title: Rescue of the highly virulent classical swine fever virus strain "Koslov" from cloned cDNA and first insights into genome variations relevant for virulence.
abstract: Classical swine fever virus (CSFV) strain "Koslov" is highly virulent with a mortality rate of up to 100% in pigs. In this study, we modified non-functional cDNAs generated from the blood of Koslov virus infected pigs by site-directed mutagenesis, removing non-synonymous mutations step-by-step, thereby producing genomes encoding the consensus amino acid sequence. Viruses rescued from the construct corresponding to the inferred parental form were highly virulent, when tested in pigs, with infected animals displaying pronounced clinical symptoms leading to high mortality. The reconstruction therefore gave rise to a functional cDNA corresponding to the highly virulent Koslov strain of CSFV. It could be demonstrated that two single amino acid changes (S763L and P968H) in the surface structural protein E2 resulted in attenuation in the porcine infection system while another single amino acid change within the nonstructural protein NS3 (D2183G) reduced virus growth within cells in vitro.
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark; Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Fahnøe
authors.affiliation: Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Anders Gorm
authors.initials: AG
authors.lastname: Pedersen
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Peter Christian
authors.initials: PC
authors.lastname: Risager
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Höper
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark. Electronic address: tbrur@vet.dtu.dk.
authors.forename: Thomas Bruun
authors.initials: TB
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D002460:Cell Line; D006691:Classical Swine Fever / Q000401:mortality / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000472:pathogenicity*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D016679:Genome, Viral; D007668:Kidney / Q000166:cytology; D013552:Swine; D013997:Time Factors; D014774:Virulence
chemical_list: D018076:DNA, Complementary
keywords: CSFV; Classical swine fever virus; Flaviviridae; Full-length cDNA clone; Koslov; Next generation sequencing; Pathogenesis; Pestivirus; Viral populations; Virulence