Winter ecology of the main mosquito vectors of West Nile virus in a human outbreak hotspot in southern Europe.

West Nile Virus (WNV), recognized as the most widespread mosquito-borne flavivirus globally, has re-emerged in Europe since 2018. The virus has become endemic in many southern European countries, although limited data exist on its persistence in mosquito populations during winter. The present study investigates the overwintering ecology, activity, and potential viral maintenance in mosquitoes in an area of southern Spain affected by two large WNV outbreaks in 2020 and 2024. Between November and March in the years 2021-2024, 1145 mosquitoes, primarily Culex pipiens, Cx. theileri, and Cx. perexiguus, were collected by aspiration while resting at urban, peri-urban, and livestock farming sites. Mosquitoes with recent blood meals were captured throughout the winter, with Culex pipiens primarily feeding on birds, an important factor in maintaining the enzootic transmission cycle of WNV. Mean maximum temperature was positively associated with the abundance of the primary WNV vectors (Culex perexiguus and Cx. pipiens), whereas accumulated precipitation during the 15 days prior to sampling showed a negative relationship. We did not find mosquito pools positive for WNV, however, a power analysis indicated that this was expected. Based on the infection rates reported during the 2020 WNV outbreak, between 3000 and 15,000 female mosquitoes would need to be tested to detect a positive pool. These results indicated year-round gonotrophic activity of the main WNV vectors and underscored the necessity for targeted winter surveillance and control in WNV-endemic regions. Enhanced understanding of mosquito overwintering behaviour and feeding dynamics can inform public health interventions to mitigate future WNV outbreaks.