Universal cloning system independent of restriction sites and DNA ligation speeds up generation of recombinant influenza A viruses by reverse genetics

Reverse genetics has become pivotal in influenza virus research. Basic research studies and vaccine development rely on the rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus segments which have been cloned using restriction endonucleases and DNA ligation. However, in some cases suitable restriction cleavage sites are not available. Therefore, we established a cloning method which is universal for any influenza A virus strain and independent of sequence information. This approach is based on an inverse PCR protocol in which the two strands of an amplicon from an influenza A gene segment serve as megaprimers. The prospective insert must contain termini homologous to the regions of the plasmid adjacent to the insertion site. In order to improve the efficiency, we modified the cloning vector by introducing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. From five influenza A virus strains (A/Thailand/1(KAN-1)/2004 (H5N1), A/Swine/Belzig/2/2001 (H1N1), A/Duck/Ukraine/1/1963 (H3N8), A/HongKong/1/1968 (H3N2), and A/Chicken/Emirates/R66/2002 (H9N2)), we cloned all eight genomic segments independent of sequence information amounting to 40 successfully cloned influenza genes. This approach allows fast cloning of all segments from any influenza A strain without knowledge of the genome sequence. If the PCR amplicon ends are homologous to plasmid annealing sites only, this approach is suitable for uniform and efficient cloning of any insert with conserved termini.