Patterns and constraints of genome segment reassortment in Bluetongue Virus

Bluetongue virus (BTV), a segmented RNA virus affecting ruminants, often evolves through genome segment reassortment, generating novel genomic constellations with potential phenotypic consequences. To investigate reassortment dynamics, we combined field genomic data with an in vitro co-infection model. Whole-genome sequencing of 47 BTV strains collected in Italy and Tunisia during 20219–2025 revealed distinct evolutionary patterns among circulating serotypes. BTV-3 and BTV-4 displayed extensive reassortment, with multiple genomic constellations arising from segment exchange between European and North African lineages. In contrast, all BTV-8 strains showed near-complete nucleotide identity with the BTV-8 FRA 2023 lineage, indicating the circulation of a single, stable genotype. Notably, despite the absence of reassortment within BTV-8, its internal genome egments were frequently detected in BTV-3 and BTV-4 reassortant backgrounds, suggesting preferential incorporation into heterologous strains. In parallel, co-infection of Vero cells with BTV-1 and BTV-16 generated 157 plaques, of which 111 were suitable for analysis. Among these, 20 (18.02%) were reassortants. The parental contribution of genome segments was balanced overall (55.50% BTV-1 and 44.50% BTV-16), although segment-specific biases were observed, with Seg-7 predominantly derived from BTV-1 and Seg-3 from BTV-16. Despite 1022 theoretically possible combinations, only 18 distinct reassortant constellations were identified, with some recurring more frequently than expected. These results highlight that reassortment in BTV is frequent but constrained, with both field and experimental data supporting non-random segment assortment and selective compatibility among genome segments.