Absence of West Nile and Usutu Viruses Persistence in Overwintering Mosquitoes of Northeastern France: Insights from Cold-Season Surveillance

Emerging arboviruses of Orthoflavivirus genus such as West Nile virus (WNV) and Usutu virus (USUV), primarily transmitted by Culex mosquitoes, pose significant public health threats due to their ability to cause severe neurological diseases in humans and animals. While studies in North America and Central Europe have shown that these viruses can persist in overwintering mosquitoes, their role in viral maintenance during the cold season in northeastern France remains unknown. This study aimed to assess whether overwintering female mosquitoes in this region could harbor WNV or USUV during the cold season, potentially maintaining viral circulation until the following transmission season. Between October 2021 and February 2024, a total of 10,617 overwintering female mosquitoes were collected in various types of habitats, across five departments in northeastern France. The most common species was Culex pipiens (88%). Mosquitoes were grouped into 1,121 pools (1–10 individuals each) and tested by real-time RT-PCR for WNV, USUV, and other flaviviruses using a pan-Flavivirus NS5-targeting assay. All pools tested negative, indicating no evidence of viral RNA in overwintering females. These results suggest that overwintering female mosquitoes in northeastern France do not act as reservoirs for WNV or USUV, and do not contribute to their overwintering maintenance.