Evolutionary Dynamics of Bluetongue virus serotypes 3, 4, and 8 circulating in Italy, 2024-2025

Bluetongue virus (BTV) continues to pose a major threat to ruminant health in Europe, where repeated introductions, the co-circulation of multiple serotypes and frequent reassortment shape its genomic diversity. During 2024-2025, Italy experienced a marked resurgence of bluetongue, driven mainly by BTV-3 and BTV-8. In this study, we performed whole-genome sequencing of BTV-3, BTV-4 and BTV-8 strains collected in Italy during 2024-2025 and integrated these data with representative genomes from Italy and Tunisia generated over the previous decade. A total of 47 BTV whole-genome sequences were analysed. Multiple reassortant genomic constellations were identified among BTV-3 and BTV-4 strains, reflecting extensive segment exchange between North African and European lineages. In contrast, all Italian BTV-8 genomes showed near-complete nucleotide identity with the contemporary BTV-8 FRA 2023 lineage, indicating nationwide circulation of a single strain. Despite its widespread diffusion, BTV-8 did not acquire heterologous genome segments, whereas its internal genes were frequently incorporated into BTV-3 and BTV-4 genomic backgrounds. These findings highlight Italy as a key convergence point for BTV lineages in the Mediterranean basin and underscore the value of whole-genome surveillance for tracking viral introductions and reassortment dynamics.