Human forebrain-specific NPCs, astrocytes, organoids and cell line cultures.

The human iPSC line (C1-2 line) was previously generated from a skin biopsy sample of a healthy male newborn and has been fully characterized and passaged on mouse embryo fibroblast (MEF) feeder layers60. The BJ line was derived from healthy control fibroblasts obtained from the American Type Culture Collection (ATCC) (CRL-2522, male, neonatal). All studies followed institutional review board (IRB) protocols approved by Florida State University, Johns Hopkins University School of Medicine and Icahn School of Medicine at Mount Sinai. Human iPSCs were differentiated into forebrain-specific hNPCs following the previously established protocol60. Briefly, human iPSC colonies were detached from the feeder layer with 1 mg/ml collagenase treatment for 1 h and suspended in embryonic body (EB) medium, consisting of fibroblast growth factor (FGF)-2-free iPSC medium supplemented with 2 μM dorsomorphin (Sigma) and 2 μM A-83 (Tocris) in nontreated polystyrene plates for 4 d with a daily medium change. After 4 d, EB medium was replaced by neural induction medium (NPC medium), which consists of Dulbecco's modified Eagle's medium with F12 nutrient mixture (DMEM/F12), N2 supplement, non-essential amino acids (NEAA), 2 μg/ml heparin (Sigma) and 2 μM cyclopamine (Tocris). Floating EBs were then transferred to Matrigel-coated 6-well plates at day 7 to form neural-tube-like rosettes. Attached rosettes were kept for 15 d with an NPC medium change every other day. On day 22, rosettes were picked mechanically and transferred to low-attachment plates (Corning) to form neurospheres in NPC medium containing B27 supplements (Invitrogen). Neurospheres were then dissociated with Accutase (Millipore) at 37 °C for 10 min and placed onto Matrigel-coated 6-well plates at day 24 to form monolayer hNPCs in NPC medium containing B27. These hNPCs expressed forebrain-specific progenitor markers60, including NESTIN, PAX6, EMX-1, FOXG1 and OTX2.

Human BJ iPSC line and forebrain NPCs were derived as described previously61,62. Human iPSC-derived forebrain NPCs were maintained at high density, grown on Matrigel (BD Bioscience) in medium consisting of DMEM/F12, 1× N2, 1× B27-RA (Invitrogen), 1 μg/ml laminin (Invitrogen) and 20 ng/ml FGF-2 (Invitrogen). hNPCs were differentiated to astrocytes and cultured on Matrigel-coated plates in astrocyte medium (ScienCell). Astrocytes were split 1:3 every week with Accutase (Millipore) and cultured up to 120 d.

Forebrain-specific organoids were generated from the C1-2 human iPSC line as previously described17. Briefly, on day 1, human iPSC colonies were detached 7 d after passage with collagenase type IV (Thermo Fisher Scientific), washed with fresh stem cell medium and transferred to an ultra-low-attachment 6-well plate (Corning Costar), containing 3 ml of stem cell medium (without FGF-2), plus 2 μM dorsomorphine and 2 μM A83-01. On days 5–6, half of the medium was replaced with induction medium consisting of DMEM/F12, 1× N2 supplement (Invitrogen), 10 μg/ml heparin, 1× penicillin–streptomycin, 1× NEAA, 1× Glutamax, 4 ng/ml WNT-3A (R&D Systems), 1 μM CHIR99021 (Cellagentech) and 1 μM SB-431542 (Cellagentech). On day 7, organoids were embedded in Matrigel (BD Biosciences) and continued to grow in induction medium for 6 more days. On day 14, embedded organoids were mechanically dissociated from Matrigel by pipetting up and down onto the plate with a 5-ml pipette tip. Typically, 10–20 organoids were transferred to each well of a 12-well spinning bioreactor (SpinΩ) containing differentiation medium, consisting of DMEM/F12, 1× N2 and B27 supplements (Invitrogen), 1× penicillin–streptomycin, 1× 2-mercaptoenthanol, 1× NEAA and 2.5 mg/ml insulin (Sigma). Day 18 forebrain organoids were exposed to ZIKV FSS-13025 in SpinΩ for 24 h and then in replaced fresh medium for an additional 9 d before fixation and immunostaining as previously described17. To test the potential toxic effect on hNPC proliferation, day 20 forebrain organoids were also treated with PHA-690509 (1 μM) or seliciclib (5 μM) for 3 d without ZIKV exposure. On day 23 (20 + 3), treated forebrain organoids were pulsed with 10 μM 5-ethynyl-2-deoxyuridine (EdU; Thermo Fisher Scientific) for 1 h and immediately fixed for analysis.

