FIG. 4.

Replication of (C19C20)VEE/SINV variants having additional AUG repeats at the 5′ termini of their genomes. (A) Computer-predicted secondary structures of the 5′ termini of the designed genomes, RNA infectivities in the infectious center assay, and virus titers at 24 h post-RNA transfection. PEP, postelectroporation. (B) Analysis of replication of the designed mutants. Two micrograms of the in vitro-synthesized RNAs was transfected into BHK-21 cells by electroporation, and one-fifth of the cells were seeded into 35-mm dishes. At the indicated times posttransfection, media were replaced, and titers of virus in the harvested samples were measured by a plaque assay of BHK-21 cells. Electroporation was used instead of a more traditional infection, because the designed variants most likely continued to evolve and accumulate quasispecies with higher numbers of repeats by late times posttransfection. The dashed line indicates the limit of detection.