Journal: Frontiers in Immunology

Article Title: Zika Virus Infection of Human Iris Pigment Epithelial Cells

doi: 10.3389/fimmu.2021.644153

Figure Lengend Snippet: (A) Light photomicrograph of human iris pigment epithelial cells immediately prior to infection. Original magnifications: 40x (main image) and 100x (inset image); Scale bars: 500 µm (main image) and 200 µm (inset image). (B) Fluorescence photomicrographs of human iris pigment epithelial cells immunolabeled to detect presence of cytokeratin 8 (CK8) and absence of alpha-smooth muscle actin (SMA), with negative control labeled with species-matched immunoglobulin (IgG). Alexa Fluor 488 (green) with DAPI nuclear counterstain (blue). Original magnification: 200x; Scale bar: 100 µm. (C) Fluorescence photomicrographs of ZIKV-infected and uninfected human iris pigment epithelial cells immunolabeled to detect flavivirus envelope protein. Alexa Fluor 488 (green) with Hoechst 33342 nuclear counterstain (blue). Original magnification: 100x; Scale bar: 100 µm. (D) Correlation plots showing cellular viral load in genome equivalents and infectious foci generated from culture supernatant for human iris pigment epithelial cells infected for 24 hours with ZIKV (multiplicity of infection = 1 and 5). Circles represent individual cell isolates from 20 donors. Line indicates trend. Data were analyzed by Pearson correlation coefficient. (E) Graph presenting normalized expression of interferon (IFN)-β transcript, calculated relative to peptidylprolyl isomerase A (PPIA) and ribosomal protein lateral stalk subunit P0 (RPLP0), in human iris pigment epithelial cells infected for 24 hours with ZIKV (multiplicity of infection = 5). Circles represent individual cell isolates from 20 donors. Crossbars indicate mean, and error bars indicate standard deviation. Data were analyzed by Wilcoxon signed-rank test.

Article Snippet: Cell monolayers were fixed with 2% paraformaldehyde, permeabilized with 0.05% IGEPAL CA-630, and labeled with mouse anti-flavivirus envelope protein (ENV) antibody (ATCC HB-112TM hybridoma [clone 4G2] culture supernatant diluted 1:10, applied overnight at 4 °C), followed by Alexa Fluor 488-tagged donkey anti-mouse immunoglobulin antibody (Thermo Fisher Scientific-Molecular Probes, Eugene, OR; catalog number A21202; working concentration, 10 μg/mL, applied for 1 hour at room temperature) with Hoechst 33342 (2’-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5’-bi-1H-benzimidazole trihydrochloride trihydrate) counterstain.

Techniques: Infection, Fluorescence, Immunolabeling, Negative Control, Labeling, Generated, Expressing, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Saliva inhibits ZIKV MR766 infection of Vero E6 cells. (a) Saliva pooled from ten donors was serially diluted, mixed 2:1 with ZIKV MR766 and incubated for 30 min at room temperature. The mixture was added to Vero E6 cells (MOI 0.3) resulting in the final indicated cell culture concentrations. 2 hours later medium was changed and 2 days later infection was quantified by immunodetection assay that enzymatically quantifies the flavivirus protein E. Measured raw data (left) were normalized to infection rates in the absence of the respective saliva sample (right). Data are represented as average values obtained from triplicate infections ± standard deviations. (b) Vero E6 cells were incubated with pooled saliva at indicated concentrations for 2 hours. Medium was then replaced and the cellular viability determined 2 days later by CellTiter-Glo® Luminescent Cell Viability Assay. Data are normalized to viability in the absence of saliva. ns not significant; **P < 0.01; ***P < 0.001 (by one-way ANOVA with Bonferroni post-test).

