Early-warning surveillance of West Nile and Usutu viruses in wetland and larval habitat waters and in mosquito excreta using digital PCR

West Nile virus (WNV) and Usutu virus (USUV) are mainly avian mosquito-borne pathogens, increasingly cause human and equine disease in temperate regions. Mostly asymptomatic, current surveillance in France is reactive, based on clinical and veterinary reports, delaying signals for vector control and preventative interventions. We implemented in Southern France, in 2024 and 2025, a proactive surveillance methodology combining molecular xenomonitoring of mosquito excreta and environmental water detection of viral RNA in wetlands and mosquito larval habitat, through a multiplex reverse transcription digital PCR workflow. The analytics process was characterised through the limits of blank, detection, and quantification. Low-level positives were confirmed by Sanger sequencing. WNV and USUV RNA repeated detections revealed previously unrecognized, cryptic circulations up to 19 weeks before the first WNV human cases and equine alert. Environmental surveillance represents a paradigm shift for early detection of emerging Culex -borne arboviruses and a key enabler of proactive and effective One Health responses.