Fig. 2

Detection of RNAs synthesised by 1/244 DI RNA and 1/244 KO DI RNA by Northern blot and primer extension and protection of mice from influenza. a. Northern blot of RNA extracted from cells 48 h after infection with 1/244 DI virus and helper virus to detect positive-sense influenza RNA transcribed from genome segment 1 and the 1/244 RNA itself. Lane 1: total RNA; lane 2: non-polyadenylated RNA; lane 3 polyadenylated mRNA. The positions of size markers (nt) are indicated. b. Viral RNAs synthesised by 1/244 DI RNA and 1/244 AUG KO DI RNA and detected by primer extension. Samples were taken 48 h after transfection with plasmids expressing Seg 1-GFP, PB1, PB2, PA and NP proteins and 1/244 and 1/244 AUG KO DI RNAs. Lane 1: a 10 nt size ladder; lane 2: RNA made in the presence of 1/244 DI RNA; lane 3: RNA made in the presence of 1/244 AUG KO DI RNA. The positions of vRNA and mRNA are indicated. 5S ribosomal RNA was measured as a loading control. c. Mice were inoculated intranasally with A/WSN alone (10 LD₅₀, 1000 f.f.u.), A/WSN + 1/244 AUG KO DI virus, A/WSN + 1/244 DI virus, A/WSN + inactivated 1/244 AUG KO DI virus, A/WSN + inactivated 1/244 DI virus, or saline alone. Each DI virus comprised 1 μg protein. d. Surviving mice were challenged with a high dose of A/WSN (10,000 LD₅₀) at 3 weeks post-infection to determine their immune status. Panels C and D show the mean clinical score. In panel C A/WSN + 1/244 DI, 1/244 KO DI only, 1/244 DI only, and mock are all hidden under A/WSN + 1/244 AUG KO DI with a clinical score of 1. The percentage of surviving mice is shown in parenthesis