PROJECT SUMMARY Trichomonas vaginalis viruses (TVVs) are double stranded RNA viruses that persist in the obligate human parasite Trichomonas vaginalis (Tvag). Tvag is the causative agent of the human disease trichomoniasis, the most common nonviral sexually transmitted infection worldwide. Infection with TVV-containing parasites is thought to increase the severity of this disease and its complications by increasing the pro-inflammatory response by the human superhost. There are five known species in genus Trichomonasvirus (TVV1, TVV2, TVV3, TVV4, and TVV5), but most experiments to date have been performed using purified TVV1 virions or TVV1-containing isolates, due in part to the greater availability of Tvag isolates singly infected with TVV1. TVVs do not have an extracellular lifecycle and instead are transmitted vertically as the parasite divides. Further, multiple TVV species can coinfect a singular trichomonad. Thus, studying the effect of TVVs on the parasite and the human superhost has been difficult, as uninfected trichomonads cannot be infected to create isogenic TVV+ vs. TVV- strains. Using the nucleoside analog 2’-C-methylcytidine (2CMC), which has been recently reported to clear trichomonads of TVV-infection, we have generated isogenic pairs of cured and uncured strains generated from the same parent isolate. With these isogenic pairs, we propose to test our hypothesis that T. vaginalis maintains a persistent viral infection, which confers a fitness advantage to the protozoan and increases virulence to the human superhost. Thus far, we have performed high throughput RNA-sequencing with one such isogenic pair to measure differential gene expression. In doing so, we have identified several candidate genes that may be involved in limiting viral replication to tolerable levels in order to maintain long-term viral persistence. These differentially expressed genes have largely not been characterized, but some encode factors with homology to known RNA exonucleases and helicases, which we hypothesize might be involved in limiting viral replication. In Aim 1 of this project, we propose to further validate these results in additional isogenic pairs in order to define factors Tvag uses to maintain a stable trichomonasvirus infection. In Aim 2, we propose to determine impacts of TVV infection on Tvag proliferation, morphology, and adherence to human cells. We hypothesize that TVVs will increase parasite survival and attachment, providing an evolutionary explanation for long-term TVV persistence. Finally, in Aim 3, we propose to compare responses of human cells to infection with TVV+ and TVV- trichomonads across TVV species. We anticipate that TVV+ Tvag strains, regardless of virus species, will elicit stronger inflammatory responses than TVV- strains. Through this work, we hope to uncover valuable information about an important human pathogen and an intriguing model system to study virus–host interactions.