Distinct ZIKV strain signatures and type I IFN modulation reveal a protective role of brain endothelial interferon signaling in vitro and in vivo

Figures

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  Figure 1. Global gene expression analysis in HBMECs infected with different ZIKV lineages. (A) Principal component analysis (PCA) showing control samples and cells infected with ZIKVMR766 and ZIKVPE243 strains. (B) Differentially expressed genes (DEGs) between ZIKVMR766 infection condition compared to ZIKVPE243 condition. (C, D) Volcano plots depicting DEGs in HBMECs infected with ZIKVMR766(C) and ZIKVPE243(D), compared to control. Three biological replicates were analyzed for each condition and significantly regulated genes according to p-values and log2 fold changes are labeled in each volcano plot. (E, F) Differentially expressed genes associated with biological pathways during ZIKV infection compared with control condition. The plot illustrates the relation between genes and enriched pathways in infections with the ZIKVMR766(E) and ZIKVPE243(F).

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  Figure 2. Replication, dissemination, and cytopathic effects of ZIKV strains in HBMECs. HBMECs were infected with ZIKVPE243 or ZIKVMR766, at a MOI of 1 or 0.1, and cell lysates and culture medium were collected at the indicated time points. (A) ZIKV mRNA copies were quantified in cell lysates by RT–qPCR. (B) Infectious viral titers were measured in the culture medium by plaque assay. (C) Ratio between PFU and viral RNA levels comparing ZIKVPE243 and ZIKVMR766. (D) Infected cells were stained with the 4G2 antibody, and the frequency of infected cells (%4G2+) was determined by immunofluorescence. (E) Cell viability was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay. (F) Correlation between viral titers (PFU) and cell viability in HBMECs infected with ZIKVPE243 and ZIKVMR766. Data represent mean ± SD of two independent experiments. *p<0.05; **p<0.01; ***p<0.001.

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  Figure 3. ZIKV induces RIG-I-dependent IFN-β production by HBMECs. (A) HBMECs were mock-treated or infected with ZIKVPE243 or ZIKVMR766 (MOI=1); poly (I:C) was used as positive control. The cells were harvested at the indicated time points and IFN-β mRNA was measured by RT-qPCR. Bars represent the mean and standard deviation of seven independent experiments; statistical analyses were performed by two-way anova and Bonferroni’s multiple comparisons test. (B-E) Depletion of TLR3 (B, C) and RIG-I (D, E) in HBMECs were performed by specific siRNA transfection; lipofectamine only (Lipo), and scramble siRNA (Scr) were used as controls. Receptor depletion was confirmed by western blotting using anti-TLR3 (B) or anti-RIG-I (D) specific antibodies, and anti-βactin as a loading control. After 48 h, the cells were mock-treated or infected with ZIKVPE243 or ZIKVMR766 and, at 24 hpi, IFN-β expression was evaluated by RT-qPCR (C, E). Statistical analysis were performed by unpaired t test; *p<0.05, **p<0.01, ***p<0.001.

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  Figure 4. ZIKV infection inhibits IFN-β response in HBMEC. (A) pISRE-HBMEC-luc reporter cells were mock-treated or infected with ZIKVPE243, ZIKVMR766 or DENV-2 at a MOI of 1. Cells were treated with IFN-β or poly I:C after virus adsorption and cellular luciferase activity was measured at 24 hpi. (B, C) HBMECs were cultured as in (A). At 24 hpi, OAS1, ISG15, MX1, and IFIT1 mRNA was assessed by RT-qPCR (B) and released viral titers was measured by plaque assay (C). The data represent the mean and standard deviation of five independent experiments, and the statistical analyses were performed by two-way ANOVA and Dunnett’s multiple comparisons test. (D, E) The expression of phosphorylated or nonphosphorylated STAT1 (pSTAT1, STAT1) (D) and of pSTAT2 and STAT2 (E) were evaluated by western blotting; β-actin expression was measured as loading control. Representative membranes are demonstrated in the left panels; the ratio of pSTAT1 in relation to STAT1, as well as pSTAT2 in relation to STAT2 are indicated in the middle panels; and total STAT1 or STAT2 expression in relation to β-actin are demonstrated in right panels. Data represent mean and standard deviation of six independent experiments, and…

