Tracking the urban spread of Usutu virus in southern France: Detection across biological and environmental matrices.

The Usutu virus, a neurotropic Orthoflavivirus transmitted by mosquitoes, was first identified in South Africa in 1959 and has progressively spread across Europe over the past two decades. This virus follows an enzootic cycle between mosquitoes and birds, leading to periodic outbreaks that have caused significant bird mortality. Although primarily an avian pathogen, Usutu virus can occasionally infect humans and other mammals who act as incidental or dead-end hosts. The repeated avian epizootics observed in Europe in the last two decades raise concerns about potential zoonotic risks, even though human infections remain rare. In most cases, human infection is either asymptomatic or results in mild symptoms. However, in some instances, Usutu virus has been linked to severe neurological conditions, including encephalitis and meningoencephalitis. The Occitanie region in the south of France is particularly vulnerable to this threat due to its ecosystem, which harbors both competent mosquito vectors and numerous avian hosts that act as amplifying hosts for the virus. We investigated the urban circulation of Usutu virus in the city of Montpellier, where the first human case of infection by this virus in France was previously identified. To assess the presence of Usutu virus, we conducted a repeated cross-sectional study using serological (ELISA, microneutralization) and molecular (RT-qPCR) analyses of captive avifauna, including a longitudinal study of captive birds at the Montpellier zoological park between 2016 and 2024. Additionally, in 2024, we completed our study with avian cloacal swabs, pigeon droppings, rat blood, mosquito faeces, and environmental water samples (dPCR). Our findings revealed active circulation of the Usutu virus in the urban environment over multiple years. Furthermore, we demonstrated the feasibility of detecting the virus in droppings and environmental waters, highlighting the potential of environmental surveillance as a non-invasive and large-scale method. This study contributes to a better understanding of Usutu virus circulation and highlights its established presence in urban areas.