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. 2018 May 15;15(1):15.
doi: 10.1186/s12987-018-0100-y.

Zika virus crosses an in vitro human blood brain barrier model

Judie B Alimonti  1 , Maria Ribecco-Lutkiewicz  2 , Caroline Sodja  2 , Anna Jezierski  3 , Danica B Stanimirovic  2 , Qing Liu  2 , Arsalan S Haqqani  1 , Wayne Conlan  1 , Mahmud Bani-Yaghoub  2
Affiliations

Zika virus crosses an in vitro human blood brain barrier model

Judie B Alimonti et al. Fluids Barriers CNS. .

Abstract

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.

Keywords: AXL; Blood–brain barrier model; Brain endothelial cells; Neural progenitors; Zika virus; iPSC.

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Figures

Fig. 1
Fig. 1
Direct ZIKV infection and AXL expression in i-NP and i-Ns. a–d ZIKV infection was detected in the cytoplasm of b i-NPs and d i-Ns by immunofluorescence staining 72 h after ZIKV infection (MOI = 4). The nuclei were stained with Hoechst dye (blue) and the virus was detected using anti-flavivirus group antibody (FV; green). i-Ns were additionally stained with the anti-MAP2 (red) antibody. Scale bar = 10 µm. e Fold difference of ZIKV (ZV) infection in i-NP, relative to i-Ns, based on cell counts of FV positive cells (mean ± SD). Cell counts are from two independent experiments, six fields counted, with at least 80 cells/field. (****p < 0.0001, Student’s T test). f A representative Western blot of putative ZIKV receptor AXL expression in i-NPs and i-Ns detected by an anti-AXL antibody. β-ACTIN was used as an internal loading control
Fig. 2
Fig. 2
ZIKV infection and AXL expression in BECs. a, b Immunofluorescence staining was conducted 24 h post ZIKV infection (MOI = 4) of i-BECs using anti-flavivirus group antibody (FV; green). ZIKV was detected in infected i-BECs (b) but not in uninfected controls (a). Tight junction marker ZO-1 was used to label cell–cell contacts (red); nuclei were stained using Hoechst dye (blue). Scale bar = 10 µm. c The presence of ZIKV in i-BECs was also determined by Western blotting at 2 or 24 h after exposure to the virus. d Representative Western blot demonstrating putative ZIKV receptor AXL expression in i-BECs using an anti-AXL antibody. β-ACTIN was used as an internal loading control. e Expression levels of SLC2A1 (GLUT-1), MERTK and AXL in human brain endothelial cell line hCMEC/D3 determined by proteomics (LC–MS). Shown are ratio of intensity-based absolute quantification values (mean ± SD from 3 independent cell preparations) calculated for each protein and β-ACTIN, as described previously [41]. f The expression of AXL, MERTK and TYRO3 in primary human and rat brain endothelial cells (BECs) or in isolated rat or mouse brain vessels (BV) or whole brain tissues (BT), determined by NGS. Shown are abundances (mean ± SD) of RNA transcripts relative to β-ACTIN from at least three cell/tissue preparations (***p < 0.001, One-Way ANOVA, Tukey’s post hoc test)
Fig. 3
Fig. 3
ZIKV crosses the human BBB and infects i-NPs. i-BECs were cultured on Transwell inserts and the cells were mock-infected (control) or infected with ZIKV. a TEER (Ω cm2) was measured on each i-BEC insert, to ensure barrier integrity was intact, before infection with ZIKV. Only i-BEC inserts with TEER values ≥ 300 Ω cm2 were used in the assay. Sodium fluorescein permeability (Pe; cm/min) were measured in infected i-BECs at 2 h (2 h), 2 h infection followed by 24 h recovery (2 + 24 h) or 24 h (24 h) post ZIKV-infection with no recovery (mean ± SD). Low sodium fluorescein impermeability (Pe) during the course of ZIKV infection, similar to mock-infected controls (ns, p > 0.1, One-way ANOVA), confirmed an intact barrier. To demonstrate i-BEC viability and continuity of membrane contacts, uninfected (b, d, f) and ZIKV-infected (c, e, g) i-BECs were co-labeled with CFDA (b, c; green) and CellMask Orange (CMO) (d, e; red); overlay images shown in (f–g), on the Transwell membranes at the end of ZIKV transport experiments (24 h). h, i ZIKV crossing of the BBB and infection of underlying i-NPs (i) was confirmed through immunofluorescence staining with an anti-flavivirus antibody (FV; green) 24 h post-infection. Scale bar = 20 µm
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