Zika virus cell tropism in the developing human brain and inhibition by azithromycin
Contributed by Joseph L. DeRisi, November 1, 2016 (sent for review October 7, 2016; reviewed by Nenad Sestan and Pei-Yong Shi)
Letter
January 31, 2017
Significance
Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly spread through the Americas and has been associated with fetal abnormalities, including microcephaly. To understand how microcephaly develops, it is important to identify which cell types of the developing brain are susceptible to infection. We use primary human tissue to show that radial glia and astrocytes are more susceptible to infection than neurons, a pattern that correlates with expression of a putative viral entry receptor, AXL. We also perform a screen of Food and Drug Administration-approved compounds, with an emphasis on drugs known to be safe in pregnancy. We identify an antibiotic, azithromycin, that reduces viral proliferation in glial cells, and compare its activity with daptomycin and sofosbuvir, two additional drugs with anti-ZIKV activity.
Abstract
The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.
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A correlation between congenital exposure to the mosquito-borne and sexually transmitted Zika flavivirus (ZIKV) and the increased incidence of severe microcephaly suggests a causal relationship between ZIKV infection and neurodevelopmental abnormalities (1, 2). However, the mechanisms of infection and specifically which cell populations are vulnerable to ZIKV during the course of human brain development remain unclear. Major insights have been drawn from in vitro models of human brain development and primary mouse tissues. In the developing mouse brain, ZIKV has been shown to infect radial glia and neurons (3), whereas studies in human pluripotent stem cell (hPSC)-derived neural cells have highlighted widespread infection and apoptosis of neural progenitor cells (4, 5). Because these models do not fully recapitulate the developmental events and cell types present during human brain development, these results may not faithfully represent ZIKV-induced pathology in vivo.
During human brain development, radial glial cells, the neural stem cells, give rise to diverse types of neuronal and glial cells, including neurons, oligodendrocytes, and astrocytes, in a temporally controlled pattern. We reasoned that identifying cell types that are especially vulnerable to viral infection would facilitate studies of the viral life cycle, including entry mechanisms and host cell requirements. Building on studies that suggested that enriched expression of the candidate entry factor AXL could confer vulnerability to ZIKV entry (6–8), we used AXL expression levels to predict that radial glia, astrocytes, microglia, and endothelial cells would be particularly vulnerable to infection (9). A recent study highlighted the utility of ex vivo models using primary human tissue samples to analyze the consequences of ZIKV infection in the human prenatal brain (7). Here we further use primary tissue samples from distinct stages of brain development corresponding to periods of peak neurogenesis and early gliogenesis.
Determining the tropism of ZIKV for specific cell types will help identify suitable cellular models for investigating potential therapeutic interventions. Although development of a vaccine could provide a long-term solution to the current ZIKV epidemic, there remains an unmet clinical need to identify drugs that can limit or prevent the consequences of congenital infection. A recent screen of a subset of Food and Drug Administration (FDA)-approved compounds against ZIKV in hepatic cells identified several anticancer, antimicrobial, antiparasitic, and antifungal drugs with anti-ZIKV activity (10). Another screen, based on human neural progenitor cells, identified an antifungal drug and several scaffold compounds for further development (11). However, the majority of compounds with anti-ZIKV activity from these screens are contraindicated or of unknown safety during pregnancy. Furthermore, two promising candidates that might be safe during pregnancy, daptomycin and sofosbuvir, showed variable effectiveness by cell type (7, 10, 12). Combining unbiased screens of approved compounds with comparisons of top candidates with known antiviral activity may quickly narrow the search for drugs that could mitigate the effects of congenital ZIKV infection.
Here we assessed ZIKV cell tropism in the developing human brain and performed a drug screen on relevant cell types targeted by the virus with an emphasis on drugs known to be safe in pregnancy. We found that radial glia and, later in development, astrocytes were especially vulnerable to ZIKV infection. By screening FDA-approved compounds for anti-ZIKV activity in a glial cell line with features of both cell types, we also found that the common antibiotic azithromycin prevented viral production and virus-mediated cell death, which we further validated in human astrocytes.
Results
To determine the cell populations most susceptible to ZIKV infection, we investigated the infectivity of ZIKV in the developing human brain using organotypic cultures from primary human tissue. We exposed human cortical tissue slices to three strains of ZIKV: Cambodia 2010 (ZIKV-CAM), Brazil 2015 (ZIKV-BR), and Puerto Rico 2015 (ZIKV-PR), cultured them for 72 h, and detected infection by immunostaining for the flavivirus envelope protein (ENV), an approach we validated in cultured cells (Fig. S1). Infection in tissue was confirmed by immunostaining for the viral RNA-dependent RNA polymerase nonstructural protein 5 (NS5), present only during viral replication. In samples from midneurogenesis [13 to 16 postconception wk (pcw)], we observed high rates of infection in the ventricular and subventricular zones (Fig. 1 and Fig. S2). We found that the virus preferentially infected both ventricular and outer radial glial cells (Fig. 1 A–F and Fig. S2). Interestingly, we observed clusters of infected radial glia (Fig. S2B), which may reflect local viral spread. A minor fraction of cells positive for ENV at these stages included postmitotic neurons (Fig. 1H) and microglia (Fig. 1I). We observed similar patterns of infection across ZIKV strains (Fig. S2). We also observed a small but significant increase in cell death of ENV+ cells compared with ENV− cells in ZIKV-infected or mock-infected tissue (Fig. S3).
