title: A plasmid-based reverse genetics system for animal double-stranded RNA viruses.
abstract: Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.
authors.affiliation: Department of Pediatrics, Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Annukka A R
authors.initials: AA
authors.lastname: Antar
authors.forename: Karl W
authors.initials: KW
authors.lastname: Boehme
authors.forename: Pranav
authors.initials: P
authors.lastname: Danthi
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Eby
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Guglielmi
authors.forename: Geoffrey H
authors.initials: GH
authors.lastname: Holm
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Johnson
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Maginnis
authors.forename: Sam
authors.initials: S
authors.lastname: Naik
authors.forename: Wesley B
authors.initials: WB
authors.lastname: Skelton
authors.forename: J Denise
authors.initials: JD
authors.lastname: Wetzel
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Wilson
authors.forename: James D
authors.initials: JD
authors.lastname: Chappell
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D016679:Genome, Viral; D008322:Mammals; D010957:Plasmids; D012328:RNA Viruses / Q000235:genetics* / Q000276:immunology; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000276:immunology; D011995:Recombination, Genetic; D012087:Reoviridae / Q000235:genetics / Q000276:immunology; D012088:Reoviridae Infections / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014614:Vaccines, Synthetic; D014765:Viral Vaccines
chemical_list: D012330:RNA, Double-Stranded; D014614:Vaccines, Synthetic; D014765:Viral Vaccines