Differential Drug Susceptibility across Trichomonasvirus Species Allows for Generation of Varied Isogenic Clones of Trichomonas vaginalis

<i>Trichomonas vaginalis</i> (Tvag) is a sexually transmitted human pathogen that is commonly infected with strains of one or more of five known species of Trichomonas vaginalis viruses (TVVs), members of genus <i>Trichomonasvirus</i>. TVVs are thought not to have an extracellular phase to their lifecycle and instead to be transmitted vertically from mother to daughter cells. As a result, generation of isogenic virus-positive and virus-negative sets of Tvag clones has been a major barrier to studying interactions between TVVs and their host. Nucleoside analog 2'-C-methylcytidine (2CMC) has been recently reported to clear trichomonads of infections with TVV1, TVV2, and TVV3. We used 2CMC to treat a panel of Tvag isolates that collectively harbor at least one representative strain of each TVV species and thereby provided evidence that infections with TVV4 and TVV5 can also be cleared by 2CMC. Furthermore, our results suggest a newly identified difference in drug susceptibility between TVV species. We took advantage of these susceptibility difference to generate isogenic sets of Tvag clones harboring different combinations of the five TVV species. These results provide both new insight into differences between these species and new avenues for generating tools to study the potential roles of TVVs in Tvag biology.