Multiple species of native mosquitoes may be involved in Usutu virus circulation and maintenance in temperate zones

<title>Abstract</title> <p> The introduction of emerging arboviruses into novel regions poses significant challenges for public, animal and ecological health. Usutu virus (USUV), first detected in the United Kingdom in 2020, has since become endemic in Southeast England. This study aimed to elucidate the role of native, temperate, mosquitoes in the transmission dynamics and maintenance of USUV. From March 2023 to February 2025 inclusive, 32,054 mosquitoes representing 21 species were collected from sites across central and southern England and screened for USUV RNA. Usutu virus RNA was detected in 116 (3.2%) of 3,637 mosquito pools across 13 species of five genera. <italic>Culex pipiens s.l.</italic> was the most frequently USUV-positive species, while the potential bridge vectors <italic>Cx. modestus</italic> and <italic>Anopheles claviger</italic> also exhibited notable prevalence. Phylogenetic analysis confirmed co-circulation of two lineages, Africa 3.2 and Europe 3, in multiple mosquito species within the same geographic regions. Finally, we show that prolonged periods of warmer weather could have promoted viral transmission and disease occurrence. These findings highlight the complexity of USUV transmission ecology in temperate zones, demonstrating the value of high-throughput vector screening for arboviruses to improve understanding of the underlying maintenance cycles of USUV circulation and better inform future risk assessments. </p>