Identification of novel CRESS-DNA viruses in the human vaginal microbiome

Introduction: Circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS-DNA) viruses are widely distributed across diverse hosts and environments, yet their diversity within the human vaginal virome remains poorly characterized. This study aimed to investigate the presence, diversity, and evolutionary relationships of CRESS-DNA viruses in the human vaginal niche. Methods: Viral metagenomic sequencing was performed on 24 pooled vaginal swab libraries derived from women with and without vaginitis. After host sequence removal and quality control, de novo assembly and viral identification were conducted. Candidate viral genomes were curated based on genomic features, followed by functional annotation, phylogenetic analysis using Rep protein sequences, and genome-wide pairwise nucleotide identity comparisons. Results: A total of five CRESS-DNA viral genomes were identified, including four complete and one nearly complete circular genomes. All genomes exhibited canonical architectures, encoding Rep and Cap proteins and containing conserved HUH endonuclease and superfamily 3 helicase motifs. Phylogenetic analysis placed these viruses within the orders Rohanvirales, Ringavirales, Cirlivirales, and Cremevirales, representing multiple distinct evolutionary lineages. Genome-wide pairwise identity analysis showed that all identified viruses fell below established species- and genus-level thresholds, indicating that they represent novel taxa. Comparative analyses further revealed substantial divergence from known environmental and vertebrate-associated viruses. Discussion: These findings expand the known diversity of CRESS-DNA viruses in the human vaginal virome and highlight their broad evolutionary diversity. The detected viruses likely represent diverse ecological origins rather than stable host-specific infections, and no clear association with vaginitis was observed. This study provides new insights into the evolutionary landscape of CRESS-DNA viruses in the human reproductive tract and underscores the need for further investigation into their biological roles and potential health implications.