Fig. 1.
Fig. S1.
Fig. S2.
Fig. S3.
At later stages of development (after 17 pcw), we observed infection and viral replication throughout the developing cortex, including the cortical plate and subplate, with production of infectious virus by 48 h postinfection (hpi) (Fig. 2 and Fig. S4). Among cortical plate cells, we observed a high rate of infection in astrocytes, as distinguished by their location, morphology, and immunoreactivity with the glial markers GFAP and SOX2 (Fig. 2 A, B, and D and Fig. S4 A–D). We also observed cells immunoreactive for both ENV and the microglial marker IBA1, indicating microglial infection or phagocytosis of other ZIKV-infected cells (Fig. 2F and Fig. S4 G and H). This ENV+/IBA1+ microglial population was quantified at 7 ± 1% of ENV+ cells, and represented 7 ± 2% of the total IBA1+ population (n = 4, 15 to 22 pcw; SI Materials and Methods). We further observed infection of oligodendrocyte precursor cells (Fig. 2G and Fig. S4I) but limited infection of neurons (Fig. 2 B and D and Fig. S4 A and J). This pattern of infectivity was consistent across ZIKV strains (Fig. S4), and matched viral tropism predicted by AXL receptor expression (9).
Fig. 2.
Fig. S4.
To test the possible role of AXL in mediating ZIKV entry into human astrocytes, we infected hPSC-derived astrocytes (13, 14) in the presence of a nonactivating antibody specific for the extracellular domain of AXL. Blocking the AXL receptor substantially reduced infection (Fig. 2 I and J and Fig. S5A). To further test the requirement of AXL for ZIKV infection of glial cells, we used the U87 glioblastoma line that expresses high levels of astrocyte marker genes and AXL (15). U87 cells were readily infected with ZIKV, with strong virus production at 48 hpi (Fig. S1) and robust cytopathic effect at 72 hpi (Fig. 3C and Fig. S6D). We then used CRISPR interference (CRISPRi) to knock down AXL in this cell line (SI Materials and Methods; validated by Western blot in Fig. S5B) and observed a substantial decrease in infection (Fig. 2K), confirming the importance of this receptor for ZIKV infection in this cell type. Given that AXL is a receptor tyrosine kinase with signaling pathways that could be involved in innate immune responses (16), we tested whether the kinase activity of AXL was relevant for the decrease in infection observed in the knockdown line. After pretreatment with a small-molecule inhibitor, R428, we observed no decrease in infection at up to 1 µM, which is >70-fold the half-maximal effective concentration (EC50) for AXL kinase inhibition (Fig. S5C) (17). Although we did observe a decrease in infection at 3 µM R428, this high concentration of >200-fold the EC50 likely created off-target effects. Together, these results suggest that AXL has an important role in glial cell infection that depends more on its extracellular domain than on its intracellular kinase activity.
Fig. 3.
Fig. S5.
Fig. S6.
There is a pressing need to identify pharmacological compounds that can diminish the effects of ZIKV infection in relevant human cell types. We performed a screen of 2,177 clinically approved compounds (2,016 unique) by monitoring inhibition of virus-dependent cell death at 72 hpi in Vero cells. Although our screen revealed compounds that rescued cell viability, including antibiotics and inhibitors of nucleotide and protein synthesis, many showed toxicity in Vero or U87 cells or are contraindicated during pregnancy (Tables S1–S4). We focused on further characterization of the macrolide antibiotic azithromycin (AZ), which rescued ZIKV-induced cytopathic effect with low toxicity in our primary screens and is generally safe during pregnancy (18). AZ dramatically reduced ZIKV infection of U87 cells at an EC50 of 2 to 3 µM at multiplicities of infection (MOIs) of 0.01 to 0.1, as evaluated by ENV staining (Fig. 3 A and B and Fig. S6A). We further established a relationship between EC50 and baseline infection rate (Fig. S6B) and showed that even at >60% infection, AZ consistently reduced infection at concentrations 10- to 20-fold below the half-maximal toxicity concentration (TC50) of 53 µM (Fig. S6 A and C). AZ treatment also rescued cell viability (Fig. 3C and Fig. S6D) and decreased viral production (Fig. 3D). Finally, we found that AZ substantially reduced infection in hPSC-derived astrocytes without toxicity at the effective concentration (EC50 15 µM at 72% baseline infection) (Fig. S6 E–G). To compare AZ with compounds identified in previous screens, we evaluated the anti-ZIKV activity of daptomycin and sofosbuvir in U87 cells (EC50 2.2 and 12.4 µM, respectively) (Fig. S6H). We observed that treatment with daptomycin was insufficient to lower the percentage of infected cells below 46% even at the highest dose in this cell type (20 µM) (Fig. S6H), whereas AZ and sofosbuvir treatment decreased ZIKV infection from 78 to below 5% infection at 20 and 50 µM, respectively. These results highlight AZ as a potential tool compound against ZIKV infection in glial cells.
