Feline leukemia virus (FeLV) is a gammaretrovirus with horizontally transmitted and endogenous forms. Domestic cats are the primary reservoir species, but FeLV outbreaks in endangered Florida panthers and Iberian lynx have resulted in mortalities. To assess prevalence and interspecific/intraspecific transmission, we conducted an extensive survey and phylogenetic analysis of FeLV infection in free-ranging pumas (n=641), bobcats (n=212) and shelter domestic cats (n=304). Samples were collected from coincident habitats across the United States between 1985-2018.  FeLV infection was detected in 3.12% puma, 6.25% domestic cat, and 0.47% bobcat samples analyzed. Puma prevalence varied by location, with Florida having the highest rate of infection.  FeLV env sequences revealed variation among isolates, and we identified two distinct clades.  Both progressive and regressive infections were identified in cats and pumas. Based upon time and location of sampling and phylogenetic analysis, we inferred 3 spillover events between domestic cats and puma; 3 puma-to-puma transmissions were inferred in Florida. An additional 14 infections in pumas likely represented spillover events following contact with reservoir host domestic cat populations. Our data provides evidence that FeLV transmission from domestic cats to pumas occurs widely across the US, and puma-to-puma transmission may occur in genetically and geographically constrained populations.

All samples in the dataset were screened for feline leukemia virus with an FeLV-A specific qPCR. The proviral load of positive samples was quantified via qPCR by normalizing against the puma or domestic cat CCR5 housekeeping gene, depending upon the species of the sample. Additionally, we used conventional PCR to isolate the FeLV env gene from FeLV positive samples, cloned the PCR product, and Sanger sequenced the clones for a phylogenetic analysis of the env gene. This dataset identifies all samples screened and which individuals tested positive. For positive animals, this dataset lists the proviral load and if FeLV isolates from the invidual were sequenced. FeLV sequences for this study are published to GenBank.