FIG 6.

CHIKV variants containing selected, mutated VLoop sequences efficiently replicate and are highly potent IFN-β inducers. (A) Schematic presentation of recombinant CHIKV genomes containing indicated substitutions in VLoop and additional mutation in nsP2 [CHIKV/RLH(A730V)/GFP], RNA infectivities in the infectious-center assay, and infectious titers in the stocks harvested at 24 h after electroporation of BHK-21 cells. (B) NIH 3T3 cells were infected with the indicated viruses at an MOI of 50 PFU/cell, and samples were harvested at 22 h p.i. Viral titers were determined by plaque assay on BHK-21 cells, and concentrations of IFN-β in the same samples were assessed by ELISA as described in Materials and Methods. (C) NIH 3T3 cells were infected with the indicated viruses at an MOI of 20 PFU/cell and harvested at 9 h p.i. The levels of degradation of RPB1 and levels of nsP2 were determined by Western blotting using specific Abs. Membranes were analyzed on an Odyssey imager (LI-COR). These experiments were reproducibly repeated more than three times, and the results of one of the representative experiments are presented.