SI Materials and Methods
Cell Lines.
Vero and U87 cell lines were cultured in DMEM containing 10% (vol/vol) FBS, 2 mM l-glutamine, 100 U/mL penicillin/streptomycin, and 10 mM Hepes buffer at 37 °C with 5% (vol/vol) CO2. Human astrocytes (after 8 mo of in vitro development) were derived from human pluripotent stem cells [NIH Human Embryonic Stem Cell Registry line WA09 (H9) at passages 30 to 35] according to a recently published protocol (13) and maintained in neural media composed of DMEM/F12 with sodium pyruvate and GlutaMAX, N2, B27, heparin, and antibiotics. Medium was either supplemented with growth factors [epidermal growth factor (10 ng/mL) and fibroblast growth factor (10 ng/mL)] or with ciliary neurotrophic factor (10 ng/mL) during the experiments. hPSC-derived astrocytes were chosen due to their high fidelity to fetal astrocytes in vivo (14), and the U87 glioblastoma line expresses high levels of astrocyte marker genes and AXL (15). All cell lines tested negative for mycoplasma using MycoAlert (Lonza).
Virus Propagation and Titering.
ZIKV strains SPH2015 (Brazil 2015; ZIKV-BR), PRVABC59 (Puerto Rico 2015; ZIKV-PR), and FSS13025 (Cambodia 2010; ZIKV-CAM) were propagated in Vero cells infected at a multiplicity of infection (MOI) of 0.01. Supernatants were collected at 72 and 96 h postinfection (hpi), clarified by centrifugation at 350 × g for 5 min, and filtered through a 0.45-μm surfactant-free cellulose acetate membrane. For mock infections, supernatant was collected from uninfected Vero cells and prepared by the same protocol used to make viral stocks. Virus was titered by plaque assay and focus assay. Briefly, plaque assays were performed using Vero cells with a 0.7% agarose overlay and processed 5 d postinfection. Focus assays were performed on Vero cells and processed 24 hpi with a mouse monoclonal antibody (mAb) specific for flavivirus group envelope proteins (1:250; EMD Millipore; MAB10216, clone D1-4G2-4-15). Titers determined by both methods were consistent. Each strain was sequence-verified using a previously published protocol (32), and all viral stocks tested negative for mycoplasma contamination by MycoAlert (Lonza). ZIKV-PR and ZIKV-CAM continued to test negative after prolonged incubation in culture (96 h). Contamination of ZIKV-BR with mycoplasma was detected at low levels after 72 to 96 h in culture. No other evidence of contamination was seen in cells infected with this viral strain.
Brain Samples.
Deidentified primary tissue samples were collected with previous patient consent in strict observance of the legal and institutional ethical regulations. Protocols were approved by the Human Gamete, Embryo and Stem Cell Research Committee (institutional review board) at the University of California, San Francisco.
Developing Brain Organotypic Slice Culture Experiments.
Human primary cortical tissue blocks were embedded in 3.5% low-melting-point agarose (Thermo Fisher) and sectioned perpendicular to the ventricle to 300 μm using a Leica VT1200S vibrating blade microtome in artificial cerebrospinal fluid containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 1.25 mM NaH2PO4. Slices were transferred to slice culture inserts (Millicell) on six-well culture plates (Corning) and cultured in medium containing 66% (vol/vol) Eagle’s basal medium, 25% (vol/vol) HBSS, 5% (vol/vol) FBS, 1% N2 supplement, and 1% penicillin/streptomycin and glutamine (Thermo Fisher). Slices were cultured in a 37 °C incubator at 5% CO2, 8% O2 overnight at the liquid–air interface created by the cell-culture insert. Virus addition was performed by dispensing 200 μL inoculum onto the air-facing surface of the slice, with the remaining 800 μL inoculum into the well under the insert. Total inoculation was 2.2 × 106 pfu ZIKV-BR, 1.1 × 107 pfu ZIKV-CAM, or 2.2 × 107 pfu ZIKV-PR, with incubation for 4 h, before replacement with fresh medium and culture for an additional 72 hpi. Tissue samples were fixed overnight in 4% (vol/vol) paraformaldehyde (PFA).
