c protein  (Novus Biologicals)

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Infection of all three cell types was confirmed by IF detection of the ZIKV C protein (green) at 48 hpi. Infected cells were identified through co-staining with cell-type specific markers (red). While all cultures demonstrated susceptibility to ZIKV infection, (A) neurons exhibited the most pronounced morphological alteration compared to uninfected controls. Notably, viral antigen is localized not only to perinuclear region but also to the axon hillock (arrow) and neurites (arrowhead). In contrast, (B) astrocytes and (C) MBECs maintained their typical cellular architecture despite infection. The highlighted square denotes a region of interest shown at higher magnification. Nuclei were counterstained with DAPI (blue). Scale bars represent 20 µm for neurons and MBECs, and 50 µm for astrocytes. Images are representative of two independent experiments, each comprising three replicates and eight fields analyzed per slide.

Article Snippet: Following this period, some wells were fixed with 4% PFA and processed by immunoperoxidase staining, using the ZIKV capsid antibody (Novus, NBP3–13200).

Techniques: Infection, Staining

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Immunoperoxidase staining for ZIKV E protein in A549 cell inoculated for 72 h with EVs or ZIKV. (A) Controls included non-infected cells (NIC), mock-treated cells (supernatants from uninfected C6/36HT cells), and supernatants of ZIKV-infected cells at a MOI of 0.1. (B) Brown peroxidase signal indicated productive infection in cells exposed to EVs-IC, while no detectable staining was observed in EVs-GlyR-treated cells. Representative images from three independent experiments, each performed in triplicate. Scale bars: 100 µm for controls and EVs-GlyR) and 50 µm for EVs-IC.

Article Snippet: Following this period, some wells were fixed with 4% PFA and processed by immunoperoxidase staining, using the ZIKV capsid antibody (Novus, NBP3–13200).

Techniques: Immunoperoxidase Staining, Infection, Staining

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Infection of all three cell types was confirmed by IF detection of the ZIKV C protein (green) at 48 hpi. Infected cells were identified through co-staining with cell-type specific markers (red). While all cultures demonstrated susceptibility to ZIKV infection, (A) neurons exhibited the most pronounced morphological alteration compared to uninfected controls. Notably, viral antigen is localized not only to perinuclear region but also to the axon hillock (arrow) and neurites (arrowhead). In contrast, (B) astrocytes and (C) MBECs maintained their typical cellular architecture despite infection. The highlighted square denotes a region of interest shown at higher magnification. Nuclei were counterstained with DAPI (blue). Scale bars represent 20 µm for neurons and MBECs, and 50 µm for astrocytes. Images are representative of two independent experiments, each comprising three replicates and eight fields analyzed per slide.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Infection, Staining

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Viral RNAs were extracted from both (A) lysates (reflecting intracellular viral load) and (B) culture supernatants (representing extracellular free virus) of primary neurons, astrocytes and MBECs at indicated times post-infection. Absolute RT-qPCR was performed to quantify ZIKV transcripts, with results expressed as “ZIKV copies/mL”. Across all cell types and time points, intracellular viral copy numbers were consistently higher than those detected in the supernatant. Among the three cell types, neurons exhibited the highest levels of both intracellular transcript production and extracellular viral release. Data represents the mean of three independent experiments each performed in duplicate. Statistical analysis was performed using one-way ANOVA followed by Dunnet’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Virus, Infection, Quantitative RT-PCR, Comparison

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: The smaller insets depict light scattering intensity profiles obtained from DLS measurements, while the larger panels present NTA data, illustrating particle concentration peaks (particles/mL) as a function of particle size (diameter) for each analyzed sample from (A) neurons, (B) astrocytes and (C) MBECs. These analyzes demonstrated that ZIKV infection altered both the size distribution and abundance of EVs produced in each culture compared to their respective NIC. Representative data from two independent experiments.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Concentration Assay, Infection, Produced

