Cell lines and virus strains
Human colorectal adenocarcinoma cells (HT-29, ATTC HTB-38), rhesus monkey kidney cells (LLCMK2, provided by the Municipal Health Services, the Netherlands), and African green monkey kidney cells (Vero, provided by the National Institute of Public Health and the Environment, RIVM, the Netherlands) were used for virus culture. All cell lines were maintained in Eagle’s minimum essential medium (EMEM, Lonza) supplemented with 8% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich), 100 U/mL penicillin/streptomycin (Pen-Strep, Lonza), 1% (v/v) non-essential amino acids (100x, ScienceCell Research Laboratories), and 0.1% (v/v) L-glutamine (Lonza). Cell lines were incubated at 37ºC, 5% CO2 and 95% humidity and passaged every seven days using trypsin.
The PeV-A1 Harris strain was obtained from the RIVM and cultured on HT-29 cells. The PeV-A3 152037 strain, a Dutch isolate from 2001 adapted to cell culture, was cultured on LLCMK2 cells. The Echovirus 11 (E11) 50473 strain, a Dutch isolate from faecal material was cultured on Vero cells. Heat inactivated (HI) controls were generated by incubating the virus stock in a water bath at 65ºC for 20 min and infection was performed as described previously.
Human induced pluripotent stem cell culture
Human induced pluripotent stem cells (hiPSCs) (IMR90-4, WiCell) were cultured on human laminin 521 (Biolamina) – coated culture treated six-well plates and maintained in mTeSR+ medium (STEMCELL Technologies) supplemented with 1% (v/v) Pen-Strep. Cells were maintained at 37°C with 5% CO2, passaged weekly with ReLeSR™, and subcultured in mTeSR+ medium with 10 µM Y-27632 Rho Kinase (ROCK) inhibitor (Cayman Chemical Company). Lines were kept in culture with removal of differentiated patches when necessary and regular testing for mycoplasma was performed.
Generation of unguided neural organoids
UNOs were generated from the IMR90 hiPSC (WiCell®) using the Cerebral Organoid Generation and Maturation kit from STEMCELLTM Technologies, that is based on the protocol described for UNOs generation by Lancaster et al28. In short, hiPSCs were detached into a single cell suspension using Gentle Cell Dissociation Reagent (STEMCELLTM Technologies) and seeded in an ultra-low attachment round bottom 96 well-plate (Corning) with embryoid body (EB) Formation Medium to obtain EBs. Hereafter, induction of neuroectoderm was obtained using Induction Medium (STEMCELLTM Technologies) followed by expansion of neuroepithelia by embedding EBs in ESC-qualified Matrigel (Corning) and culturing in Expansion Medium (STEMCELLTM Technologies). On day ten the organoids were placed on an orbital shaker (66 rpm) in Maturation Medium (STEMCELLTM Technologies) and medium was refreshed every 3-4 days until infection at day 67.
Infection of unguided neural organoids
UNOs from three independent batches were infected in technical triplicates with 105 50% tissue culture infectious dose (TCID50) per mL of the different viruses. Individual organoids were placed on a round bottom 96-well plate coated with Anti-Adherence Rinsing Solution (STEMCELLTM Technologies) and 100 µL of the virus inoculum were added. Organoids were incubated for 2 h at 37ºC with 5% CO2, washed three times with phosphate buffer saline (PBS, Lonza), and moved to a freshly coated 48-well plate with 500 µL of Maturation Medium (STEMCELLTM Technologies). After 10 min incubation the 0 h time-point was collected, and medium was replenished. Collection with full medium replenishment was repeated at 1-, 3-, 5-, 7-, and 10-days post infection (dpi).
RT-qPCR
RNA was isolated from 25 µL of the collected supernatant using the Bioline Isolate II RNA mini kit (Meridian Bioscience®) following the manufacturer´s instructions. Equal volumes of the eluted RNA were used for reverse-transcription and 5 µL of the cDNA was used for reverse-transcription quantitative PCR (RT-qPCR). qPCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad), and Cq values were transformed into viral genome copies using a standard curve with known concentrations of the viral genomes. For RT-qPCR primers see Supplementary Table 1.
