Extended Data Fig. 6: The role of AaVCP in EV biogenesis of Ae. aegypti and the knockdown of AaVCP abolished flavivirus infection under acidic conditions.
From: Mosquito–capsid interactions contribute to flavivirus vector specificity
a, b, Knockdown efficiency of AaVCP in Ae. aegypti. Mosquitoes were microinjected with AaVCP dsRNA. GFP dsRNA served as a control. AaVCP abundance was assessed by RT-qPCR (a) and western blotting (b) at 3 days postinjection. a, Each dot represents data from an individual mosquito. The horizontal line represents the median. Statistical significance was evaluated by two-tailed Mann–Whitney test. c-e, Knockdown of AaVCP did not influence the number of EVs in the mosquito haemolymph. Mosquitoes were microinjected with AaVCP dsRNA. GFP dsRNA served as a control. A mixture containing fresh human blood (50% v/v) and supernatant from (c) uninfected Vero cells, (d) DENV2- or (e) ZIKV-infected Vero cells (50% v/v) was used for in vitro blood feeding. EVs were isolated from the haemolymph of Ae. aegypti mosquitoes at 4 days post bloodmeal. The concentration and size distribution of EVs were analysed by NTA. The graph (left) shows the fold change in the number of EVs isolated from mosquito haemolymph. Each dot represents data from a pool of EVs collected from the haemolymph of at least 60 mosquitoes. Statistical significance was evaluated by two-tailed t test. n.s., not significant. Data are presented as the mean ± s.e.m. Representative results from 3 independent experiments are shown. f, Generation of a mouse anti-AaVCP polyclonal antibody by immunizing mice with purified recombinant AaVCP protein expressed in E. coli. The antibody was validated by probing mosquito EVs purified from the Aag2 or Cxq-1 cell culture supernatants. The samples probed with mouse pre-immune serum served as a negative control. Representative results from 2 independent experiments are shown. g-i, Silencing mosquito VCP abolished flavivirus infection under acidic conditions. Growth curves of DENV2 (g) and ZIKV (h) in Aag2 cells transfected with GFP or AaVCP dsRNA under either normal or acidic conditions (pH=6.1). Growth curve of JEV in Cxq-1 cells transfected with GFP or CqVCP dsRNA under either normal or acidic conditions (pH=6.1) (i). The cells were transfected with the indicated dsRNA and then infected with DENV2 or ZIKV at an MOI of 0.1 or JEV at an MOI of 0.01 at 48 h post-transfection. The cell lysates were collected at 24, 48, 72, and 96 h postinfection. Total RNA was extracted, and infectivity was detected by RT-qPCR. The viral load was normalized to that of Ae. aegypti actin (AAEL011197) or Cx. quinquefasciatus actin (CPIJ012570). Statistical significance was evaluated by two-tailed t test. n.s., not significant. Data are presented as the mean ± s.e.m. Representative results from 2 independent experiments are shown.