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: For each cell type, the upper panel displays a coverage map of ZIKV C11541 genomic regions detected in EVs-GlyR via reference-based assembly of small RNA-seq data. The lower panel illustrates the full-length viral genome (10.8 kb), with blue, purple or green segments indicating regions flanked by sequenced reads. Metrics include percentage genome coverage (CV) and normalized viral nucleotide counts per million total nucleotides (NCPM). No ZIKV sequences were detected in EVs-NIC. To calculate NCPM, we considered as total the number of nucleotides remaining after excluding low-quality and host reads (Neurons: 16’553,501; Astrocytes: 498’011,206; MBECs: 45’213,763). Viral nucleotides were identified as those within this filtered dataset that aligned with the ZIKV C11541 genome (Neurons: 3,907; Astrocytes: 754; MBECs: 9,196). Scale bar: 1 kb. Representative data from three independent experiments.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: RNA Sequencing

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Immunoperoxidase staining for ZIKV E protein in A549 cell inoculated for 72 h with EVs or ZIKV. (A) Controls included non-infected cells (NIC), mock-treated cells (supernatants from uninfected C6/36HT cells), and supernatants of ZIKV-infected cells at a MOI of 0.1. (B) Brown peroxidase signal indicated productive infection in cells exposed to EVs-IC, while no detectable staining was observed in EVs-GlyR-treated cells. Representative images from three independent experiments, each performed in triplicate. Scale bars: 100 µm for controls and EVs-GlyR) and 50 µm for EVs-IC.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Immunoperoxidase Staining, Infection, Staining

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: A549 monolayers were treated with EVs-Gly or EVs-GlyR. (A) Absolute quantification by RT-qPCR revealed detectable viral RNA in cells treated with EVs-Gly. Statistical analysis was performed using a Mann-Whitney test. (B) Only cells treated with EVs-GlyR derived from neurons or MBECs contained measurable ZIKV RNA, whereas no viral RNA were detected in cells treated with EVs-Gly or GlyR from astrocytes. Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s multiple comparisons test as the post hoc test. (C) ddPCR quantified ZIKV RNA copies directly within EVs preparations (EVs-IC, EVs-Gly, and EVs-GlyR) isolated from infected neurons and MBECs. Dotted lines indicate the ZIKV-positive control (virus diluted 1:10,000, red) and EVs from non-infected control (EVs-NIC, Orange). Data represents the mean of two independent experiments performed in duplicate. Statistical significance: ****p < 0.0001.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY, Derivative Assay, Isolation, Infection, Positive Control, Virus, Control

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: Brains from EVs-inoculated mice were analyzed to assess ZIKV infection. (A) Western blot analysis of whole brain lysates confirmed ZIKV C protein (~12 kDa) in mice inoculated with EVs- GlyR derived from neurons (EVs-N) or MBECs (EVs-EC), as well as in ZIKV-infected positive controls. C protein signal was absent in mock-treated, or EVs-NIC–inoculated mice. β-actin (~42 KDa) served as loading control. (B) ZIKV RNA copies quantified by RT-qPCR in whole-brain lysates revealed significantly higher viral RNA loads in mice receiving neuron-derived EVs-GlyR compared to MBECs-derived EVs-GlyR. Representative data from three independent experiments performed in duplicate. Statistical analysis was performed using a Mann-Whitney test (p < 0.05 threshold), but no significant differences were detected.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques: Infection, Western Blot, Derivative Assay, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLOS One

Article Title: Neural and endothelial cell-derived extracellular vesicles mediate Zika virus genome dissemination and productive infection in vivo

doi: 10.1371/journal.pone.0337609

Figure Lengend Snippet: IF analysis localized ZIKV C protein (red) in MAP2-positive neurons and GFAP-positive astrocytes in brains of EVs-GlyR-inoculated mice. Activated astrocytes (arrowheads) and ZIKV antigen in capillary-associated endothelial cells (arrows) were observed across all treatment groups. Scale bar: 20 µm. Images are representative of four inoculated animals per group.

