Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At embryonic day (E) 10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV, Brazil ZIKV, or media (mock). (A) At 48 hours post infection (hpi), dams were euthanized, and fetal viability was determined as the percentage of fetuses within the inoculated uterine horn (n = total number of fetuses from 11–16 dams per group from two independent replicates). (B) Nigeria ZIKV RNA copies were quantified from placenta or spleen collected 48 hpi. Data represent mean ± standard error of the mean from two independent replications (n = 7–9/group). (C) Placentas collected 48 hpi from either mock (upper panel) or Nigeria ZIKV (lower panel) infected dams were paraformaldehyde fixed and immunostained for ZIKV (red) and 4′6-diamidino-2-phenylindole (DAPI, blue) to label nuclei. Representative images taken at 20× magnification (left) and further zoomed 5.5 fold (right) are shown. (D) ZIKV RNA (green) was measured in fetuses of Brazil ZIKV infected dams by in situ hybridization (ISH) at 48 hpi. Samples were also imaged without RNA probe incubation as a control (left) to remove background and a representative image at 5× magnification is shown for fetuses of ZIKV infected dams (right). Significant differences (*p<0.05) were determined by χ2 test (A) or unpaired two tailed t-test (B). Limit of detection (LOD) indicated with a dashed line. Scale bar: 100 or 50 μm (IHC, C),1000 μm (ISH, D)

Article Snippet: Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Infection, In Situ Hybridization, Incubation, Control, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi and placentas were paraformaldehyde-fixed. (A) Representative H & E images were taken at 5× magnification (upper panels) and 20× magnification (lower panels). (B) Representative TUNEL+ (green) stained placentas with DAPI to label nuclei taken at 100× magnification (left) and quantification of TUNEL+ cells as a percentage of DAPI+ cells. Data represent mean ± standard error of the mean (n=3–4 dams/group, each dot indicates 1 placenta and is the mean quantification of 10 fields of view). Significant differences (*p<0.05) were determined by unpaired two tailed t-test. Scale bar: 1 mm (H&E, A),100 or 50 μm (TUNEL, B).

Article Snippet: Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, TUNEL Assay, Staining, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi, and placentas were paraformaldehyde-fixed and immunostained for cytokeratin (A, red) to mark trophoblasts or vimentin (B, red) to mark endothelial cells and DAPI (blue) to label nuclei. Representative images taken at 20×magnification (left) and further zoomed (3.7- and 2.4- fold for trophoblasts and endothelial cells, respectively, right) are shown. Quantification of the percentage positive area for each marker is shown. Data represent mean ± standard error of the mean (n=3–4 dams/group, each dot indicates 1 placenta and is the mean quantification of 6 fields of view). Significant differences (*p<0.05) were determined by unpaired two tailed t-test. Scale bar: 200 μm.

Article Snippet: Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Marker, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock).Placentas (A, C, D, E, F) and spleens (B) were collected at 3 hpi (C, E) or 6 hpi (D, F), homogenized, and analyzed by ELISA (A-B) or Western blot for phosphorylated (p)NFκB p65, total NFκB p65, and NLRP3 or cleaved-caspase 1 (20 kDa cleavage fragment) and pro-caspase 1. GAPDH is shown as the loading control. Placentas from two independent experiments were analyzed together to avoid gel to gel variability (C-D). Quantified protein expression (E-F) of pNFκB p65, NLRP3, and pro-caspase 1 in the placenta is shown as the fluorescence signal for each protein normalized to GAPDH. Phosphorylation of NFκB p65 is also shown as a proportion of total NFκB p65 per placenta. As a positive control for caspase-1 expression, RAW-ASC cells were primed and stimulated and cell lysate collected for Western blot analysis (G) Data represent mean ± standard error of the mean [n =5–12/group (ELISA), 6–7/group (WB)]. Significant differences were determined (*p<0.05, ns=not significant) were determined by unpaired two tailed t-test (WB) or two-way ANOVA with Bonferroni post hoc test (ELISA).

Article Snippet: Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Expressing, Fluorescence, Phospho-proteomics, Positive Control, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi, placentas collected, and RNA extracted. mRNA libraries for NGS were prepared with Tecan Genomics Universal Plus mRNA Seq kit and sequenced with a 200 cycle (2×100bp) SP run on an Illumina NovaSeq 6000 and analyzed in Partek® Flow® (A) and QIAGEN Ingenuity Pathway Analysis (B-F). A Volcano Plot showing the 928 genes that were differentially expressed between ZIKV and mock placentas using cut-offs of p value ≤ 0.05 and fold change −2< or >2. Red indicates upregulation and green indicates downregulation; individual dots indicate individual genes (A). The top 5 networks associated with the 928 differentially expressed genes, were identified using Ingenuity Pathway Analysis. Red indicates significant upregulation and green indicates downregulation with the shade indicating the magnitude of the fold change (B).

