related variant. The implications of our results are discussed.