Detection and genomic characterization of West Nile virus lineage 2 in captive harbor seals in southern Italy

Introduction West Nile virus (WNV) is a mosquito-borne flavivirus maintained in bird–mosquito cycles, with occasional spillover events causing neuroinvasive disease in humans and animals. This study describes two fatal cases of WNV lineage 2 infection in captive harbor seals housed in a zoological park in southern Italy, together with genomic characterization and phylogenetic analysis of the detected strains. Methods Harbor seals were tested using multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) for the simultaneous detection of WNV lineage 1 and lineage 2. Selected extracts were further analyzed by droplet digital PCR (ddPCR). Next-generation sequencing was performed, and the resulting sequences were subjected to phylogenetic analysis. A Centers for Disease Control and Prevention (CDC) light trap was used for targeted mosquito collection near the fence of the dead harbor seals, and female mosquitoes were screened for WNV. Histopathological examination was also performed in one seal. Results WNV lineage 2 RNA was detected in central nervous system tissues of both seals and in a pool of female Culex pipiens collected near the fence. Near-complete WNV genomes clustered with contemporary southern Italian strains and were consistent with local viral circulation. Discussion These findings document WNV lineage 2 infection associated with fatal neurological disease in harbor seals, expanding current knowledge of the host range of this virus and providing complementary in situ entomological and genomic evidence. Overall, this study highlights the value of an integrated One Health surveillance approach in zoological parks.