Genome Constellations Recovered Following In Vitro Co-Infection and Serial Passage of Bluetongue Virus Serotypes 1 and 16

Abstract Bluetongue virus (BTV) is a segmented RNA virus capable of genome reassortment during co-infection. We characterized genome constellations recovered after co-infection of Vero cells with western BTV-1 and eastern BTV-16 strains followed by three serial passages. Of 157 plaques, 46 showed simultaneous detection of BTV-1 and BTV-16 Seg-2 and were excluded because a single parental background could not be established. Among 111 Seg-2-monotypic plaques, 20 were identified as reassortants, representing 18.0% of the evaluable plaques and 12.7% of all plaques collected. Across the 20 recovered reassortants, 111 of 200 segment assignments derived from BTV-1 and 89 from BTV-16. BTV-1-derived Seg-7 was observed in 18 of 20 plaques and BTV-16-derived Seg-3 in 14 of 20 plaques; 18 distinct genome constellations were observed. Because all plaques originated from a single co-infection and were sampled after three serial passages without parallel single-virus passage controls, the observed segment distributions integrate reassortment and post-reassortment selection and cannot separate these processes. These data demonstrate recovery of diverse BTV-1/BTV-16 reassortant genomes under the experimental conditions but do not establish the true reassortment frequency or reproducible segment-association patterns.