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 10(4) 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 10(7)-10(8) 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.
de Wit
Emmie
E
National Influenza Center and Department of Virology, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Spronken
Monique I J
MI
Bestebroer
Theo M
TM
Rimmelzwaan
Guus F
GF
Osterhaus
Albert D M E
AD
Fouchier
Ron A M
RA
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary