Generation of influenza A viruses entirely from cloned cDNAs

TLDR

The study presents a reverse‑genetics system enabling efficient generation of influenza A viruses entirely from cloned cDNAs. The method uses 293T cells transfected with eight plasmids encoding viral RNAs and supporting proteins, plus additional plasmids for structural proteins, to produce viruses and generate reassortants. The system produced >10³ pfu/ml, rising to 3×10⁴–5×10⁷ pfu/ml with full structural plasmids, and enabled creation of reassortant and epitope‑modified viruses, making it valuable for mutagenesis, vaccine, and gene therapy vector production.

Abstract

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 10 3 plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 10 4 –5 × 10 7 pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.