Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention (original) (raw)
Figure 2
mosGCTL-3 facilitated DENV infections.
(A–B) Silencing efficiency of mosGCTL-3 on both RNA level and protein level. 2 ug GFP or mosGCTL-3 dsRNA was inoculated into mosquitoes thorax respectively. At 3, 6 and 9 days after the treatment, the mosquitoes were sacrificed to determine silencing effect by qPCR and normalized by A. aegypti actin (A). The primers of dsRNA synthesis and qPCR detection were described in Table S3. The mosGCTL-3 silenced or mock mosquitoes were grinded in lysis buffer and mosGCTL-3 was detected by immunoblotting (B). (C–F) mosGCTL-3 suppression impairs the infection of dengue serotypes. 10 M.I.D.50 DENV-1 Hawaii strain (C), DENV-2 New Guinea C strain (D), DENV-3 Guangdong strain (E) and DENV-4 H241 strain (F) viruses were inoculated into mosGCTL-3 silenced mosquitoes by microinjection respectively. The viral load was determined at 6 days post-infection by qPCR and normalized by A. aegypti actin. The result was pooled from 3 independent experiments. (G) Diagram of mosGCTL-3 gene. An extra exon (Exon 1) of mosGCTL-3, which encoded a peptide with 20 amino acid signal sequence, was identified by the 5′-RACE. (H) Expression and purification of mosGCTL-3 by a Drosophila expression system. mosGCTL-3 gene was cloned into pMT/BiP/V5-His-A DNA vector. The recombinant mosGCTL-3 was expressed and purified by a Cobalt-His column (Left panel). The expression was probed by anti-V5 mAb (Right panel). Mock was the concentrated supernatant of empty vector transfected Drosophila S2 cells. (I) Inoculation of mosGCTL-3 purified protein benefited DENV-2 infection in A. aegypti. 50 pg or 500 pg purified S2-expressed mosGCTL-3 protein was microinjected into each mosquito with 10 M.I.D.50 DENV-2. The infected mosquitoes were sacrificed at 3 and 6 days post infection. The same amount of BSA was inoculated with DENV-2 as mocks. The viral load was determined by qPCR and normalized by A. aegypti actin. This experiment was repeated 3 times individually. One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. We used the Mann-Whitney test for statistical analysis.