Integrated human seroprevalence study and entomological surveillance of West Nile virus in Cádiz, Spain
9 affiliations
- 1Centro Nacional de Microbiología, Instituto de Salud Carlos III, CNM-ISCIII, Madrid, Spain
- 2Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
- 3Escuela de Doctorado, Universidad Autónoma de Madrid, Madrid, Spain
- 4Hospital Universitario Puerto Real, Cádiz, Spain
- 5Centro Nacional de Epidemiología, Instituto de Salud Carlos III, CNE-ISCIII, Madrid, Spain
- 6Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain
- 7Estación Biológica de Doñana, EBD-CSIC, Seville, Spain
- 8Servicio de Salud Ambiental, Consejería de Sanidad, Presidencia y Emergencias, Junta de Andalucía, Seville, Spain
- 9Hospital Universitario Puerta del Mar de Cádiz, Cádiz, Spain
Abstract
Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Graphical Abstract:
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13071-026-07696-4.
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BibTeX
@article{guillencalvo2026integrated,
title = {{Integrated human seroprevalence study and entomological surveillance of West Nile virus in Cádiz, Spain}},
author = {Laura Guillén-Calvo and Carolina Freyre-Carrillo and Laura Herrero and Beatriz Fernández-Martínez and Esther Calonge and Mayte Pérez-Olmeda and Nuria Labiod and Francisca Molero and Arantxa Potente and María José Ruiz-López and Carolina Sánchez-Peña and Jordi Figuerola and Manuel Rodriguez-Iglesias and Mª Paz Sánchez-Seco and Ana Vázquez},
journal = {Parasites \& Vectors},
year = {2026},
month = oct,
doi = {10.1186/s13071-026-07696-4},
url = {https://doi.org/10.1186/s13071-026-07696-4},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Integrated human seroprevalence study and entomological surveillance of West Nile virus in Cádiz, Spain
AU - Laura Guillén-Calvo
AU - Carolina Freyre-Carrillo
AU - Laura Herrero
AU - Beatriz Fernández-Martínez
AU - Esther Calonge
AU - Mayte Pérez-Olmeda
AU - Nuria Labiod
AU - Francisca Molero
AU - Arantxa Potente
AU - María José Ruiz-López
AU - Carolina Sánchez-Peña
AU - Jordi Figuerola
AU - Manuel Rodriguez-Iglesias
AU - Mª Paz Sánchez-Seco
AU - Ana Vázquez
PY - 2026
DA - 2026/10/21
JO - Parasites & Vectors
DO - 10.1186/s13071-026-07696-4
UR - https://doi.org/10.1186/s13071-026-07696-4
AB - Background: 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. Methods: 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. Results: 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. Conclusions: 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. Graphical Abstract: Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1186/s13071-026-07696-4.
SN - 1756-3305
ER -