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. 2019 Oct 17;8:e41244. doi: 10.7554/eLife.41244

Figure 1. Piwi4 is a restriction factor in vivo, upregulated after blood meal.

(A) Multi step growth curves of SINV, DENV-2 and CHIKV in Aag2 cells (MOI 0.1) treated with dsRNA targeting Ago2 or Piwi4 measured by plaque assay. Error bars depict standard deviation of four replicates. Significant changes over controls are marked with asterisks (p<0.05, Mann-Whitney U test). (B) Piwi4 mRNA measured by RT-qPCR from pools of either blood- or sugar- fed Ae. aegypti mosquitoes. The mean and standard deviation of four biological replicates of pools of five mosquitoes in carcass, midgut and ovary are shown. Significant changes over controls are marked with asterisks (p<0.05, Mann-Whitney U test). (C) Replication of DENV2 as measured by RT-qPCR or virus plaque assays. Mosquitoes were infected by intrathoracic injection with either dsPiwi4 or control dsRNA prior to infection. Error bars correspond to standard error of 20 (RT-qPCR) or 30 (plaque assays) biological replicates of individual mosquitoes. Significant changes over controls are marked with asterisks (p<0.05, Mann-Whitney U test).

Figure 1.

Figure 1—figure supplement 1. Piwi4 knockdown in cell culture and adult female Ae. aegypti mosquitoes.

Figure 1—figure supplement 1.

(A) RNAi screen against the seven Aedes aegypti Piwi genes and other selected RNAi factors in SINV-eGFP infected Aag2 cells. Viral replication was measured as GFP intensity per cell. (B) Piwi4 mRNA measured by qPCR from pools of dissected tissues from blood-fed female Ae. aegypti mosquitos injected with dsRNA against Firefly luciferase (dsFluc, negative control) or against Piwi4 (dsPiwi4). The error bars represent standard deviations of four biological replicates of pools of five of the respective tissue. Significant changes over control are marked with asterisks (p≤0.05, Mann-Whitney U test).