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Supplemental Figures - Nature Research · Supplemental Figures . Supplemental Figure 1. Axial MRI...
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Supplemental Figures Supplemental Figure 1. Axial MRI Images from a ZIKV Infected Pigtail Macaque 43 Days After Inoculation Serial axial images from a ZIKV infected pigtail macaque at the final time point in the study (43 days after inoculation; 162 days gestation, ~38 weeks human pregnancy). Scale: 0-40mm.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 2. Sagittal MRI Images from a ZIKV Infected Pigtail Macaque 43 Days After Inoculation Serial sagittal images from a ZIKV infected pigtail macaque at the final time point in the study (43 days after inoculation; 162 days gestation, ~38 weeks human pregnancy). Scale: 0-40mm.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 3. Coronal MRI Images from a ZIKV Infected Pigtail Macaque 43 Days After Inoculation Serial coronal images from a ZIKV infected pigtail macaque at the final time point in the study (43 days after inoculation; 162 days gestation, ~38 weeks human pregnancy). Scale: 0-40mm.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 4. Tissue Segmentation by MRI for Volume and Surface Area Estimation Tissue segmentations by time point are shown as a color overlay of tissues (T2 hyperintense lesion=light pink, light maroon=white matter, dark blue=cortical gray matter, yellow=deep gray matter, purple=cerebellum, light blue=sulcal cerebrospinal fluid, light purple=ventricular cerebrospinal fluid, green=brainstem). The surface mesh used for surface area estimation is shown as a yellow contour line where the surface mesh for that brain image intersects with the displayed slice plane. A: +10 days post-inoculation/129 days gestation, B: +17 days post-inoculation/136 days gestation, C: +31 days post-inoculation/150 days gestation, D: +43 days post-inoculation/162 days gestation. Scale: 0-8mm.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 5. Surface Rendering of the Fetal Brain from a ZIKV-Infected Fetus Over Time Surface rendering of the inner cortical surface extracted from each time point (A: +10 days post-inoculation/129 days gestation, B: +17 days post-inoculation/136 days gestation, C: +31 days post-inoculation/150 days gestation, D: +43 days post-inoculation/162 days gestation).
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 6. MR Spectroscopy Magnetic resonance spectroscopy was performed during MRI immediately prior to Cesarean section (+43 days post-inoculation; 162 days gestation). In the posterior brain near the T2 hyperintense focus, there was a lower relative concentration of N-acetyl aspartate, a marker for neuronal integrity, compared to the anterior brain.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 7. Ependymal Injury and Subependymal Caspase 3 Expression in the Zika virus-infected fetus and control. The ependymal epithelial cell lining of the lateral ventricle was intact in controls with relatively sparse surrounding glial cells (A). In contrast, large portions of the ependymal epithelium were missing in the ZIKV-infected fetal cerebrum, and the underlying stroma densely packed with glial cells (B). The arrow (B) indicates a transition point between normal ependymal epithelium to a denuded section with a hypercellular stroma. Activated caspase 3-immunoreactive cells were generally rare in the subependymal region of the control with sparse foci of more concentrated cells as shown (C). In the ZIKV-infected fetus, the fraction of cells with cleaved caspase 3 immunoreactivity was dramatically increased in the exposed subependymal zone of the denuded ventricular surface (D).
Nature Medicine: doi:10.1038/nm.4193
Supplemental Figure 8. MRI of a Child with Congenital ZIKV Syndrome and a ZIKV-infected Fetal Pigtail Macaque
A case of congenital ZIKV syndrome is presented to illustrate an analogous pattern of fetal brain injury to that seen in ZIKV-infected fetal pigtail macaque. A Brazilian boy was born at term with a head circumference of 29 cm (-4.5 standard deviations below the mean). He had early global developmental delay and hypotonia. His mother reported a fever and rash at 4 months gestation. A T2-weighted axial MRI (A) at the level of the thalami at four months showed severe volume loss with enlarged extra-axial spaces and ventricles, dramatically reduced white matter volume, and severe undersulcation suggesting a cortical malformation. Intrauterine MRI (B) of a ZIKV-infected fetal pigtail macaque at 162 days gestation shows a periventricular T2 hyperintense focus most prominent in the right posterior brain. While the boy has more severe brain abnormalities than the macaque fetus, the pattern is similar.
Nature Medicine: doi:10.1038/nm.4193
Tables
Supplemental Table 1. Changes in Volumes and Surface Area of Brain Layers Days Post-Inoculation
Days Gestation
Gray Matter Volume
White Matter Volume
Deep Gray Matter Volume
T2 Hyperintense Focus Volume
Cerebellum Volume
Inner Cortical Surface Area
+10 129 11.5 16.4 3 0.71 1.4 121 +17 136 14.8 18.1 3.4 0.63 1.5 171 +31 150 18.8 18 4.1 0.63 2.1 200 +43 162 21.2 18.7 4.8 0.9 2.8 220
Volumes are shown as cubic centimeters (cc) and area as centimeter squared (cm2).
Nature Medicine: doi:10.1038/nm.4193
Supplemental Table 2. Flow Cytometry of ZIKV Infected NHP Maternal and Fetal Brain
CELL TYPE MATERNAL BRAIN
FETAL BRAIN
Cell Subset No.
(% of Total Cells)
Total Single Cells 23937 12586
Astrocytes (CD56-, GFAPhigh)
353 (1.5)
517 (4.1)
Single cell suspensions of maternal and fetal brain tissue were stained to determine astrocyte numbers. Samples were gated for neural cell adhesion marker (CD56) negative, intracellular glial fibrillary acidic protein (GFAP) high single cells (CD56-, GFAPhigh). Note that the fetal brain tissue is enriched for astrocytes compared to the maternal brain tissue.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Table 3. ZIKV-Specific IgG Serology Over Time
Day (Post-Inoculation)
Zika IgG
Maternal (Dam) 0 Neg 4 Neg 8 Neg
10 Neg 14 Pos 21 Pos 24 Pos 31 Pos 35 Pos 42 Pos 43 Pos
Fetal 43 Pos
Day 0 = day of inoculation; neg, negative; pos, positive.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Table 4. Detection of ZIKV RNA in Organs or Tissues
Organ or Tissue ZIKV RNA Detection by qPCR
Fetus Brain + Eyes - Spinal Cord - Testes (Gonad) - Lungs - Spleen - Liver + Kidney - Adrenal -
Placenta Chorionic Villi + Chorioamniotic Membrane
-
Mother (Dam) Brain + Eyes + Ovary (Gonad) - Spleen + Liver + Kidney - Uterus - Lymph Node -
RNA was extracted from the tissues shown and 400 ng of total RNA was used to generate cDNA. This cDNA was used in the qPCR reaction to determine viral loads in the tissues listed. The copy number sensitivity, as determined using a standard curve from diluted known quantities of ZIKV genome, was 25 copies/qPCR reaction. Organs and tissues displaying Ct values below copy number detection are listed as not detected and denoted as (–) in the table. Tissues and organs in which ZIKV RNA was detectable, defined as greater than 25 copies/PCR reaction, are noted as positive (+) in the table. For samples with ZIKV viral loads above the limit of detection, mean viral RNA load was further calculated per mg of tissue as shown in Fig. 2 (panel I, analyzed in triplicate).
Nature Medicine: doi:10.1038/nm.4193
Supplemental Table 5. Immunohistochemistry antibodies and conditions Antibody Source (cat#) Species Dilution Immunostainer
Settings
Target
CD3 Dako
(A0452)
Rabbit 1:200 Mild, 32 min, 37ºC T-lymphocytes
CD4 Dako
(M0755)
Mouse 1:200 Mild, 32 min, 37ºC B-lymphocytes
CD163 Leica (NCL-
L-CD163)
Mouse 1:600 Mild, 16 min, 37ºC Histiocytes,
microglia
GFAP Dako
(M0761)
Mouse 1:400 Mild, 28 min, 37ºC Astrocytes
Neurofilament Dako MO762 Mouse 1:400 “CC1 short” Axons
Cleaved
Caspase 3
Cell Signal
Technology
Rabbit 1:250 Mild, 32 min, 37ºC Apoptotic cells
For immunohistochemistry, formalin-fixed paraffin embedded samples was used. The antibody, source, species of origin, dilution and protocol conditions are specified.
Nature Medicine: doi:10.1038/nm.4193
Supplemental Table 6. ZIKV Gene-Specific Primers
Primer and Nucleotide Position Sequence
ZIKV 1193 5'-CCGCTGCCCAACACAAG
ZIKV 1214 FAM (Probe) 5'-AGCCTACCTTGACAAGCAGTCAGACACTCAA
ZIKV 1269c 5'-CCACTAACGTTCTTTTGCAGACAT
Nucleotide positions correspond to ZIKV strain FSS13025 (GenBank number: KU955593.1).
Nature Medicine: doi:10.1038/nm.4193