Replication of recombinant CVB3/TDs in HeLa cell culture and RT-PCR. (A) Each deleted 5′-NTR sequence was cloned into pCMVCVB3/28 (CVB3/TD8, CVB3/TD13, CVB3/TD18, CVB3/TD31, and CVB3/TD50). Transfection of cDNA in HeLa cell cultures maintained a noncytopathic phenotype and the respective deletions through five passages of HeLa cells by supernatants. Monolayers were fixed and stained with crystal violet. (B) Viral RNA from each CVB3/TD (pass 5) was analyzed by RT-PCR with primers specific for sequences at or near the 5′ end. Primers used are indicated. The arrow indicates a 600-bp marker. Lane M, 100-bp DNA ladder; lane +, CVB3/28; lane 1, CVB3/TD8; lane 2, CVB3/TD13; lane 3, CVB3/TD18; lane 4, CVB3/TD31; lane 5, CVB3/TD50. (C) Map of primer positions on CVB3 genome.