A) 2 days post-infection (dpi). B) 4 dpi. C) 9 dpi. Top panel shows mean mapped reads and count distribution along DENV2 genome for a representative library at each time point. Bottom panel shows mean viRNA distribution by sRNA size group. Blue and red bars indicate sense P505-15 mw and anti-sense viRNAs, respectively. Host sRNA Profiles To identify host factors that
are differentially regulated by SRRPs during DENV2 infection, we asked whether sRNA profiles mapping to host RNAs change in DENV2-infected mosquitoes compared to un-infected controls. sRNA profiles were categorized by the target RNA to which they mapped, as well as by sRNA size group. Changes to ncRNAs were also measured, because NVP-BSK805 recent evidence suggests that they are regulated by RNAi pathway activity [28, 32]. For this line of inquiry, we did not distinguish between 20-23 nt siRNAs, endo-siRNAs, or microRNAs. We reason that enriched sRNA profiles for a given target represent the product of enhanced target cleavage, in the absence of concomitant transcriptional repression or mRNA decay [28, 33]. Conversely, depleted sRNA profiles among the DENV2-infected pools would be indicative of fewer degraded mRNAs. We defined a single sRNA profile as the number of reads mapped to a single target transcript. The presence of sRNAs aligning to a given transcript would be expected to change
sporadically across the three biological replicates if they arose through MYO10 non-specific decay events. Moreover, non-specific decay events should produce sRNAs across all size groups in similar frequency. Therefore, we expect that sRNA levels with statistically significant enrichment or depletion represent altered RNAi pathway activity. The RNA-seq
program edgeR was used determine the significance of sRNA profile enrichment or depletion for sense and anti-sense sRNAs across all three replicates for each timepoint . Sense strand reads were more abundant than anti-sense reads. All target transcripts are categorized by read orientation in Additional File 2. A cut-off value of 0.05 False Discovery Rate (FDR) was used to determine whether changes were statistically significant . sRNA populations mapping to mRNAs and ncRNAs were grouped into functionally similar categories. Figure 3 shows functional categories for which sRNA profiles were modulated over the course of infection. At 2 dpi, 555 unique targets showed enriched sRNA profiles compared to controls, whereas at 4 dpi, only 67 targets had significantly enriched sRNA profiles (Figure 3A and Additional File 2). Under-represented sRNA profiles were much less abundant; 43 unique targets showed depleted sRNAs in DENV2-infected mosquitoes at 2 dpi, and 44 targets showed depleted sRNAs at 4 dpi (Figure 3B). Very few differentially abundant profiles were observed at 9 dpi, therefore they were excluded from further analysis (Additional File 2).