Integrated human seroprevalence study and entomological surveillance of West Nile virus in Cádiz, Spain

In Europe, West Nile virus (WNV) and Usutu virus (USUV) are emerging mosquito-borne orthoflaviviruses, which pose a growing threat to public health. Although outbreaks have been reported across the Mediterranean basin, data on population-level immunity and viral diversity in Spain remain limited. This study aimed to evaluate temporal changes in human exposure to WNV and to characterize circulating orthoflaviviruses in Cádiz, southern Spain, using an integrated seroprevalence in humans and entomological surveillance. The study was conducted in Cádiz, a recognized area of WNV transmission, combining a human seroprevalence study with genomic surveillance in mosquitoes. Human serum samples collected in 2015 and 2022 from 10 municipalities were screened for WNV antibodies by enzyme-linked immunosorbent assay. Positive samples were confirmed through viral neutralization tests against WNV and other related orthoflaviviruses, such as Usutu, dengue, tick-borne encephalitis and Zika viruses. Simultaneously, mosquitoes were trapped in the same region between 2022 and 2024 and analyzed by RT–PCR to detect and characterize circulating orthoflaviviruses. Viral neutralization tests confirmed the presence of neutralizing antibodies against WNV in the local population, indicating exposure and activation of a specific immune response. We detected specific WNV antibodies in three municipalities in 2015 and nine in 2022 with seroprevalence increasing from 0.2% (2015) to 0.9% (2022). WNV-positive mosquitoes were detected in some areas, where human and animal cases have been reported. In addition, two USUV-positive mosquito pools belonging to the Africa-3 lineage were detected. This study provides the first integrated serological and genomic evidence of WNV transmission and USUV circulation in Cádiz. The combined detection of WNV-specific antibodies in humans and viral RNA in mosquitoes confirms the endemicity of WNV, and it reveals the co-circulation with USUV, a related orthoflavivirus, described for the first time in this area. These findings highlight the importance of One Health-based surveillance programs using mosquitoes and characterizing local immune responses to better understand the orthoflavivirus transmission dynamics in endemic regions.