Article Figures & Data
Figures
- FIG 1
Zika virus infection of primary hBMECs. (A) Primary hBMECs were infected with ZIKV (PRVABC59) at an MOI of 10, and 12 to 72 hpi ZIKV antigen-positive cells were detected by anti-DENV4 HMAF. (B to D) Titers of ZIKV-infected hBMEC supernatants were determined in an FFU assay (B) and analyzed for cellular ZIKV RNA levels by qRT-PCR (C) and for infected cells (D). (E to G) hBMECs and Vero E6 cells were pretreated with IFN-α (1,000 U/ml) for 3 h prior to ZIKV infection (MOI, 10) (E), or IFN-α was added at the indicated time postinfection and infected Vero E6 (F) or hBMECs (G) were immunostained and quantitated 24 later.
- FIG 2
hBMECs are viable after ZIKV infection. (A) Vero E6 or hBMECs were infected with ZIKV (MOI, 10), and costained 3 dpi with calcein-AM (green [live cells])/propidum iodide (red [dead cells]). Following calcein-AM/PI staining, monolayers were fixed and immunostained for ZIKV antigen. (B) Viability of ZIKV-infected Vero E6 cells and hBMECs was assessed via CyQuant NF uptake 3 dpi, and results were compared to those for mock-infected controls.
- FIG 3
ZIKV persistently infects viable hBMECs 9 dpi. (A) hBMECs were infected with ZIKV as described for Fig. 2A, and titers present in cell supernatants were compared 2 to 9 dpi. (B and C) hBMECs were infected as described above, and cell lysates were analyzed for ZIKV RNA by qRT-PCR (B) and for ZIKV envelope protein (anti-Env) by Western blotting (C). Results were compared to those for the GAPDH controls 1 to 9 dpi. (D and E) Vero E6 cells or hBMECs were analyzed 9 dpi for ZIKV antigen and via calcein-AM/PI stain (D) or in CyQuant assays (E) for cell viability.
- FIG 4
ZIKV-infected hBMECs are viable and productive following cellular passage. (A and B) ZIKV-infected Vero E6 cells or hBMECs (MOI, 10) were trypsinized and passaged (1:3) 3 dpi and every 3 days thereafter. ZIKV-infected passaged hBMECs were detected by immunostaining. (C) Cells were infected and passaged as for panels A and B, and cell viability was assessed via calcein-AM/PI staining and fluorescent image overlay. (D) ZIKV titers in supernatants of hBMECs consecutively passaged 1 to 3 times (every 3 days).
- FIG 5
Analysis of cellular protein expression in ZIKV-infected hBMECs. (A to C) hBMECs were mock infected or infected with ZIKV, and 1 to 9 dpi supernatants were analyzed in an ELISA (R&D Systems) for CCL/RANTES (A), IFN-β (B), and IFN-λ (C) levels relative to antigen standards. As an hBMEC IFN-β response control, we transfected hBMECs with poly(I/C) (1 µg/ml) and Fugene6 at 3:1 and evaluated secreted IFN-β levels in supernatants via ELISA (36 h posttransfection) (D) Western blot analysis of MXA and IFIT1 genes, and GAPDH controls, in lysates from mock-infected or ZIKV-infected hBMECs (1 to 9 dpi). IFIT and MxA protein levels in ZIKV-infected hBMECs 9 dpi versus results 6 h post-IFN-α treatment (1,000 U/ml).
- FIG 6
ZIKV-infected hBMECs release ZIKV basolaterally. (A) Polarized hBMECs, grown for 5 days in Transwell plates, were apically or basolaterally infected with ZIKV (MOI, 5) in triplicate, and TEER was measured 1 to 3 dpi. To demonstrate monolayer barrier function, EDTA was added (10 mM for 10 min) to hBMEC monolayers; this resulted in an ~100-Ω reduction in TEER. (B) hBMECs apically or basolaterally infected with ZIKV were assayed for permeability to FITC-dextran (40 kDa), which was added to apical medium at 3 dpi; fluorescence over time was measured in the lower chambers. (C) hBMECs grown on Transwell inserts for 5 days were evaluated for TEER. Cells were apically or basolaterally infected (MOI, 5) with ZIKV, and titers present in apical and basolateral supernatants were quantitated at 1 dpi. (D) Potential model of the spread of ZIKV systemically and to neuronal compartments from hBMECs.
