TR339-eGFP virus infection transiently increased in mosquitoes receiving a Ago2, Dcr2, or TSN dsRNA injection. (A) qRT-PCR analysis. Percent reduction in cognate Dcr2, and TSN mRNA levels, relative to actin, in midguts following Ago2, Dcr2 or TSN dsRNA treatment. "ND", none detected. Asterisks indicate statistical significance (Mann Whitney U test P ≤ 0.05). Error bars indicate standard error of three experimental replicates. (B) Immunoblot. Evidence of Ago2 protein silencing following dsRNA injection. "A" indicates Ago2 dsRNA-injected; "C" indicates βGAL injected controls. "0" days post-infection corresponds to female midguts harvested prior to virus meal. In each lane, equivalent amounts of protein extract from a pool of six midguts were separated by SDS-PAGE, blotted and probed with anti-Ago2 antibody. The loading control is a 19 kDa anti-Ago-2 cross-reacting band. Blot shown is from a single experiment and is representative of three independent replicates. (C) Two-step qRT- PCR showed that TR339 positive strand (+) RNA significantly increased in midguts of Ago2 dsRNA-injected mosquitoes over βGAL dsRNA controls at 1 dpi (P ≤ 0.05, Mann-Whitney U test). Pools of five midguts per group were used. Error bars depict standard error of three independent feedings. Viral (+) strand RNA copy numbers were calculated using the standard curve method. Asterisk indicates statistical significance, Mann-Whitney U test p ≤ 0.05. (D) TR339-eGFP viral titers of individuals at 4 dpi in Ago2, Dcr2, and TSN dsRNA-injected mosquitoes, with βGAL dsRNA controls. "**" indicates infection rate significantly higher than βGAL controls. "^^" above the graph indicates viral titers significantly higher than βGAL controls. Closed squares, Ago2 dsRNA; closed circles, βGAL dsRNA; closed diamonds, DCR2 dsRNA; closed triangles, TSN dsRNA; open squares, nsP3. Horizontal line indicates the median titer; no line indicates a median value of zero. "()" indicates (number of mosquitoes in each group per number positive). (E) TR339-eGFP viral titers at 7 dpi.