To analyse cytokine expression UNOs were harvested in RLY lysis buffer (Bioline Isolate II RNA mini kit (Meridian Bioscience®)) and stored at -80°C until RNA isolation. The sample was thoroughly homogenized by vortexing and resuspension by pipetting before RNA was isolated. The same protocol as described previous was used for RNA extraction, cDNA synthesis, and RT-qPCR. Cytokine upregulation was measured using primer sets (see Supplementary Table 1, Biolegio) where expression of the target gene was normalized to reference genes. The combination of RPLP0 and RPLP2 was chosen as most stably expressed set of reference genes under both mock and virus infected organoids using Normfinder69. Gene expression was normalized using the method70 using the geometric mean of both reference genes. Infected samples were normalized to the uninfected control to visualize the effect of infection on cytokine expression in the UNOs.
TCID50
Supernatant samples (25 µL) of multiple time-points were titrated for each virus, where PeV-A1 was titrated on HT-29, PeV-A3 was titrated on LLCMK2 and E11 was titrated on Vero cells. Briefly, ten-fold dilutions of each sample were performed and seeded in a 96-well plate (50 µL), the appropriate cells were added (200 µL) and incubated for 10 days until readout. For the readout, the cells were examined for the appearance of cytopathic effect and the TCID50 was calculated using the Reed and Muench Method71 and normalized to the 0 h time-point to determine the increase of infectious particles over time.
Immunofluorescence staining
Organoids were fixed at 5 and 10 dpi with 4% (v/v) formaldehyde (Sigma-Aldrich) in PBS for 30 min at room temperature (RT). After fixation organoids were washed three times with PBS and incubated in 30% (w/v) sucrose (Merck) by overnight incubation at 4°C. The organoids were embedded in optimal cutting temperature compound (OCT, Tissue Tek) snap frozen on dry ice, and stored at -80°C until sectioning. 20 µm sections were cut using a cryostat (NX71, Thermo Fisher Scientific) and collected on SuperFrost Plus slides (Thermo Scientific). Sections were stored at -80°C until staining. For immunostaining, sections were blocked for 2 h at RT in a blocking solution consisting of 10% (v/v) SeaBlock Blocking Buffer (Thermo Scientific) with 1% (v/v) Triton X-100 (Sigma) in PBS. After blocking, primary antibodies (Supplementary Table 2) were added in 1:1 blocking solution:PBS and incubated overnight at 4°C. Sections were washed three times with PBS for 5 min, and incubated with secondary antibody (Supplementary Table 2) solution and Hoechst (Thermo Fisher Scientific) at RT for 1 h. Samples were quenched using ReadyProbes Tissue Autofluorescence Quenching kit (Invitrogen, kit) and incubated for 5 min, followed by 3 PBS washes. Finally, slides were mounted with glass coverslips using ProLong Gold Antifade Mounting Medium (Invitrogen). UNOs were imaged using Leica TCS SP8-X microscope and Leica LAS AF Software (Leica Microsystems). Z-stacks were also taken, and 3D reconstructions were made using the LAS-X 3D software (Leica Microsystems).
Ruxolitinib treatment
UNOs were pretreated with 5 µM or vehicle dimethyl sulfoxide (DMSO, Santa Cruz Biotechnology) and incubated for 1 h before infection at 37°C. After pre-treatment organoids were stimulated with 500 ng interferon (IFN) β (R&D Systems), or IFN-λ3 (R&D Systems), or infected as described previously with PeV-A1, PeV-A3, or E11. Treatment with 5 µM Rux/vehicle was continued throughout the 10 days post infection with every medium change at 1-, 3-, 5-, 7-, and 10dpi.
Procartaplex Multiplex Immunoassay
To detect cytokines, present in supernatant samples of (un)infected brain organoids, a customized 10-plex Luminex® assay was used (Procartaplex Multiplex Immunoassay, Invitrogen). Samples were lysed with 12.5% (v/v) Cell Lysis Buffer (Invitrogen) to inactivate viruses and the measurement was performed following the manufacturer’s instructions. Fluorescence was measured using a Luminex (R&D) and from this cytokine concentrations were calculated using the provided standard curve in the kit. Values that were below the lower limit of detection (LLOD) were replaced by the LLOD/72.
Statistical analysis
All statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc.). experiments were performed in three independent organoid batches in triplicates (unless otherwise indicated). Data are presented as geometric mean ± geometric SD. The specific statistical tests performed for each analysis are indicated in the correspondent figure legend. Differences were considered significant when the p-value was <0.05.