Background: Leishmania RNA Virus-1 (LRV-1) is a double stranded RNA virus identified in strains of the Viannia subgenus in Latin America. Virulence factors (VFs) are gene products that augment pathogenicity of an organism, and are mostly constitutively expressed. Both LRV-1 and VFs are known to modulate the host immune response to Leishmania infection. We aimed to quantify RNA transcripts of known VFs in Leishmania to understand the influence, if any, of LRV-1. Methods & Materials: Five clinical cultures of Leishmania Viannia spp. housed in our biobank were species identified by PCR, RFLP analysis, and Sanger sequencing. Cultures were inoculated into a macrophage model and supernatants were collected at 24- and 48-hrs. Total cellular RNA was extracted at baseline and post-macrophage infection, and cDNA was reverse transcribed. LRV-1 status was confirmed by qPCR and quantified using the 2-ΔΔ CT method. qPCR assays were performed using the 18S gene as a reference for the following RNA transcripts: zinc-metalloproteainse (gp63), mannose phosphate isomerase (mpi), cysteine proteinase B (cpb), and heat shock proteins 23, 70, 83, and 100 (hsp23, hsp70, hsp83 and hsp100, respectively). Results: Cultured isolates were derived from male patients with a median age of 22 years (range 9-80 years) and were species identified as: L. V. braziliensis (n = 1, 20%) and L. V. panamensis(n = 4, 80%). Of 5 cultured isolates, 3 (60%) were positive for LRV-1. RNA transcript expression between LRV-1 positive strains did not differ from LRV-1 negative strains for the following VFs at baseline: pooled VFs (p = 0.20), cpb (p = 0.40), gp63 (p = 0.20), mpi (p = 0.20), hsp23 (p = 0.20), hsp70 (p = 0.20), hsp83 (p = 0.20), and hsp100 (p = 0.20). Similarly, transcript expression did not differ by LRV-1 status post macrophage infection for the following: pooled VF at 24- and 48-hrs, respectively (p = 0.80 and p = 0.40), CPB at 48 hrs (p = 0.20) and MPI at 48 hrs (p > 0.99). Conclusion: VF RNA transcript expression did not differ significantly between LRV-1 positive and negative clinical strains of Leishmania at baseline and post-macrophage infection. Our findings suggest that LRV-1 does not contribute to enhanced virulence in our analyzed VFs, however further exploration of additional VFs, particularly those specific to New World or the Viannia subgenus is warranted.