Extended Data Fig. 9: Regulation of chimeric virus DENV2-CJEV infection and assessment of DENV2-CJEV dissemination in Culex.
From: Mosquito–capsid interactions contribute to flavivirus vector specificity
a,b, Infection with the chimeric virus DENV2-CJEV was inhibited by GW4869 in Cxq-1 cells but not by the neutralizing anti-DENV-E antibody. Cxq-1 cells were infected at an MOI of 0.1 with DENV2-CJEV mixed with neutralizing antibodies (a) or a series of concentrations of GW4869 (b). The cell lysates were harvested at 24, 48, and 72 h postinfection. Total RNA was extracted, and the viral load was assessed by RT-qPCR. c,d, Silencing CqVCP impaired the chimeric virus DENV2-CJEV infection in Cxq-1 cells. Cxq-1 cells were transfected with GFP or CqVCP dsRNA, followed by infection at an MOI of 0.1 with the chimeric virus DENV2-CJEV at 48 h posttransfection. The cell lysates were harvested at 24, 48, 72, and 96 h post-infection. Infectivity was assessed by RT-qPCR (c) and fluorescence microscopy (d) at 72 h post-infection. d, DENV2-CJEV was stained with an anti-JEV-C antibody conjugated to Alexa Fluor 488, and CqVCP was stained with an anti-AaVCP polyclonal antibody conjugated to Alexa Fluor 546. Nuclei were stained with DAPI. Scale bar, 20 μm. e,f, Assessment of chimeric virus DENV2-CJEV dissemination in Cx. quinquefasciatus. Mosquitoes were intrathoracically inoculated with 3 FFU of the chimeric virus DENV2-CJEV. The parental DENV2 16681 strains served as a control. Viral load in heads (e) and salivary glands (f) was determined by RT-qPCR at the indicated time points after virus inoculation. g, Assessment of chimeric virus DENV2-CJEV infection in Aedes and Culex by oral feeding. A mixture containing fresh human blood (50% v/v) and supernatant from DENV2-CJEV-infected Vero cells (50% v/v) was used to feed Ae. aegypti or Cx. quinquefasciatus via an in vitro blood feeding system. A total of 3 × 106 FFU/ml DENV2-CJEV were used for oral infection. Mosquito infectivity was determined by RT-qPCR at 8 days after a bloodmeal. a-c, The viral load was normalized to that of Cx. quinquefasciatus actin (CPIJ012570). These data are presented as the mean ± s.e.m. Statistical significance was evaluated with a two-tailed t test. Representatives from 2 independent experiments are shown. e-g, The number at the top of each column represents the number of infected mosquitoes/total number of mosquitoes. Each dot represents data from a mosquito tissue (e, f) or a mosquito sample (g). The horizontal line represents the median and the limit of detection is illustrated by black dotted lines. The data were pooled from 2 independent biological replicates. a-c,e-g, Statistical significance was evaluated with a two-tailed t test (a-c) or a two-tailed Mann–Whitney test (e-g).
