Extended Data Fig. 5: Detection of flaviviral genomes in EVs from infected mosquitoes and propagation of the DENV2 sub-viral repliconΔprM-E in Ae. aegypti.
From: Mosquito–capsid interactions contribute to flavivirus vector specificity
a-c, Flaviviral genome in EVs was detected by reverse transcription-polymerase chain reaction (RT-PCR). The EVs were isolated from haemolymph of (a) DENV2- or (b) ZIKV-infected Ae. aegypti, or (c) JEV-infected Cx. quinquefasciatus. Total RNA was extracted. The cDNA derived from total RNA was used as template. The viral genome was detected by RT-PCR with specific primers (Supplementary Table 3). Five fragments generated by PCR demonstrated the amplification of the viral genome. The regions of each fragment in viral genome are shown: DENV2 fragment 1 (1-2228), DENV2 fragment 2 (2130-4456), DENV2 fragment 3 (4323-6635), DENV2 fragment 4 (6505-8839), and DENV2 fragment 5 (8736-10723); ZIKV fragment 1 (1-2243), ZIKV fragment 2 (2184-4436), ZIKV fragment 3 (4348-6560), ZIKV fragment 4 (6502-8816), and ZIKV fragment 5 (8744-10675); JEV fragment 1 (1-2282), JEV fragment 2 (2215-4495), JEV fragment 3 (4426-6650), JEV fragment 4 (6579-8822), and JEV fragment 5 (8760-10976). NC, uninfected groups; I, infected groups; Mock, ultrapure water; M, marker. The results are representative images of 2 independent biological replicates. d, Propagation of the DENV2 sub-viral repliconΔprM-E in Ae. aegypti. Mosquito Aag2 cells were transfected with DENV2 repliconΔprM-E RNA, and the supernatant was collected at 72 h post transfection. EVs were collected by ultracentrifugation and then intrathoracically microinjected into Ae. aegypti mosquitoes. Mosquito infectivity was determined by RT-qPCR at indicated time points. Each dot represents data from a mosquito sample. The horizontal line represents the median. The number at the top of each column represents the number of infected mosquito/total number of mosquitoes. The limit of detection is illustrated by black dotted lines. Statistical significance was evaluated with a two-tailed Mann–Whitney test. The data were pooled from 2 independent biological replicates.
