title: Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus.
abstract: Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics approaches for engineering the BTV genome rely upon in vitro synthesis of capped RNA transcripts from cloned cDNA corresponding to viral genome segments. In an effort to expand the utility of BTV reverse genetics, we constructed a reverse genetics vector containing a T7 RNA polymerase promoter, hepatitis delta ribozyme sequence and T7 RNA polymerase terminator sequence. Viable virus was recovered following transfection of mammalian cells, expressing T7 RNA polymerase, with 10 plasmid constructs representing the cloned BTV-1 genome. Furthermore, the plasmid-based reverse genetics system was used successfully to isolate viable cross-serotype reassortant viruses and a mutant virus containing a defined mutation in the replicating viral genome. The new reverse genetics platform established here for BTV is likely applicable to other orbiviruses.
authors.affiliation: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Jakobus M
authors.initials: JM
authors.lastname: Pretorius
authors.affiliation: Department of Genetics, University of Pretoria, Pretoria 0002, South Africa.
authors.forename: Henk
authors.initials: H
authors.lastname: Huismans
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: D000818:Animals; D001820:Bluetongue virus / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D016679:Genome, Viral; D010957:Plasmids / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods*; D014779:Virus Replication
keywords: Bluetongue virus; DsRNA; Genome modification; Orbivirus; Reassortment; Reverse genetics; Reverse genetics plasmid; T7 RNA polymerase