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: FIGURE 1 | In utero exposure to ZIKV causes placental infection, vertical transmission, and fetal loss. 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 h 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 to 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), 1,000 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: In Utero, Infection, Transmission Assay, Injection, 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: FIGURE 2 | ZIKV infection induces placental damage and cell death. 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 and 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 (HandE, 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: Infection, 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: FIGURE 3 | ZIKV infection disrupts the trophoblast and endothelial layer of the labyrinth zone. 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: Infection, 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: FIGURE 4 | Intrauterine ZIKV infection transiently increases the IL-1β concentration but does not induce NLRP3 inflammasome upregulation or activation in placenta. At E10, pregnant mice underwent intrauterine injection of 106 TCID50 units of Nigeria ZIKV or media (mock). Placentas (A,C–F) and spleens (B) were collected at 3 hpi (Continued)

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

Techniques: Infection, Concentration Assay, Activation Assay, 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: FIGURE 5 | ZIKV infection downregulates host mRNA expression in the placenta, including genes associated with cellular function and development. 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 × 100 bp) SP run on an Illumina NovaSeq 6,000 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 five 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: Infection, Expressing, Cell Function Assay, Injection

Journal: Frontiers in Microbiology

Article Title: A Peptide-Based Virus Inactivator Protects Male Mice Against Zika Virus-Induced Damage of Testicular Tissue

doi: 10.3389/fmicb.2019.02250

Figure Lengend Snippet: Z2 inhibited infection of ZIKV strains with Asian and African lineages in TM4 cells. (A) Sequence and location of Z2 in ZIKV E dimer. Side view of ZIKV E protein in the dimeric conformation is shown [Protein Data Bank: 5IRE ]; DI, DII, DIII, stem and transmembrane domain are shown in red, yellow, blue, orange, and wheat, respectively; the fusion peptide is shown in cyan, and Z2 (421–453) is shown in green. TM4 cells were infected with ZIKV strain SZ01 (B) , MR766 (C) and FLR (D) , treated with Z2 at different concentration, and then viral copies in the supernatant at 48 h were measured by qRT-PCR. Data were presented as means ± SD. (E) Z2-treated ZIKV of different strain lost infectivity on TM4 cells irreversibly. After incubation with Z2 at 37°C for 2 h, ZIKV particles were separated from the unbound Z2 by PEG 8000 to measure their infectivity on TM4 cells. Each sample was tested in triplicate and the experiments were repeated at least once. The data from two independent experiments were presented as mean ± SD.

Article Snippet: ZIKV strains MR766 (#VR1838) and FLR (#VR1844) were obtained from ATCC.

Techniques: Infection, Sequencing, Concentration Assay, Quantitative RT-PCR, Incubation

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: Image-based screening on Zika virus (ZIKV)-infected cells identifies TH3289 and TH6744 as active small molecule inhibitors against ZIKV. ( A ) Experimental workflow of phenotypic antiviral testing of small molecule compounds. Nuclei count was used as an indicator of cytotoxicity, viral antibody staining was quantified to assess the percentage of virus infected cells and virus titer was quantified by end-point dilution assay. ( B ) U87 cells were infected with ZIKV (MOI 10) and treated with structural analogs of TH3289 at 10 µM concentration for 48 h. Infected cells were stained for DAPI and ZIKV NS1 and analyzed by high-throughput imaging. Virus titers from supernatants were determined by end-point dilution assay and inhibition of viral titer was calculated relative to dimethyl sulfoxide (DMSO). Data are presented as a mean of two technical replicates per compound performed in n = 1 biological replicate. Representative images from ZIKV-infected U87 cells treated with DMSO or TH3289 (upper image panels; Scale bar equals 100 µm) and corresponding example of titrated ZIKV from the end-point dilution assay (lower image panels) are shown. DAPI in grey, ZIKV NS-1 in magenta. ( C ) The inhibition of viral titers of Hazara virus (HAZV) and ZIKV from both assays was correlated. In the HAZV assay, SW13 cells were infected with HAZV (MOI 10) and treated with 10 μM of compounds from the in-house library for 24 h. Virus titer from supernatant was determined by end-point dilution assay. Cells were stained for DAPI (in blue) and HAZV NP (in green) and analyzed by high-throughput imaging. Data are presented as a mean of two technical replicates per compound performed in n = 1 biological replicate. ( D ) Chemical structures of top hits from ZIKV and HAZV antiviral screenings and their respective inhibition on viral titers.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Virus, Infection, Staining, End-point Dilution Assay, Concentration Assay, High Throughput Screening Assay, Imaging, Inhibition

