Widespread circulation of West Nile and Usutu viruses in sedentary and migratory avifauna: A two-year study (2024-2025) of active surveillance in South of France.

West Nile virus (WNV) and Usutu virus (USUV) are neurotropic Orthoflaviviruses sharing a similar enzootic transmission cycle primarily involving Culex pipiens mosquitoes as vectors and birds as amplifying hosts. First identified in Africa, both viruses established endemicity across Europe over the past two decades, most likely introduced and spread by migratory bird species along Mediterranean flyways. In avian species, infection outcomes range from subclinical to fatal neuroinvasive disease, varying by viral strain, host immunity, and species sensibility. Southern France emerges as a key hotspot for the circulation of these viruses, supported by diverse avian habitats conducive to year-round viral maintenance. This study investigated the RNA prevalence of WNV and USUV in more than 2500 sedentary and migratory wild birds from this region during 2024-2025 using molecular surveillance. Samples were collected using mists net and bird boxes, across multiple passerine and non-passerine taxa, spanning wetlands, urban fringes, and agricultural zones. Our analyses revealed widespread viral detection across diverse species, mainly among passerines such as great tits, house sparrows, and barn swallows with USUV detected at higher rates than WNV in both study years. Overall RNA prevalence was markedly higher in 2024 than in 2025, potentially reflecting climatic or ecological drivers. These results highlight the pivotal role of mixed avifauna in arbovirus dynamics within Mediterranean Europe and emphasize the necessity for integrated, year-round surveillance targeting high-risk species and habitats.