Synthetic transcripts of double-stranded Birnavirus genome are infectious.
We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.
Mundt
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Federal Research Center for Virus Disease of Animals, Friedrich-Loeffler-Institutes, Insel Riems, Germany.
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D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D003588:Cytopathogenic Effect, Viral; D018076:DNA, Complementary / Q000235:genetics; D005814:Genes, Viral; D007243:Infectious bursal disease virus / Q000235:genetics* / Q000472:pathogenicity; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015678:Viral Structural Proteins