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: Series of antiviral compounds have a therapeutic window and rescue ZIKV-induced CPE in cellular models. ( A – E ) U87 cells were infected with ZIKV (MOI 1) and treated with increasing doses of ( A ) TH3289, ( B ) TH5264, ( C ) TH6744, or ( D ) TH5487 for 48 h. Cell viability was determined by nuclei count (in blue), infected cells by ZIKV NS1 staining (in orange) and virus titer by an end-point dilution assay (in black). Data are presented as mean ± SD from n = 3 independent replicates. ( E ) Curve fitting was performed to calculate EC 50 values for living cells, infected cells and viral titer. ( F ) Vero cells were infected with ZIKV (MOI 10) and treated with 12.5 μM TH6744 or DMSO for 48 h. Cells were stained for ZIKV NS1 (in green), ZIKV Capsid (in red) and DAPI (in blue). Images of n = 1 biological replicate. In the overview image, scale bar equals 100 μm and in the close-up 25 μm. ( G ) U87 cells were infected with ZIKV (MOI 1) and treated with 10 μM TH6744 or DMSO for 24, 48, or 72 h. Cells were stained for Pan-Flavi (in green), Golgi marker GM130 (in red), ER marker KDEL (in yellow), and DAPI (in blue). Images of n = 1 biological replicate. Scale bar equals 50 μm. ( H ) Vero cells were infected with ZIKV (MOI 0.1) and treated with increasing doses of TH6744 for 24 h. Viral titer was measured from the supernatants by end-point dilution assay on Vero cells and the development of CPE were monitored by visual assessment. Data presented as mean ± SD from n = 2 biological replicates.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Staining, Virus, End-point Dilution Assay, Marker

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: TH6744 and TH5487 treatment reverses ZIKV-induced neurotoxicity and limits ZIKV propagation in three-dimensional (3D) cerebral organoids. ( A ) Experimental workflow of ZIKV infection on brain organoid model. ( B – F ) Organoids were infected with ZIKV (2 × 10 4 particles/organoid) followed by treatment with 5 μM TH6744, 5 μM TH5487 or DMSO. ( B ) Organoids were dissociated to single cells at 7 or 10 dpi and cell viability was measured by Resazurin assay. Viability is shown relative to uninfected DMSO-treated control at respective timepoints. One datapoint represents one organoid. Data are presented as a mean ± SD of n = 2 biological replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01, ( C ) ZIKV-infected and uninfected whole organoids treated with TH6744 or DMSO for 7 or 10 dpi, stained by Hoechst and imaged by high-content confocal microscopy. Representative images of n = 2 biological replicates per condition. The scale bar equals 500 μm. Hoechst in blue. ( D ) ZIKV-infected organoids that were treated with TH6744 or DMSO were fixed at 7 dpi, cryosectioned and stained with Hoechst and ZIKV NS1 protein antibody. ZIKV NS1 intensity was quantified using Cellprofiler software. Data are presented as single values and mean ± SD from n = 2 biological replicates. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01. ( E ) ZIKV-infected organoids treated with TH6744 or DMSO were fixed at 7 dpi, cryosectioned, stained with Hoechst and ZIKV NS1 protein antibody and imaged by confocal microscopy. Representative images of n = 2 biological replicates per condition. Scale bar equals 500 μm in overview image and 100 μm in close-up. ZIKV NS1 protein in red, Hoechst in blue. ( F ) Viral titer from ZIKV-infected and compound treated organoids at indicated time points was determined by an end-point dilution assay. Viral titer is presented as relative to ZIKV-infected DMSO-treated control at respective timepoints. Data are presented as a mean ± SD from n = 2 biological replicates. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparison analysis. * p < 0.05, ** p < 0.01.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Resazurin Assay, Control, Comparison, Staining, Confocal Microscopy, Software, End-point Dilution Assay

Journal: Viruses

Article Title: Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids

doi: 10.3390/v13010037

Figure Lengend Snippet: TH6744 disturbs late replication cycle steps in ZIKV life cycle. ( A – D ) U87 cells were infected with ZIKV (MOI 1) and treated with indicated compounds for 24, 48 or 72 h. ( A ) ZIKV RNA levels inside the cells were quantified by qPCR. ( B ) Number of ZIKV-infected cells was quantified by virus infectivity assay. ( C ) ZIKV RNA levels in the supernatant were quantified by one-step qRT-PCR. ( D ) ZIKV titers were quantified by end-point dilution assay. ( E ) Schematic overview of time-of-addition experimental setup in U87 cells. ( F ) U87 cells were infected with ZIKV (MOI 10) for a total of 24 h and treated with DMSO control, 10 μM TH6744 or 100 μM Ribavirin for indicated periods during early (entry), late (budding) or throughout replication cycle (post-inoculation). ZIKV titers were quantified by end-point dilution assay. ( G ) Schematic overview of budding experimental setup. ( H ) U87 cells were infected with ZIKV (MOI 10) for 22 h and treated with indicated TH6744 doses for 2 h. Intracellular ZIKV particles were obtained by mechanical cell lysis and extracellular ZIKV particles from the supernatants and both were quantified by end-point dilution assay. ( I ) Average fractions of intracellular and extracellular infectious ZIKV particles of the total particles quantified in ( H ). ( A – D , F , H ) Data are expressed as a mean ± SD from at least n = 3 biological replicates. Statistical significance was determined by using one-way ANOVA with Dunnett’s multiple comparison analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For supernatants, the obtained Ct values were converted to the number of ZIKV RNA molecules using a standard curve that was created using Quantitative Synthetic ZIKV RNA from ATCC (ATCC ® VR-3252SDTM).

Techniques: Infection, Virus, Quantitative RT-PCR, End-point Dilution Assay, Control, Lysis, Comparison

Journal: Nutrients

Article Title: Novel Therapeutic Nutrients Molecules That Protect against Zika Virus Infection with a Special Note on Palmitoleate

doi: 10.3390/nu15010124

Figure Lengend Snippet: List of novel nutrient molecules and mechanism of protection against ZIKV infection.

Article Snippet: Palmatine , ATCC VR-1843 , Vero cells , Prevents ZIKV binding and entry , Virucidal effects , [ ] .

Techniques: Infection, Activity Assay, Derivative Assay, Binding Assay, Inhibition, Glycoproteomics, Virus, Isolation, In Vitro, In Vivo, Blocking Assay, Membrane, Recombinant, Expressing, Concentration Assay

Journal: Emerging microbes & infections

Article Title: Extracellular acidosis enhances Zika virus infection both in human cells and ex-vivo tissue cultures from female reproductive tract.

doi: 10.1080/22221751.2021.1932606

Figure Lengend Snippet: Figure 2. Acidosis enhances ZIKV infection in cells derived from the female reproductive tract and other target cells. Cells were incubated with ZIKV (A1C1-V2) at different pH values for 1 h at 37°C. Then, cells were washed and infection was assessed 72 h later. Infection of genital tract-derived cells VK2/E6E7 (A), Ect1/E6E7 (B), and End1/E6E7 (C) was quantified by measuring the extra- cellular concentration of NS-1 by Luminex. Infection of human endometrial fibroblasts (T-HESC) was quantified by qPCR (D). Infec- tion of monocyte-derived macrophages (MDM) was quantified by measuring the extracellular concentration of NS-1 by Luminex (E). Primary culture-derived human astrocytes (NHA) infection was measured by qPCR (F). Human ectocervical (G) and endocervical (H) tissue cultures were dissected into 2 mm [3] pieces and infected with ZIKV (VR-1844 moi 0.1) either at pH 7.3 or 6.2 for 1 h at 37°C. The viral production was evaluated at days 3, 6, 10, 13, 17, and 21 by measuring ZIKV NS1 in the culture supernatant. Results in A, B, C, D, and F represent data of two independent experiments. Data in E comprises the results of four different donors. Panels H and G were constructed with data of three independent experiments per explant type, and data were analysed using paired two-way ANOVA. Results are presented as mean ± SEM.

Article Snippet: The standard curve was obtained by serial dilution of recombinant NS1 protein (Fitzgerald, cat no. 30-1931).

