The Brazilian Zika virus strain causes birth defects in experimental models methods
Aim. Evidence-backed execution summary for The Brazilian Zika virus strain causes birth defects in experimental models methods from The Brazilian Zika virus strain causes birth defects in experimental models.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Virus titration
reagent used in the protocol.
- Use
- Titration (in PFU/mL) of each C6/36 subculture was obtained by plaque assay to determine the amount of infectious viral particles (PFU). The virus titration was performed in porcine kidney epithelial (PS) cells and in L15 medium with 5% FBS. Briefly, the virus titration was done using 200 µL of L15 medium (5% F...
Methods
reagent used in the protocol.
- Use
- A lyophilized ZIKV isolate from a clinical case in Brazil (ZIKV BR ), gently provided by the Evandro Chagas Institute in Belém, Pará, was reconstituted in 0.5 mL of sterile DEPC water. The African-lineage MR-766 (ZIKV AF ), a reference strain isolated in Uganda in 1947 and the Yellow Fever Vaccine strain (...
Real-time PCR
reagent used in the protocol.
- Use
- RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologie...
NPCs, neurons, neurospheres and organoids
reagent used in the protocol.
- Use
- We used 3 human and 2 chimpanzee iPSC clones that were previously characterized in the Beltrão-Braga and in the Muotri lab - plus H9 human embryonic stem cells (hESC) for all the experiments using pluripotent stem cells. All the cell lines tested negative for mycoplasma contamination. Briefly, high passag...
In vitro infection
reagent used in the protocol.
- Use
- NPCs, neurons, neurospheres and organoids were infected with ZIKV BR, ZIKV AF, YFV and mock (culture supernatant from uninfected C6/36 cells). NPCs were seeded in plates in 24 well plates and after 24 hours viral samples were diluted to the desired MOI (0.1; 1 or 10) and added to the cells. For viral adsorption, c...
Immunofluorescence
reagent used in the protocol.
- Use
- Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added:...
Cerebral organoids analyses
reagent used in the protocol.
- Use
- Human and Chimp organoids were infected with MOI of 0.1 and analyzed after 24 and 96 hours p.i. Organoids were cryosectioned at 20 um. Immunofluorescence was performed after blocking sections in a solution with 0.1% Triton and 3% BSA (Gemini) for 1 hour at room temperature. The primary antibodies were diluted in a s...
Transmission Electron Microscopy (TEM)
reagent used in the protocol.
- Use
- Cell pellet was fixed using a 3% glutaraldehyde solution (Merck) at 4°C for 2 hours, rinsed in 3 changes of PBS for 1 hour, and incubated for 16 hours at 4°C. The next day, post-fixation was performed with 1% of osmium tetroxide for 30 minutes at room temperature. Dehydration was carried out gradually with...
Real-time PCR
RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologie...
- Use
- RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologie...
Cell death pathway analysis
One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target genes using the RT 2 Profiler™ PCR Array Mouse Cell Death PathwayFinder (Cat# PAMM-212ZA-Qiagen®) according to manufacturer'...
- Use
- One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target genes using the RT 2 Profiler™ PCR Array Mouse Cell Death PathwayFinder (Cat# PAMM-212ZA-Qiagen®) according to manufacturer'...
Immunofluorescence
Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added:...
- Use
- Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added:...
Transmission Electron Microscopy (TEM)
Cell pellet was fixed using a 3% glutaraldehyde solution (Merck) at 4°C for 2 hours, rinsed in 3 changes of PBS for 1 hour, and incubated for 16 hours at 4°C. The next day, post-fixation was performed with 1% of osmium tetroxide for 30 minutes at room temperature. Dehydration was carried out gradually with...
- Use
- Cell pellet was fixed using a 3% glutaraldehyde solution (Merck) at 4°C for 2 hours, rinsed in 3 changes of PBS for 1 hour, and incubated for 16 hours at 4°C. The next day, post-fixation was performed with 1% of osmium tetroxide for 30 minutes at room temperature. Dehydration was carried out gradually with...