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Infection, Incubation, Cell Culture, Immunodetection, Cell Viability Assay

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Saliva has broad anti-ZIKV activity. (a) Saliva pooled from ten donors was serially diluted, mixed 2:1 with ZIKV GWUH or PRV and incubated for 30 min at room temperature. The mixtures were then added to Vero E6 cells (MOI 0.6) resulting in the indicated concentrations. 2 hours later medium was changed and 2 days later infection was quantified by immunodetection assay that enzymatically quantifies the flavivirus protein E. (b and c) Pooled saliva was mixed with ZIKV MR766 and incubated for 30 min at room temperature. The mixture was added to Vero E6, HFF, A548 and HeLa cells resulting in the indicated concentrations and medium was changed 2 hours after inoculation. 2 days later infection was determined by b) immunodetection assay or c) immunofluorescent staining and fluorescence microscopy; scale bar: 500 µM. Data in a) and b) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Activity Assay, Incubation, Infection, Immunodetection, Staining, Fluorescence, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Saliva inhibits ZIKV attachment. (a) For virus treatment ZIKV MR766 was mixed with pooled saliva (concentrations: 0.18, 0.35, 0.7, 1.4, 2.8, 5.6, 11.25, 22.5, 45, 90% saliva) and incubated for 30 min at room temperature before the mix was diluted 10-fold onto Vero E6 cells (MOI 0.3). For cell treatment Vero E6 cells were incubated with saliva for 30 min at 37°C and then inoculated with ZIKV MR766 (concentrations: 0.018, 0.035, 0.07, 0.14, 0.28, 0.56, 1.125, 2.25, 4.5, 9% saliva). 2 hours later, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. (b) Vero E6 cells in Ibidi slides were inoculated with ZIKV MR766 (MOI 35) in the presence of saliva and incubated for 2 hours at 4°C. The inoculum was removed and immunofluorescent staining directed against flavivirus protein E was performed. Cell nuclei were stained with Hoechst 33342. Attached virus particles were imaged by confocal microscopy (see Fig. S1) and quantified using ImageJ (Fiji). (c) Vero E6 cells were inoculated with pooled saliva and infected with different dilutions of ZIKV MR766 resulting in MOI 36.5, 9.1, 2.3, 0.57 and 0.14. Thereafter, it was proceeded as in a). The relative infection for saliva titration (left) and for 10% saliva (right) is shown. Data in a) and c) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Incubation, Infection, Immunodetection, Staining, Confocal Microscopy, Titration

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Stability of the anti-ZIKV factor in saliva. (a) Pooled saliva which was incubated at 4°C, 22°C, 37°C or 90°C for 1 hour was serially diluted, mixed 2:1 with ZIKV MR766 and incubated for 30 min at room temperature. The mix was added onto Vero E6 cells (MOI 0.3) resulting in indicated concentrations. 2 hours later, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. (b) Saliva from five individual donors was boiled at 99°C for 20 min and centrifuged for 15 min with 20,000 × g. Supernatants and untreated saliva samples were serially diluted and added onto Vero E6 cells, which were subsequently infected with ZIKV MR766 (MOI 0.15). Then, it was proceeded as in a). The figure shows the average of the quantified infection rates from the five donors. (c) Pooled saliva which was fresh or has been frozen at −20°C or −80°C for 3 hours was serially diluted, mixed 10:1 with ZIKV MR766 and incubated for 30 min at room temperature. The mix was added onto Vero E6 cells (MOI 0.3) and it was proceeded as in a). Data in a)-c) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Incubation, Infection, Immunodetection

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: SaEVs inhibit ZIKV infection through inhibiting viral attachment. (a) Vero E6 cells were inoculated with serially diluted saliva or saEVs, of which the particle numbers were determined, and infected with ZIKV MR766 (MOI 0.15). 2 hours post-infection, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. (b) Vero E6 cells were inoculated with (i) first saEVs and then, after a washing step, ZIKV 2 hours later (SaEVs→ZIVK); (ii) saEVs and ZIKV simultaneously (SaEVs+ZIKV) or first ZIKV and then, after a washing step, saEVs 2 hours later (ZIKV→SaEVs). After another 2 hours, medium was changed and 2 days later infection rates measured by the immunodetection assay. (c) Vero E6 cells in Ibidi slides were inoculated with ZIKV MR766 (MOI 35) in the presence of saEV preparations and incubated for 2 hours at 4°C. The inoculum was removed and immunofluorescent staining directed against flavivirus protein E was performed. Cell nuclei were stained with Hoechst 33342. Attached ZIKV virions were imaged by confocal microscopy (see Fig. S2) and quantified using ImageJ (Fiji). (d) SaEVs were boiled at 99°C for 20 min and centrifuged for 15 min with 20,000 × g. One sample was additionally incubated with 300 µg/ml proteinase K for 2 hours at 37°C, following another boiling and centrifugation step. These supernatants and the untreated saEV preparation were serially diluted and added onto Vero E6 cells, which were subsequently infected with ZIKV MR766 (MOI 0.15). Then, it was proceeded as in a). (e) The saEV samples from d) were separated by SDS-PAGE and proteins were stained with GelCode Blue stain for 1 hour. Image was taken on a LI-COR near-infrared imager. f) Pooled saliva was ultrafiltrated (cut-off: 100 kDa) and the particle concentrations of flowthrough (< 100 kDa), retentate (reconstituted in PBS, > 100 kDa) as well as corresponding untreated saliva was determined performing NTA (left). These samples were added onto Vero E6 cells and cells were infected with ZIKV MR766 (MOI 0.15). Then, it was proceeded as in a) (right). Data in a), b), d) and f) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Infection, Immunodetection, Incubation, Staining, Confocal Microscopy, Centrifugation, SDS Page

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: TFF/BE-SEC purified saEVs inhibit ZIKV. (a) TFF/BE-SEC purified saEVs (left) and the corresponding saliva pool used for EV preparation (right) were analysed by NTA to determine the concentration and the size distribution of EVs/free-floating particles. (b) Detection of EV (CD9, flotillin-1, Alix) and salivary protein (lysozyme, α-amylase) markers in saliva and saEVs by western blot. (c) Characterization of the saEV surface protein composition was performed by multiplex bead-based flow cytometry using a mixture of anti-CD9, anti-CD63, and anti-CD81 detection antibodies (see Fig. S3 for single stainings). Background-subtracted median fluorescence APC intensity values are shown for 37 candidate EV markers and two internal isotype controls (mIgG1 and hIgG1 (REA)). (d) SaEVs were left untreated, boiled at 99°C for 20 min and centrifuged for 15 min with 20,000 × g, or treated with 300 µg/ml proteinase K for 2 hours at 37°C, following another boiling and centrifugation step and used for further experiments. Samples were serially diluted and added onto Vero E6 cells, which were subsequently infected with ZIKV MR766 (MOI 0.15). 2 hours post-infection, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. (e) Saliva and the differently treated saEVs (d) were separated by SDS-PAGE and proteins were stained with GelCode Blue stain for 1 hour. Image was taken on a LI-COR near-infrared imager. (f) Untreated and boiled saEVs were adsorbed on glow discharged carbon-coated copper grids for 1 min at room temperature and negatively stained with 2% (w/v) uranyl acetate in H 2 O. Dried samples were imaged with a transmission electron microscope. Scale bar: 200 nm. (g) SaEVs were incubated with 25 U Benzonase® Nuclease for 2 h at room temperature with mild shaking. Samples were then serially diluted in PBS and added onto Vero E6 cells, which were subsequently infected with ZIKV MR766 as described in d). Data in d) and g) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Purification, Concentration Assay, Western Blot, Multiplex Assay, Flow Cytometry, Fluorescence, Centrifugation, Infection, Immunodetection, SDS Page, Staining, Transmission Assay, Microscopy, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Primary gingival fibroblasts are productively infected by ZIKV, but infection is inhibited by saEVs. a) Primary gingival fibroblasts were infected with ZIKV MR766 or GWUH (MOI 8) and supernatant was collected on 0, 1, 3, 5 and 7 dpi. By performing TCID 50 endpoint titration TCID 50 /ml was determined according to Reed and Muench. (b) Primary gingival fibroblasts were inoculated with serially diluted saEVs, of which the particle number was determined, and infected with ZIKV MR766 (MOI 5). 2 hours post-infection, medium was changed and 2 days later infection rates were determined by the cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. Data in b) are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Infection, Titration, Immunodetection

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Anti-ZIKV activity of saliva is donor-dependent. (a) Vero E6 cells were inoculated with serially diluted saliva from 11 donors and infected with ZIKV MR766 (MOI 0.3). 2 hours post-infection, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. Data are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations. (b) Vero E6 cells were incubated with the saliva samples from a) at indicated concentrations for 2 hours. Medium was then replaced and the cellular viability determined 2 days later by MTT assay. Data are normalized to metabolic activity in the absence of saliva. (c) Vero E6 were inoculated with serially diluted saliva from donors 6 and 8 (as in taken at different times of day and infected with ZIKV MR766 (MOI 0.15). 2 hours post-infection, medium was changed and 2 days later infection rates were determined as described in a). (d) The samples from c) were assessed for effects on cellular metabolic activity by MTT assay.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Activity Assay, Infection, Immunodetection, Incubation, MTT Assay

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: EVs from urine, vaginal lavage, and urine inhibit ZIKV but are less potent than saEVs. EVs were purified from saliva, semen and vaginal lavage by TFF/BE-SEC (left) or enriched from saliva and urine by F-UF (middle). These EV preparations, of which the particle number was determined, as well as DOPC liposomes (right), were used to inoculate Vero E6 cells, which were subsequently infected with ZIKV MR766 (MOI 0.15). 2 hours post-infection, medium was changed and 2 days later infection rates were determined by the cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. Data are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Purification, Infection, Immunodetection

Journal: Journal of Extracellular Vesicles

Article Title: Salivary extracellular vesicles inhibit Zika virus but not SARS-CoV-2 infection

doi: 10.1080/20013078.2020.1808281

Figure Lengend Snippet: Saliva and saEVs inhibit ZIKV but not SARS-CoV-2 infection. (a) Serially diluted saliva from 10 donors was mixed 1:1 with ZIKV MR766 and incubated for 30 min at 37°C. Then, mixture was used to inoculate Vero E6 cells (MOI 0.15). 3 hours post-infection, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the flavivirus protein E. (b and c) Serially diluted saliva from 10 donors was mixed 1:1 with SARS-CoV-2 isolates from the Netherlands or France and incubated for 30 min at 37°C. Then mixture was used to inoculated Caco-2 cells (MOI 0.003 or 0.009). 3 hours post-infection, medium was changed and 2 days later infection rates were determined by a cell-based immunodetection assay that enzymatically quantifies the spike protein S. (d) Serially diluted saEVs, which have been purified by TFF/BE-SEC, were added onto Caco-2 cells and subsequently infected with a French SARS-CoV-2 isolate (MOI 0.005). Medium was changed 2 hours later, and infection was quantified 2 days post-infection performing the immunodetection assay that quantifies the spike protein S. Data are normalized to infection rates in the absence of the respective sample and represented as average values obtained from triplicate infections ± standard deviations.

Article Snippet: Viruses were stained with 1:10,000 diluted mouse anti-flavivirus protein E antibody 4G2 (Absolute Antibody #Ab00230-2.0) in PBS with 1% (v/v) BSA for 45 min. After three washing steps with PBS, samples were incubated with 1:1,000 diluted goat anti-mouse secondary antibody conjugated to Alexa Fluor 647 (ThermoFisher Scientific #A21235) for 45 min.

Techniques: Infection, Incubation, Immunodetection, Purification

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Recovery of infectious rZIKV-RGN from the BAC cDNA clone. ( A ) Virus rescue. Vero cells at 90% of confluence (6-well plate format; triplicates) were mock-transfected (Mock) or transfected with 4 µg/well of the ZIKV BAC cDNA clone (pBAC-ZIKV-RGN) and at the indicated times post-transfection, virus titers in the tissue culture supernatant of transfected cells were determined by plaque assay. Error bars represent standard deviations of the means from three experiments. ( B ) Analysis of the cytopathic effect (CPE) induction and ZIKV E protein expression. Vero cells were mock-infected (left) or infected (right) with 0.5 PFU/cell of the rescued virus (rZIKV-RGN) and at 48 h post-infection (hpi) analyzed for the induction of CPE by light microscopy (top) and for viral E protein expression by immunofluorescence assay (IFA) using the pan-flavivirus E protein mAb 4G2 (bottom). Bars, 20 µm.

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Virus, Transfection, Plaque Assay, Expressing, Infection, Light Microscopy, Immunofluorescence

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Viral growth kinetics and plaque phenotype of rZIKV-RGN. ( A ) Growth kinetics. Vero and A549 cells at 90% confluence (24-well plate format; triplicates) were infected at a multiplicity of infection (MOI) of 1 PFU/cell and at the indicated hpi, virus titers in the tissue culture supernatants were determined by plaque assay on Vero cells. Error bars represent standard deviations of the mean from three experiments. ( B ) Plaque morphology. Vero cells at 90% confluence (6-well plate format) were infected with 50 PFU of rZIKV-RGN and at four days post-infection viral plaques were visualized by staining with crystal violet (left) or immunostaining (right) using the pan-flavivirus E protein mAb 4G2.

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Infection, Virus, Plaque Assay, Staining, Immunostaining

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Pathogenesis of rZIKV-RGN in A129 mice. ( A ) Weight loss and mortality. Female 4-to-6-week-old A129 mice (five mice per group) were mock-infected (PBS) or infected s.c. in the footpad with the indicated PFU of rZIKV-RGN, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during 14 days. Mice that lost more than 20% of their initial body weight or presented hind limb paralysis were humanely euthanized. Error bars represent standard deviations of the mean for each group of mice. Asterisks indicate that the differences between viral doses of 10 3 and 10 4 are statistically significant when data are compared using the unpaired t test (*, P < 0.05; **, P < 0.01). ( B ) Viral titers in mice sera. Female 4-to-6-week-old A129 mice (six mice per group) were infected with the indicated PFU of rZIKV-RGN as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus E protein mAb 4G2. Symbols represent data from individual mice and bars the geometric means of viral titers. Asterisks indicate that the differences in viral titers between experimental samples are statistically significant when data are compared using the unpaired t test (**, P < 0.01; ***, P < 0.001). &: virus not detected in two mice; ND: virus not detected. The detection limit of the assay (200 PFU/mL) is indicate as a dashed line.

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Infection, Plaque Assay, Immunostaining, Virus

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Rescue and growth properties of rZIKV-RGN-mNS2A in Vero cells. ( A ) Virus rescue. Vero cells at 90% of confluence (6-well plate format; triplicates) were transiently transfected with 4 µg/well of the infectious clones pBAC-ZIKV-RGN or pBAC-ZIKV-RGN-mNS2A, and at the indicated times post-transfection, virus titers in tissue culture supernatants were determined by plaque assay. Error bars represent standard deviations of the means from three experiments. ( B ) Plaque morphology. Vero cells at 90% confluence (6-well plate format) were infected with 25 PFU of rZIKV-RGN or rZIKV-RGN-mNS2A and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus E protein mAb 4G2. ( C ) Growth kinetics. Vero cells at 90% confluence (24-well plate format; triplicates) were infected with rZIKV-RGN or rZIKV-RGN-mNS2A at high (2 PFU/cell, left) or low (0.05 PFU/cell, right) MOI, and at the indicated hpi virus titers were determined by plaque assay. Error bars represent standard deviations of the mean from three experiments. ( D ) Analysis of viral RNA synthesis. Vero cells at 90% confluence (12-well plate format; triplicates) were infected (MOI of 0.5 PFU/cell) with rZIKV-RGN or rZIKV-RGN-mNS2A and at the indicated hpi, viral RNA levels were quantified by RT-qPCR. Error bars represent standard deviations of the mean from three experiments. ( E ) Analysis of viral E protein expression. Vero cells were infected (MOI of 0.5 PFU/cell) with rZIKV-RGN or rZIKV-RGN-mNS2A and at the indicated hpi, viral E protein expression was analyzed by IFA using the pan-flavivirus E protein mAb 4G2. Bars, 20 µm. Asterisks in panels A, C, and D indicate that the differences between rZIKV-RGN and rZIKV-RGN-mNS2A are statistically significant when data are compared using the unpaired t test (***, P < 0.001).

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Virus, Transfection, Clone Assay, Plaque Assay, Infection, Immunostaining, Quantitative RT-PCR, Expressing

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Pathogenesis of rZIKV-RGN-mNS2A in A129 mice. ( A ) Weight loss and mortality. Female 4-to-6-week-old A129 mice (five mice per group) were mock-infected (PBS) or infected s.c. in the footpad with 10 5 PFU of rZIKV-RGN or rZIKV-RGN-mNS2A, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during 14 days. Mice that lost more than 20% of their initial body weight or presented hind limb paralysis were humanely euthanized. Error bars represent standard deviations of the mean for each group of mice. ( B ) Viral titers in mice sera. Female 4-to-6-week-old A129 mice (six mice per group) were infected with 10 5 PFU of rZIKV-RGN (WT) or rZIKV-RGN-mNS2A (MUT) as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus E protein mAb 4G2. Symbols represent data from individual mice and bars the geometric means of viral titers. Asterisks indicate that the differences between rZIKV-RGN and rZIKV-RGN-mNS2A are statistically significant when data are compared using the unpaired t test (***, P < 0.001). ND: virus not detected. The detection limit of the assay (200 PFU/mL) is indicate as a dashed line. ( C ) Plaque phenotype. Vero cells at 90% confluence (6-well plate format) were infected with 25 PFU of rZIKV-RGN (left) or rZIKV-RGN-mNS2A (right) recovered from infected mice at day two post-infection and the plaque size evaluated by plaque assay and immunostaining using the pan-flavivirus E protein mAb 4G2.

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Infection, Plaque Assay, Immunostaining, Virus

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Protection efficacy of rZIKV-RGN-mNS2A. ( A ) Weight loss and mortality. Female 4-to-6-week-old A129 mice (five mice per group) were mock-vaccinated (PBS) or vaccinated s.c. in the footpad with 10 5 PFU of rZIKV-RGN-mNS2A. At 21 days after vaccination, mice were challenged with 10 5 PFU of rZIKV-RGN and the body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during 14 days. Mice that lost more than 20% of their initial body weight or presented hind limb paralysis were humanely euthanized. Error bars represent standard deviations of the mean for each group of mice. ( B ) Induction of humoral response. One day before challenge with rZIKV-RGN, sera samples were collected from mock-vaccinated (PBS) and rZIKV-RGN-mNS2A vaccinated mice, and total IgG antibodies against ZIKV-RGN were evaluated by ELISA. OD, optical density. Error bars represent standard deviations of the mean for each group of mice. ( C ) Viral titers in mice sera. Female 4-to-6-week-old A129 mice (six mice per group) mock-vaccinated (PBS) or vaccinated with 10 5 PFU of rZIKV-RGN-mNS2A (MUT) were challenged with 10 5 PFU of rZIKV-RGN as described above, and viral titers in sera were determined at days two and four after challenge (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus E protein mAb 4G2. Symbols represent data from individual mice and bars the geometric means of viral titers. †: virus not detected in one mouse; ND: virus not detected. The limit of detection of the assay (200 PFU/mL) is indicate as a dashed line.

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Plaque Assay, Immunostaining, Virus

Journal: Viruses

Article Title: An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

doi: 10.3390/v10100547

Figure Lengend Snippet: Stability of rZIKV-RGN-mNS2A in cultured cells. Vero cells growth in 6-well plates at 90% of confluence were infected with 0.1 PFU/cell of rZIKV-RGN or rZIKV-RGN-mNS2A. At 72 hpi, cell culture supernatants were collected and used to infect fresh Vero cells. This process was repeated four more times and virus stocks of passages 1 to 5 (P1 to P5) were generated. ( A ) Plaque size. Vero cells were infected with the different passages (P1 to P5) of rZIKV-RGN or rZIKV-RGN-mNS2A, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus E protein mAb 4G2. ( B ) Growth kinetics. Vero cells at 90% confluence (24-well plate format; triplicates) were infected (MOI of 1 PFU/cell) with P1 and P5 of rZIKV-RGN or rZIKV-RGN-mNS2A and at the indicated hpi, virus titers were determined by plaque assay. Error bars represent standard deviations of the mean from three experiments. Asterisks indicate that the differences between rZIKV-RGN-mNS2A P1 and the experimental samples, rZIKV-RGN P1, rZIKV-RGN P5 and rZIKV-RGN-mNS2A P5, are statistically significant when data are compared using the unpaired t test (***, P < 0.001).

Article Snippet: At selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mM Hepes pH 7.4 during 20 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After that, cells were treated for 1 h at room temperature with blocking solution (10% FBS in PBS) and incubated with 1 μg/mL of the pan-flavivirus E protein mAb 4G2 (BEI Resources; NR-50327) in blocking solution for 2 h at room temperature.

Techniques: Cell Culture, Infection, Virus, Generated, Immunostaining, Plaque Assay

Journal: bioRxiv

Article Title: Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors

doi: 10.1101/2024.05.21.595181

Figure Lengend Snippet: ( A ) Schematic illustrating survival studies in which mice underwent either intracranial (i.c.) ZIKV or subcutaneous (s.c.) WNV infections. ( B-E ) Survival and weight measurements in mice of indicated genotypes following i.c. ZIKV-MR766 infection (B), i.c. ZIKV-Fortaleza infection (C) infection, s.c. WNV-WN02-Bird114 infection (D), or i.c. ZIKV-MR766 infection (E). n=5-14 mice/group. ( F ) qRT-PCR analysis of ZIKV-MR766 genome copies in whole brain homogenates derived from mice of indicated genotypes, two or four days following i.c. ZIKV-MR766 infection. Data are expressed as plaque forming unit equivalents (PFU eq)/gram of brain tissue. ( G ) Multistep growth curve analysis of ZIKV-MR766 replication in primary cortical astrocytes derived from mice of indicated genotypes. Viral titers were assessed via plaque assay. N=4 replicates/group. ( H ) Immunohistochemical (IHC) staining of NeuN (magenta), DAPI (cyan), and pan-flavivirus E protein (yellow) staining in cortical brain tissue derived from mice of indicated genotypes five days following i.c. ZIKV-MR766 infection. Images are 2×2 tiled composites at 20x magnification. Scale bar = 20μm. ( I ) Quantification of cells exhibiting colocalization of NeuN and pan-flavivirus E protein in cortical brain tissue derived from mice described in (H). (J) IHC staining of GFAP (magenta), DAPI (cyan), and pan-flavivirus E protein (yellow) in mice as described in (H). Scale bar = 20μm. Images are at 40x magnification. *p<0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Article Snippet: Antibodies used were NeuN (1:500, Synaptic systems, 266-004), Pan-flavivirus E protein antibody (1:100, VWR Enzo, 76285-044), GFAP (1:250, Invitrogen, 13-0300), Claudin-5 (1:200, Invitrogen, 34-1600), and Zo-1 (1:200, Millipore, MABT11).

Techniques: Infection, Quantitative RT-PCR, Derivative Assay, Plaque Assay, Immunohistochemical staining, Immunohistochemistry, Staining