Fig. 9

Compatibility of H1N1 virus replication with the optimal level of H3N2 M2. a A549 cells were infected with the H1N1 WSN strain (WT) or with chimeric H1N1 RG viruses incorporating either WT 55T (H1N1 + H3wt) or mutant 55C (H1N1 + H3mut) H3N2 M segments, at a MOI of 0.001. The virus titer was determined at 12, 24, 36, 48, and 60 h post-infection (hpi) through plaque assay. The data represent means ± standard deviations (error bars) of three independent biological replicates. NS, not significant; *P < 0.05. Statistical analysis was performed using paired t-tests. WSN verses WSN + H3mut groups, t = 2.435, df = 4 and WSN verses WSN + H3wt groups, t = 2.002, df = 4. (t = t-value, df = degrees of freedom). b Plaque formation was visualized through crystal violet staining, and plaque size was determined using ImageJ. c Total RNA was collected at the indicated time points. 1F and 1R primers were used to detect different M transcripts, and 3F and 1R primers were used to determine the expression of all M transcripts. d Total protein extracts were analyzed by western blotting using specific primers. e C57BL/6 mice were intranasally infected with WT, mutant H1N1 (55T), H1N1 + H3wt, and H1N1 + H3mut, or administrated control vehicle (mock). Survival rate and body weight were assessed daily. All data were normalized to the initial weight of each mouse. Data are expressed as mean ± standard error of the mean (n = 5 mice per group) and three independent biological replicates