FIG 6.

Improvement of PIV_NS2A production through adaptive mutations from selection I. (A) IFA of naive BHK-21 cells transfected with the genome-length RNA of the WT or mutants that contain NS2A K188-del and the mutations derived from selection I. The transfected cells were monitored for E protein expression (green) using MAb 4G2. (B) IFA of NS2A-expressing BHK-21 cells transfected with the genome-length RNA of the WT or mutants that contain NS2A K188-del and different mutations rescued from selection I. The E protein was stained in red using MAb 4G2. (C) Quantification of PIV_NS2A titers generated from NS2A-expressing BHK-21 cells transfected with the indicated genome-length RNAs. The detection limit of IFA quantification was 10 IFU/ml. Mean values from three independent experiments are shown, and the error bars represent standard deviations. (D) Replicon analysis. WT or mutant replicon RNAs containing single or combined mutations were electroporated into naive BHK-21 cells. An NS4B lethal mutation, K143A (45), was included as a negative control (NC). The means and standard deviations from the results of three independent experiments are presented.