Brain Tissue Immunohistochemistry.
Heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6) at 95 °C for 20 min. Slices were incubated in blocking buffer containing 10% (vol/vol) donkey serum, 1% Triton X-100, and 0.2% gelatin diluted in PBS at pH 7.4 for 1 h. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C: mouse anti-flavivirus group antigen mAb (1:100; EMD Millipore; MAB10216), goat anti-SOX2 (1:250; Santa Cruz; SC17320), rabbit anti-SATB2 (1:200; Abcam; SC81376), chicken anti-GFAP (1:500; Abcam; ab4674), rabbit anti-IBA1 (1:200; Wako; 019-19741), rabbit anti-OLIG2 (1:200; Millipore; AB9610), rabbit anti-cleaved caspase-3 (1:100; Cell Signaling Technologies; 9661), rabbit anti-PAX6 (1:200; BioLegend, previously Covance; PRB-278P), rabbit anti-CD31 (1:200; Abcam; ab28364), or rabbit anti-NS5 pAb (1:600; Novus Biologicals; NBP2-42900). Binding was revealed using an appropriate Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, and Alexa Fluor 647 fluorophore-conjugated secondary antibody (Thermo Fisher) diluted 1:1,000. Slices were incubated with secondary antibodies overnight at 4 °C, and cell nuclei were counterstained using DAPI (Thermo Fisher). All washes were performed using PBS without calcium/magnesium containing 0.5% Triton X-100. Slides were mounted with Fluoromount (SouthernBiotech). Images were collected using a Leica TCS SP5 X or Leica TCS SP8 confocal microscope. Three-dimensional rendering was performed using Imaris v. 8.2 (Bitplane).
Immunoblotting.
Whole-cell lysates for Western blot analysis were prepared by lysing cells in RIPA or 2× Laemmli buffer boiled at 95 °C for 5 min. Lysates were separated by standard SDS/PAGE protocol and transferred to nitrocellulose membranes for subsequent immunoblot analysis. Immunoblots were incubated in the following primary antibodies diluted in 5% milk: anti-AXL (Cell Signaling; 8661; 10 ng/mL), anti-NS3 (GeneTex; GTX133309; 1:1,000), anti-vinculin (Sigma; V4505), and anti-GAPDH (Cell Signaling Technologies; 2118S; 1:5,000). Primary antibody signal was visualized with secondary antibodies IR800 (Bio-Rad; 1:15,000).
Virus Yield Assays.
Brain organotypic slice infections were performed as above. After removing the inoculum, slices were washed once, and then samples were collected by combining conditioned media and tissue in the well and douncing to homogenize tissue at 4, 48, and 96 hpi. Samples were immediately used in focus-forming assays to quantify virus as described above. For testing AZ effect on virus yield, U87 cells were pretreated with increasing concentrations of AZ or vehicle for 1 h before infection with ZIKV-PR at an MOI of 0.1 or 0.01. The conditioned media of infected cells were collected at 1 h following removal of the inoculum and one wash and at 24, 48, or 72 hpi. Quantification of virus was performed by focus-forming assay as described above.
Quantification of Staining in Human Cortical Sections.
Images were quantified using Imaris v. 8.2 (Bitplane). For quantification of cell type and distribution of infected cells at 72 hpi, one (13, 14, 20 pcw) or two (22 pcw) representative ZIKV-infected slices were chosen from each biological replicate. On each slice, one rectangle was drawn, spanning from the ventricular zone (VZ) to the pia and 1 mm in width. This rectangle was portioned into five counting boxes with a height of either 1 mm or one-fifth the distance from the ventricular surface to the pia, whichever was smaller, and evenly distributed across the full height. With the location of each box defined this way, box 1 was directly adjacent to the ventricular surface, boxes 2 and 3 were typically found in the outer subventricular zone (OSVZ), box 4 typically covered the subplate (SP), and box 5 was located in the cortical plate (CP) directly adjacent to the pia. First, cells positive for anti-flavivirus envelope protein (ENV) were identified based on cytoplasmic staining around a clear nucleus positive for DAPI. Then, ENV+ cells were scored for the nuclear expression of SOX2 or SATB2 (ENV+ cells for 13 pcw, n = 259 cells; 14 pcw, n = 347 cells; 20 pcw, n = 192 cells; 22 pcw, n = 450 cells). The percentage of ENV+ cells that were also positive for SOX2 or SATB2 was calculated by combining cells in all five boxes. In addition, the percentage of ENV+ cells in each box relative to the total number of ENV+ cells in the entire rectangle was displayed for each box and biological replicate. For quantification of cell death at 5 d postinfection (dpi), representative mock- and ZIKV-PR–infected slices paired from each biological sample were chosen. Fields spanning the VZ, OSVZ, and CP were chosen for counting, with total cell number determined by DAPI nuclei and ENV+ cells determined as described above: 13 pcw mock (1 slice, 5,171 cells); 13 pcw ZIKV (2 slices, 77,323 cells, including 817 ENV+); 15 pcw mock (1 slice, 44,647 cells); 15 pcw ZIKV (2 slices, 182,636 cells, including 443 ENV+); 17 pcw_1 mock (1 slice, 43,993 cells); 17 pcw_1 ZIKV (1 slice, 88,803 cells, including 417 ENV+); 17 pcw_2 mock (1 slice, 224,775 cells); 17 pcw_2 ZIKV (2 slices, 57,224 cells, including 619 ENV+); 19 pcw mock (1 slice, 53,074 cells); and 19 pcw ZIKV (1 slice, 85,555 cells, including 465 ENV+). Cells were scored for cleaved caspase-3 positivity independently, and then the overlap between ENV and cleaved caspase-3 was determined. For quantification of microglia, fields spanning the VZ, OSVZ, and CP were chosen from samples at 15, 18, and 22 pcw infected with ZIKV-PR, and a sample at 22 pcw infected with ZIKV-BR, all fixed at 72 hpi. Cells positive for ENV were identified as above (n = 102, 737, 587, and 859 ENV+ for each respective sample), cells were independently scored for IBA1 (n = 220, 1,039, 472, and 687 IBA1+ cells, respectively), and then the overlap between ENV and IBA1 was determined.