The glioblastoma SNB-19 cell line (part of the National Cancer Institute 60 human tumor cell lines) was a gift from Dr. David Meckes (Florida State University, Tallahassee, FL). SNB-19 cells were maintained at 37 °C in 5% CO 2 in RPMI-60 medium, 1× penicillin–streptomycin and 10% FBS (Invitrogen). The Aedes albopictus C6/36 cell line (ATCC) was maintained at 28 °C in 5% CO 2 .

Preparation of ZIKV and cell infection.

The MR766-ZIKV stock with the titer of 1 × 105 tissue-culture-infective dose (TCID)/ml, in the form of culture fluid from an infected rhesus Macaca cell line LLC-MK2, was originally obtained from ZeptoMetrix (Buffalo, NY). The FSS13025-ZIKV strain was obtained from Drs. Robert Tesh and Pei-Yong Shi (University of Texas Medical Branch, Galveston, Texas). The PRVABC59 strain was obtained from ATCC (Manassas, VA). Original viral stocks were then amplified in Aedes albopictus clone C6/36 cells. Briefly, C6/36 cells were inoculated with viral inoculum for 1 h at 28 °C in a low volume of medium (3 ml per T-75 flask), with rocking every 15 min, before the addition of 17 ml medium. Virus-inoculated cells were incubated at 28 °C for 6–7 d before harvesting the supernatant. C6/36-amplified ZIKV titer was determined by infecting Vero cells for 48 h with a methylcellulose overlay and analyzed for focus-forming units per ml (FFU/ml). In mock infections, an equal volume of spent uninfected C6/36 culture medium was used. For infections, cells were seeded into 12-well plates 1 d before virus addition. For all cell types, compound was added 1 h before viral addition unless otherwise specified. Cells were harvested at 24–72 h after infection.

Compound libraries.

The Library of Pharmacologically Active Compounds (LOPAC), which consists of 1,280 compounds, was purchased from Sigma-Aldrich. The NCATS pharmaceutical collection63, a collection consisting of 2,816 clinically approved and investigational drugs, was established in 2011. All compounds were dissolved in DMSO as 10-mM stock solutions, then diluted in DMSO at a 1:3 ratio in 384-well plates, followed by reformatting into 1,536-well compound plates for use in high-throughput screening (HTS).

Caspase-3 assay.

Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity. Reagents were reconstituted as described in the protocol from the manufacturer. Polystyrene plates (384-well and 1,536-well; regular tissue-culture-treated and PDL-coated) were purchased from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- and 1536-well assay plates and cultured at 37 °C with 5% CO 2 for 16 h. ZIKV solution was added to cells, followed by incubation at 37 °C with 5% CO 2 for 6 h. Caspase-Glo-3/7 reagent was added to each well, unless otherwise specified, and incubated at room temperature for 30 min. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase-3 activity) and wells containing ZIKV infected cells that induced caspase-3 activity were used as a positive control (100% induction of caspase 3 activity).

ATP content assay for cell viability and compound cytotoxicity.

The ATPlite luminescence assay system assay kit (catalog number 6016731; PerkinElmer) was used to determine cell viability. The reagent was reconstituted and prepared as described by the manufacturer. In order to measure the cell death caused by ZIKV infection, cells were cultured for 16 h at 37 °C with 5% CO 2 in assay plates, followed by addition of ZIKV solution and incubation at 37 °C with 5% CO 2 for 72 h. ATPlite, the ATP-monitoring reagent, was then added to the assay plates, which were then incubated for 15 min. The resulting luminescence was measured using the ViewLux plate reader. Data were normalized using wells without cells as a control for 100% cell killing, and cell-containing wells without ZIKV infection as full cell viability (0% cell killing). For analysis of potential toxicity of select compounds, cells were seeded in 96-well plates. One day later, cells were treated with the indicated compounds and concentrations for 24–48 h before the addition of Cell Titer-Glo substrate (Promega) and measurement, according to manufacturer's instructions.

Large-scale compound screening.