Flow Cytometric Analysis (FAC)
The cells were infected under a MOI of 10 and 1, prepared using supernatants from infected C6/36, and equal volume of mock. Cellular infection occurred at 4°C for 1 hour with cell homogenization every 10 minutes. After that, the cells were washed once and then maintained at 37°C in CO 2 incubators with med...
- Use
- The cells were infected under a MOI of 10 and 1, prepared using supernatants from infected C6/36, and equal volume of mock. Cellular infection occurred at 4°C for 1 hour with cell homogenization every 10 minutes. After that, the cells were washed once and then maintained at 37°C in CO 2 incubators with med...
Computed Tomography (CT)
Mice were properly anesthetized with isoflurane and immobilized on their right side on the bed with a piece of gauze and positioned with the whole body in the field of view (FOV). CT images were acquired using small animal imaging equipment (Triumph ™ Trimodality Gamma Medica Ideas) with 45 kVp, 0.4 mA and 2.1...
- Use
- Mice were properly anesthetized with isoflurane and immobilized on their right side on the bed with a piece of gauze and positioned with the whole body in the field of view (FOV). CT images were acquired using small animal imaging equipment (Triumph ™ Trimodality Gamma Medica Ideas) with 45 kVp, 0.4 mA and 2.1...
Cell death pathway analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target genes using the RT 2 Profiler™ PCR Array Mouse Cell Death PathwayFinder (Cat# PAMM-212ZA-Qiagen®) according to manufacturer'...
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Virus titration
Titration (in PFU/mL) of each C6/36 subculture was obtained by plaque assay to determine the amount of infectious viral particles (PFU). The virus titration was performed in porcine kidney epithelial (PS) cells and in L15 medium with 5% FBS. Briefly, the virus titration was done using 200 µL of L15 medium (5% FBS, 1% penicillin-streptomycin, and 1% glutamine) in a 24-well plate. Then, a serial dilution of each virus stock from ZIKV BR, ZIKV AF and YFV-17D in L15 medium was performed, from 10 -1 to 10 -11.Then, 200 µl of each dilution was added in each 24-well plate. After this, 1 × 10 6 PS cells were seeded in the each 24-well plate for at least 3 hours at 37°C to allow virus adsorption and PS cells adherence. Later, each well was overlaid with complete carboxymethyl cellulose (CMC) medium (0.6% in L15 supplemented with 3% FBS). After 5 days of incubati...
Methods
A lyophilized ZIKV isolate from a clinical case in Brazil (ZIKV BR ), gently provided by the Evandro Chagas Institute in Belém, Pará, was reconstituted in 0.5 mL of sterile DEPC water. The African-lineage MR-766 (ZIKV AF ), a reference strain isolated in Uganda in 1947 and the Yellow Fever Vaccine strain (YFV-17D), both used here as controls, were gently provided by the Institute Pasteur in Dakar, Senegal. Aedes albopictus mosquito cells (C636 cells) were previously prepared to culture the three viruses. C6/36 cell culture was maintained using Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) (Gibco ™ ), 1% non-essential amino acids (Gibco ™), 1% sodium pyruvate (Gibco ™ ), 1% penicillin/streptomycin (Gibco ™ ), 0.05% of Amphotericin B (Gibco ™ ) and kept at 27°C in the absence of CO 2. After reaching an approxi...
In vivo infection
Pregnant mice, 6-8 weeks of age, C57BL/6 or SJL (JAX), were infected intra-venously with 200µL of ZIKV BR -infected C6/36 cell supernatant containing 10 3, 4 x 10 10 or 1 x 10 12 PFUs/mL of virus on day 10-13 of gestation. The animals were observed daily. All the experiments were performed with the approval of the Institute of Biomedical Sciences Ethics Committee protocol number 05/2016.
Real-time PCR
RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologies) and stored at -80°C. The set of primers/probes specific for ZIKV were synthesized by Sigma Life Science, with 5- FAM as the reporter dye for the probe. The set of primers/probes ZIKV 835, ZIKV 911c and ZIKV 860-FAM were previously described. The real-time reaction was performed with 10 µL of each sample and 10 µL of the AgPath-IDTM One-Step RT-PCR reagents (Applied Biosystems). The amplification was done in an Applied Biosystems 7500 real-time PCR system, and involved activation at 45°C for 15 min, 95°C for 15 min followed by 40 amplificat...
