FIG 5.

Role of AeCullin-3 during CHIKV infection in A. aegypti. (a) A. aegypti mosquitoes were fed with either uninfected blood (control group) or CHIKV-spiked blood (10⁶ PFU/mL), a pool of n = 5 mosquitoes was collected at indicated time points in TRIzol for RNA extractions. AeCullin-3 gene expression was quantified in the two groups by qRT-PCR. The experiments were performed a minimum of three times, with each experiment set in triplicates. Fold change in expression in CHIKV-spiked blood-fed mosquitoes is compared to that in uninfected blood-fed mosquitoes. (b) Experimental plan for dsRNA-mediated knockdown of AeCullin-3 in A. aegypti mosquitoes. (c) Mosquitoes were intrathoracically injected with either dsRNA against GFP (dsGFP, control group) or dsRNA against AeCullin-3 (dsCUL3 group), and the mosquitoes (n = 5) from both groups were then collected and AeCullin-3 gene expression was estimated in each individual mosquito using qRT-PCR. (d) dsGFP- and dsCUL3-injected mosquitoes were subjected to CHIKV-spiked blood feeding 24 h post nanoinjection. Mosquitoes (n = 5) were then collected and CHIKV genomic RNA levels were analyzed in each individual mosquito. (a to d) Rps17 served as endogenous control, and qRT-PCR analysis for experiments indicated in this figure was done using 2^−ΔCT method. Fold change in expression in dsCUL3-injected mosquitoes is compared to that in dsGFP-injected mosquitoes. Data are expressed as mean ± SD. ***, P < 0.001; **, P < 0.01; *, P < 0.1 (versus control).