title: Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus.
abstract: 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.
authors.affiliation: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Andelé M
authors.initials: AM
authors.lastname: Conradie
authors.affiliation: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Liesel
authors.initials: L
authors.lastname: Stassen
authors.affiliation: Department of Genetics, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Henk
authors.initials: H
authors.lastname: Huismans
authors.affiliation: Deltamune (Pty) Ltd., Lyttelton, Centurion, South Africa; Department of Biochemistry, Centre for Human Metabonomics, North-West University, Potchefstroom, South Africa.
authors.forename: Christiaan A
authors.initials: CA
authors.lastname: Potgieter
authors.affiliation: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa. Electronic address: jacques.theron@up.ac.za.
authors.forename: Jacques
authors.initials: J
authors.lastname: Theron
mesh_terms: D000356:African Horse Sickness Virus / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary; D015870:Gene Expression; D023061:Gene Order; D016679:Genome, Viral; D010957:Plasmids / Q000235:genetics*; D012367:RNA, Viral; D059386:Reverse Genetics; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral
keywords: African horse sickness virus; Orbivirus; Reassortment; Reoviridae; Reverse genetics; T7 RNA polymerase; Virus rescue; dsRNA