Figure 1.
The role of mosGCTL genes in DENV-2 infection of A. aegypti.
The phenotype, of which the 9 mosGCTL genes exhibited susceptible effect in DENV-2 (New Guinea C strain) infection (p<0.05, Table S1), was reproduced. There were no less than 15 mosquitoes in each group. The quantification of DENV-2 E gene was normalized with A. aegypti actin (AAEL011197). The primers of dsRNA synthesis were shown in Table S3. All data were presented as mean ± standard error(SEM. The Mann-Whitney test was used for statistical analysis. The results were combined from 2 independent experiments.
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.
Figure 3.
The interaction between mosGCTL-3 and DENV-2.
(A) mosGCTL-3 interacted with DENV-2 Envelop protein in a co-immunoprecipitation (Co-IP) assay. The purified S2-expressed mosGCTL-3 and DENV-2 E proteins (2 ug each) were mixed for 2 h at 4°C. The protein complex was pulled down by anti-V5 mouse monoclonal antibody and probed by anti-FLAG rabbit polyclonal antibody. The experiment was repeated twice. (B) mosGCTL-3 captured DENV-2 virions by ELISA. The purified S2-expressed mosGCTL-3 was used in this experiment. The interaction was determined by a flavivirus E mAb 4G2. Data was expressed as mean ± standard error, and the experiment was reproduced three times. (C–D) The co-localization of mosGCTL-3 and DENV-2 in the tissues of A. aegypti. The mosquitoes were infected by intrathoracic microinjection. The salivary glands and hemocytes were then collected on 6 days after 1,000 M.I.D.50 DENV-2 infection for immunofluorescence staining. The tissues of PBS inoculated mosquitoes were used as mocks. mosGCTL-3 was stained with anti-rabbit IgG Alexa-488 (Green), and DENV-2 E protein was probed by anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide. The arrow represented the associated area between mosGCTL-3 and DENV-2 in hemocytes (C) and salivary glands (D). Images were examined by a Zeiss LSM 780 meta confocal with a Multi-Track mode.
Figure 4.
mosGCTL-3 antisera interrupted DENV-2 infection of A. aegypti.
(A) Diagram of dengue life cycle and a transmission-blocking strategy for dengue prevention. (B) Inoculation of mosGCTL-3 antisera impaired DENV-2 infection of A. aegypti. The serial dilutions of mosGCTL-3 antisera or pre-immune sera with 10 M.I.D.50 DENV-2 were inoculated into mosquitoes by microinjection. The infected mosquitoes were scarified at 3 days and 6 days post inoculation. DENV-2 was determined by qPCR and normalized by A. aegypti actin. One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. The Mann-Whitney test was used for statistical analysis. The result was combined from 2 independent experiments. (C–D) mosGCTL-3 antisera interrupted DENV-2 infection by a blood meal. The mosGCTL-3 antisera or pre-immune sera were diluted 100-, 1,000- and 5,000-fold with fresh human blood and Vero cells-generated DENV-2. The mosquitoes were allowed to ingest the blood mixture by a membrane feeding. The fed mosquitoes were sacrificed 8 days later and DENV-2 infectivity was assessed by qPCR. The number of infected mosquitoes/total mosquitoes was presented on the top of each column (C). The mosquito infective ratio (D) was then interpreted from the Figure (C). One dot represented a mosquito. The result was pooled from 2 independent experiments.
Figure 5.
Feeding the combination of 9 mosGCTLs antisera blocked DENV-2 uptake by a membrane blood feeding.
(A) The interaction of the susceptible mosGCTLs and DENV-2 E protein. The mosGCTL genes exhibiting susceptible role in DENV-2 infection (p<0.05) were cloned into pMT/Bip/V5-His A DNA vector. The recombinant mosGCTLs with a V5 tag in C-terminus were expressed in Drosophila S2 cells. mosGCTL-26 (AAEL017265) was failed to be expressed in Drosophila S2 cells. The interaction was determined by ELISA. An anti-V5 mouse mAb was used to probe the binding activity. The experiment was reproduced twice. (B–D) Feeding the combination of 9 mosGCTL antisera blocked DENV-2 infection. The pre-immune sera, mosGCTL-3 antisera and combined mosGCTLs antisera (1/9 mosGCTL antiserum each) were serially diluted 100- (B), 1,000- (C), or 5,000- (D) fold with fresh human blood and Vero cells-generated DENV-2. The fed mosquitoes were sacrificed on 8 days post infection to determine the infectivity by qPCR. The number of infected mosquitoes/total mosquitoes was presented on the top of each column (Left panel of each subfigure). The mosquito infective ratio (Right panel) was then interpreted from the left panel. One dot represented a mosquito. The result was pooled from 4 independent experiments.
Figure 6.
Transmission-blocking effect of mosGCTLs antisera in the infection of low-passage DENV-2 strains.
(A–F) Two low-passage DENV-2 strains, which were isolated from patients' sera, were employed to test transmission-blocking effect of mosGCTL antisera in A. aegypti. The pre-immune sera, mosGCTL-3 antisera and combined mosGCTLs antisera (1/9 mosGCTL antiserum each) with Vero cells-generatedDENV-2 AF2014178 strain (A to C)/JX470186 strain (D to F) and human blood were used for mosquitoes feeding. The antisera were serially diluted 100- (A and D), 1,000- (B and E) and 5,000- (C and F) fold for the investigation. The fed mosquitoes were sacrificed on 8 days post infection to determine the infectivity by qPCR. The numbers of infected mosquitoes/total mosquitoes are presented on the top of each column (Left panel of each sub-figure). The mosquito infective ratio (Right panel) indicates the ratio of DENV positive to total mosquitoes. One dot represents a mosquito. The results are pooled from 3 independent experiments.