Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells

Abstract

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-β and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments.

Keywords: IFN-β regulation; ISG15 induction; Zika virus; basolateral release; cell survival; chemokine CCL5; human brain endothelial cells; innate immune regulation; persistent infection; transcriptome analysis.

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.