Preparation of human fetal organotypic brain slice cultures
Human fetal brain tissues from legally terminated second trimester pregnancies (17–20 weeks) were obtained by the Human Immune System (HIS)-Mouse Facility of Academic Medical Center (AMC; Amsterdam, The Netherlands), after written informed consent of the mothers for the use of tissues in research and with approval of the Medical Ethical Review Board of the AMC (MEC: 03/038) and Erasmus MC (MEC-2017-009). Study procedures were performed according to the Declaration of Helsinki, and in compliance with relevant Dutch laws and institutional guidelines. The tissues obtained were anonymized and non-traceable to the donors. On request by the researchers, only gender and gestational age is provided. Upon removal of exterior blood vessels and meninges, brain tissue fragments (~ 0.5 x 0.5 cm) were cut into 350 µm-thick slices using a vibratome (Leica; type VT1200S) in artificial cerebrospinal fluid (aCSF) under constant oxygenation (95% O2, 5% CO2) as described previously [47]. Slices were transferred to 12 mm transwell plates with polyester membrane inserts (4 µm pore size; Corning) to recuperate the tissue in recovery medium composed of a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium (both Life Technologies) supplemented with 20% heat-inactivated fetal bovine serum (FBS) and antibiotics. Following 1 hour (hr) incubation in a CO2 incubator at 37°C, the recovery medium was replaced with optimized hfOBSC serum-free culture medium containing a 7:3 (v/v) mixture of Neurobasal media and advanced DMEM/F12 culturing medium with essential growth factors. To ensure fluidic flow in the transwell system, 750 µl medium was added to the basolateral compartment and 50 µl medium added to the apical compartment. The culture medium of the hfOBSCs was refreshed every 48 hrs. Detailed information on the development, characterization and culture conditions of the hfOBSC has been described recently [13].
Virus strains and culturing
All viruses were grown and passaged on Vero cells (African green monkey kidney epithelial cells, ATCC CCL-81) at a multiplicity of infection of 0.01 for 5–6 days in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) with 2% FBS (Sigma-Aldrich), 100U/ml penicillin, 100ug/ml streptomycin (Lonza) and 2mM L-glutamine (Lonza). Supernatants were harvested at the indicated times post-infection, spun down at 4,000 g for 10 minutes and aliquoted and frozen at -80oC. The virus strains used in this study were USUV (lineage Africa 3, GenBank accession MH891847.1), WNV (lineage 2, GenBank accession OP762595.1) and ZIKV (Suriname 2016, KU937936). The USUV and WNV strains used represent prevalent strains currently circulating in Europe [48, 49], thereby modelling the current risk situation in Europe. The ZIKV strain used represents the Asian lineage responsible for the large scale outbreaks of congenital disease in the Americas[50]. All virus stocks were used at passage 3 and sequenced to ensure no amino acid changes resulting from passaging, compared with the original isolate.
Flavivirus infection and antiviral treatment of human fetal organotypic brain slice cultures
Brain slice cultures were used for experiments after day 3 post-sample acquisition and culture establishment. All experiments were performed on tissues from 3 independent donors, which were obtained on different days, for which 3 to 4 hfOBSC cultures per condition were used in each experiment. In case of antiviral treatment, media from both the apical and basolateral compartments was removed and replaced with culture medium supplemented with 50 ng/ml recombinant human interferon beta (IFN-β; Peprotech), 25 µM 2′-C-Methylcytidine (2CMC), (which was kindly provided by Johan Neyts; Lab of Virology, Antiviral Drug & Vaccine Research, KU Leuven, Belgium) a nucleoside analogue which acts to inhibit viral RNA dependent RNA polymerases, or the respective vehicle consisting of PBS plus 0.025% DMSO. Antiviral dosage and toxicity was determined on Vero cells (Table S1.). Drug treated hfOBSCs were incubated overnight at 37°C prior to infection and treatment was maintained throughout the infection course. On the day of infection, all culture medium of the apical compartment was removed before addition of 106 TCID50 virus inoculum. As determination of the exact cell number in each hfOBSC culture used for infection was not possible, we standardized the inoculation dose to input of viral titer, rather than cell number. This relatively high inoculation dose was determined to maximize the chance of infection, and based on previous literature using ZIKV on ex vivo fetal brain[3]. The hfOBSCs were returned to the incubator at 37°C for 1 hour and subsequently washed 2–3 times with PBS to remove the inoculum, allowing for detection of virus release into the supernatant with time. The hfOBSC transwells were transferred to a clean culture plate and 750 µl of culture medium was added to the basolateral compartment and 50 µl added to the apical compartment. The complete volume of medium in both compartments was replaced every 24 hours. The harvested supernatants were stored at -80oC for virus titration.
Virus titration
Tenfold serial dilutions of culture supernatants were inoculated onto a semiconfluent monolayer of Vero cells in a 96-well plate (2.3 × 104 cells/well) in 3 technical replicates. Cytopathic effect (CPE) was used as read out and determined at 6 days post-infection (6 dpi). Virus titers were calculated as the 50% tissue culture infective dose (TCID50) using the Spearman-Kärber method [51]. An initial 1:10 dilution of supernatant resulted in a detection limit of 31.6 TCID50/ml.