AXL Inhibition.
Cells were treated with AXL-blocking antibody (R&D Systems; AF154) or goat IgG control (R&D Systems; AB108C) at 100 μg/mL for 1 h before infection with ZIKV at an MOI of 10, washed once, and cultured for 24 h before fixation and immunostaining against flavivirus envelope protein (1:100; EMD Millipore; MAB10216, clone D1-4G2-4-15) and DAPI. Two fields of view for each condition were imaged at 10× using a BZ-X700 microscope (Keyence), and the percentage of ENV+ cells was quantified using Imaris v. 8.2 (Bitplane). For AXL kinase inhibition, cells were pretreated with 1 or 3 μM R428 (ApexBio; A8329) or vehicle (<0.1% DMSO) for 1 h before infection at an MOI of 20, and then cultured for 48 h before immunostaining for envelope protein and DAPI.
CRISPRi-Mediated Knockdown of AXL.
U87 cells stably expressing dCas9-KRAB (15) were transduced with lentiviral particles expressing a pool of single-guide (sg)RNAs targeting AXL: sgRNA AXL-1, 5′-GCTGCCTGGCACAGCGGCAG-3′; sgRNA AXL-2, 5′-GACTGAGAGAGGAACTGAA-3′; sgRNA AXL-3, 5′-GTGCTGAGAAGGCGGCTGCT-3′; sgRNA AXL-4, 5′-GCGTGGCGGTGCCCCAGGAT-3′; and sgRNA AXL-5, 5′-GGCAGGCAGTGCCAAATCCG-3′. As a nontargeting control, we transduced cells with lentiviral particles expressing the sgRNA-GFP.NT2 5′-GACCAGGATGGGCACCACCC-3′ specific for GFP. After transduction, cells were selected with 1 µg/mL puromycin for 7 d. Knockdown of AXL was confirmed by immunoblot using anti-AXL (Cell Signaling; 8661). Cells were infected with ZIKV-PR at an MOI of 3. At 24 hpi, cells were immunostained for envelope protein as described above, and the percentage of ZIKV-infected cells was assayed by flow cytometry using a BD LSR II instrument with analysis by FlowJo v. 8.
High-Throughput Drug Screen.
For each screen, Vero or U87 cells were seeded onto 384-well plates at a density of 1,000 cells per well in cell-culture media and the conditions described above. After 18 h of incubation, each well was treated with 1 of 2,177 FDA-approved compounds (2,016 unique) at a final concentration of 2 µM or left untreated. Two hours after drug addition, the cells were infected with ZIKV-BR at an MOI of 1, 3, or 10 in 10 µL DMEM or mock-infected with DMEM alone. Cell viability was assessed at 72 hpi by adding 50 µL CellTiter-Glo 2.0 reagent (Promega) and measuring the luminescence of each well using the Promega GloMax-Multi plate reader in relative luminescence units (RLUs). The RLU value for each drug-treated well was compared with the mean RLU value for untreated ZIKV-infected wells to determine compounds that reduced ZIKV-induced cytopathic effect (CPE). Compounds of interest were selected on the basis of having a greater than 2.5-fold increase in viability from untreated wells in all three Vero cell screens. We noted minor carryover between plates of a dye affecting well A17 and removed these compounds from the candidate list (four compounds removed), and also noted minor edge effects causing us to discard candidates in row A (three further compounds removed, of which one was repeated in a different well/position that also met criteria). Of the remaining 21 candidates, 9 were chosen for further validation (see methods below) on the basis of a known mechanism of action (positive controls) or their potential to be used safely in pregnancy.
