Figure 2. AaMCR restricts flaviviral infections.

(A-B) Knockdown of the AaMCR gene. The mosquitoes were microinjected with AaMCR or GFP dsRNA. The inoculated mosquitoes were sacrificed, and AaMCR abundance was assessed via qPCR (A) and immuno-blotting with an AaMCR antibody (B) at 6 days post microinjection. 50 μg of protein from mosquito lysates was loaded into each lane (B). (C-G) Silencing of AaMCR enhanced DENVs and YFV (17D) infections in A. aegypti. 10 MID₅₀ DENVs or YFV was inoculated at 3 days post-AaMCR silencing. The viral load was assessed at 6 days post-infection through qPCR and normalized against A. aegypti actin (AAEL011197). The primers and probes used for qPCR are described in Table S2. The experiment was repeated three times with similar results. One dot represents 1 mosquito and the horizontal line represents the median of the results. The data were analyzed statistically by the non-parametric Mann-Whitney test. (H) Immuno-blockade of AaMCR enhanced the DENV-2 infection of A. aegypti. The AaMCR-N antibody, in 10-fold serial dilutions, was premixed with 10 MID₅₀ DENV-2 for thoracic co-microinjection. The treated mosquitoes were sacrificed to examine the viral load at 3 (i) and 6 (ii) days post-infection via qPCR and normalized against A. aegypti actin. The results were pooled from 3 independent experiments. One dot represents 1 mosquito and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric Mann-Whitney test. (I) The expression of AaMCR fragment peptides in Drosophila S2 cells. Three fragments of the AaMCR (a, 30–601 aa; b, 590–1,240 aa; c, 1,200–1,793 aa) with a C-terminal HA tag were cloned into the pMT/BiP/V5-His A vector and expressed in the S2 cell supernatant. The supernatant from empty vector-transfected S2 cells was used as a mock. The recombinant peptides were detected with an anti-HA antibody via western blotting. (J) The fragments of AaMCR do not directly interact with DENV-2 E protein in ELISA. The cell supernatant, including the AaMCR fragments were collected at 48 hrs after transfection. The DENV-2 E protein was expressed and purified from a Drosophila expression system. Each plate well was coated with 2 ug of DENV-2 E. The interaction was determined using an anti-HA antibody. The empty DNA vector-transfected S2 cell supernatant was used as a negative control. The data are expressed as the mean ± standard error from 3 independent experiments.