Co-circulation of multiple arboviruses in acute febrile patients in Yunnan, China, identified by metagenomic sequencing

mosquitoes and mainly circulate in tropical and subtropical regions. With global warming, their geographic range is expanding, increasing their threat to public health. Yunnan Province, China, bordering Southeast Asia, is a hotspot for viral importation due to intensive cross-border mobility. However, systematic surveillance for these arboviruses among acute febrile patients remains insufficient. We performed metagenomic sequencing on serum specimens from 990 acute febrile patients at the China-Myanmar border between 2017 and 2023. The pathogens were confirmed by PCR and viral isolation. Phylogenetic and spatiotemporal analyses were used to infer viral origins and transmission dynamics. In this study, a CHIKV outbreak was confirmed in 2019, with strains closely related to those from Myanmar and Thailand. Four DENV serotypes 1-4 were identified, with the predominant serotype varying annually. ZIKV was detected and closely related to strains from Myanmar. Co-infections were identified, including one case each of CHIKV with DENV-1, CHIKV with DENV-3, CHIKV with ZIKV, and DENV-1 with DENV-2. Bayesian spatiotemporal analysis of CHIKV reconstructed global transmission routes, indicating that the 2019 outbreak in China likely originated in India and spread sequentially through Bangladesh, Thailand, and Myanmar. In addition, we also detected enterovirus, hepatitis virus, Saffold virus, and rhinovirus. This study reveals a comprehensive spectrum of pathogens, including the co-circulation of DENV, CHIKV, and ZIKV, and underscores the potential risk of arbovirus importation into China, highlighting the need for strengthened border surveillance. IMPORTANCE: Arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV), are expanding their range and threatening global public health. Yunnan, situated along the China-Southeast Asia border, is highly susceptible to viral introduction. By applying viral metagenomic sequencing to acute febrile patients, this study uncovered a comprehensive spectrum of pathogens and the co-circulation of DENV, CHIKV, and ZIKV. Phylogenetic analyses revealed that arboviruses were closely related to strains from Myanmar and Thailand, indicating possible frequent cross-border viral introductions. Meanwhile, we reconstructed the global transmission pathways of CHIKV through Bayesian spatiotemporal analysis, providing valuable insights for regional prevention and control of arboviruses. These findings demonstrate that Yunnan serves as a critical interface for viral importation and underscore the urgent need to strengthen border surveillance and early warning systems to mitigate the spread of arboviruses.