Metatranscriptomic Survey of the Virome of Culicoides Biting Midges in Scenic Areas of the Beijing-Tianjin-Hebei Region, China

Biting midges are important medical and veterinary vector insects that can transmit a range of zoonotic viruses. The Beijing-Tianjin-Hebei (BTH) region, as the core area in northern China, is rich in tourism resources, but the characteristics of the virome of biting midges in this region remain unclear. A total of 12,152 biting midges individuals were collected, and 7 species of Culicoides were identified. Culicoides punctatus (67.02%) and Culicoides morisitai (19.01%) were the dominant species. All specimens were grouped according to sampling sites and midge species, and a total of 49 sample pools were constructed. Each pool contained 50 individual midges as the standard, and specimens with fewer than 50 individuals were excluded from the analysis. Each sample pool only contained female midges of the same species collected from a single sampling site. Metatranscriptomic sequencing yielded 45,369 preliminary virus-like contigs. After ORF prediction, sequence clustering, and subsequent annotation, 208 predicted viral sequences were obtained for downstream virome analysis. Virome analysis revealed that RNA viruses predominated in the virome, with positive-sense single-stranded RNA ((+)ssRNA) viruses being the most abundant group. Both geographic location and host species significantly influenced viral community composition (PERMANOVA, P < 0.05). Phylogenetic analysis based on curated sequences identified 22 viral species, including 4 known viruses and 18 putative novel viral lineages. This study is the first to systematically reveal the diversity characteristics of the Culicoides virome in the BTH region and discover a variety of putative novel viral lineages, which provides an important scientific basis for the monitoring and prevention of regional arboviruses. The results emphasize the ecological importance of Culicoides as virus hosts, suggesting the need to strengthen ongoing monitoring of vector-borne viruses in this region.