Plasmid-only rescue of infectious influenza virus. Twelve plasmids are introduced into mammalian cells: four plasmids lead to expression of the viral proteins required for viral RNA replication (PA, PB1, PB2, and NP), and the eight transcription plasmids express precise copies of the eight viral RNA segments (PA, PB1, PB2, HA, NP, NA, M, and NS). The resulting viral RNAs are replicated and transcribed by the reconstituted influenza virus RNA-dependent RNA polymerase. Recombinant infectious influenza virus is generated 48–72 hours after transfection of cells (17).