Figure 7. Stable introduction of a reporter gene into the PB2-segements results in severe attenuation.

(A) Viral growth of SC35M_WT and the indicated reporter viruses in MDCK-II cells. Virus titers in the supernatant of MDCK-II cells infected at an MOI of 0.001 were determined by plaque assay at the indicated time points. Error bars indicate standard error of the mean from three independent experiments. The cartoon schematically represents the modified PB2 segments. The fluorescent live cell image in the upper left corner depicts the GFP-positive A549 cells infected at an MOI of 1 of SC35M_{PB2_2A_GFP} 24 hours post infection. (B) Luciferase activity in the supernatant and the lysate of MDCK-II cells infected the indicated viruses at an MOI of 0.01, 24 hours post infection. Error bars indicate standard deviation from three independent experiments. (C and D) Viral growth of SC35M_WT, SC35M_{PB2mod_2A_GFP} (C) and SC35M_PB2mod (D) was determined as described above. The cartoons schematically represent the modified PB2 segments. Shaded boxes indicate silent mutations introduced into the last 129 nucleotides of the PB2 ORF.