Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.

Department of Biomedical Sciences College of Medicine Chang Gung University Guishan District Taoyuan 333 Taiwan

Department of Computer Science and Information Engineering College of Engineering Chang Gung University Taoyuan 333 Taiwan

Department of Medical Laboratory Science College of Medical Science and Technology 1 Shou University Kaohsiung 824 Taiwan

Department of Parasitology College of Medicine Chang Gung University Taoyuan 333 Taiwan

Department of Parasitology Faculty of Science Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec Průmyslová 595 252 42 Vestec Czech Republic

Genomic Medicine Core Laboratory Chang Gung Memorial Hospital Linkou Branch Taoyuan 333 Taiwan

Molecular Infectious Disease Research Center Chang Gung Memorial Hospital Linkou Branch Taoyuan 333 Taiwan

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