Drug Validation by Cell-Viability Assay.
U87 cells were treated with specified concentrations of azithromycin [Selleck Chemicals, S1835; Sigma, 75199 (analytical standard); Sigma, PZ0007 (HPLC-purified)], the clinical preparation Zithromax (Apotex; NDC 60505-6076-4), or vehicle (PBS, ethanol, or DMSO, final concentration <0.1%). One hour posttreatment, cells were infected with ZIKV-PR or ZIKV-BR at an MOI of 10 or left uninfected. Cell viability was assayed using the CellTiter-Glo 2.0 luminescence assay (Promega) following the manufacturer’s recommendations. The percentage of viable cells in the presence of azithromycin was calculated as a fraction of uninfected/untreated cells.
Drug Validation by Flow Cytometry and Imaging for Dose–Response.
U87 cells were treated with specified concentrations of azithromycin (see catalog numbers above), clindamycin (Selleck Chemicals; S2457), cycloheximide (Acros Organics; AC357420250), daptomycin (Sigma, D2446; Selleck Chemicals, S1373), doxorubicin (Sigma; D1515), lincomycin (Sigma, 62143; Selleck Chemicals, S2479), moxifloxacin (Sigma; Y0000703), mycophenolate (Selleck Chemicals; S2487), sofosbuvir (Selleck Chemicals; S2794), or vehicle for at least 1 h, and then infected with ZIKV-PR at MOIs ranging from 0.01 to 3. At 48 hpi, cells were fixed with 3.7% PFA for 15 min and permeabilized with 90% methanol for at least 2 h. Fixed and permeabilized cells were stained with clarified mouse hybridoma supernatant against flavivirus envelope protein (1:100; clone D1-4G2-4-15) and secondary goat anti-mouse Alexa Fluor 488 (1:2,000; Thermo Fisher Scientific; A-11029). Stained fixed cells were analyzed by flow cytometry using a BD LSR II instrument. The percentage of envelope-positive, ZIKV-infected cells was determined by analysis of the data by FlowJo v. 8.
For immunofluorescence, U87 and hPSC-derived astrocytes were treated and infected using the same conditions as above for flow cytometry. Cells were fixed with 3.7% PFA at 48 hpi, permeabilized with 1% Triton X-100 where indicated, and stained with clarified mouse hybridoma supernatant against flavivirus envelope protein (1:100; clone D1-4G2-4-15) and secondary goat anti-mouse Alexa Fluor 488 (U87 cells) (1:2,000; Thermo Fisher Scientific; A-11029) or secondary goat anti-mouse Alexa Fluor 594 (astrocytes) (1:2,000; Thermo Fisher Scientific; A-11032), and DAPI (Thermo Fisher). At least two fields of view for each sample were imaged at 10× using an Eclipse Ti-E microscope (Nikon Instruments) with an automated stage for high-throughput imaging.
Discussion
The rapid spread of ZIKV and its link to fetal abnormalities, including microcephaly, have created a global health crisis. Understanding viral tropism for specific cell types in the developing brain furthers our understanding of the pathophysiology of ZIKV-associated microcephaly and provides a basis for investigating antiviral drugs in a relevant cell type. Our findings offer several novel aspects. In particular, we show ZIKV tropism for astrocytes in addition to radial glia in the primary developing human brain, demonstrate the importance of AXL for ZIKV infection of glial cells, and identify a common antibiotic with anti-ZIKV activity, AZ, which we compare with two other drugs with anti-ZIKV activity that may be safe in pregnancy.
Our finding that radial glia are preferentially infected during early neurogenesis is consistent with experiments in cultured primary human brain cells (19), developing mouse cortex (3, 20), and primary human organotypic brain slice culture (7). These studies also reported overall survival of infected radial glia, in contrast to in vitro derived neural stem cells that undergo apoptotic cell death following infection (4, 5, 21). Cell lines derived from primary neural progenitors have variably shown infection with substantial apoptosis (7) or persistence (19). In our organotypic slice culture, we observe a small increase in apoptosis of infected cells. The discrepancy in levels of apoptosis in dissociated versus tissue cell culture may reflect differences in gene expression, maturation, or experimental conditions. Besides causing cell death, ZIKV infection could also affect cell-cycle progression (3, 21), differentiation, or the migration and survival of newborn neurons—mechanisms thought to underlie genetic causes of microcephaly and lissencephaly (22). Tissue disorganization in organotypic slice culture suggests these non–cell death-mediated mechanisms may contribute to clinical phenotypes (7), but this remains to be confirmed by directly analyzing cell behavior.
