title: Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.
abstract: 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.
authors.affiliation: National Influenza Center and Department of Virology, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
authors.forename: Emmie
authors.initials: E
authors.lastname: de Wit
authors.forename: Monique I J
authors.initials: MI
authors.lastname: Spronken
authors.forename: Theo M
authors.initials: TM
authors.lastname: Bestebroer
authors.forename: Guus F
authors.initials: GF
authors.lastname: Rimmelzwaan
authors.forename: Albert D M E
authors.initials: AD
authors.lastname: Osterhaus
authors.forename: Ron A M
authors.initials: RA
authors.lastname: Fouchier
mesh_terms: 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
chemical_list: D018076:DNA, Complementary