Genomic epidemiology and phylogeographic reconstruction of West Nile virus 2 in Italy from 2011 to 2023

Since its introduction to Europe in 2004, West Nile Virus Lineage 2 (WNV-2) has become endemic, with Italy reporting the highest number of cases each season. In 2022, WNV infections in Italy exceeded those recorded during the major 2018 outbreak-the largest ever reported in Europe. This study investigates the genomic epidemiology of WNV during the 2022 and 2023 transmission seasons. We analyzed 123 environmental samples from wild birds and mosquito pools collected between May and October 2022-2023 in northwestern Italy. All but one sample belonged to Lineage 2; lineage 1 was detected in two bird samples, with one showing co-infection. A total of 98 complete genomes were sequenced. Phylogeographic reconstruction indicated the origin of the main European clade in Hungary in 2004, with introduction into Italy between 2009 and 2010. Most Italian genomes clustered within a single highly supported subclade, with one sampled in the Marche region in 2011 as the outgroup. Continuous phylogeographic analysis suggested the Italian WNV-2 clade originated in 2009 in the area between Emilia-Romagna and Lombardy, followed by east-west spread during 2022-2023. Several mutations were identified, including F49L in the NS2A gene linked to neuronal tropism, and M184V in the NS4B gene, associated with increased pathogenicity. Our results highlight how integrated genomic surveillance of WNV, combining whole genome sequencing and phylogenetic analyses to environmental samples, can support One Health approaches for early detection and risk assessment of arboviral transmission.