P212S and V178I mutation in NS1 of PR8 virus enhances its virulence in vivo. A Shown is the body weight change of mice mock infected or infected with PR8-WT, PR8-S212 or PR8-I178 (1 × 104 PFU/mouse). Body weight was measured daily. The results are shown as mean percentage weight changes from three independent experiments. B Survival rate of mice infected with PR8-WT, PR8-S212 or PR8-I178 (n = 10 mice per group). Mice were monitored for up to 14 days. During this period, mice were sacrificed when they displayed severe unrelieved distress, hind limb paralysis or excessive weight loss (25% weight loss from initial body weight). C Shown is the viral load measured on day 5 post-infection in the lungs of mice infected with PR8-WT, PR8-S212 or PR8-I178. D Mice were mock infected or infected with PR8-WT, PR8-S212 or PR8-I178 for 5 days. Shown are representative micrographs of mouse lungs stained with hematoxylin and eosin (HE). Bars, 200 μm. E, F Three groups of mice (five mice per group) were mock infected or infected with PR8-WT and PR8-S212 for 5 days. Then the mRNA expression level of RIG-I (E) and IFN-β (F) was detected by quantitative real-time PCR. In each experiment, the value observed in mock-infected cells was normalized to 1. The error bars represent the s.e.m. G A549 cells were infected with PR8-WT and PR8-I178 for 16 h, then the cells were treated with CHX (100 μg/mL) for indicated times, followed by Western blotting to detect the NS1 protein levels. The results are representative of three independent experiments. **P < 0.01.