NPCs, neurons, neurospheres and organoids
We used 3 human and 2 chimpanzee iPSC clones that were previously characterized in the Beltrão-Braga and in the Muotri lab - plus H9 human embryonic stem cells (hESC) for all the experiments using pluripotent stem cells. All the cell lines tested negative for mycoplasma contamination. Briefly, high passages of iPSC/hESC colonies on feeder-free plates were maintained for 5 days with mTSeR media (Stem Cell Technologies). On the fifth day, the medium was changed to N2 media (DMEM/F12 medium supplemented with 1X N2 supplement (Invitrogen) and the dual SMAD inhibitors, 1µM dorsomorphin (Tocris) and 1 µM SB431542 (Stemgent), for 48 hours. Further, the colonies were detached from the plate and cultured in suspension as Embryoid Bodies (EBs) for 5 days at 90 rpm in N2 media with the dual SMAD inhibitors. The EBs were plated on matrigel-coated plates with NBF media compose...
In vitro infection
NPCs, neurons, neurospheres and organoids were infected with ZIKV BR, ZIKV AF, YFV and mock (culture supernatant from uninfected C6/36 cells). NPCs were seeded in plates in 24 well plates and after 24 hours viral samples were diluted to the desired MOI (0.1; 1 or 10) and added to the cells. For viral adsorption, cells in monolayer were incubated for 1 hour at 4°C with gentle agitation every 10 min. Next, the inoculum was removed and cells were washed once with PBS (USB Corporation). Culture medium was added to each well, and cells were incubated at 37°C and 5% CO 2 for the duration of the experiment. For neurospheres, NPCs were kept in constant shaking. For neuronal infection, NPCs were previously differentiated for 28 days and then neurons were infected with the desired MOI. For organoids, the number of cells available for infection was estimated to be 2.5 x 10 4 cells, a...
Immunofluorescence
Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) ( ). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa...
Cerebral organoids analyses
Human and Chimp organoids were infected with MOI of 0.1 and analyzed after 24 and 96 hours p.i. Organoids were cryosectioned at 20 um. Immunofluorescence was performed after blocking sections in a solution with 0.1% Triton and 3% BSA (Gemini) for 1 hour at room temperature. The primary antibodies were diluted in a solution with 0.1% Triton and 3% BSA, and the sections were incubated with following antibodies: anti-ZIKV, anti-flavivirus, anti-MAP2, anti-cleaved-caspase-3 and anti-Sox2, all mentioned above, and anti-PAX6 (rabbit, Covance PRB-278P, 1:100), anti-TBR1 (rabbit, Abcam ab31940, 1:300), CTIP2 (rat, Abcam ab18465, 1:100) and Ki67 (rabbit, Abcam ab15580, 1:100). The sections were blocked with 0.1% Triton (Sigma-Aldrich) and 3% BSA for 30 min at room temperature and the secondary antibodies previously diluted, the same mentioned above, were added. The nuclei were stained with DAP...
Measurement outputs
What raw and processed outputs should exist?
RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All R...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target g...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in P...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The cells were infected under a MOI of 10 and 1, prepared using supernatants from infected C6/36, and equal volume of mock. Cellular infection occurred at 4°C for 1 hour wi...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target genes using the RT 2 Profiler™ PCR Array Mouse Cell Death PathwayFinder (Cat# PAMM-212ZA-Qiagen...
from paperScoring or quantification
Quantify the primary readouts for this experiment: RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All R...; One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target g...; Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in P...; The cells were infected under a MOI of 10 and 1, prepared using supernatants from infected C6/36, and equal volume of mock. Cellular infection occurred at 4°C for 1 hour wi....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target g...; a, Scheme of the in vitro experiments using hPSCs. The cells were differentiated into NPCs, neurons, neurospheres and cerebral organoids to test the impact of ZIKV BR over time...; a, Representative image of an entire cross-section of a cerebral human organoid infected with the ZIKV BR (MOI = 0.1, 24 hours p.i.). Scale bar = 200 µm. b, Detail of the...
