Introduction of a European Central-South-Eastern West Nile Virus Lineage 2 Strain in Italy in 2023: Evidence from the First Locally Acquired Neuroinvasive Case in the Calabria Region

West Nile virus lineage 2 (WNV-2) is a growing public health concern in Europe causing West Nile fever or West Nile neuroinvasive disease (WNND) with substantial morbidity and mortality; however, genomic data from southern Italy are limited despite recent expansion of autochthonous transmission. The aim of the study was to characterize the phylogenetic and molecular features of the WNV-2 strain responsible for the first autochthonous human infection reported in Calabria (2023), and two more additional WNND cases detected in 2024. Full WNV-2 genomes were generated from the three cases. Phylogenetic analysis was performed using all publicly available WNV sequences up to September 2025. Amino acid changes in the polyprotein were compared with known WNV-2 lineage and sub-lineage signatures. The three sequences formed a monophyletic group within sub-lineage WNV-2a, clustering with strains circulating in Central-South-Eastern Europe and showing closest affinity to Hungarian sequences. Non-synonymous substitutions characteristic of the Hungary 578/10 strain (NS2B-119I, NS4B-14G, NS4B-49A, and NS5-298A) were identified and were absent from Central-Northern-Western European and previously reported Italian sequences. Additional substitutions (E-159T, E-399R, and NS3-249P) corresponded to signatures from a fatal WNV-2 infection in a Great Grey Owl in Slovakia. Our study provides the first report of Central-South-Eastern European WNV-2 circulation outside Eastern Europe, supporting its likely spread through the Balkans into Italy by 2022. These findings underscore the rapid spread of WNV-2 in newly affected areas and highlight the critical need for sustained molecular surveillance.