Article Snippet: Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At embryonic day (E) 10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV, Brazil ZIKV, or media (mock). (A) At 48 hours post infection (hpi), dams were euthanized, and fetal viability was determined as the percentage of fetuses within the inoculated uterine horn (n = total number of fetuses from 11–16 dams per group from two independent replicates). (B) Nigeria ZIKV RNA copies were quantified from placenta or spleen collected 48 hpi. Data represent mean ± standard error of the mean from two independent replications (n = 7–9/group). (C) Placentas collected 48 hpi from either mock (upper panel) or Nigeria ZIKV (lower panel) infected dams were paraformaldehyde fixed and immunostained for ZIKV (red) and 4′6-diamidino-2-phenylindole (DAPI, blue) to label nuclei. Representative images taken at 20× magnification (left) and further zoomed 5.5 fold (right) are shown. (D) ZIKV RNA (green) was measured in fetuses of Brazil ZIKV infected dams by in situ hybridization (ISH) at 48 hpi. Samples were also imaged without RNA probe incubation as a control (left) to remove background and a representative image at 5× magnification is shown for fetuses of ZIKV infected dams (right). Significant differences (*p<0.05) were determined by χ2 test (A) or unpaired two tailed t-test (B). Limit of detection (LOD) indicated with a dashed line. Scale bar: 100 or 50 μm (IHC, C),1000 μm (ISH, D)

Article Snippet: Viruses and cells Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Infection, In Situ Hybridization, Incubation, Control, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi and placentas were paraformaldehyde-fixed. (A) Representative H & E images were taken at 5× magnification (upper panels) and 20× magnification (lower panels). (B) Representative TUNEL+ (green) stained placentas with DAPI to label nuclei taken at 100× magnification (left) and quantification of TUNEL+ cells as a percentage of DAPI+ cells. Data represent mean ± standard error of the mean (n=3–4 dams/group, each dot indicates 1 placenta and is the mean quantification of 10 fields of view). Significant differences (*p<0.05) were determined by unpaired two tailed t-test. Scale bar: 1 mm (H&E, A),100 or 50 μm (TUNEL, B).

Article Snippet: Viruses and cells Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, TUNEL Assay, Staining, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi, and placentas were paraformaldehyde-fixed and immunostained for cytokeratin (A, red) to mark trophoblasts or vimentin (B, red) to mark endothelial cells and DAPI (blue) to label nuclei. Representative images taken at 20×magnification (left) and further zoomed (3.7- and 2.4- fold for trophoblasts and endothelial cells, respectively, right) are shown. Quantification of the percentage positive area for each marker is shown. Data represent mean ± standard error of the mean (n=3–4 dams/group, each dot indicates 1 placenta and is the mean quantification of 6 fields of view). Significant differences (*p<0.05) were determined by unpaired two tailed t-test. Scale bar: 200 μm.

Article Snippet: Viruses and cells Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Marker, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock).Placentas (A, C, D, E, F) and spleens (B) were collected at 3 hpi (C, E) or 6 hpi (D, F), homogenized, and analyzed by ELISA (A-B) or Western blot for phosphorylated (p)NFκB p65, total NFκB p65, and NLRP3 or cleaved-caspase 1 (20 kDa cleavage fragment) and pro-caspase 1. GAPDH is shown as the loading control. Placentas from two independent experiments were analyzed together to avoid gel to gel variability (C-D). Quantified protein expression (E-F) of pNFκB p65, NLRP3, and pro-caspase 1 in the placenta is shown as the fluorescence signal for each protein normalized to GAPDH. Phosphorylation of NFκB p65 is also shown as a proportion of total NFκB p65 per placenta. As a positive control for caspase-1 expression, RAW-ASC cells were primed and stimulated and cell lysate collected for Western blot analysis (G) Data represent mean ± standard error of the mean [n =5–12/group (ELISA), 6–7/group (WB)]. Significant differences were determined (*p<0.05, ns=not significant) were determined by unpaired two tailed t-test (WB) or two-way ANOVA with Bonferroni post hoc test (ELISA).

Article Snippet: Viruses and cells Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Expressing, Fluorescence, Phospho-proteomics, Positive Control, Two Tailed Test

Journal: Frontiers in virology

Article Title: Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes

doi: 10.3389/fviro.2022.782906

Figure Lengend Snippet: At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Dams were euthanized at 48 hpi, placentas collected, and RNA extracted. mRNA libraries for NGS were prepared with Tecan Genomics Universal Plus mRNA Seq kit and sequenced with a 200 cycle (2×100bp) SP run on an Illumina NovaSeq 6000 and analyzed in Partek® Flow® (A) and QIAGEN Ingenuity Pathway Analysis (B-F). A Volcano Plot showing the 928 genes that were differentially expressed between ZIKV and mock placentas using cut-offs of p value ≤ 0.05 and fold change −2< or >2. Red indicates upregulation and green indicates downregulation; individual dots indicate individual genes (A). The top 5 networks associated with the 928 differentially expressed genes, were identified using Ingenuity Pathway Analysis. Red indicates significant upregulation and green indicates downregulation with the shade indicating the magnitude of the fold change (B).

Article Snippet: Viruses and cells Zika virus (ZIKV) strain IB H 30656 (Nigeria, 1968) was purchased from the American Type Culture Collection (ATCC, # VR-1839).

Techniques: Injection