Figure 3.

CVB3 replicates, but does not induce, CPE in the indicator HeLa cell monolayers following sequential infection of replication-restricted primary cells. (A) Single-step growth curves of CVB3/28 in primary cell cultures NPF-1, MHC, and HCF, and in immortal cell lines HeLa and Panc-1 (inoculated with an MOI of 20 TCID50 per cell). Error bars show standard deviations. (B) Supernatants of CVB3/28 passaged in primary cell cultures lose the ability to cause CPE in HeLa cell monolayers. P1 to P5, HeLa cells infected with supernatants of passages 1 to 5; TCC, uninfected HeLa cell control. Cell cultures were fixed in acetic acid/acetone and then stained with crystal violet. Titers at each passage were determined either by CPE on HeLa cell monolayers or by real-time quantitative RT-PCR. (C) Titers from serial passages shown in panel B are plotted. (D) Relative positions in the CVB3 genome of primers used in this study. (E) CVB3/28 RNAs in primary cell cultures detected by RT-PCR. Lane M, Hi-Lo DNA marker (Minnesota Molecular, Inc., Minneapolis, MN); lanes P1 to P5, passages 1 to 5. Arrows indicate 750 bp bands. Reprinted with permission from [61], 2008, American Society for Microbiology.