Techniques: Infection, Derivative Assay, Incubation, Concentration Assay, Luminex, Construct

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 1 ZIKV production, titration, and endothelial infection in vitro. (A) Schematic diagram of ZIKV production. Zika virus strain R103451 (Honduras) was propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and complete DMEM was added to the cells. Supernatants were collected four days post-infection (dpi). HEPES buffer was added to the stock to prevent the acidification of the virus following freeze and thawing. (B) Cytopathic effects of ZIKV infection in Vero cells at 4 dpi. Mock infection serves as a comparison. Note changes in cell shapes and floating cells, indicating cell death. (C) ZIKV was tittered (10− 1 to 10− 6) by plaque assay using Vero cells with a 0.7% aga rose overlay. As a control, mock-infected Vero cells were subjected to the same procedure. Foci of plaques were detected at 3 dpi, fixed with methanol: acetic acid (3:1 ratio), and stained with 0.1% crystal violet. (D) The expression of ZIKV mRNA in mock and ZIKV-infected (MOI 0.01) wild-type or Bmal1- silenced brain endothelial cells at 2 dpi. NS, non-specific. (E) Representative images of ZIKV-specific NS1 protein (green) in control and/or Bmal1-silenced brain endothelial cells infected as in (D). Blue, nuclear staining with Hoechst 33,342. (F) Bmal1 protein expression in Bmal1-silenced or NS-transfected brain endothelial cells infected as in (D). Upper panel, immunoblots of Bmal1 protein. Lower panel, quantitative protein expression data. The results were normalized to GAPDH levels. The immunoblot images were linearly adjusted for brightness and contrast to enhance visibility. Values are mean ± SEM; n = 3. *p < 0.05. Scale bar: 20 μm

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Titration, Infection, In Vitro, Virus, Incubation, Comparison, Plaque Assay, Control, Staining, Expressing, Transfection, Western Blot

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 2 Impact of Bmal1 silencing and/or infection with ZIKV on the expression of TJ genes. Brain endothelial cells were transfected with 75 nM Bmal1-spe cific or non-specific (NS) siRNA, followed by infection with ZIKV as in Fig. 1D. Claudin-5, occludin, JAM-3, ZO-1, and β-catenin mRNA levels were measured by RT-qPCR, with GAPDH mRNA expression serving to normalize the results. Values are mean ± SEM; n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Expressing, Transfection, Quantitative RT-PCR

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 3 Impact of the Bmal1 silencing and/or infection with ZIKV on the expression of TJ protein and endothelial barrier integrity. Cells were treated and infected as in Fig. 1D. (A) Claudin-5, occludin, JAM-3, ZO-1, and B-catenin protein levels were measured by immunoblotting and normalized to GAPDH levels. Upper panels, representative immunoblots of TJ proteins. Lower panels, quantified protein expression data. Values are mean ± SEM; n = 6; *p < 0.05. (B-C) Endothelial barrier function. Brain endothelial cells were grown to confluency on the 0.4 μm membrane in the upper compartment of the CellQART 24-well culture system. Cell culture inserts without cells were constructed as a blank. Cells were transfected with Bmal1 or NS siRNA, and infected with ZIKV. Data was monitored and analyzed using the provided CellZscope2 software. (B) The relative TEER values (% of control) at 2 dpi. Left panel, schematic representation of the method; Right panel, quantified data. (C) The transendothelial passage of 20 kDa FITC-dextran measured for 90 min at 2 dpi. Fluo rescence intensity (A.U.) of FITC-dextran in the lower chamber was measured as a marker of endothelial integrity. Left panel, schematic representation of the method; Right panel, quantified data. The immunoblot images were linearly adjusted for brightness and contrast to enhance visibility. Values are mean ± SEM; n = 5–6; *p < 0.05, **p < 0.01

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Expressing, Western Blot, Membrane, Cell Culture, Construct, Transfection, Software, Control, Marker

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 4 Impact of the Bmal1 silencing and/or infection with ZIKV on mitochondrial dynamics. Brain endothelial cells were treated and infected as in Fig. 1D. (A) Immunoblotting analysis for mitochondrial fission protein DRP1, pDRP1s616, MFF, FIS1, mitochondrial fusion protein OPA1, and mitochondrial marker TOM20. Upper panels, representative immunoblots. Lower panels, quantitative protein expression data. (B) Immunoblotting analysis of the ex pression of the mitochondrial respiratory chain complexes. Left panel, representative immunoblots. Right panel, quantitative protein expression data. The results were normalized to GAPDH levels. The immunoblot images were linearly adjusted for brightness and contrast to enhance visibility. Values are mean ± SEM; n = 5–6 per group. *p < 0.05 and **p < 0.01

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Western Blot, Marker, Expressing

