Jorge Rojas-Vargas, University of Western Ontario

Jorge A. Rojas-Vargas1,2, Ainslie C. Shouldice1, Hanna Wilcox1, Bern Monari3,4, Jacques Ravel4, Jessica L. Prodger1,5 1Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, Canada; 2Department of Biology, Western University, Canada; 3Center for Advanced Microbiome Research and Innovation, Institute for Genome Sciences, University of Maryland School of Medicine, USA; 4Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, USA; 5Department of Epidemiology and Biostatistics, Schulich School of Medicine and Dentistry, Western University, Canada

Discovering & Evolving

The viral component of the neovaginal microbiome in transfeminine individuals with penile inversion vaginoplasty remains largely uncharacterized, limiting our understanding of virus-host dynamics in this unique anatomical niche. We performed shotgun metagenomic sequencing on 44 neovaginal samples to investigate viral diversity and its association with reconstructed bacterial genomes. After host read removal, quality control and de novo assembly with MEGAHIT, viral sequences were identified using VirSorter2 and quality-assessed with CheckV. Taxonomic annotation of the medium and high-quality contigs with viral genes was performed against the NCBI Viral database (2024) using BLASTN and MEGAN6. In parallel, metagenome-assembled genomes (MAGs) were reconstructed using CONCOCT, MetaBAT, and MaxBin, curated with CheckM, and phylogenetically resolved using UBCG2 and IQ-TREE2. Eight contigs corresponding to Human Papillomavirus (types 15, 39, 42, 43, 84, and 89) were detected in four participants, highlighting the presence of clinically relevant eukaryotic viruses. In addition, 72 prophage contigs were identified, predominantly affiliated with Siphoviridae (n=31) and Microviridae (n=29) families. A total of 620 high-quality MAGs (completeness >70%, contamination <10%) were reconstructed, largely representing Peptoniphilus, Prevotella, Porphyromonas, Mobiluncus, Anaerococcus, Campylobacter, Hoylesella, and Varibaculum genera. Notably, the prophage repertoire mirrored the dominant bacterial taxa reconstructed as MAGs, suggesting structured virus-host associations within the neovaginal ecosystem. These findings reveal a diverse and ecologically structured virome embedded within a defined bacterial community and provide a genomic framework for exploring viral evolution and host interactions in transfeminine health.