Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon.
This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products could be a valuable instrument for virus rescue, conservation and further characterization.
Rasmussen
Thomas Bruun
TB
National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
Reimann
Ilona
I
Hoffmann
Bernd
B
Depner
Klaus
K
Uttenthal
Ase
A
Beer
Martin
M
D000818:Animals; D001882:Border Disease / Q000821:virology; D018083:Border disease virus / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D016679:Genome, Viral; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep; D012757:Sheep Diseases / Q000821:virology
D018076:DNA, Complementary