Introduction and Spread of West Nile Virus in Lazio, Italy, 2025: Molecular Epidemiology and Genomic Characterization

West Nile virus (WNV) is a flavivirus whose cycle involves mosquitoes and avian hosts that occasionally may infect horses and humans as "dead-end hosts." Human infection is mostly asymptomatic, sometimes leading to febrile or neuroinvasive disease. In 2025, Italy experienced its largest epidemic with an unprecedented geographic spread (79 provinces; 52% of cases in central Italy). Here we report whole‑genome sequencing and phylogeographic analysis of 39 WNV lineage 2 samples collected in Lazio and Campania regions between July and September 2025 from humans and animal samples. All 2025 strains belonged to a Southern Italian clade that diversified around 2020, with evidence of earlier circulation in 2024. All the 2025 genomes shared a substitution that lies in envelope protein domain III (K399R), potentially influencing interactions with the viral membrane. The same mutation was previously detected in Eastern European strains, leading to the hypothesis of introductions in Italy via migratory birds from these regions. Phylogeography identified strong viral dispersal routes within Central Italy supporting a pattern of short‑range spread and local overwintering. Overall, results highlight the roles of ecological conditions and cross‑border transmission dynamics in shaping the 2025 Italian epidemic, underscoring the need for integrated genomic/ecological surveillance.