Virome Diversity and Host-Associated Heterogeneity in Mosquitoes and Biting Midges Across Yunnan, China

Mosquitoes and biting midges harbor diverse viruses, but the factors shaping their viromes remain incompletely understood. We analyzed 35 pooled libraries from seven host groups across 15 locations in Yunnan, China, sampled in June 2025, using total-RNA sequencing, viral annotation, phylogenetics, and community analyses. We retained 1295 viral contigs grouped into 221 annotation-defined viral taxonomic units (VTUs) spanning 24 families; 107 VTUs were supported only by protein homology. Banna virus-related sequences, an Aedes-associated flavivirus, and four putative components of a divergent Jingmenvirus-related candidate were identified. Host group was associated with family-level virome composition after adjustment for location and sequencing depth (R2 = 0.213, p = 0.0052), and the association persisted across alternative feature definitions. Culex tritaeniorhynchus showed the greatest cumulative VTU richness, although its ranking changed when library number was equalized. Most VTUs detected in this host occurred at one sampled site, without a significant monotonic distance-decay pattern. This study expands knowledge of mosquito-associated and biting midge-associated viral diversity in Yunnan, identifies representative lineages for further characterization, and documents host-associated differences in the detected viromes.