Viral load dependent cellular heterogeneity in Trichomonas vaginalis at the single cell level

Trichomonasvirus (TVV) is a double-stranded RNA virus that persistently infects Trichomonas vaginalis, a protozoan parasite responsible for the most common non-viral sexually transmitted infection. While TVV has been implicated in modulating parasite biology and drug sensitivity, the extent and significance of viral heterogeneity remain unclear. Using ultra-deep single-cell RNA sequencing (scRNA-seq) on three TVV-positive T. vaginalis isolates, we reveal that viral distribution is highly heterogeneous and dynamic, with marked variability in infection status and viral load across individual cells. Time-resolved scRNA-seq datasets confirm that this variability is not static but fluctuates across replicates. By integrating bootstrap-based pseudobulk analysis with viral load-stratified trend analysis, we uncover infection-associated transcriptional changes involving stress response, adhesion, and chromatin regulation. Notably, MLF-like and histone genes are selectively upregulated in cells with high viral loads. These findings redefine current models of TVV-host interactions and lay a foundation for future mechanistic studies of virus-mediated pathogenesis in protozoan parasites.