Pathogenicity assessment of Spanish West Nile virus isolates of lineages 1 and 2 in a Swiss mouse model

West Nile virus (WNV) is one of the most widespread emerging arboviruses in the world. Recently, Europe has undergone a significant increase in WNV incidence and geographic extension, with two genetic lineages, lineage 1 and lineage 2, actively circulating. In Spain, lineage 1 was first identified in 2007, and subsequently spread through the southwest and central regions of the country. Since 2010, it has affected both horses and humans (mainly in southern Spain), including two large human outbreaks of WNV meningoencephalitis in 2020 and 2024. Lineage 2 was first identified in Catalonia (northeastern Spain) in 2017, spreading through this region, primarily affecting birds, but with low incidence in humans and horses. In this study, the infectivity and disease severity of Spanish WNV isolates obtained between 2007 and 2020, representing both the southwestern and northeastern variants, was examined by in vivo inoculation in mice, with the aim of inferring their pathogenicity in mammalian hosts. The results demonstrated that the analysed isolates from northeastern Spain consistently induced lower virulence profiles in mice compared to the isolates from the southwest. Differences in mortality rates, median survival times, and survival curves, the latter being statistically significant allowed the classification of northeastern (lineage 2) and southwestern (lineage 1) Spanish isolates as moderately virulent and highly virulent, respectively. In vitro replication assays did not reveal significant differences between the Spanish isolates. Although biological and genetic differences between species could limit the extrapolation of mice data to other mammals, our findings are consistent with virulence patterns observed in humans and horses in the geographic regions where the examined isolates originated.