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  Figure 5. Comparison of genomic and amino acid sequences by the 5’UTR, NS5 and capsid protein from ZIKVMR766 (NC_012532.1) and ZIKVPE243 (GenBank KX197192.1). (A, B) Sequence and predicted secondary structure of stem-loop A in the 5′UTRs from ZIKVPE243(A) and ZIKVMR766(B). The black arrows indicate sequence variations in the 5′-UTR. (C) Schematic representation of ZIKV NS5 from ZIKVMR766 indicating the amino acid substitutions in the NS5 from ZIKVPE243. MTase and RdRp domains are colored purple and gray, respectively. (D) Ribbon representation showing the MTase (light pink) and RdRp (gray) domains of ZIKVMR766 NS5. The locations of residues that differed in NS5 from ZIKVPE243 in the context of ZIKVMR766 NS5 structure (PDB, 5U0B) are shown in orange sticks. The residues Y25, R327, D734 and H855 are highlighted in blue sticks, and the K252 residue (green stick) is labeled in orange. (E) Ribbon representation showing the structure of the ZIKV capsid protein (PDB, 6C44), with the α-helix pairs indicated: α1/α1’ (purple), α2/α2’ (red), α3/α3’ (brown), and α4/α4’ (blue). (F) Schematic representation of the capsid protein from ZIKVPE243 indicating the amino acid substitutions from ZIKVMR766. The…

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  Figure 6. IFN-β treatment before ZIKV infection reduces viral replication. (A, B) HBMECs were treated with IFN-β (1000U/mL) and, after 24 h, cells were mock-treated or infected with ZIKVPE243 or ZIKVMR766 (MOI=1). Cells were harvested and mRNA expression of OAS1, ISG15, MX1, and IFIT1 were measured by RT-qPCR (A). Viral titers were measured by plaque assay (B). (C, D) HBMECs were treated with different concentration (10-3–104 U/mL) of IFN-β for 24 hours and were then infected with ZIKVPE243(C) or ZIKVMR766(D). At 24 hpi, the supernatants were harvested, virus titration was performed by plaque assay, and the IFN concentration inhibiting 50% and 90% of virus titer (IC50 and IC90) were calculated with GraphPad prism. The data represent mean and SD of three independent experiments. Bars indicate average and SD from three independent assays and statistical analysis were performed by two-way anova and Tukey’s multiple comparison test; *p<0.05; **p<0.01 ***p<0.001.

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  Figure 7. Type I IFN limits ZIKV replication, dissemination and preserves HBMECs barrier integrity. (A) HBMECs were infected with ZIKV strains (MOI=0.1) in the presence or absence of a combination of neutralizing anti-IFN-α and anti-IFN-β antibodies (αIFN-α/β). At 24 and 48 hpi, virus replication was evaluated by plaque assay; graph bars represent the mean and SD of four (24 h) and two (48 h) independent assays, and statistical analyses were performed by paired t-test. (B) Cells were cultured as in A, and viability was evaluated at the indicated time points using a Cell Titer assay. Data indicates the mean and SD of four independent experiments, and statistical analyses were performed by two-way ANOVA and Tukey’s multiple comparison test. (C–E) Cells were cultured as in (A) and, at 24 and 48hpi, the cells were stained with 4G2 antibody. Representative figures are shown in (C) and the average and SD of the frequency of ZIKV-infected cells (% 4G2+) is depicted in (D) and (E). Data was calculated from two experiments, including 20 images. Statistical analyses were performed by one-way ANOVA. (F, G) HBMECs were cultured onto transwell plates and the cells were mock-treated or infected with…

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  Figure 8. IFNAR deficiency conditioned to endothelial cells partially protects mice from ZIKV-mediated disease. Fully IFNAR-deficient mice (IFNAR-/-) and mice deficient in IFNAR only in endothelial cells (CDH5cre IFNARflox/flox), as well as IFNARflox/flox (IFNARflox/flox) control mice were intravenously infected with 2x105`PFU of ZIKVMR766 or inoculated with mock control. Mice were followed for 21 days. (A) Survival curve showed statistical significance (**p<0.01) as evaluated by log-rank (Mantel-Cox) tests. (B) Mice body weight was followed until the sacrifice and the results were statistically analyzed using two-way Anova and Tukey’s posttest; ****p<0.0001 in relation to IFNARflox/flox (mock and ZIKVMR766), CDH5-cre+IFNARflox/flox (mock and ZIKVMR766 survived). (C, D) Viral titer in the spleens (C) and the brains (D) were evaluated by plaque assay; statistical analyses were performed at 6 dpi by one-way anova and Tukey’s posttest. *p<0.05; **p<0.01 ***p<0.001; ****p<0.0001.