A quantitative high-throughput screening (qHTS, ref. 64), in which each compound was assayed in four concentrations (0.37, 1.84, 9.2 and 46 μM), was performed in singlet for the primary compound screen. While a single-compound concentration (in singlet) has been traditionally used for HTS of large compound collections (such as 1 to 3 million compounds), the qHTS format with multiple compound concentrations has recently been used for medium or small compound collections, such as an approved drug library. Specifically, SNB-19 cells and hNPCs were seeded onto PDL-coated 1,536-well assay plates at 250 cells per 3 ml per well and incubated at 37 °C in 5% CO 2 for 16 h. Test compounds, dissolved in DMSO, were transferred to assay plates at a volume of 23 nl/well by an automated pintool workstation (Wako Automation, San Diego, CA). Compounds were incubated with cells for 30 min at 37 °C in 5% CO 2 , immediately followed by the addition of 2 ml/well of ZIKV (2 FFU/cell). Incubation time of compound-treated cells with ZIKV varied based on assay format. Experiments measuring virus-induced caspase-3/7 activity required a 6-h incubation of ZIKV in the presence of compounds at 37 °C in 5% CO 2 . Following this incubation, 3.5 μl/well of caspase-3/7 reagent mixture was added to the assay plates. The plates were incubated for 30 min at room temperature, and the resultant luminescence signal was measured using a ViewLux plate reader (Perkin Elmer). Experiments measuring virus-induced cell death required a 72-h incubation of ZIKV in the presence of the compounds, at 37 °C in 5% CO 2 . Following this incubation, 3.5 μl/well of ATP content detection reagent was added to assay plates. The plates were incubated for 30 min at room temperature, and the resultant luminescence signal was measured in a ViewLux plate reader. Step-by-step assay protocols are listed in Supplementary Tables 1 and 2.

Confirmation of primary hits and counter-screen.

The primary hits were selected with criteria of IC 50 values ≤ 30 μM, and maximal inhibition ≥ 50%. The hit compounds were diluted 1:3 in DMSO to 11 concentrations for the confirmation experiments. The same caspase-3 activity assay was first used to confirm the activity of the primary-hit compounds (n = 3). In order to further confirm the activity of these compounds, the protective effect of these primary hits on the cell death caused by incubation of cells with ZIKV for 3 d was also determined using an ATP content cell viability assay (n = 3).

Because compound cytotoxicity could nonspecifically reduce the caspase activity induced by ZIKV, we also used the ATP content assay to measure compound cytotoxicity in the absence of ZIKV infection. Cells were seeded in the same way as described above in 1,536-well assay plates. After a 6-h incubation with compounds in the absence of ZIKV, 3.5 μl/well of ATP content reagent mixture was added to the assay plates, which were then incubated for 30 min at room temperature. The luminescence signal in the assay plates was measured using a ViewLux plate reader (Supplementary Table 3). Any compounds that exhibited cytotoxicity were eliminated as false-positive compounds.

Western blot analysis.

Cells were harvested by trypsinization, pelleting and subsequent lysis in 1× Laemlli buffer, and the lysates were then boiled, or the cells were directly lysed in 1× Laemlli buffer and boiled. Antibodies used were anti–ZIKV NS1 (1:2,000; BF-1225-36, BioFront Technologies, Tallahassee, FL) or anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Texas). The screening of two subsets of compounds for antiviral activity (Supplementary Fig. 2a and Supplementary Fig. 4b) was performed in a blinded manner, whereas all other experiments were performed in a nonblinded manner.

Immunocytochemistry.

Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min at room temperature. Samples were permeabilized and blocked with 0.25% Triton X-100 (Sigma) and 10% donkey or goat serum in phosphate-buffered saline (PBS) for 20 min as previously described60. Samples were then incubated with primary antibodies at 4 °C overnight, followed by multiple PBST (PBS plus Tween-20) washes and incubation with secondary antibodies for 1 h at room temperature. Slides were mounted using VECTASHIELD with 4′,6-diamidino-2-phenylindole (DAPI; Vectorlabs, Burlingame, CA). The following primary antibodies were used: anti–flavivirus group antigen (clone D1-4G2-4-15; mouse; 1:500; Millipore), anti–cleaved caspase-3 (Asp15; rabbit; 1:500; Cell Signaling Technology) and anti–phospho-histone-H3 (PH3; 9701S; rabbit; 1:300; Cell Signaling Technology) as previously described17. Antibodies were prepared in PBS containing 0.25% Triton X-100 and 10% donkey serum. For EdU assay, the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) was used according to the manufacturer's protocol. Images were taken by Zeiss LSM 700 and 880 confocal microscopes, an Olympus BX61 microscope or a Zeiss Axiovert 200M microscope.

