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

An integrated study of human blood samples and mosquitoes found evidence of West Nile virus exposure and circulation in Cádiz, southern Spain, alongside the first reported detection of Usutu virus in the area. Researchers confirmed West Nile virus-specific neutralizing antibodies in human serum and detected West Nile virus RNA in mosquitoes collected in locations where human and animal cases had previously been reported. The mosquito surveillance also identified two Usutu virus-positive pools belonging to the Africa-3 lineage.

The observational study examined human sera collected in 2015 and 2022 from 10 municipalities in Cádiz. Samples were first screened for West Nile virus antibodies with enzyme-linked immunosorbent assays. Positive samples then underwent viral neutralization tests against West Nile virus and related orthoflaviviruses, including Usutu, dengue, tick-borne encephalitis and Zika viruses.

The investigators used the neutralization results to identify evidence of a West Nile virus-specific immune response in the tested population. West Nile virus-specific antibodies were confirmed in samples from three municipalities in 2015 and nine municipalities in 2022. The reported seroprevalence increased from 0.2% in 2015 to 0.9% in 2022.

These estimates describe the proportion of tested individuals with evidence of exposure, rather than identifying the time, place or circumstances of individual infections. In parallel, investigators trapped mosquitoes in Cádiz between 2022 and 2024 and analyzed the pools using reverse-transcription polymerase chain reaction to detect and characterize circulating orthoflaviviruses. West Nile virus-positive mosquitoes were detected in some sampled areas, including places where human and animal cases had been reported.

Two additional mosquito pools tested positive for Usutu virus, and genetic characterization placed both in the Africa-3 lineage. The authors described the Usutu finding as the first report of that virus in Cádiz, demonstrating circulation of a related mosquito-borne virus in the same regional environment. The combined findings support the authors’ conclusion that West Nile virus is endemic in Cádiz and that Usutu virus is co-circulating locally.

The human and mosquito data address different aspects of transmission: the serology indicates prior immune exposure among sampled residents, while the molecular detections document viral RNA in captured mosquitoes. The components were not sampled over identical periods, however, with human sera collected in two years and mosquitoes sampled across three years, so they do not constitute a continuous year-by-year record. The study also does not establish the precise routes, timing or frequency of human infection.

The authors propose that One Health surveillance combining mosquito monitoring, human immune-response testing and characterization of circulating viruses can help researchers understand orthoflavivirus transmission dynamics in endemic regions.

Subject of Research: Human seroprevalence and mosquito surveillance of West Nile and Usutu viruses in Cádiz, Spain

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

Article References: Guillén-Calvo, L., Freyre-Carrillo, C., Herrero, L., Fernández-Martínez, B., Calonge, E., Pérez-Olmeda, M., Labiod, N., Molero, F., Potente, A., Ruiz-López, M. J., Sánchez-Peña, C., Figuerola, J., Rodriguez-Iglesias, M., Sánchez-Seco, M. P., & Vázquez, A. (2026). Integrated human seroprevalence study and entomological surveillance of West Nile virus in Cádiz, Spain. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07696-4

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07696-4

Keywords: West Nile virus, Usutu virus, Cádiz, seroprevalence, neutralizing antibodies, mosquito surveillance, RT-PCR, orthoflaviviruses, Africa-3 lineage, One Health, Integrated, human