title: Analysis of classical swine fever virus RNA replication determinants using replicons.
abstract: Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark.
authors.forename: Peter Christian
authors.initials: PC
authors.lastname: Risager
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Fahnøe
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Gullberg
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark.
authors.forename: Thomas Bruun
authors.initials: TB
authors.lastname: Rasmussen
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave 4771, Denmark.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
mesh_terms: D000818:Animals; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012093:Replicon; D012756:Sheep; D013194:Staining and Labeling / Q000379:methods; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D008156:Luciferases