Life-history traits associated to West Nile virus seroprevalence in wild birds: Lessons from the recent outbreaks in Spain.

West Nile virus (WNV) is a vector-borne zoonotic pathogen maintained in a bird-mosquito cycle, occasionally infecting humans and horses. It is considered a re-emerging pathogen in Europe, with recurrent outbreaks recently reported in Spain. However, the ecological and evolutionary factors driving spatio-temporal variation in WNV incidence remain unclear. To address this, we analysed a long-term dataset of the presence of WNV neutralizing antibodies in 19,105 serum samples from 48 avian species. Blood samples were collected from birds in southern Spain over two decades, between 2003 and 2024, encompassing periods with varying human WNV incidence. Using phylogenetically informed models, we analysed the ecological and life-history traits associated with WNV seroprevalence in wild birds. Age, phylogeny, locality, and year explained significant variation in seroprevalence. Among first-year individuals, urban-associated species showed higher WNV seroprevalence than species inhabiting forest or shrubland habitats, and it varied significantly based on primary lifestyle. In adults, seroprevalence also correlated positively with body mass and negatively with beak length. Notably, WNV seroprevalence in urban birds increased significantly during years with human outbreak, whereas no such association was found in birds from non-urban habitats. These results suggest that the complexity of WNV cycles may be partly unravelled by incorporating data on avian ecology and phylogeny. In addition, we provide evidence of active virus circulation inside the cities during WNV human outbreaks and suggest the potential of urban bird species as epidemiological sentinels for human outbreaks. Overall, this study underscores the importance of a One Health approach to understand vector-borne zoonotic pathogen dynamics and to inform effective surveillance and control strategies.