Abstract
West Nile Virus (WNV) lineage 2, primarily endemic to parts of Africa and Europe, has recently emerged as a public health concern in new geographic regions. In 2024, the first autochthonous human case of neuroinvasive disease caused by WNV lineage 2 was identified in Andalusia, Southern Spain. Molecular testing and whole-genome sequencing confirmed WNV lineage 2 as the causative agent. Phylogenetic analysis revealed a close relationship with strains circulating in Central Europe, distinct from previous WNV lineage 2 detections in Spain. Concurrently, WNV lineage 2 RNA was detected in an imperial eagle near the case location, suggesting local viral circulation. This case marks a significant shift in WNV epidemiology in Spain, where lineage 1 has historically been dominant. The findings underscore the expanding range of WNV lineage 2 and the necessity for enhanced vector surveillance, genomic monitoring, and strengthened One Health strategies to mitigate future outbreaks and protect public health.