FIGURE 1.

Mini-genome system for assessing the intrinsic influenza polymerase mutation rate and mutational spectra. A mini-genome system was applied to assess the RdRP activity in human 293T cells using the reporter plasmid driven by human Polymerase I promoter encoding firefly luciferase flanked by the 3′- and 5′-noncoding regions of influenza segment seven. Human 293T cells were cotransfected with the PB2, PB1, PA, NP, and the firefly luciferase and the cells were lysed for total RNA isolation or for luciferase activity measurements at 24 h post-transfection. The negative control transfection contained no IAV PB1 plasmid. (A) PCR amplification of the luciferase reporter gene driven by the RdRPs derived from Wuhan (Wu) or VN1203 (VN) viruses. Transfections were performed in triplicates and the negative controls (without PB1) were included in parallel. (B) Real-time PCR assay for firefly luciferase mRNA quantification and firefly luciferase activity as a proximate for the polymerase activity of Wuhan and VN1203 RdRPs. The results shown (mean ± SD) were from triplicated wells in one out of two independently performed experiments.