Mosquito- and biting-midge-borne arboviruses in Western Yunnan’s border region, China

BACKGROUND: Arboviruses that infect humans and other mammals pose significant public health challenges worldwide, particularly in developing countries. The border region of Yunnan Province, a key entry point for imported arboviruses into China, is characterized by a subtropical climate and a rich diversity of mosquito and biting midge species, making it a high-risk area for arbovirus transmission. While some studies have focused on mosquito-borne viruses in this region, research on midge-borne viruses, particularly those with zoonotic relevance, remains limited. This study aimed to systematically investigate the diversity of arboviruses in both vector groups and to address critical gaps in the assessment of spillover risk. METHODS: In July 2018, mosquitoes and biting midges were collected from Mangshi, Yingjiang, Lushui, and Tengchong in the border region of western Yunnan Province, China. Following morphological classification, specimens were pooled for virus isolation using BHK-21 and C6/36 cells. Viral isolates were identified by reverse transcription polymerase chain reaction (RT-PCR) using virus-specific primers, and sequence analyses were performed using Clustal X, DNASTAR, and MEGA-X. RESULTS: A total of 8581 mosquitoes (8 species in 4 genera) and 18,298 biting midges (19 species) were collected. Culex tritaeniorhynchus dominated the mosquito populations in Mangshi (92.11%) and Yingjiang (97.23%), whereas Anopheles sinensis was predominant in Lushui (44.01%) and Tengchong (59.23%). Biting midge composition varied among regions, with Culicoides tainanus predominating in Mangshi (59.79%) and C. arakawai in Lushui (48.11%). In total, 189 viral strains representing 10 virus species in 8 families were isolated. Tibet orbivirus (TIBOV) was the most widely distributed virus (83 strains), occurring in all four regions, with biting midges accounting for most isolates (50/83 strains). Biting midges also contributed substantially to arbovirus diversity, yielding 13 Yunnan orbivirus (YUOV) strains, 4 Banna virus (BAV) strains, and the only Akabane virus (AKAV) isolate. In contrast, mosquitoes mainly harbored insect-specific viruses (ISVs), including 53 Manglie virus (MAV) strains. Novel viruses such as Culex-originated Tymoviridae-like virus (CuTLV) and Armigeres iflavirus (ArIFV) were identified in both vectors. These findings highlight biting midges as important yet understudied vectors, harboring distinct zoonotic and vector-associated viruses in this border region. CONCLUSIONS: This study demonstrates the circulation of diverse arboviruses in both mosquitoes and biting midges in the border region of western Yunnan Province. Notably, biting midges harbored multiple arboviruses, including several viruses with zoonotic potential or known pathogenicity, such as Tibet orbivirus (TIBOV), Yunnan orbivirus (YUOV), Banna virus (BAV), and Akabane virus (AKAV). These findings emphasize the important role of biting midges, alongside mosquitoes, in maintaining and potentially spreading these viruses in the local arboviral ecosystem. The study underscores the importance of including midge-borne viruses in future surveillance efforts, as they contribute substantially to the diversity and epidemiology of arboviral transmission in the region, with potential implications for public health.