Serosurvey Reveals Exposure to West Nile Virus in Asymptomatic Horse Populations in Central Spain Prior to Recent Disease Foci

West Nile fever/encephalitis (WNF) is an infectious disease affecting horses, birds and humans, with a cycle involving birds as natural reservoirs and mosquitoes as transmission vectors. It is a notifiable disease, re-emerging in Europe. In Spain, it first appeared in horses in the south (Andalusia) in 2010, where outbreaks occur every year since. However, in 2014, an outbreak was declared in horses in central Spain, approximately 200km away from the closest foci in Andalusia. Before that, evidence of West Nile virus (WNV) circulation in central Spain had been obtained only from wildlife, but never in horses. The purpose of this work was to perform a serosurvey to retrospectively detect West Nile virus infections in asymptomatic horses in central Spain from 2011 to 2013, that is before the occurrence of the first outbreaks in the area. For that, serum samples from 369 horses, collected between September 2011 and November 2013 in central Spain, were analysed by ELISA (blocking and IgM) and confirmed by virus neutralization, proving its specificity using parallel titration with another flavivirus (Usutu virus). As a result, 10 of 369 horse serum samples analysed gave positive results by competitive ELISA, 5 of which were confirmed as positive to WNV by virus neutralization (seropositivity rate: 1.35%). One of these WNV seropositive samples was IgM-positive. Chronologically, the first positive samples, including the IgM-positive, corresponded to sera collected in 2012 in Madrid province. From these results, we concluded that WNV circulated in asymptomatic equine populations of central Spain at least since 2012, before the first disease outbreak reported in this area.