(A) Constructing CVV from EVV and WT. (B and C) Attenuated vTk expression and GFP expression in CVV-infected cells (*p <0.0001). (D) Viral replication assay of WT, EVV and CVV in Sk-Hep-1. CVV deficient of vTk replicated at lower level at 24 h post-infection, but showed higher replication rates than EVV (*p < 0.005). (E) Cytotoxicity of CVV, JX594 and WT in HepG2. CVV showed higher cytotoxicity than another VV deficient of vTK such as JX594 (*p < 0.0001). VV, vaccinia virus; CVV, cancer-favoring engineered vaccinia virus; EVV, evolved vaccinia virus; WT, wild type; HCC hepatocellular carcinoma; vTK, viral thymidine kinase.