from paperReporting output
Report representative outputs alongside summary comparisons for RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All R..., One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target g..., Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in P..., The cells were infected under a MOI of 10 and 1, prepared using supernatants from infected C6/36, and equal volume of mock. Cellular infection occurred at 4°C for 1 hour wi....
inferred from protocolStructured statistical methods
One microgram of total RNA from brains of 4 pups, 2 pooled mock and 2 pooled ZIKV BR infected from SJL mothers were submitted to gene expression analysis for cell death target g...; a, Scheme of the in vitro experiments using hPSCs. The cells were differentiated into NPCs, neurons, neurospheres and cerebral organoids to test the impact of ZIKV BR over time...; a, Representative image of an entire cross-section of a cerebral human organoid infected with the ZIKV BR (MOI = 0.1, 24 hours p.i.). Scale bar = 200 µm. b, Detail of the...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Titration (in PFU/mL) of each C6/36 subculture was obtained by plaque assay to determine the amount of infectious viral particles (PFU). The virus titration was performed in porcine kidney epithelial (PS) cells and in L15 medium with 5% FBS. Briefly, the virus titration was done using 200 µL of L15 medium (5% FBS, 1% penicillin-streptomycin, and 1% glutamine) in a 24-well plate. Then, a serial dilution of each virus stock from ZIKV BR, ZIKV AF and YFV-17D in L15 medium was performed, from 10 -1 to 10 -11.Then, 200 µl of each dilution was added in each 24-well plate. After this, 1 × 10 6 PS cells were seeded in the each 24-well plate for at least 3 hours at 37°C to allow virus adsorption and PS cells adherence. Later, each well was overlaid with complete carboxymethyl cellulose (CMC) medium (0.6% in L15 supplemented with 3% FBS). After 5 days of incubation at 37°C, the plaque visualization was made using blue black staining solution. The most appropriate viral dilution was estimated to determine the amount of infected cells visible (PFU per mL). For ZIKV BR, the first C6/36 subculture had a titer of 6 x 10 8. The following subcultures had, r...
A lyophilized ZIKV isolate from a clinical case in Brazil (ZIKV BR ), gently provided by the Evandro Chagas Institute in Belém, Pará, was reconstituted in 0.5 mL of sterile DEPC water. The African-lineage MR-766 (ZIKV AF ), a reference strain isolated in Uganda in 1947 and the Yellow Fever Vaccine strain (YFV-17D), both used here as controls, were gently provided by the Institute Pasteur in Dakar, Senegal. Aedes albopictus mosquito cells (C636 cells) were previously prepared to culture the three viruses. C6/36 cell culture was maintained using Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) (Gibco ™ ), 1% non-essential amino acids (Gibco ™), 1% sodium pyruvate (Gibco ™ ), 1% penicillin/streptomycin (Gibco ™ ), 0.05% of Amphotericin B (Gibco ™ ) and kept at 27°C in the absence of CO 2. After reaching an approximately 70% confluent monolayer, 50µL of each viral samples were inoculated into C6/36 with an hour of adsorption, with gentle shaking every 10 minutes to allow the homogeneous adsorption of the viruses. At the end of the adsorption period, 5mL of the culture media were added, plus 2% FBS, 1% no...
Pregnant mice, 6-8 weeks of age, C57BL/6 or SJL (JAX), were infected intra-venously with 200µL of ZIKV BR -infected C6/36 cell supernatant containing 10 3, 4 x 10 10 or 1 x 10 12 PFUs/mL of virus on day 10-13 of gestation. The animals were observed daily. All the experiments were performed with the approval of the Institute of Biomedical Sciences Ethics Committee protocol number 05/2016.
RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologies) and stored at -80°C. The set of primers/probes specific for ZIKV were synthesized by Sigma Life Science, with 5- FAM as the reporter dye for the probe. The set of primers/probes ZIKV 835, ZIKV 911c and ZIKV 860-FAM were previously described. The real-time reaction was performed with 10 µL of each sample and 10 µL of the AgPath-IDTM One-Step RT-PCR reagents (Applied Biosystems). The amplification was done in an Applied Biosystems 7500 real-time PCR system, and involved activation at 45°C for 15 min, 95°C for 15 min followed by 40 amplification cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. The real-time data were analyzed using SDS software from Applied Biosystems. For the detection and quantification of viral RNA, the real time PCR of each sample was compared with threshold cycle (CT) value with a ZIKV plas...
We used 3 human and 2 chimpanzee iPSC clones that were previously characterized in the Beltrão-Braga and in the Muotri lab - plus H9 human embryonic stem cells (hESC) for all the experiments using pluripotent stem cells. All the cell lines tested negative for mycoplasma contamination. Briefly, high passages of iPSC/hESC colonies on feeder-free plates were maintained for 5 days with mTSeR media (Stem Cell Technologies). On the fifth day, the medium was changed to N2 media (DMEM/F12 medium supplemented with 1X N2 supplement (Invitrogen) and the dual SMAD inhibitors, 1µM dorsomorphin (Tocris) and 1 µM SB431542 (Stemgent), for 48 hours. Further, the colonies were detached from the plate and cultured in suspension as Embryoid Bodies (EBs) for 5 days at 90 rpm in N2 media with the dual SMAD inhibitors. The EBs were plated on matrigel-coated plates with NBF media composed of the following: DMEM/F12 media supplemented with 0.5X N2, 0.5X B27 supplement (Gibco ™ ), 20 ng/mL of FGF2 and 1% penicillin/streptomycin. The emerged rosettes containing the NPCs were manually picked, dissociated and plated in a double-coated plate with poly-ornithine (10 µg/mL, Sigm...
NPCs, neurons, neurospheres and organoids were infected with ZIKV BR, ZIKV AF, YFV and mock (culture supernatant from uninfected C6/36 cells). NPCs were seeded in plates in 24 well plates and after 24 hours viral samples were diluted to the desired MOI (0.1; 1 or 10) and added to the cells. For viral adsorption, cells in monolayer were incubated for 1 hour at 4°C with gentle agitation every 10 min. Next, the inoculum was removed and cells were washed once with PBS (USB Corporation). Culture medium was added to each well, and cells were incubated at 37°C and 5% CO 2 for the duration of the experiment. For neurospheres, NPCs were kept in constant shaking. For neuronal infection, NPCs were previously differentiated for 28 days and then neurons were infected with the desired MOI. For organoids, the number of cells available for infection was estimated to be 2.5 x 10 4 cells, as calculated by dividing the average surface area of a typical organoid by the average area of a typical cell ( i.e., a fibroblast). This calculus was used to estipulate the desired 0.1 MOI. For mock controls, the same volume of supernatant was added to each experiment, and the same procedures were...
Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) ( ). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa Fluor 555 and anti-rabbit Alexa Fluor 555 (Invitrogen). The nuclei were stained with DAPI (Invitrogen, 1:10,000) diluted in a PBS 1x solution for 5 minutes and mounted with DPX (Sigma). Images were acquired with Nikon Eclipse 80i. Analysis of data was performed using software NIS Elements 3.22 (Tok...
Human and Chimp organoids were infected with MOI of 0.1 and analyzed after 24 and 96 hours p.i. Organoids were cryosectioned at 20 um. Immunofluorescence was performed after blocking sections in a solution with 0.1% Triton and 3% BSA (Gemini) for 1 hour at room temperature. The primary antibodies were diluted in a solution with 0.1% Triton and 3% BSA, and the sections were incubated with following antibodies: anti-ZIKV, anti-flavivirus, anti-MAP2, anti-cleaved-caspase-3 and anti-Sox2, all mentioned above, and anti-PAX6 (rabbit, Covance PRB-278P, 1:100), anti-TBR1 (rabbit, Abcam ab31940, 1:300), CTIP2 (rat, Abcam ab18465, 1:100) and Ki67 (rabbit, Abcam ab15580, 1:100). The sections were blocked with 0.1% Triton (Sigma-Aldrich) and 3% BSA for 30 min at room temperature and the secondary antibodies previously diluted, the same mentioned above, were added. The nuclei were stained with DAPI, as mentioned above and slides were mounted with DPX (Sigma-Aldrich).
