Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase.
A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.
van Gennip
H G
HG
Institute for Animal Science and Health (ID-DLO), Virology Department, Lelystad, Netherlands.
van Rijn
P A
PA
Widjojoatmodjo
M N
MN
Moormann
R J
RJ
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D006691:Classical Swine Fever / Q000821:virology; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000302:isolation & purification*; D018076:DNA, Complementary; D004279:DNA, Viral / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D010957:Plasmids; D013552:Swine; D014162:Transfection; D014764:Viral Proteins
D018076:DNA, Complementary; D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases