In vitro ribonucleoprotein (RNP) reporter assay. All transfections and measurements were performed in triplicate. A, Mean fluorescence intensity of GFP -positive cells in transfections measured by flow cytometry. The NY470 (clade B′) virus RNP showed significantly more activity than did the NY198 and NY32 (clade B) RNP, as well as the NY26 and NY33 (clade A) RNP, exhibiting greater GFP expression in H1299 cells. Error bars represent 95% confidence intervals. B, When the NY470 PA segment is substituted for the NY32 PA segment, increased GFP expression is seen, similar to that of the wild-type NY470 RNP. When each of 2 key mutations are made at position 62 and 256 in the NY470 PA segment to revert to a more clade B–like PA, a significant decrease in GFP expression is observed, and when both mutations are present, activity is most similar to that of the wild-type NY32 RNP. Error bars represent 95% confidence intervals.