Retrospective Whole-Genome Sequencing and Molecular Epidemiological Analysis of Austrian WNV-2 Veterinary Cases (2008–2025)

The West Nile virus of the lineage 2 (WNV-2) has become endemic in large parts of Europe; however, comprehensive whole-genome sequence datasets documenting long-term national circulation are still sparse. This study aimed for the retrospective genetic characterization of WNV-2 detected in animals in Austria between the first introduction in 2008 and 2025 using whole-genome sequencing. Phylogenetic analyses of the sequences allowed the viral diversity, lineage dynamics and molecular epidemiology to be investigated within a broader European context. Twenty-four Austrian WNV-2-positive samples originating from birds (22), one horse and one alpaca were subjected to in-house whole-genome amplification and Illumina sequencing. The generated genomes were analyzed using maximum-likelihood phylogenies and a European WNV-2 Nextstrain build comprising approximately 1000 publicly available whole-genome sequences. Whole-genome sequences were successfully recovered for all selected samples, indicating a reliable whole-genome amplification method for the chosen Austrian strains. The phylogenetic analysis identified three different WNV-2 subclades circulating in Austria (D2, E1 and E2) and revealed multiple periods of subclade co-circulation. Furthermore, Austria and Hungary were inferred as important geographic locations in the diversification and dissemination of WNV-2 in Europe. These findings demonstrate the dynamic circulation as well as the repeated introduction of WNV-2 subclades into Austria and highlight the value of whole-genome sequencing surveillance for monitoring lineage turnover and improving the understanding of WNV-2 evolution across Europe.