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case_images: Placentae histopathology. (A-C) Placentae from non-ZIKV patients stained with H&E and presenting normal features: maternal decidua (Dec) and chorionic villi (CV). (D-R) Placentae from ZIKV infected patients that presented a range of different alterations such as VC, vascular congestion; IC, intervillous congestion; MO, macrophage infiltrate; Ly, lymphocytic infiltrate villous; He, hemorrhage; Th, endothelial thickening; Im, immature chorionic villi; Hf, Hofbauer cells; Ca, calcification; Fi, fibrin areas; IFT, infarct, and Is, ischemia.
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case_images: The histopathological analysis of control samples showed a regular arrangement of the decidual layer and normal chorionic villi that included syncytiotrophoblasts, cytotrophoblasts and endothelial cells (Figures 1A-C), which suggested that the collection and fixation of placental samples was adequate. In infected placentae, we observed relevant damage in the decidua and chorionic villi (Figures 1D-R). T CD8+ cells were found in the same areas (Figures 4J-M), few CD4+ cells were detected within the tissues (Supplementary Figure S1).
case_images: Electron microscopy analysis of placental sections showed alterations and virus-like particles in ZIKV infected samples. (A-D) Electron microscopy images of ultrathin sections of placental tissue from a single, non-ZIKV infected mother that exhibited regular cytotrophoblasts (CTB), syncytiotrophoblasts (STB), nucleus (N), mitochondria (M), and collagen filament structures (COL). (E-H) Electron micrographs of ultrathin sections of placental tissue from different ZIKV-infected mothers showing CTB with alterations in the cytoplasm, nuclear variation (N) and swollen mitochondria with a loss of cristae and membrane rupture. The identified STB presented an enlargement of vesicles and apoptotic bodies (asterisks) along with an absence of normal membrane extensions and evidence of microvesicle secretion. The extracellular matrix (EM) did not present collagen filaments. (I-K) Identification of clusters of virus-like particles that were positioned near the endoplasmic reticulum (ER) of CTB and ~25 nm in diameter, which is consistent with the dimensions of ZIKV.
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case_images: No evidence of ultrastructural changes was observed in placental cells from control patients, represented in images form a single control 1 (Figures 2A-D), which again suggests the collection and treatment of samples maintained their structural integrity. The cytotrophoblasts presented normal aspects for all organelles, including the mitochondria and the nucleus, which was heterochromatic (Figures 2A,B). Syncytiotrophoblasts presented an electron dense cytoplasm, as expected, with heterochromatic nuclei (Figure 2C). In the extracellular matrix, collagen filament structures were readily identified in transverse and longitudinal sections (Figure 2D). In contrast, the analysis of infected placentae showed cytotrophoblasts with little nuclear variation and an electron lucid cytoplasm containing swollen mitochondria showing a loss of cristae and ruptured membranes (Figures 2E,F). The syncytiotrophoblasts aspects were extensively modified with an enlargement of vesicles and apoptotic bodies along with an absence of their normal membrane extensions and secretions of microvesicles (Figures 2E,G). An investigation of the extracellular matrix across the images of placenta samples from the different donors did not reveal collagen filaments, which suggests a decrease in this matrix component (Figure 2H). These particles were measured to have a diameter of ~25 nm in diameter, which is consistent with the dimensions of ZIKV (Figures 2I-K).
case_images: Detection of viral antigens. Tissue sections were probed by immunohistochemistry for the E (A-D) and NS1 (E-H) antigens of ZIKV as well as by in situ hybridization for the genome of ZIKV (I-L). Representative images of control placentae (A,E,I) showing the absence of reactivity for the antigens and genome of ZIKV. Representative images of placenta from ZIKV+ placenta, independent of the cephalic circumference of the infant, show: (B-D) the presence of E protein in decidual cells (DC) of decidua (Dec), mesenchymal cells (MS), endothelial cells (En), and syncytiotrophoblast (STB) of chorionic villi (CV) that was independent of the cephalic circumference of the infant; (F-H) NS1 protein detected in decidual cells (DC) of decidua (Dec), mesenchymal cells (MS), Hofbauer (Hf), syncytiotrophoblast (STB) and cytotrophoblast (CTB) of chorionic villi (CV); and (J-L) Detection of ZIKV RNA negative strand by in situ hybridization in decidual cells (DC) of decidua (Dec), mesenchymal cells (MS), syncytiotrophoblast (STB) and cytotrophoblast (CTB) of chorionic villi (CV).
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case_images: These viral antigens were detected in all samples obtained from infected patients, while immunostaining was negative in samples of control placentae (Figures 3A,E). The E structural protein was detected in decidual cells and in syncytiotrophoblasts as well as endothelial and mesenchymal cells of chorionic villi (Figures 3B-D). The NS1 protein was also detected in cytotrophoblasts, syncytiotrophoblasts and mesenchymal cells, moreover in Hofbauer cells of chorionic villi and in decidual cells (Figures 3F-H). Additionally, the replication was also confirmed by in situ hybridization using a probe that anneals only to the negative strand of the ZIKV RNA, which revealed the presence of this RNA in decidual cells, syncytiotrophoblasts, cytotrophoblasts and villous mesenchymal cells (Figures 3J-L). All controls were negative for the immunohistochemistry and in situ hybridization (Figure 3I).
case_images: Increased cellularity of mononuclear cell subpopulations in ZIKV-infected placental tissues. CD68 and CD8 were detected in placenta samples by immunohistochemistry. (A-B) CD68+ cells in decidua and chorionic villi of control placenta. (C-F) CD68+ cells in decidua and chorionic villi of ZIKV infected placentae. (G) Quantification of CD68+ cells showing a 5 or 6-fold increase in placentae of ZIKV+MIC- and ZIKV+MIC+ groups, respectively. (H,I) CD8+ cells in decidua and chorionic villi of control placentae. (J-M) CD8+ cells in decidua and chorionic villi of ZIKV infected placentae. (N) Quantification of CD8+ cells showing a 7 or 8-fold increase in the tissues of ZIKV+MIC- and ZIKV+MIC+ groups, respectively. (O,P) Immunofluorescence for the presence of ZIKV NS1 protein (green) and CD163 (red; biomarker for macrophages) showing colocalization. Nuclei were stained using DAPI (blue). (O) ZIKV NS1 antigen was not detected in the control placenta. Data are represented as mean +- SDM. Asterisks indicate differences that are statistically significant between groups (***p < 0.001).
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case_images: A significant upsurge in the number of CD68+ cells were detected in both groups of infected placentae (ZIKV+MIC- and ZIKV+MIC+), which suggested a recruitment of macrophages and hyperplasia caused by the infection in the basal decidua and Hofbauer cells (Figures 4C-F). The increase of macrophages were quantified to be 5- and 6-fold in placentae of ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 4G). T CD8+ cells were found in the same areas (Figures 4J-M), few CD4+ cells were detected within the tissues (Supplementary Figure S1). The T CD8+ lymphocytes were increased 7- and 8-fold in the tissues from ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 4N). The control placentae showed a low number of positive cells for both markers (Figures 4A,B,H,I). Additional evidence for the replication of ZIKV in macrophages was observed by the colocalization of NS1 protein with the CD163 marker for differentiated macrophages in dual stained immunofluorescent images (Figure 4P). As expected, no signals were observed for NS1 in control tissue (Figure 4O).
case_images: Cytokine-producing cell profile. Detection of TNF-alpha, IFN-gamma, VEGFR-2 and RANTES/CCL5 by immunohistochemistry show (A) TNF-alpha in cells of chorionic villi in control placentae (Left panel) and ZIKV infected placentae (Right panel). (C) Production of IFN-gamma in macrophages as well as endothelial cells in chorionic villi and decidual cells of the decidua of control placentae (Left panel) and ZIKV infected placentae (Right panel). (E) VEGFR-2 was expressed in endothelial cells of decidua and chorionic villi in control placentae (Left panel) and ZIKV infected placentae (Right panel). (G) RANTES/CCL5 present mainly in the endothelium and Hofbauer cells located within the chorionic villi and decidual cells and syncytiotrophoblasts of the decidua in control placentae (Left panel) and ZIKV infected placentae (Right panel). Quantification of the cells positive for (B,D,F,H) Quantification of the number of cells expressing TNF-alpha (B), IFN-gamma (D), VEGFR2 (F) and RANTES/CCL5 (H) showed an increased expression of local pro-inflammatory cytokines and mediators in ZIKV positive placentae compared to controls. Data are represented as mean +- SDM. Asterisks indicate differences that are statistically significant between groups (**p < 0.01) or (***p < 0.001).