Machine-readable layer
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{
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"@type": "HowTo",
"name": "The Brazilian Zika virus strain causes birth defects in experimental models methods",
"description": "Evidence-backed execution summary for The Brazilian Zika virus strain causes birth defects in experimental models methods from The Brazilian Zika virus strain causes birth defects in experimental models.",
"totalTime": "PT29020M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Virus titration",
"text": "Titration (in PFU/mL) of each C6/36 subculture was obtained by plaque assay to determine the amount of infectious viral particles (PFU). The virus titration was performed in porcine kidney epithelial (PS) cells and in L15 medium with 5% FBS. Briefly, the virus titration was done using 200 µL of L15 medium (5% FBS, 1% penicillin-streptomycin, and 1% glutamine) in a 24-well plate. Then, a serial dilution of each virus stock from ZIKV BR, ZIKV AF and YFV-17D in L15 medium was performed, from 10 -1 to 10 -11.Then, 200 µl of each dilution was added in each 24-well plate. After this, 1 × 10 6 PS cells were seeded in the each 24-well plate for at least 3 hours at 37°C to allow virus adsorption and PS cells adherence. Later, each well was overlaid with complete carboxymethyl cellulose (CMC) medium (0.6% in L15 supplemented with 3% FBS). After 5 days of incubati..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Methods",
"text": "A lyophilized ZIKV isolate from a clinical case in Brazil (ZIKV BR ), gently provided by the Evandro Chagas Institute in Belém, Pará, was reconstituted in 0.5 mL of sterile DEPC water. The African-lineage MR-766 (ZIKV AF ), a reference strain isolated in Uganda in 1947 and the Yellow Fever Vaccine strain (YFV-17D), both used here as controls, were gently provided by the Institute Pasteur in Dakar, Senegal. Aedes albopictus mosquito cells (C636 cells) were previously prepared to culture the three viruses. C6/36 cell culture was maintained using Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) (Gibco ™ ), 1% non-essential amino acids (Gibco ™), 1% sodium pyruvate (Gibco ™ ), 1% penicillin/streptomycin (Gibco ™ ), 0.05% of Amphotericin B (Gibco ™ ) and kept at 27°C in the absence of CO 2. After reaching an approxi..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "In vivo infection",
"text": "Pregnant mice, 6-8 weeks of age, C57BL/6 or SJL (JAX), were infected intra-venously with 200µL of ZIKV BR -infected C6/36 cell supernatant containing 10 3, 4 x 10 10 or 1 x 10 12 PFUs/mL of virus on day 10-13 of gestation. The animals were observed daily. All the experiments were performed with the approval of the Institute of Biomedical Sciences Ethics Committee protocol number 05/2016."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Real-time PCR",
"text": "RNA was extracted from each sample (cells, supernatant of cell culture or mouse tissue) using the QIAamp UltraSens Virus Kit (Qiagen) or TRIzol® reagent (Invitrogen). All RNA pellets were resuspended in 30 µl of RNase-free distilled water, quantified using a NanoDrop spectrophotometer (NanoDrop Technologies) and stored at -80°C. The set of primers/probes specific for ZIKV were synthesized by Sigma Life Science, with 5- FAM as the reporter dye for the probe. The set of primers/probes ZIKV 835, ZIKV 911c and ZIKV 860-FAM were previously described. The real-time reaction was performed with 10 µL of each sample and 10 µL of the AgPath-IDTM One-Step RT-PCR reagents (Applied Biosystems). The amplification was done in an Applied Biosystems 7500 real-time PCR system, and involved activation at 45°C for 15 min, 95°C for 15 min followed by 40 amplificat..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "NPCs, neurons, neurospheres and organoids",
"text": "We used 3 human and 2 chimpanzee iPSC clones that were previously characterized in the Beltrão-Braga and in the Muotri lab - plus H9 human embryonic stem cells (hESC) for all the experiments using pluripotent stem cells. All the cell lines tested negative for mycoplasma contamination. Briefly, high passages of iPSC/hESC colonies on feeder-free plates were maintained for 5 days with mTSeR media (Stem Cell Technologies). On the fifth day, the medium was changed to N2 media (DMEM/F12 medium supplemented with 1X N2 supplement (Invitrogen) and the dual SMAD inhibitors, 1µM dorsomorphin (Tocris) and 1 µM SB431542 (Stemgent), for 48 hours. Further, the colonies were detached from the plate and cultured in suspension as Embryoid Bodies (EBs) for 5 days at 90 rpm in N2 media with the dual SMAD inhibitors. The EBs were plated on matrigel-coated plates with NBF media compose..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "In vitro infection",