Tables
- TABLE 1
Global transcriptional responses of ZIKV-infected hBMECs
Category and
proteinFold induction versus controls at: Confirmed by
qRT-PCR12 hpi 1 dpi 2 dpi 3 dpi 9 dpi Interferons IFN-β 142 7 4 X IFN-λ1 238 212 4 X IFN-λ2 11 10 IFN-λ3 23 23 2 ISGs IFIT1 62 734 1,086 650 2,071 X IFIT2 17 1126 869 68 58 X IFIT3 6 226 434 130 282 IFITM1 3 5 5 8 MX1 36 515 1,081 575 209 X OAS2 6 271 961 475 737 X RSAD2a 57 193 106 616 ISG15 and related ISG15 28 64 43 90 X HERC5 5 451 292 52 46 X HERC6 36 43 20 26 USP18 3 4 7 13 X USP41 3 6 7 13 IRFs IRF1 114 24 IRF7 5 9 6 10 IRF9 6 7 3 3 Transcription factors ATF3 4 301 101 7 7 EGR1 7 168 155 20 TRAF1 86 39 3 JUNB 4 14 15 Apoptosis regulatory factors BIRC3 IAP2 35 43 5 6 X XAF1 5 62 201 86 50 X Permeability Rnd1 3 169 80 5 X ARHGAP26 RhoAct 12 8 2 Chemokines and related CCL5/RANTES 9 2,327 1,914 352 40 X CXCL10 247 179 35 60 X CXCL11 305 244 57 7 CCL20 385 126 8 IL-1 20 11 5 3 IL-6 10 11 3 2 ↵ a MX2 and OAS1 were similarly induced.
FIG S1
(A) Vero E6 cells were infected with ZIKV (PRVABC59) at an MOI of 10, treated with IFN-α (1,000 U/ml) at 3 to 12 hpi, and immunoperoxidase stained for ZIKV antigen at 24 hpi. (B) A telomerase-immortalized human cerebral microvascular EC cell line (hCMEC/D3) was infected with ZIKV (MOI, 5) and at 36 hpi, ZIKV antigen-positive cells were detected by immunoperoxidase staining. (C) hCMEC/D3 cells were infected with ZIKV (MOI, 5), and titers of infectious virus in supernatants were determined at 12 to 48 hpi. (D) hCMEC/D3 cells were pretreated with IFN-α (1,000 U/ml) for 3 h, or untreated, prior to ZIKV infection (MOI, 5). ZIKV antigen-positive cells were detected by immunoperoxidase staining at 24 hpi. Download FIG S1, TIF file, 30.7 MB.
Copyright © 2017 Mladinich et al.
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FIG S2
(A) hCMEC/D3 cells were mock or ZIKV infected (MOI, 10) and at 3 dpi or 3 days after hCMEC/D3 passage, cells were stained for ZIKV antigen or costained with calcein-AM/propidium iodide. (B) HUVECs and hCMEC/D3 cells were infected with ZIKV (MOI, 10) and analyzed at 9 dpi via immunoperoxidase staining. (C) Titers from supernatants of ZIKV-infected HUVECs and hCMEC/D3 cells were determined 3 days following cellular passage. Download FIG S2, TIF file, 23.8 MB.
Copyright © 2017 Mladinich et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
FIG S3
hBMECs were ZIKV infected as described for Fig. 1A. RNAs were purified from cell lysates at 1 to 9 dpi, and the induction of the cellular genes identified as induced by Affymetrix arrays (Table 1) (GEO GSE98889) were assayed by qRT-PCR and compared to RNA from mock-infected hBMECs harvested at the same time points. Download FIG S3, TIF file, 50.8 MB.
Copyright © 2017 Mladinich et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