Article Snippet: Following inoculum removal, cells were maintained in fresh medium for 48 h. Infection was confirmed via IF assay using the ZIKV capsid-specific antibody (Novus, NBP3–13200) as described before, and absolute RT-qPCR.

Techniques:

Journal: Communications biology

Article Title: Inhibition of sterol O-acyltransferase 1 blocks Zika virus infection in cell lines and cerebral organoids.

doi: 10.1038/s42003-024-06776-4

Figure Lengend Snippet: Fig. 6 | Lack of SOAT1 activity impairs virion production and ZIKV specific infectivity. a Scheme of experimental setup to decipher the dependency of different ZIKV replication steps onSOAT1.Hmc3cells were pre-treated withSOAT1-specific inhibitors (9.72 µM ATR-101 or 20 µM K604) or DMSO 24 h prior to infection with ZIKV. After 1 h, the inoculum was removed and replaced by fresh media supple- mented with the respective inhibitors and different steps of virus replication were analyzed. b To determine viral entry, cells were infected with ZIKV (MOI 5) 24 h after initial treatment. 4 hpi, total RNA was isolated and ZIKV GE copies were determined by qRT-PCR. Shown is the relative quantification of ZIKV RNA (mean ± SEM, n = 3, no asterisk = not significant, Welch´s t-test). c Hmc3 ZIKV EGFP reporter cells were pre-treated with 9.72 µM ATR-101, 20 µM K604, or DMSO for 24 h, infected with ZIKV (MOI 0.0025) and fixed 48 hpi. Cells were stained using a dsRNA-specific antibody and analyzed by confocal microscopy. ZIKV-infected cells were randomly selected by the nuclear translocation of the EGFP reporter.

Article Snippet: The following antibodies and reagents used in this study were obtained commercially: Flavivirus group antigen antibody (D1-4G2-4-15 (4G2), NBP2-52666, Novus Biologicals), Flavivirus NS1 antibody ((D/2/D6/B7), ab214337, abcam), Zika virus capsid protein antibody (GTX133317), SOAT1antibody (GTX32890) (allGeneTex),GAPDHantibody (cloneG-9, sc-365062), DGAT1 antibody (clone H-255, sc-32861) (all Santa Cruz Biotechnology), J2 dsRNA antibody (Scicons/Jena Biosciences RNT-SCI10010200), PLIN2 antibody (610102, Progen), PLIN3 antibody (HPA006427, Sigma), beta-actin antibody (clone AC-74, A2228-200UL, Sigma), FLAG antibody (F7425, Sigma), Strep-tag II antibody (ab184224, abcam), HRP-labelled secondary antibodies (Jackson ImmunoResearch), Anti-Rabbit IgGHRP conjugated (Rabbit TrueBlot) (clone eB182, 18-8816- 33, Rockland Immunochemicals), Alexa647-conjugated secondary antibody (donkey, IgG (H+ L), A-31571), BODIPY493/503 (D-3922), BODIPY 655/676 (B-3932), Hoechst 33342 (H1399), Cholesteryl BODIPY FL C12 (C3927MP) (all Invitrogen), filipin III (F4767), K-604 (SML1837), ATR-101 (SML2802), DGAT1 inhibitor (PF-04620110), DGAT2 inhibitor (PF-06424439), NITD 008 (SML2409), cholesteryl oleate (C9253), cholesterol-water soluble (~ 40mg of cholesterol per gram; balanced with methyl-β-cyclodextrin) (C4951), oleic acid-BSA complex (O3008) (all Sigma).

Techniques: Activity Assay, Infection, Virus, Isolation, Quantitative RT-PCR, Staining, Confocal Microscopy, Translocation Assay

Journal: Communications biology

Article Title: Inhibition of sterol O-acyltransferase 1 blocks Zika virus infection in cell lines and cerebral organoids.

doi: 10.1038/s42003-024-06776-4

Figure Lengend Snippet: Fig. 9 | Inhibition of SOAT1 and DGAT2 reduces ZIKV infection of human iPSC-derived cerebral organoids. a Differentiation of hIPSCs into cerebral organoids. Two different hIPS cell lines (HMGU#1 and TISSUi006A) were used for differentiation with the StemDiff Cerebral organoid kit. b Scheme of the experimental setup. After maturation (day ~35), organoids were pre-treated with DGAT inhibitors (each 5 µM), SOAT1-specific inhibitors (ATR- 101 = 9.72 µM, K604 = 20 µM), or DMSO for 24 h, followed by inoculation with 105 TCID50 per orga- noid of ZIKV for 24 h. After removal of the inocu- lum, organoids were cultured in media supplemented with inhibitors for 3 days. Individual organoids were harvested and infection was ana- lyzed by qRT-PCR and immunoblot. c Organoids were lysed and total RNA was isolated at 3 dpi. ZIKV genome copies were analyzed by qRT-PCR. Each dot represents an individual organoid from 3 inde- pendent experiments using 2 different cell lines (mean ± SEM, n = 3, *p ≤0.05, no asterisk = not significant, Welch´s t-test). d Individual organoids from two independent experiments were lysed at 3 dpi and ZIKV E and C protein level were analyzed by immunoblot. GAPDH served as loading control. Mock-infected and pan-orthoflavivirus inhibitor (NITD008)-treated organoids were used as control. Shown is one representative immunoblot. e Band signal intensities were quantified using ImageLab. Box-and-whisker-plot shows ZIKV E and C protein levels as log2 fold change over DMSO normalized to GAPDH (center line: median, box limits: upper and lower quartiles, whiskers: 1.5 x interquartile range, points: outliers, n = 4, *p ≤0.05, **p ≤0.01, no asterisk = not significant, one-sample t-test).

Article Snippet: The following antibodies and reagents used in this study were obtained commercially: Flavivirus group antigen antibody (D1-4G2-4-15 (4G2), NBP2-52666, Novus Biologicals), Flavivirus NS1 antibody ((D/2/D6/B7), ab214337, abcam), Zika virus capsid protein antibody (GTX133317), SOAT1antibody (GTX32890) (allGeneTex),GAPDHantibody (cloneG-9, sc-365062), DGAT1 antibody (clone H-255, sc-32861) (all Santa Cruz Biotechnology), J2 dsRNA antibody (Scicons/Jena Biosciences RNT-SCI10010200), PLIN2 antibody (610102, Progen), PLIN3 antibody (HPA006427, Sigma), beta-actin antibody (clone AC-74, A2228-200UL, Sigma), FLAG antibody (F7425, Sigma), Strep-tag II antibody (ab184224, abcam), HRP-labelled secondary antibodies (Jackson ImmunoResearch), Anti-Rabbit IgGHRP conjugated (Rabbit TrueBlot) (clone eB182, 18-8816- 33, Rockland Immunochemicals), Alexa647-conjugated secondary antibody (donkey, IgG (H+ L), A-31571), BODIPY493/503 (D-3922), BODIPY 655/676 (B-3932), Hoechst 33342 (H1399), Cholesteryl BODIPY FL C12 (C3927MP) (all Invitrogen), filipin III (F4767), K-604 (SML1837), ATR-101 (SML2802), DGAT1 inhibitor (PF-04620110), DGAT2 inhibitor (PF-06424439), NITD 008 (SML2409), cholesteryl oleate (C9253), cholesterol-water soluble (~ 40mg of cholesterol per gram; balanced with methyl-β-cyclodextrin) (C4951), oleic acid-BSA complex (O3008) (all Sigma).

Techniques: Inhibition, Infection, Derivative Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Isolation, Control, Whisker Assay