"text": "NPCs, neurons, neurospheres and organoids were infected with ZIKV BR, ZIKV AF, YFV and mock (culture supernatant from uninfected C6/36 cells). NPCs were seeded in plates in 24 well plates and after 24 hours viral samples were diluted to the desired MOI (0.1; 1 or 10) and added to the cells. For viral adsorption, cells in monolayer were incubated for 1 hour at 4°C with gentle agitation every 10 min. Next, the inoculum was removed and cells were washed once with PBS (USB Corporation). Culture medium was added to each well, and cells were incubated at 37°C and 5% CO 2 for the duration of the experiment. For neurospheres, NPCs were kept in constant shaking. For neuronal infection, NPCs were previously differentiated for 28 days and then neurons were infected with the desired MOI. For organoids, the number of cells available for infection was estimated to be 2.5 x 10 4 cells, a..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Immunofluorescence",
"text": "Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) ( ). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Cerebral organoids analyses",
"text": "Human and Chimp organoids were infected with MOI of 0.1 and analyzed after 24 and 96 hours p.i. Organoids were cryosectioned at 20 um. Immunofluorescence was performed after blocking sections in a solution with 0.1% Triton and 3% BSA (Gemini) for 1 hour at room temperature. The primary antibodies were diluted in a solution with 0.1% Triton and 3% BSA, and the sections were incubated with following antibodies: anti-ZIKV, anti-flavivirus, anti-MAP2, anti-cleaved-caspase-3 and anti-Sox2, all mentioned above, and anti-PAX6 (rabbit, Covance PRB-278P, 1:100), anti-TBR1 (rabbit, Abcam ab31940, 1:300), CTIP2 (rat, Abcam ab18465, 1:100) and Ki67 (rabbit, Abcam ab15580, 1:100). The sections were blocked with 0.1% Triton (Sigma-Aldrich) and 3% BSA for 30 min at room temperature and the secondary antibodies previously diluted, the same mentioned above, were added. The nuclei were stained with DAP..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Real-time PCR"
},
{
"@type": "HowToTool",
"name": "Cell death pathway analysis"
},
{
"@type": "HowToTool",
"name": "Immunofluorescence"
},
{
"@type": "HowToTool",
"name": "Transmission Electron Microscopy (TEM)"
},
{
"@type": "HowToTool",
"name": "Flow Cytometric Analysis (FAC)"
},
{
"@type": "HowToTool",
"name": "Computed Tomography (CT)"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Virus titration"
},
{
"@type": "HowToSupply",
"name": "Methods"
},
{
"@type": "HowToSupply",
"name": "Real-time PCR"
},
{
"@type": "HowToSupply",
"name": "NPCs, neurons, neurospheres and organoids"
},
{
"@type": "HowToSupply",
"name": "In vitro infection"
},
{
"@type": "HowToSupply",
"name": "Immunofluorescence"
},
{
"@type": "HowToSupply",
"name": "Cerebral organoids analyses"
},
{
"@type": "HowToSupply",
"name": "Transmission Electron Microscopy (TEM)"
}
],
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"headline": "The Brazilian Zika virus strain causes birth defects in experimental models",
"datePublished": "2016",
"author": [
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"name": "Fernanda R. Cugola"
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"name": "Isabella R. Fernandes"
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"name": "Fabiele B. Russo"
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"name": "Beatriz C. Freitas"
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"name": "Sarah Romero"
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"name": "Isabela Cunha"
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"name": "Carla L. Freitas"
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"name": "Wesley N. Brandão"
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"name": "Cristiano Rossato"
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"name": "David G. Andrade"
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"name": "Daniele de P. Faria"
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"name": "Alexandre T. Garcez"
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"name": "Carlos A.. Buchpigel"
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"name": "Carla T. Braconi"
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"name": "Erica Mendes"
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"name": "Jean Pierre S. Peron"
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