FIG. 4.

PCR and sequence analysis of CVB3-PL2-Ad2L1. pCVB3-PL2-Ad2L1 was transfected into HeLa cells, and the resultant progeny virus (CVB3-PL2-Ad2L1, pass 1) was subsequently serially passaged in HeLa cell cultures (passes 2 to 10). Viral RNA was isolated from virus stocks at each passage, and the presence of the inserted Ad2 sequence was analyzed by PCR using primers flanking the insertion site in the CVB3 genome. (a) Amplimers were separated by agarose gel electrophoresis. CVB3-PL2-Ad2L1, RT-PCR amplimer using chimeric viral RNA as the template; pCVB3-PL2-Ad2L1, PCR amplimer using the chimeric plasmid DNA as the template; CVB3/0, RT-PCR amplimer using the parental CVB3/0 RNA as template; neg., RT-PCR using RNA as template from uninfected HeLa cells; Marker, 100-bp DNA ladder. (b and c) The sequence of the Ad2 insert-containing 446-bp amplimer (CVB3-PL2-Ad2L1) (b) and the sequence of the 225-bp Ad2 fragment-deleted amplimer (CVB3-PL2-Ad2L1del) (c) were obtained after isolation of the DNA fragments from agarose gels. Sequence analysis was performed with the same primers as for the RT-PCR analysis. Numbering is based on the CVB3/0 genome (Genbank accession no. M88483).