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 5 ZIKV infection of endothelial cell-specific conditional Bmal1-deficient mice (Bmal1ECKO). (A) Representative confocal images of Bmal1 (red) and CD31 (green) immunoreactivity, and Hoechst 33342 (blue) staining in WT and Bmal1ECKO brain microvessels. (B) ZIKV mRNA levels in infected, brain RT- qPCR positive WT and Bmal1ECKO mouse brains. Mice were infected with ZIKV in 105 PFU via retro-orbital injection. At 2 dpi, mice were sacrificed, and RNA was extracted from the brain tissues for RT-qPCR measuring. The results were normalized to HBB levels. Values are mean ± SEM; n = 3–5. Scale bar: 20 μm

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Staining, Quantitative RT-PCR, Injection

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 6 Impact of ZIKV infection on the expression of TJ proteins in WT and Bmal1ECKO mice. Mice were infected as in Fig. 5. Claudin-5, occludin, JAM-3, ZO-1, and β-catenin protein levels were measured by immunoblotting and normalized to β-tubulin levels. Upper panels, representative immunoblots. Lower panels, quantitative protein expression data. The immunoblot images were linearly adjusted for brightness and contrast to enhance visibility. Val ues are mean ± SEM; n = 3–5; *p < 0.05, and **p < 0.01

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Expressing, Western Blot

Journal: Fluids and barriers of the CNS

Article Title: Circadian misalignment potentiates blood-brain barrier disruption and mitochondria dysregulation in Zika virus infection.

doi: 10.1186/s12987-025-00669-2

Figure Lengend Snippet: Fig. 7 Impact of ZIKV infection on the mitochondrial dynamics in WT and Bmal1ECKO mice. Mice were infected as in Fig. 5. (A) Immunoblotting analysis of mitochondrial fission proteins pDRP1s616 and DRP1, MFF, and FIS1, mitochondrial fusion protein OPA1, and mitochondrial marker TOM20. Upper panels, representative immunoblots. Lower panels, quantitative protein expression data. (B) Protein expressions of the mitochondrial respiratory chain complexes. Left panel, representative immunoblots. Right panel, quantitative protein expression data. The results were normalized to β-tubulin levels. The immunoblot images were linearly adjusted for brightness and contrast to enhance visibility. Values are mean ± SEM; n = 3–5 per group. *p < 0.05

Article Snippet: Zika virus strain R103451 (Honduras 2015) was obtained from ATCC (#VR-1848) and propagated in Vero cells at a multiplicity of infection (MOI) of 0.01 in serum-free DMEM for 2 h. After incubation, the virus inoculum was removed, and pre-warmed complete DMEM was added to the cells.

Techniques: Infection, Western Blot, Marker, Expressing

Journal: Viruses

Article Title: A Rat Model of Prenatal Zika Virus Infection and Associated Long-Term Outcomes.

doi: 10.3390/v13112298

Figure Lengend Snippet: Figure 2. (A,C) groups separated by sex; (B,D) groups collapsed across sex. There is no significant effect of infection or sex in rotarod task in the juvenile offspring (A,B). At the adult time point (P60), there is a significant main effect of infection, where the rats prenatally exposed to ZIKV had a significantly shorter latency to fall compared to diluent control rats (F(1,30) = 4.502, p = 0.019); (D). When sex is included as a variable, there is a trend toward an infection x sex interaction (F(1,29) = 1.767, p = 0.057) (separated by sex: n = 8–10 animals in each group, collapsed across sex: n = 16–20 per group). * Denotes p < 0.05 relative to controls, # denotes p < 0.07 relative to diluent controls.

Article Snippet: Viral presence was assessed by comparing samples to a standard curve generated using serial 10-fold dilutions of synthetic ZIKV RNA (ATCC).

Techniques: Infection, Control

Journal: Viruses

Article Title: A Rat Model of Prenatal Zika Virus Infection and Associated Long-Term Outcomes.

doi: 10.3390/v13112298

Figure Lengend Snippet: Figure 5. Hippocampus gene expression. There was a main effect of poly(I:C) for IL-4 (A) and Iba1 (B), where poly(I:C) exposure resulted in significantly lower gene expression compared to saline controls (IL-4: F(1,37) = 5.422, p = 0.025; Iba1: F(1,35) = 9.596, p = 0.004). There was an infection x poly(I:C) interaction for COX-2 gene expression (C) where rats that received prenatal ZIKV infection, poly(I:C) injection, or both, had significantly lower levels of COX-2 compared to controls (COX-2: F(1,38) = 18.695, p = 0.048); n = 10–12 per group. * denotes p < 0.05.

Article Snippet: Viral presence was assessed by comparing samples to a standard curve generated using serial 10-fold dilutions of synthetic ZIKV RNA (ATCC).

Techniques: Gene Expression, Saline, Infection, Injection