Improved dual promotor-driven reverse genetics system for influenza viruses.

Reverse genetic systems for influenza A virus (IAV) allow the generation of genetically manipulated infectious virus from a set of transfected plasmid DNAs encoding the eight genomic viral RNA segments (vRNA). For this purpose, cDNAs representing these eight vRNA segments are cloned into specific plasmid vectors that allow the generation of vRNA-like transcripts using polymerase I (Pol I). In addition, these plasmids support the transcription of viral mRNA by polymerase II (Pol II), leading to the expression of viral protein(s) encoded by the respective transcripts. In an effort to develop this system further, we constructed the bi-directional vector pMPccdB. It is based on pHW2000 (Hoffmann et al., 2000b) but contains additionally (i) the ccdB gene whose expression is lethal for most Escherichia coli strains and therefore used as a negative selection marker and (ii) more efficient AarI cloning sites that flank the ccdB gene on either side. Furthermore, we used a modified one-step restriction/ligation protocol to insert the desired cDNA into the respective pMPccdB vector DNA. Both the use of a negative selection marker and an improved cloning protocol were shown to facilitate the generation of genetically engineered IAV as illustrated in this study by the cloning and rescue of the 2009 pandemic isolate A/Giessen/6/2009 (Gi-H1N1).