Figure 6. CHIKV E2 K200R has no impact on vector competence or viral fitness in mosquitoes.
(A) Ae. aegypti mosquitoes were fed a blood meal containing 1.1 × 106 PFU/mL of CHIKV or CHIKV E2 K200R, and the head, legs, and saliva were collected at three dpi. Samples initially found to be positive for virus were evaluated by focus formation assay to quantify infectious virus. Mean ± SD. N = 50, one experiment. Mann-Whitney test; p>0.05. (B) Ae. albopictus C6/36 cells were infected in triplicate at an MOI of 1 PFU/cell with a 1:1 mixture of CHIKV marked with an ApaI restriction site (CHIKV-ApaI) and WT CHIKV or CHIKV E2 K200R, and 1/10th of the supernatant was serially passaged onto new C6/36 cells every 24 hr. RNA was extracted from the inoculum and supernatant of passage 5, cDNA was generated, and PCR amplified. Digestion of the PCR product was used to identify ratios of ApaI marked to unmarked virus, and the percent band intensity is displayed. Mean ± SD. N = 3, one experiment. (C and D) Ae. aegypti mosquitoes were microinjected with 138 PFU of 1:1 mixtures of CHIKV-ApaI and CHIKV, or CHIKV-ApaI and CHIKV E2 K200R. Bodies (C) and saliva (D) were collected at seven dpi. Ratios of each virus present in the input and in samples at day seven were evaluated as described in (B). Mean ± SD. N = 20, one experiment. Two-way ANOVA with Bonferroni’s correction; p>0.05 for all comparisons.