Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments
A reverse genetics system for the generation of influenza virus A/PR/8/34 (NIBSC vaccine strain) from plasmid DNA was developed. Upon transfection of eight bidirectional transcription plasmids encoding the gene segments of A/PR/8/34 into 293T cells, virus titers in the supernatant were about 104 TCID50/ml. The production of A/PR/8/34 in 293T cells was compared to that of A/WSN/33, for which virus titers in the supernatant were 107–108 TCID50/ml. Time-course analysis of virus production indicated that the differences in virus titers were due to reinfection of 293T cells by A/WSN/33 but not A/PR/8/34. Indeed, virus titers of A/PR/8/34 comparable to those of A/WSN/33 were achieved upon addition of trypsin to the culture medium of transfected cells. The production of chimeric viruses revealed that the difference in virus titers between A/PR/8/34 and A/WSN/33 are determined primarily by differences in the surface glycoproteins hemagglutinin and neuraminidase and the polymerase protein PB1. In conclusion, high-titer virus stocks of recombinant influenza A/PR/8/34 virus can be produced as well as virus stocks with much lower titers, but without the requirement of virus amplification through replication.
|Keywords||*Transcription, Genetic, Animals, Cell Line, DNA, Complementary/*genetics/metabolism, Dogs, Humans, Influenza A virus/*genetics/*physiology, Plasmids, Recombination, Genetic, Transfection|
|Persistent URL||dx.doi.org/10.1016/j.virusres.2004.02.028, hdl.handle.net/1765/3958|
de Wit, E., Spronken, M.I., Bestebroer, T.M., Rimmelzwaan, G.F., Osterhaus, A.D.M.E., & Fouchier, R.A.M.. (2004). Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Research, 103(1-2), 155–161. doi:10.1016/j.virusres.2004.02.028