The high rate of infection in astrocytes at later developmental ages, many of which contact microcapillaries, could link our understanding of initial infection with clinical findings of cortical plate damage. For example, after prolonged infection, viral production in astrocytes could lead to a higher viral load in the cortical plate, causing infection of additional cortical cell types, and astrocyte loss could lead to inflammation and further damage, even in uninfected cells. Widespread cell death in vivo, which may take days to weeks to occur and is therefore outside the time frame of our experimental paradigm, is expected, given clinical reports of band-like calcifications in the cortical plate, cortical thinning, and hydrocephalus (2, 23). On the basis of their susceptibility to ZIKV infection and a central role in brain tissue homeostasis, human astrocytes provide a good cellular model for further investigation of mechanisms of viral entry and a platform for testing the efficacy of candidate therapeutic compounds.
Our observation that blocking or knocking down the AXL receptor prevents infection of human astrocytes, but that blocking intracellular kinase activity does not, suggests that the extracellular domain of AXL contributes to ZIKV infection whereas AXL signaling is dispensable. This extends comparable findings in endothelial cells to a cell type relevant for understanding microcephaly (6, 8) but does not address other viral receptors that may be important for ZIKV infectivity in other cell types or rule out a role for AXL signaling in the context of a full immune response in vivo. Although AXL knockout mice can be readily infected with ZIKV, disruption of the blood–brain barrier in these mice could lead to atypical routes for infection of the brain (24).
In addition to characterizing brain cell tropism, we also sought to identify possible therapeutic candidates with known safety profiles, especially in pregnancy. Several compounds expected to inhibit ZIKV were identified by our drug screen. These positive controls include the protein synthesis inhibitor cycloheximide, nucleic acid synthesis inhibitors such as mycophenolate derivatives, and intercalating compounds such as doxorubicin and homidium bromide. We additionally identified compounds that are known to be safe in pregnancy, including AZ. AZ is recommended for the treatment of pregnant women with sexually transmitted infections or respiratory infections due to AZ-susceptible bacteria (25, 26). Adverse events have not been observed in animal reproduction studies, and studies in pregnant women show no negative effects on pregnancy outcome or fetal health associated with AZ (18, 27). Orally administered AZ has been shown to reach concentrations of ∼2.8 µM in the placenta, and is rapidly transported to amniotic fluid and umbilical cord plasma in humans (28, 29). Moreover, AZ accumulates in fetal tissue and in the adult human brain at concentrations from 4 to 21 µM (30, 31). Together, these pharmacokinetic studies suggest that AZ could rapidly accumulate in fetal tissue, including the placenta in vivo, at concentrations comparable to those that inhibit ZIKV proliferation in culture. Nonetheless, it remains unknown whether these in vitro results would be recapitulated in humans.
We further compared AZ with two promising drug candidates that might be safe in pregnancy and have reported anti-ZIKV activity in cell culture: daptomycin and sofosbuvir. Our dose–response curves are in agreement with the documented activity of sofosbuvir in human neuroepithelial stem cells (7), and extend the activity of daptomycin previously seen in HuH-7 and HeLa cells (10) to glial cells. We noted that daptomycin would not have been highly ranked in our initial screen due to the limited maximum effect of the drug as observed in dose–response curves. Unlike sofosbuvir, which likely targets the ZIKV RNA-dependent RNA polymerase (NS5) based on its mechanism against hepatitis C virus, daptomycin and AZ have unknown mechanisms of action against ZIKV. Nonetheless, the difference in in vitro dose–response between AZ and daptomycin is intriguing, and suggests different mechanisms of inhibition. Another important factor for a drug candidate for ZIKV treatment is accessibility. Access to sofosbuvir and its derivatives may be limited by its current price whereas AZ and daptomycin are available as generic forms, although daptomycin is not available in oral formulation due to poor oral bioavailability. Our comparison adds new data to consider alongside other antiviral activity data, safety, cost, and accessibility in moving forward with further exploration of these and related compounds. In parallel with direct comparisons in vitro, follow-up studies in animal models can be useful for prioritizing candidates. However, as with in vitro studies, there are caveats in interpreting animal models, such as substantial differences between human and mouse immune systems, placental structure, and fetal brain development.
Together, our work identifies cell type-specific patterns of ZIKV infection in second-trimester human developing brain, provides experimental evidence that AXL is important for ZIKV infection of relevant human brain cell types, and highlights a common antibiotic with inhibitory activity against ZIKV in glial cells. Ongoing studies will be required to determine whether AZ, daptomycin, sofosbuvir, and other inhibitors or combinations are capable of reducing ZIKV infection in the critical cell types identified here in vivo. Although preventative measures such as mosquito abatement and a ZIKV vaccine are imperative for long-term control of this pathogen, the study of ZIKV infection of primary human tissues and identification of inhibitors with therapeutic potential remain important components of a global response to this emerging threat.
Materials and Methods
Detailed materials and methods are available in SI Materials and Methods.
Cells and Viruses.