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case_images: In addition, Additionally, the markers VEGFR-2 and RANTES/CCL5 were included as they have been implicated with an alteration in vascular permeability. In control samples, all markers were detected at low levels (Figures 5A-H). TNF-alpha expression was diffuse in Hofbauer and mesenchymal cells in the chorionic villi and in decidual cells (Figure 5A). Its expression was 12-fold higher in the ZIKV+MIC- group and 16-fold higher in the placenta from the ZIKV+MIC+ group (Figure 5B). IFN-gamma was found mostly in decidual cells and decidual macrophages (Figure 5C), with a 3- and 5-fold increase in the ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 5D). The expression of VEGFR-2 was found in endothelial and mesenchymal cells in chorionic villi as well as in circulating macrophages within the vessels and in endothelial cells in decidua (Figure 5E). This receptor had an increased expression level of 11- and 13-fold in the ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 5F). RANTES/CCL5 was detected mainly in the endothelium and in Hofbauer cells located within the chorionic villi and in decidual cells and syncytiotrophoblasts of the decidua (Figure 5G). RANTES/CCL5 was expressed 4- and 5-fold upward in ZIKV+MIC- and ZIKV+MIC+ groups, respectively, compared to control group (Figure 5H). The statistical analysis of the expression of all these markers determined that they were significantly increased in the ZIKV+ patient placentae compared to ZIKV- control tissues (Figures 5B,D,F,H).
case_images: Detection and quantification of collagen, MMP-2 and MMP-9 collagenases expression. (A) Collagen detection by Picro Sirius Red staining in placental tissues. (B) The percent collagen area was quantified in all cases that showed a decrease in the expression of collagen in infected placentae. (C-E) Detection of MMP-2 and MMP-9 in decidual cells and cells located within the chorionic villi in both control and ZIKV infected placentae. (D-F) Quantification of the number of cells expressing MMP-2 and MMP-9 showed an increased expression in ZIKV infected placental tissues. Data are represented as mean +- SDM. Asterisks indicate differences that are statistically significant between groups (**p < 0.01) or (***p < 0.001).
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case_images: The absence of collagen in the electron micrographs was confirmed by its specific staining with Picro Sirius Red, which showed that a ZIKV infection led to a drastic decrease in placental collagen (Figure 6A). The reduction was 5- and 9-fold in the tissues from ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 6B). However, their expression was substantially elevated in the placenta from ZIKV+ mothers and displayed a diffuse pattern (Figures 6C,E). MMP-2 levels were 6- and 8-fold greater in ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 6D). MMP-9 increased 11- and 10-fold in ZIKV+MIC- and ZIKV+MIC+ groups, respectively (Figure 6F).
case_images: BDNF detection and quantification in placental tissues. (A-F) Detection of BDNF in cells of chorionic villi and maternal decidua of controls and ZIKV infected placentae by immunohistochemistry. (G) A quantification of the number of cells expressing BDNF showed a decreased in the expression of this hormone in ZIKV+ MIC+ cases. Data are represented as mean +- SDM. Asterisks indicate differences that are statistically significant between groups (**p < 0.01).
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case_images: In control samples, BDNF was readily detected by an intense and diffuse signal in cells of decidua and chorionic villi (Figures 7A,B). BDNF expressing cells was considerably lower (Figures 7C-F). By quantification, there were 12.35 positive cells for BDNF/mm2 in the control group and 4.25 positive cells for BDNF/mm2 in the ZIKV + MIC group, which had no statistical difference. In the ZIKV+MIC+ group, only 0.7 BDNF positive cells/mm2 were detected, which was significantly different (Figure 7G).