Logo analysis of DENV2 viRNA from mosquitoes and cell culture and CFAV viRNAs from cell culture.

<p>Logo analysis was performed on DENV2 and CFAV viRNAs using Weblogo 3. (A) Aag2 DENV2 (5 dpi) viRNA logo. (B) <i>A. aegypti</i> DENV2 (9 dpi) viRNA logo. (C) C6/36 DENV2 (5 dpi) viRNA logo. (D) Aag2 CFAV viRNA logo. (E) C6/36 CFAV viRNA logo.</p

viRNA size distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and <i>Aedes aegypti</i> mosquitoes.

<p>Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, <i>A. aegypti</i> (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.</p

Northern blot hybridization to detect expression of <i>dicer2</i> mRNA in cultured mosquito cells.

<p>Total RNA from Aag2 or C6/36 cell cultures was fractionated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized to either an <i>A. albopictus dcr2</i> probe (left) or an <i>A. aegypti dcr2</i> probe (right). The biotinylated probes were detected with the BrightStar BioDetect Kit.</p

viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and <i>Aedes aegypti</i> mosquitoes.

<p>viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, <i>A. aegypti</i> (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.</p

DENV2 strain Jamaica 1409 grows to higher titers in C6/36 than in Aag2 mosquito cell lines.

<p>Cell cultures were infected at a MOI of 0.001 and aliquots of medium were removed at 24 hour intervals and titrated by plaque assay. The assays were performed in triplicate and titers are expressed as means ± SEM.</p

Primers used for the generation of dsRNAs targeting RNAi pathway genes.

<p>T7 promoter (not shown) was added to the 5′ end of each primer for in vitro transcription of dsRNA.</p

viRNA size and genome coverage distributions vary among CFAV-infected Aag2 and C6/36 cell cultures.

<p>(A) viRNA size distribution. (B) viRNA coverage across CFAV genome for each library. Shown are Aag2 (5dpi) library, C6/36 (5dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note difference in Y-axes among graphs.</p

DENV2 viRNAs from whole mosquito and mosquito cell culture libraries.

<p>DENV2 viRNAs from whole mosquito and mosquito cell culture libraries.</p

DENV2 infection of Aag2 cells.

<p>(A) Growth curve of DENV2 in Aag2 cells. Virus titers in cell culture medium were determined by plaque assay. (B) Detection of DENV2 genomic RNA in Aag2 cells by northern blot analysis (5 µg total RNA/lane). Stained ribosomal RNA is shown as loading control. (C) Detection of dsRNA in mock infected (a) and DENV2 infected (b) Aag2 cells using the dsRNA-specific J2 antibody in an indirect immunofluorescence assay (IFA). (D) Detection of DENV2 genome-derived small RNAs consistent in size with siRNAs on successive days after infection. Small RNAs were detected using sense (a) and antisense (b) riboprobes from the prM gene of DENV2.</p

Transmission of DENV2 by <i>Ae. aegypti</i> at 7, 10, and 12 days post infection.

<p>Groups of 200 mosquitoes were non-injected or injected with PBS or 500 ng dsRNA.βGAL, dsRNA.ago2, dsRNA.r2d2, or dsRNA.dcr2. Two days later all mosquito groups were orally infected with DENV2. At 7 (A), 10 (B) and 12 (C) dpi multiple batches of 10 mosquitoes were allowed to probe artificial feeding solutions. The feeding solutions were assayed for virus titer (* indicate P<0.05).</p