Figure 2
Recovery of infectious viruses from mutant clones. (a) Detection of infectivity by mutant viruses. Vero cells grown in monolayers on coverslips in 12-well plates were cultured in medium alone or infected with EMEM containing wt CVB3, pBRCVB3, Mt 2, Mt 3, Mt 4, Mt 8, or Mt 10, then incubated for 12 h at 37 °C. Cells were fixed and incubated with anti-CVB3 serum, and a secondary FITC-conjugated IgG was added to detect viral antigens. The coverslips were washed and mounted, then examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60×. (b) Comparison of viral titers. Monolayers of Vero cells were grown to 80–90% confluence in 12-well plates. Triplicate wells were infected at an MOI of 0.1 or 3.0 with passage 2 viruses of wt CVB3, pBRCVB3, Mt 2, Mt 3, Mt 4, Mt 8, or Mt 10. After noting the CPE, the respective viral supernatants were harvested, and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3). Mann–Whitney test and Kruskal–Wallis test were used to determine significance between wt CVB3/pBRCVB3 and other mutant viruses at different times post-infection. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.
