sars cov 2  (Sino Biological)


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    Name:
    SARS CoV SARS CoV 2 Nucleocapsid Antibody HRP Rabbit MAb
    Description:
    The unconjugated product is a recombinant monoclonal antibody expressed from HEK293 cells Then it been conjugated with horseradish peroxidase HRP
    Catalog Number:
    40143-r040-h
    Price:
    None
    Applications:
    ELISA
    Host:
    Rabbit
    Immunogen:
    Recombinant SARS-CoV Nucleoprotein / NP Protein (Catalog#40143-V08B)
    Category:
    Primary Antibody
    Antibody Type:
    MAb
    Isotype:
    Rabbit IgG
    Reactivity:
    SARS
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    Structured Review

    Sino Biological sars cov 2
    The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and <t>SARS-CoV-2</t> to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.
    The unconjugated product is a recombinant monoclonal antibody expressed from HEK293 cells Then it been conjugated with horseradish peroxidase HRP
    https://www.bioz.com/result/sars cov 2/product/Sino Biological
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "A human monoclonal antibody blocking SARS-CoV-2 infection"

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16256-y

    The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.
    Figure Legend Snippet: The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Expressing

    47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: 47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Expressing, Immunofluorescence, Negative Control, Neutralization, Infection, Luciferase, Incubation, Transduction, Strep-tag

    2) Product Images from "Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development"

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    Journal: Biomedical Journal

    doi: 10.1016/j.bj.2020.06.003

    Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.
    Figure Legend Snippet: Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.

    Techniques Used: Transduction, Generated

    Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.
    Figure Legend Snippet: Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.

    Techniques Used: Expressing, Plasmid Preparation, Derivative Assay, Transfection, Generated

    Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.
    Figure Legend Snippet: Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.

    Techniques Used:

    Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.
    Figure Legend Snippet: Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.

    Techniques Used: Transduction, Generated

    Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.
    Figure Legend Snippet: Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.

    Techniques Used: Transduction, Generated, Fluorescence, Expressing

    3) Product Images from "SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2"

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    Journal: Cell

    doi: 10.1016/j.cell.2020.09.033

    SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Isolation, Inhibition, Binding Assay, Recombinant, Flow Cytometry

    ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .
    Figure Legend Snippet: ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .

    Techniques Used: Binding Assay, Western Blot, Over Expression, CRISPR, Knock-Out, Clone Assay, Expressing, Flow Cytometry

    SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .
    Figure Legend Snippet: SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .

    Techniques Used: Recombinant, Binding Assay, Protein Binding, Staining, Electron Microscopy, Incubation

    Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
    Figure Legend Snippet: Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Techniques Used: Binding Assay, Flow Cytometry

    Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.
    Figure Legend Snippet: Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.

    Techniques Used: Infection, Luciferase

    Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .
    Figure Legend Snippet: Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

    Techniques Used: Infection, Flow Cytometry, Staining, Incubation

    SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .
    Figure Legend Snippet: SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .

    Techniques Used: Binding Assay, Titration, Recombinant, Protein Binding, Staining, Flow Cytometry

    Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.
    Figure Legend Snippet: Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.

    Techniques Used: Recombinant, Binding Assay, SDS Page, Produced, Transmission Assay, Size-exclusion Chromatography

    SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .
    Figure Legend Snippet: SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

    Techniques Used: Infection, Binding Assay, Flow Cytometry, Western Blot, Expressing, Imaging, Luciferase

    Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.
    Figure Legend Snippet: Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.

    Techniques Used: Binding Assay

    Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .
    Figure Legend Snippet: Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .

    Techniques Used: Binding Assay, Sequencing

    Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.
    Figure Legend Snippet: Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.

    Techniques Used: Binding Assay, Transmission Assay

    4) Product Images from "Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2"

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2

    Journal: Viruses

    doi: 10.3390/v12070699

    Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.
    Figure Legend Snippet: Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.