For organoid studies, quantitative analyses were conducted on randomly selected cortical structures captured by confocal microscope in a blinded fashion (example as shown in Fig. 1c). Cell death and cell proliferation were quantified by counting activated caspase-3+ and PH3+ nuclei, respectively, relative to the total number of nuclei stained by DAPI in the ventricular structures in at least six samples, as previously described17. We determined our sample sizes based on previous studies conducted in our lab17.

NS1 enzyme-linked immunosorbent assay (ELISA).

The anti-ZIKV NS1 ELISA kit (ZKV-NS1-EK) was obtained from BioFront Technologies (Tallahassee, FL) and used according to the manufacturer's protocol.

Viral titer by focus-forming unit (FFU) assay.

ZIKV titers from cell supernatants were determined by infecting Vero cells for 48 h with a methylcellulose overlay and analyzing the plates for focus-forming units per ml (FFU/ml). Briefly, cell supernatant was titrated in triplicates onto a monolayer culture of Vero cells in 96-well plates and incubated at 37 °C for 2 h. Virus inoculum was removed and replaced with a methylcellulose overlay. Vero cells were incubated for an additional 48–72 h before fixation and incubated with anti–flavivirus group antigen overnight at 4 °C. The next day, fixed cells were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody for 1 h at room temperature, washed again three times with PBS and incubated with DAB peroxidase substrate for 10 min (Vector Labs).

Antiviral compound analysis.

SNB-19 cells, human astrocytes or hNPCs were seeded in 12-well plates at approximately 3 × 105 cells/well. The next day, cells were treated with compound at 1- to 10-fold of the IC 50 value, or the indicated concentration, for 1 h before inoculation with ZIKV (multiplicity of infection (MOI) = 0.5–1). Cells and supernatant were harvested 24–48 h post infection and analyzed by western blot, immunostaining or quantitative RT–PCR analyses. Western blot bands were quantified, and immunostaining images were counted using ImageJ (NIH, Bethesda, MD). For qRT–PCR analysis, total cellular RNA was purified using the RNeasy Mini Plus kit (Qiagen). About 500–1,000 ng total RNA was reverse-transcribed into cDNA using random hexamers according to manufacturer's instructions (Superscript III First Strand Synthesis System, Invitrogen). Quantitative PCR was performed using SYBR Green PCR master mix (Invitrogen), gene specific primers (ZIKV-NS1-Forward: TGGAGTTCAACTGACGGTCG; ZIKV-NS1-Reverse: TACCCCGAACCCATGATCCT; Gapdh-Forward: TCACTGCCACCCAGAAGACTG; and Gapdh-Reverse: GGATGACCTTGCCCACAGC), and an Applied Biosystems 7500 Fast real-time PCR system. A 60 °C to 95 °C melt curve analysis following PCR was performed using default settings. Relative quantification was performed using the ΔΔC T method with Gapdh as the endogenous control, and the relative fold change was calculated by normalizing to the levels in the control cells.

Data analysis and statistics.

The primary-screen data and concentration response curves were analyzed using software developed internally65. The IC 50 values of compound confirmation data were calculated using the Prism software (GraphPad Software, Inc. San Diego, CA). All values are expressed as the mean ± s.d. unless specified otherwise. Western blots were quantified using ImageJ (NIH, Bethesda, MD). We did not exclude any samples. Experiments were not blinded, except for those that were specified. The sample sizes were estimated according to previous studies and the known variability of the assays. Statistical analyses were carried out with the one-way ANOVA using the Prism software. The Z′ factor, a measure of statistical effect size and an index for assay quality control, was calculated by: Z′ = 1 − ((3 × s.d. signal ) + (3 × s.d. basal )/(Mean signal − Mean basal )).

Accession codes.

PubChem Bioassay Database: all of the results from the primary screen of the approved drug collection and hit confirmation were deposited under assay IDs 1224859, 1224857, 1224858, 1224853, 1224855, 1224861, 1224862, 1224860, 1224856, 1224854 and 1224852.