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Effect of the number of RNA segments on the efficiency of virion production. 293T cells were transfected with protein expression plasmids for nine structural proteins and plasmids for the production of eight, seven, or six different vRNA segments. The plasmid for NS vRNA production possesses two mutations to eliminate NS2 protein production (12) such that the resultant virus does not undergo multiple cycles of replication. The plasmids for HA and NA vRNA production contain mutations that eliminate protein production, thus avoiding the effects of these proteins expressed from vRNA. To produce a virus with seven segments, we eliminated the plasmid for HA (−HA) or NA (−NA) vRNA production, whereas for the production of a six-segment virus we omitted the plasmids for both the HA and NA vRNA. Released virions were harvested at 24 (black bar) or 48 h (white bar) posttransfection and titrated by immunostaining virus-infected MDCK cells with antiserum to the influenza WSN strain.