Fig 1.
Strong temporal and inter-individual variation in midgut viral RNA load following oral DENV exposure.
Time course of viral RNA load measured by RT-qPCR in individual midguts of Ae. aegypti females following a DENV infectious blood meal. Midguts were harvested immediately after the blood meal (n = 9), at 6 hours (n = 7), 1 day (n = 25), 2 days (n = 25), 3 days (n = 25), 4 days (n = 24), 7 days (n = 10) and 10 days (n = 13) post virus exposure. The color gradient scale indicates the percentage of midguts containing blood at each time point. Solid and open dots represent infected and uninfected midguts, respectively, as determined by RT-qPCR detection threshold. Letters above the graph (a, b, c) indicate statistical differences in the mean viral RNA load between time points according to multiple pairwise t-tests with Holm correction for multiple testing. Viral RNA load is not significantly different (P > 0.05) between time points sharing the same letter.
Fig 2.
Candidate host factors identified by pairwise comparison and correlation analysis of transcriptome-wide midgut gene expression.
(A) Venn diagram showing the number of candidate genes differentially expressed between DENV-infected midguts and uninfected controls (blue) or whose expression was linearly correlated to midgut viral RNA load (red) at a false discovery rate of 0.1, across time points. Common candidate genes between pairwise and correlation analyses are shown (yellow). (B) Time-specific representation of DENV-infected midgut transcriptome plotted against control midgut transcriptome (log10 [normalized RNA-Seq counts +1]) at day 1 and day 4 post exposure. Candidate transcripts identified by pairwise comparison (blue), correlation analysis (red) or both (yellow) are indicated.
Fig 3.
SREBP is a candidate host factor during early midgut infection.
(A) SREBP expression levels measured by RNA-Seq (log2-transformed normalized counts) on day 1 and day 4 post DENV exposure in the midgut of control (n = 6) and DENV-infected (n = 16) individuals. P-values of the pairwise t-tests are indicated. (B) Correlation of SREBP expression level measured by RNA-Seq (log2-transformed normalized counts) and viral RNA load (log10 RNA copies / midgut) in DENV-infected midguts on day 1 and day 4 post virus exposure. Black lines represent the linear regression and light orange shaded areas represent the 95% confidence intervals. Pearson's coefficients of determination (r) and P-values of the linear regression coefficient are indicated.
Fig 4.
SREBP knockdown results in ∼50% reduction of midgut viral RNA load.
(A) Time line of gene knockdown assays in vivo. (B) Impact of target gene knockdown on midgut viral RNA load. Boxplots represent the viral RNA load measured by RT-qPCR on day 4 post exposure in individual midguts following injection with a single dsRNA. Data represent two (for SREBP and Ago2) or three (for Cactus) separate experiments combined. Viral RNA load was adjusted for differences between experiments and expressed in arbitrary units. On day 4 post exposure, viral RNA load was reduced by 53.8% following SREBP knockdown. The P-value above the graph indicates statistical significance of the treatment assessed with an ANOVA accounting for the experiment effect. (C) Survival curves following dsRNA injection. Mosquito survival (%) is represented as a function of day post dsRNA injection for individuals injected with dsRNA targeted against SREBP (dsSREBP, left), Ago2 (dsAGO2, center) or Cactus (dsCACTUS, right). Mosquitoes injected with dsRNA against GFP (dsGFP) or non injected (non inj.) were used as controls. Each graph of the panel represents a separate experiment. Individuals were exposed to a DENV infectious blood meal 3 days post dsRNA injection. No significant difference in mortality was detected between dsGFP- and dsSREBP-injected mosquitoes according to a Cox model (P = 0.3) whereas survival of dsAGO2- and dsCACTUS-injected mosquitoes was impaired relative to the dsGFP controls (Cox model, P < 0.0001 for both genes). (D) Effect of target gene knockdown on DENV infection prevalence. Bar plots show the percentage of midguts positive for DENV infection by RT-qPCR on day 4 post virus exposure following injection with a single dsRNA. Data from two (for SREBP and Ago2) or three (for Cactus) separate experiments were combined after verifying the lack of a detectable experiment effect. Vertical bars represent 95% confidence intervals of the percentages. The P-value above the graph indicates statistical significance of the treatment effect assessed with a logistic regression.