Cell lines were Vero cells, U87 cells, and human astrocytes derived from human pluripotent stem cells (13). ZIKV strains were SPH2015 (Brazil 2015; ZIKV-BR), PRVABC59 (Puerto Rico 2015; ZIKV-PR), and FSS13025 (Cambodia 2010; ZIKV-CAM).
Brain Samples.
Deidentified primary tissue samples were collected with previous patient consent in strict observance of the legal and institutional ethical regulations. Protocols were approved by the Human Gamete, Embryo and Stem Cell Research Committee (institutional review board) at the University of California, San Francisco (UCSF). Slices in organotypic culture were inoculated with ZIKV or mock-infected, fixed at 72 hpi or 5 d postinfection, and processed for immunohistochemistry. Quantification was performed on 13- to 22-pcw slices.
AXL.
For 1 h before infection, cells were treated with AXL-blocking antibody or goat IgG control at 100 μg/mL, or with 1 to 3 μM R428 or vehicle (<0.1% DMSO). For AXL knockdown, U87 cells stably expressing dCas9-KRAB (15) were transduced with lentiviral particles expressing a pool of gRNAs targeting AXL or a gRNA targeting GFP as a control.
Drug Screen.
A collection of 2,177 FDA-approved compounds, provided by the UCSF Small Molecule Discovery Center, was tested at 2 µM in Vero cells infected with ZIKV-BR (MOIs of 1, 3, and 10) and in U87 cells (MOI of 3). Toxicity screens in uninfected cells were performed in parallel. Cells were pretreated for 2 h before addition of ZIKV-BR or media, and cell viability was assessed at 72 hpi using the CellTiter-Glo 2.0 assay (Promega). Candidates with cell viability >2.5-fold that of untreated cells in every Vero cell screen were identified for follow-up.
Drug Validation.
U87 cells or hPSC-derived astrocytes were treated with azithromycin, daptomycin, sofosbuvir, or vehicle for >1 h, and then infected with ZIKV-PR. Cell-viability assays were performed using CellTiter-Glo as above. To assess viral envelope production, cells were fixed and stained at 48 hpi using anti-flavivirus envelope protein, and then quantified by plate imaging with automated cell counting or by flow cytometry.
Acknowledgments
We thank Marc and Lynne Benioff for their financial support of these studies. We also thank Susan Fisher (UCSF), Robert Tesh (UTMB), Nikos Vasilakis (UTMB), Julio Rodriguez-Andres (CSIRO), Graham Simmons (BSRI), Charles Chiu (UCSF), Dan Lim (UCSF), John Liu (UCSF), Max Horlbeck (UCSF), Shaohui Wang (UCSF), Diego Acosta-Alvear (UCSF), and the Small Molecule Discovery Center at UCSF for providing reagents and advice. This work was supported by NIH/NINDS Grants R01NS075998 and U01 MH105989 as well as a gift from Bernard Osher (to A.R.K.), Howard Hughes Medical Institute (J.L.D.), NIMH Grant R01MH099595-01 and Paul G. Allen Family Foundation Distinguished Investigator Award (to E.M.U.), and Damon Runyon Cancer Research Foundation Postdoctoral Fellowship DRG-2166-13 (to A.A.P.).
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Cover image: Pictured are droplets in a turbulent cloud, illuminated by a thin sheet of laser light. The long time exposure allows the velocity field to be visualized through the streaks left by moving cloud droplets. Kamal Kant Chandrakar et al. created turbulent clouds in the laboratory, and found that both the mean diameter and the width of the size distribution of cloud droplets increase with decreasing aerosol concentration. The findings provide insight into the effect of turbulence and aerosol concentration on precipitation and cloud radiative properties. See the article by Chandrakar et al. on pages 14243–14248. Image courtesy of Kamal Kant Chandrakar.
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Published online: November 29, 2016
Published in issue: December 13, 2016
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Acknowledgments
We thank Marc and Lynne Benioff for their financial support of these studies. We also thank Susan Fisher (UCSF), Robert Tesh (UTMB), Nikos Vasilakis (UTMB), Julio Rodriguez-Andres (CSIRO), Graham Simmons (BSRI), Charles Chiu (UCSF), Dan Lim (UCSF), John Liu (UCSF), Max Horlbeck (UCSF), Shaohui Wang (UCSF), Diego Acosta-Alvear (UCSF), and the Small Molecule Discovery Center at UCSF for providing reagents and advice. This work was supported by NIH/NINDS Grants R01NS075998 and U01 MH105989 as well as a gift from Bernard Osher (to A.R.K.), Howard Hughes Medical Institute (J.L.D.), NIMH Grant R01MH099595-01 and Paul G. Allen Family Foundation Distinguished Investigator Award (to E.M.U.), and Damon Runyon Cancer Research Foundation Postdoctoral Fellowship DRG-2166-13 (to A.A.P.).
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The authors declare no conflict of interest.
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