Sequences information in Fig 2.

<div><p>We conducted a large-scale survey of arboviruses in mosquitoes and biting midges to assess the presence and spread of mosquito-borne pathogens currently circulating on Hainan Island, China. RT-PCR assays were used to detect the arbovirus species, distribution, and infection rates in mosquitoes and biting midges. Cell inoculation and high throughput sequencing were performed to isolate the viruses and assemble full viral genomes. Phylogenetic analysis was conducted to identify the viral genotypes and evolutionary relationships with known viruses. During 2019–2023, 32,632 mosquitoes and 21,000 biting midges were collected from 14 of 18 cities/counties on Hainan Island. Japanese encephalitis virus (JEV) was detected in <i>Culex</i> mosquitoes from five cities/counties, where the minimum infection rate (MIR) was 1.6 (0.6–2.6) per 1,000 females tested. Tembusu virus (TMUV) was detected in <i>Culex</i> mosquitoes from three cities/counties with MIR1.0 (0–2.2) per 1,000. Getah virus (GETV) was detected in <i>Armigeres</i> mosquitoes from Qionghai city with MIR 7.1 (0–15.2) per 1,000. Oya virus (OYAV) and Bluetongue virus (BTV) were detected in biting midges from Wanning city with MIRs of 0.4 (0–1.2) and 0.1 (0–10.2) per 1,000, respectively. Three JEV strains were isolated and clustered within the genotype I group, which is presently the dominant genotype in China. Three TMUV strains were isolated for the first time on Hainan Island that belonged to Cluster 3. Three isolated GETVs were identified as Group 3. BTV was reported for the first time on Hainan Island, and the complete genome for one BTV strain was successfully assembled, which was classified as serotype 1 based on the sequences of segment 2. These results stress the need to develop adequate surveillance plan measures to better control the public health threat of arboviruses carried by mosquitoes and biting midges in local regions.</p></div>