    Techniques Used: Cell Culture, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker

    5) Product Images from "A human monoclonal antibody blocking SARS-CoV-2 infection"

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16256-y

    The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.
    Figure Legend Snippet: The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Expressing

    47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: 47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Expressing, Immunofluorescence, Negative Control, Neutralization, Infection, Luciferase, Incubation, Transduction, Strep-tag

    6) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    7) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    8) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    9) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    10) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    11) Product Images from "Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples"

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

    Antibody affinity screening. (A) Illustration of the assay mechanism, which uses a single-step ELISA. The sample-to-answer time of this assay is 8 minutes. (B)-(C) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2 (B) and SARS-CoV (C). The solid lines are the linear fit of the data in the log-log scale.
    Figure Legend Snippet: Antibody affinity screening. (A) Illustration of the assay mechanism, which uses a single-step ELISA. The sample-to-answer time of this assay is 8 minutes. (B)-(C) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2 (B) and SARS-CoV (C). The solid lines are the linear fit of the data in the log-log scale.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    S1 protein detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 20 minutes. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3×standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein and SARS-CoV S1 is 0.4 ng/mL and 0.2 ng/mL, respectively. (C) Calibration curves for S1 proteins between 0.78 and 200 ng/mL. The error bars are generated from duplicate measurements.
    Figure Legend Snippet: S1 protein detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 20 minutes. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3×standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein and SARS-CoV S1 is 0.4 ng/mL and 0.2 ng/mL, respectively. (C) Calibration curves for S1 proteins between 0.78 and 200 ng/mL. The error bars are generated from duplicate measurements.

    Techniques Used: Generated

    Detection of anti-S1 IgG. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 15 minutes. (B)-(D) Detection of S1 specific IgG in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (B), D001 in (C), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. See also Figure S3 for the entire dynamic range of CR3022, D001, and D006, and their respective lower limits of detection.
    Figure Legend Snippet: Detection of anti-S1 IgG. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 15 minutes. (B)-(D) Detection of S1 specific IgG in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (B), D001 in (C), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. See also Figure S3 for the entire dynamic range of CR3022, D001, and D006, and their respective lower limits of detection.

    Techniques Used: Generated

    12) Product Images from "COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2"

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

    Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.
    Figure Legend Snippet: Classification and structure of coronavirus. (A) Classification of coronaviruses: the 7 known HCoVs are shown in green and red. HCoVs in red bind the host receptor ACE2. (B) Schematic of the SARS-CoV-2 structure; the illustration of the virus is adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . (C) Cartoon depicts key features and the trimeric structure of the SARS-CoV-2 S protein. (D) Schematic of SARS-CoV-2 genome (top) and S protein (bottom); annotations are adapted from NCBI (NC_045512.2) and Expasy ( https://covid-19.uniprot.org/uniprotkb/P0DTC2 ), respectively. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; E, envelope; HCoV, Human Coronavirus; HR1/2, heptad repeat 1/2; M, membrane; N, nucleocapsid; Nsp, nonstructural protein; NTD, N-terminal domain; orf, open reading frame; RBD, receptor-binding domain; RBM, receptor-binding motif; RdRp, RNA-dependent RNA polymerase; S protein, spike protein; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; UTR, untranslated region.

    Techniques Used: Binding Assay

    Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Structure of the SARS-CoV-2 S protein alone and in complex with ACE2 receptor. (A) Side view of the trimeric SARS-CoV-2 S ectodomain in the prefusion state (PDB: 6VSB). The protomer in green is in the “up” conformation, and the other 2 protomers in red and cyan are in “down” conformation. (B) Top view of the trimeric S protein showing RBDs in red, blue, and green on each protomer. (C) Structure of a single protomer showing the receptor-binding subunit S1 (blue) and the membrane-fusion subunit S2 (green). The furin-like protease site at the boundary of S1/S2 subunits is depicted. (D) The S1 subunit showing the RBM in the CTD region (blue) and the NTD region (brown). The S2 subunit showing the fusion peptide (red), second cleavage site S2′ (black), and HR1 (pink). (E) Structure of the RBD, core subdomain (green), and RBM (blue) (PDB: 6LZG). (F) SARS-CoV-2-RBD:ACE2 receptor polar interface shown by specific residues. (G) Structure of the SARS-CoV-2-RBD in complex with ACE2 receptor (PDB: 6LZG). (H) Structural similarity between the SARS-CoV-RBD:hACE2 (green) and SARS-CoV-2-S-CTD:hACE2 (yellow) complexes. (I) Crystal structure of the SARS-CoV-2-RBD (green) in complex with a monoclonal antibody CR3022 (orange). The RBM and CR3022 binding sites do not overlap and are distantly located on the RBD (PDB: 6W41). The figures were prepared using Pymol. ACE2, angiotensin-converting enzyme 2; CTD, C-terminal domain; hACE2, human ACE2; HR1, heptad repeat 1; NTD, N-terminal domain; PDB, