The Brazilian Zika virus strain causes birth defects in experimental models methods
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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...
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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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"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..."
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"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..."
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"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..."
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"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..."
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"headline": "The Brazilian Zika virus strain causes birth defects in experimental models",
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