FIG 2.

Generation of higher titer VEEV-ΔC-CHIKV virus in Vero cells by extensive passaging. (A) Growth curves comparison of different passages (P0, P10, P20, P30, P40, P50) of VEEV-ΔC-CHIKV in Vero cells. Vero cells were infected at an MOI of 0.01, and the cell supernatants were collected at the indicated times for determination of virus titers by plaque assay using BHK-21 cells. The data are representative of three independent experiments, and error bars indicate the SD. (B) IFA analysis of CHIKV-E2 and Capsid in WT or the passaged VEEV-ΔC-CHIKV infected Vero cells. (C) Plaque morphology comparison between CHIKV-WT and VEEV-ΔC-CHIKV at P0, P30, and P50. BHK-21 cells were infected with indicated viruses, and plaques were developed after 72 h. (D) Plaque diameter comparison of VEEV-ΔC-CHIKV virus at P0, P30, and P50 in BHK-21 cells at 72 hpi. ns, not significant. (E) Immunogenicity comparison between CHIKV-WT and P50 passaged VEEV-ΔC-CHIKV viruses. Two independent experiments were performed in triplicate. Data represent the mean ± SD of triplicate measurements in a representative experiment. The asterisks denote statistical differences between the indicated groups. ns, not significant. (F) Infectivity of VEEV-ΔC-CHIKV in Huh-7 (hepatoma), A549 (lung adenocarcinoma), and MRC-5 (lung fibroblast cell line) cell lines. The above all cell lines were infected with VEEV-ΔC-CHIKV at an MOI of 1. At 36 h postinfection, CHIKV-E2 expression was detected using anti-CHIKV E2 polyclonal antibodies.