Effect of multiple CS mutations on growth kinetics of recombinant virus. (A) Schematic representation of WNV RNA structure. The nucleotide sequences of CS and coding amino acid sequences are indicated next to their names. Nucleotide substitutions at 5′ CS and 3′ CS and substituted amino acid in the C-coding region are indicated in lower case. Boxes indicate the eight nucleotides that are 100% conserved among all mosquito-borne flaviviruses. (B) Growth curves of WT and mutant virus on BHK, Vero, and C7/10 cells. Monolayers of cells were infected with the indicated virus at an MOI of 0.05 for BHK and Vero cells and an MOI of 0.2 for C7/10 cells. At each time point, the media were removed and frozen for subsequent titration and fresh media were added. Virus titers in cell culture media were determined on Vero cells. (C) Photograph of monolayers BHK, Vero, and C7/10 cells infected with WT or m4/7 recombinant virus, overlaid with semisolid media, fixed at 40 h postinfection for BHK and Vero cells and 72 h postinfection for C7/10 cells, and immunostained with anti-WNV antibody.