BHK cell monolayers were transfected with in vitro-transcribed D2/IC RNA, and viral titers were assessed at 72 hours post-infection by plaque assay. (A) Sequence and structure of cHP in virus supernatant obtained from D2/IC-HP3.4-transfected BHK cells. A compensatory mutation at position 132 (A132C) was detected in four separate virus batches at 96 and 120 hours post-transfection and in virus passaged through C6/36 cells. ΔG values in kcal/mol. (B) Viral titers from infectious clone-transfected BHK cells. In experiments where variants were below the limit of detection, 1.06 LOG10 PFU/ml, titer was recorded as the limit of detection. Data represent an average of 3 experiments and error bars indicate SD. *, p < 0.003 relative to WT by Wilcoxon signed rank test. **, p < 0.003 relative to HP3.4 by Wilcoxon signed rank test.