Minigenome and plaque assays to determine the role of adaptive mutations in the qa-mall/178 polymerase genes. Polymerase activity was measured using a minigenome assay (A and B) and a plaque assay (C and D) at 41°C and 37°C. (A and B) For the minigenome assay, 293T cells were transfected at 41°C (A) or 37°C (B) with PB2, PB1, PA and NP plasmids from either the mall/178 or the qa-mall/178 virus, along with a luciferase reporter plasmid and a pCMV/SEAP plasmid encoding a secreted alkaline phosphatase (to normalize the transfection efficiency). The negative control included an empty vector instead of the plasmid encoding PB1. Luciferase activity was assayed in cell supernatants at the indicated hours posttransfection (hpt). Each result is the average fold change (luciferase/SEAP ratio) ± the standard error of the mean from three independent transfections. A lowercase letter “a” above a bar indicates that the difference between mall/178 and qa-mall/178 is statistically significant (P, < 0.05 by Student's t test). (C and D) The RG mall/178 and RG qa-mall/178 viruses were used to infect CEK cells for 1 h at 37°C. Cells were then washed, overlaid with 1.8% agar and MEM (1:1), and incubated at either 41°C (C) or 37°C (D). After 48 h, overlays were removed, and plaques were stained using anti-NP antibodies. Dilutions (−6, 10−6; −4, 10−4) are shown at the bottom.