Figure 1.
DENV2 strain Jamaica 1409 grows to higher titers in C6/36 than in Aag2 mosquito cell lines.
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.
Table 1.
DENV2 viRNAs from whole mosquito and mosquito cell culture libraries.
Figure 2.
viRNA size distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.
Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.
Figure 3.
viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.
viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.
Figure 4.
C6/36 cells are unable to produce a Dicer-2-like product.
A) In vitro dicing assay. Biotinylated 500 bp β-gal dsRNA was added to Aag2 and C6/36 cell lysates, with (as indicated by +) or without human recombinant Dicer (reDicer), and aliquots were collected immediately (0) or after 18 hours. Lysate RNA was separated by electrophoresis on a polyacrylamide gel and transferred to a membrane for detection using the BrightStar detection system. C lane contains product of in vitro reaction of reDicer with 500 bp dsRNA, as size marker. (B) Analysis of dicing activity in Aag2 (a-d) and C6/36 (e-h) cell cultures. Aag2 cells were transfected with (a) pEGFP + EGFP siRNA, (b) pEGFP + WNV siRNA, (c) pEGFP + EGFP dsRNA, (d) pEGFP + WNV dsRNA. C6/36 cells were transfected with (e) pEGFP + EGFP siRNA, (f) pEGFP + WNV siRNA, (g) pEGFP + EGFP dsRNA, (h) pEGFP + WNV dsRNA. All cells were analyzed by fluorescent microscopy at 48 hr after transfection. (C) Immunoblot shows EGFP protein silencing. 1, pEGFP + EGFP siRNA, 2, pEGFP + WNV siRNA, 3, pEGFP + EGFP dsRNA, 4, pEGFP + WNV dsRNA.
Table 2.
CFAV viRNAs from mosquito cell culture libraries.
Figure 5.
viRNA size and genome coverage distributions vary among CFAV-infected Aag2 and C6/36 cell cultures.
(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.
Figure 6.
Logo analysis of DENV2 viRNA from mosquitoes and cell culture and CFAV viRNAs from cell culture.
Logo analysis was performed on DENV2 and CFAV viRNAs using Weblogo 3. (A) Aag2 DENV2 (5 dpi) viRNA logo. (B) A. aegypti DENV2 (9 dpi) viRNA logo. (C) C6/36 DENV2 (5 dpi) viRNA logo. (D) Aag2 CFAV viRNA logo. (E) C6/36 CFAV viRNA logo.
Figure 7.
Northern blot hybridization to detect expression of dicer2 mRNA in cultured mosquito cells.
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 A. albopictus dcr2 probe (left) or an A. aegypti dcr2 probe (right). The biotinylated probes were detected with the BrightStar BioDetect Kit.