At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells.

(A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIKICnsP3mC, CHIKICnsP3mC-FGN, CHIKICnsP3mC-FGC, or CHIKICnsP3mC-FGNC. Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIKICnsP3mC or CHIKICnsP3-FGNC either individually or co-transfected with in vitro transcribed RNA of CHIKIC. Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIKrep or CHIKrep-FGNC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKVIC, CHIKVIC-FGN and CHIKVIC-FGC on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID50/ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKVIC, CHIKVIC-FGN and CHIKVIC-FGC on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID50/ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.