In situ analysis of brain slices
Formalin-fixed paraffin-embedded (FFPE) brain slices were serially sectioned at 4µm thickness. Heat-induced antigen retrieval was performed using conventional citric acid buffer (pH 6.0). Consecutive sections from three different levels of the brain slices (i.e. apical, middle and basal level) were immunohistochemically stained (IHC) with the following primary antibodies: mouse anti-SOX2 (stem cell marker, R&D, 1:100), rabbit anti-Iba1 (microglia marker; Wako, 1:500), rabbit anti-GFAP (glial fibrillary acidic protein, astrocyte marker; Dako, 1:500), rabbit anti-Olig2 (oligodendrocyte transcription factor 2, oligodendrocyte marker; clone EPR2673, Abcam, 1:200) or guinea pig anti-MAP2 (microtubule-associated protein 2, neuron marker; Synaptic Systems, 1:300). Next, sections were washed and incubated with the appropriate secondary antibody including rabbit anti-mouse Ig biotinylated (Dako, 1:200), goat anti-rabbit Ig biotinylated (Dako, 1:200), or rabbit anti-guinea-pig IgG (H + L) horseradish peroxidase (HRP)-conjugated (Invitrogen, 1:200), respectively. The HRP-labeled streptavidin (Dako, 1:300) was applied to sections with biotinylated antibodies, followed by 3-amino-9-ethylcarbazole substrate. Sections were counterstained with hematoxylin, mounted with Kaiser’s glycerol, and scanned using the Hamamatsu NanoZoomer 2.0 HT (Hamamatsu).
3D tissue clearing and immunofluorescent staining
Whole brain slices were fixed in 4% paraformaldehyde (PFA) for a minimum of 24 hrs before undergoing tissue clearing and immunofluorescent (IF) staining with the 3D Cell Culture Clearing Kit (Abcam) as per the manufacturers protocol. Primary antibodies used were: mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, USA, 1:250), rabbit anti-MAP2 (Millipore, 1:200), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-SOX2 (Abcam, 1:100). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/555, donkey anti-rabbit AF488/555 and donkey anti-guinea-pig AF647 (Invitrogen). ToPro (DNA staining; Thermo Fisher Scientific, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope.
Cryosectioning and immunofluorescent staining
The PFA-fixed whole brain slices were incubated in 30% sucrose at 4°C for 24 hrs and subsequently placed in Optimal cutting temperature compound (Agar Scientific) before snap freezing on dry ice. Frozen tissue was then cut into 5µm thick sections using a cryostat (Thermo Fisher Scientific, type HM525nx). Slides were permeabilized and blocked with 0.5% triton-X100 (Sigma) and 5% BSA (Aurion) before addition of the primary antibodies including mouse anti-flavivirus envelope protein (D1-4G2-4-15 hybridoma; ATCC, 1:250), guinea-pig anti-MAP2 (Synaptic Systems, 1:200) and rabbit anti-GFAP (Millipore, 1:200). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-mouse IgG AF488/AF555 or donkey anti-mouse IgG AF488/AF555 (Invitrogen). Hoechst 33342 (DNA staining; Invitrogen, 1:1000) was used to stain cell nuclei. Images were obtained using a Zeiss LSM 700 laser scanning microscope.
Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay
The TUNEL assay was performed using the ApopTag® Plus in situ apoptosis fluorescein S7111 detection kit (Sigma) according to the manufacturer’s protocol, following instructions for a combined IF staining. Sections were treated with TrueBlack® (Biotium) after antigen retrieval to decrease autofluorescence, followed by staining with the following primary antibodies: polyclonal rabbit anti-CC3 (cleaved caspase 3 protein; Cell Signaling Technology, 1:300 dilution), polyclonal rabbit anti-pMLKL (phosphorylation of mixed lineage kinase domain-like protein; Abcam, 1:250) and monoclonal mouse anti-GSDMD (gasdermin D protein; Abnova, 1:250). Next, sections were washed and incubated with the appropriate secondary antibody including donkey anti-rabbit AF555 (1:250) or goat anti-mouse IgG2a AF647 (1:250) (all from Invitrogen).
Lactate dehydrogenase assay (LDH)
The viability of the hfOBSC was determined by lactate dehydrogenase (LDH) assay. The LDH assays were performed on conditioned medium of hfOBSC cultures using the LDH-Cytoxicity Assay kit (Abcam) according to the manufacturer’s protocol. LDH levels of brain slices incubated in lysis buffer according to the manufacturer’s instructions were used as 100% cell death positive control.
Image processing
Images were subjected to processing and file-type conversion using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, MD). Processed images were then rendered in 3D using Dragonfly software (Version 2021.1 for [Windows]; Comet Technologies Canada Inc., Montreal, Canada). This software is available at https://www.theobjects.com/dragonfly. Quantitative analysis of infected cell area and infection percentages of whole slices were done using automatic thresholding and batch-processing in QuPath 0.3.2 software [52].
Statistical analysis
Quantitative data were analyzed and statistics was carried out using Prism 8.0.2 (GraphPad).