Figure 1.
Transcriptional regulation of genes in the Aag2 cell line in response to live dengue virus (DENV) and heat-inactivated dengue virus (HIA DENV) infection.
A. Venn diagram showing the numbers of unique and commonly regulated genes in DENV- and HIA DENV-infected cells and DENV-infected mosquito carcass. Arrows indicate the direction of gene regulation. B. Functional classification of significantly regulated genes in DENV and HIA DENV infection; arrows indicate the direction of gene regulation. Functional group abbreviations are as follows: UNK, unknown functions; DIV, diverse functions; MET, metabolism; RTT, replication, transcription, and translation; TRP, transport; CS, cytoskeletal and structural; PROT, proteolysis; DIG, blood and sugar food digestive; CSR, chemosensory reception; RSM, redox, stress and mitochondrion; IMM, immunity. C. Comparative analysis of the DENV infection-responsive cell line transcriptome and the Toll-, IMD-, and JAK-STAT pathway-regulated mosquito transcriptomes. Venn diagrams show the numbers of unique and commonly regulated genes in DENV-infected Aag2 cells and Cactus-, Caspar-, and PIAS-silenced A. aegypti mosquitoes. Arrows indicate the direction of gene regulation. D. Cluster analysis of 238 genes that were regulated in at least two of three treatments: DENV infection in the cell line, HIA DENV infection in the cell line, Cactus silencing in A. aegypti mosquitoes. All genes presented in this cluster analysis are listed in Table S6. E. Detection of DENV in Aag2 cells by indirect immunofluorescence assay using mouse hyperimmune ascitic fluid specific for DENV2 (CDC) and an AlexaFluor488-conjugated goat anti-mouse secondary antibody. Blue: DAPI, Green: FITC.
Figure 2.
DENV-infected cells are less able to produce antimicrobial peptides in response to secondary bacterial challenge.
Aag2 cells were DENV- or mock-infected, then challenged or mock-challenged with heat-killed E. coli or S. aureus. The bar charts show the -fold change in (A) cecropin and (B) defensin gene expression levels relative to levels at the 0-h time point for each sample, as measured by semi-quantitative PCR. Error bars indicate the standard error of the mean; ND, non-detectable.
Figure 3.
DENV-infected cells are less able to inhibit the growth of Gram-negative bacteria in co-culture.
Eight 10-fold dilutions of Gram-positive or Gram-negative bacteria were inoculated into the cell culture medium in 96-well plates containing DENV- or mock-infected Aag2 cells, or cell culture medium alone. The graphs show OD595 as a measure of bacterial growth after a 12-h incubation at 28°C for (A) E. coli, (B) S. aureus, and (C) M. luteus. * represents p<0.05 in a Student's t-test comparing OD595 of bacteria incubated with DENV- and mock-infected cells. Error bars indicate the standard error of the mean; note that in many cases the error bars are obscured by the data point.
Figure 4.
Effect of pre-immune stimulation on DENV titers in the cell line.
Aag2 cells were pre-immune stimulated by the addition of heat-killed E. coli and S. aureus 24 h prior to DENV infection. The graph shows log10 DENV titers over 7 days as determined by plaque assay for cells pre-stimulated with heat-killed E. coli or S. aureus, or mock-stimulated with PBS. † represents p<0.05 in a Student's t-test comparing DENV titers in S. aureus- and mock-stimulated cells; * represents p<0.05 in a Student's t-test comparing DENV titers in E. coli- and mock-stimulated cells. Error bars indicate the standard error of the mean.