Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus

- An entirely plasmid-based reverse genetics system was developed for AHSV.
- Novel improvements were made that increases flexibility of AHSV plasmid-based reverse genetics.
- Virus recovery efficiency was increased by reducing plasmids required for rescue from 10 to 5.
- T7 RNA polymerase encoded by rescue plasmid backbone allows virus recovery in different cell lines.
- Recombinant wild-type AHSV, mutant and reassortant viruses were rescued from plasmid cDNA only.

In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.