Figure 4.

Schematic illustration of the strategy for generating a non-toxic CVB for oncolytic purpose. (A) Viral RNA is prepared by RNA extraction from cell cultures. (B) Viral cDNA is generated via reverse transcription using poly T primer, followed by PCR amplification with a primer pair containing cloning sites (CS) and T7 promoter sequence (red) in the forward primer. (C) Viral cDNA is then rescued into a bacterial plasmid (e.g., pUC18/19). (D) Viral genome is modified through insertion of tumor-suppressive and/or organ-specific miRNA target sequences into the 5′UTR and/or 3′UTR of viral genome or by genetic mutagenesis of the viral nucleotides or amino acid codon. (E) Finally, the viral RNA is prepared by in vitro transcription with T7 RNA polymerase, and subsequently transfected into CVB susceptible cells to prepare the viral particles for further propagation. CDS, coding sequence.