Figure 3.

Signal Pathways involved in Dengue Virus (DENV) Replication and Transmission in Aedes aegypti. (A) Development of a thioester-containing protein 1 (TEP1) loss-of-function transgenic mosquito line with a blood meal-inducible carboxypeptidase (CPA) promoter. Based on the mariner transposon system, the midgut-specific blood meal-inducible CPA promoter (AaCPA) was used for expressing the downstream synthetic miRNAs. AaCPA promoter, Ae. aegypti carboxypeptidase A promoter; AaTEP1_2miR, anti-TEP1 miRNA. (B, C) miRNA-mediated TEP1 silenced transgenic mosquitoes. The whole bodies of female wild type or transgenic mosquitoes (AaCPA>2miR-TEP1) were collected 6, 12, 24, 48, and 72 hours after a normal blood meal. Total RNA was extracted and quantified, followed by cDNA synthesis and subjected to quantitative real-time PCR analysis with a specific primer for TEP1 (B) or GFP (C). Values are mean ± S.E. (error bars) of the ratio of each gene to ribosomal protein S7. At least three biological cohorts from each time point were used for analysis. Ribosomal protein S7 was used for normalization of the relative target gene mRNA expression. (D) Anti-DENV phenotype of transgenic mosquitoes. The whole bodies of female mosquitoes were collected 0, 1, 2, 3, 4, 6, 8, and 10 days after an infectious DENV blood meal. Total protein was extracted and western blot analysis was performed with the antibody against DENV NS1 protein. Anti-GAPDH antibody was used as the loading control. PE means post-eclosion.