Emergence of Usutu virus in Southern Italy: phylogenetic profiling of potential introduction and dissemination routes.

Usutu virus (USUV) is an emerging mosquito-borne flavivirus closely related to West Nile virus (WNV), sharing ecological niches and transmission cycles involving Culex mosquitoes and wild birds. In Italy, USUV was first detected in 1996 and has become endemic in northern regions, whereas, to date, no cases have been reported in southern Italy. In August 2025, a yellow-legged gull (Larus michahellis) from the provinces of Salerno for passive surveillance and a Eurasian jay (Garrulus glandarius) during active surveillance from Caserta (southern Italy), were examined. Heart, spleen, kidney, and brain tissues were collected for virological investigation. Real-time RT-PCR assays targeting West Nile virus and Usutu virus were performed and USUV RNA was detected in both avian specimens, while samples tested negative for WNV. Next, entomological surveillance was conducted on both sites and a mosquito pool collected near the yellow-legged gull carcass tested positive. Positive samples were subjected to Sanger sequencing of the NS5 gene. BLAST and phylogenetic analyses revealed strain belonged to lineage Europe 2, sub-group 2-A showing 99.6% nucleotide identity with northern Italian strains circulating between 2018 and 2023 and 99.8% identity with northern European strains circulating between 2016 and 2024. These findings suggest two possible scenarios: local southward spread of an autochthonous Italian strain or a recent introduction from other European countries mediated by migratory wild birds. This study provides the first molecular evidence of USUV circulation in southern Italy and underscores the critical role of molecular epidemiology and phylogenetic analysis in tracing routes of pathogen introduction and spread.