A DNA transfection system for generation of influenza A virus from eight plasmids

We have developed an eight-plasmid DNA transfection system for the rescue o f infectious influenza A virus from cloned cDNA, In this plasmid-based expr ession system, viral cDNA is inserted between the RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation sit e. The orientation of the two transcription units allows the synthesis of n egative-sense viral RNA and positive-sense mRNA from one viral cDNA templat e. This pol I-pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumabl y in different compartments of the nucleus. The interaction of all molecule s derived from the cellular and viral transcription and translation machine ry results in the generation of infectious influenza A virus. The utility o f this system is proved by the recovery of the two influenza A viruses: A/W SN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1), Seventy-two hours after the tran sfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 x 10(5) and 2 x 10(7) infectious viruses per milliliter, We also used this e ight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the p ol I-pot II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recov ery of other RNA viruses entirely from cloned cDNA.