title: Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus.
abstract: Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain. Furthermore, we showed that the sequences at the 5' and 3' termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins Erns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.
authors.affiliation: Virology Department, Institute for Animal Science and Health, Lelystad, The Netherlands.
authors.forename: R J
authors.initials: RJ
authors.lastname: Moormann
authors.forename: H G
authors.initials: HG
authors.lastname: van Gennip
authors.forename: G K
authors.initials: GK
authors.lastname: Miedema
authors.forename: M M
authors.initials: MM
authors.lastname: Hulst
authors.forename: P A
authors.initials: PA
authors.lastname: van Rijn
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000472:pathogenicity; D003001:Cloning, Molecular; D004279:DNA, Viral; D016679:Genome, Viral; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral