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: The pre-concentrated sample was subsequently loaded onto BE-SEC columns (HiScreen Capto Core 700 column, GE Healthcare Life Sciences), connected to an ÄKTAstart chromatography system (GEHealthcare Life Sciences).

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: 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: The pre-concentrated sample was subsequently loaded onto BE-SEC columns (HiScreen Capto Core 700 column, GE Healthcare Life Sciences), connected to an ÄKTAstart chromatography system (GEHealthcare Life Sciences).

Techniques: Purification, Liposomes, 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: The pre-concentrated sample was subsequently loaded onto BE-SEC columns (HiScreen Capto Core 700 column, GE Healthcare Life Sciences), connected to an ÄKTAstart chromatography system (GEHealthcare Life Sciences).

Techniques: Infection, Incubation, Immunodetection, Purification