Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.
Article Details
- CitationCopy to clipboard
de Wit E, Spronken MI, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA
Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.
Virus Res. 2004 Jul;103(1-2):155-61. doi: 10.1016/j.virusres.2004.02.028.
- PubMed ID
- 15163504 [ View in PubMed]
- 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.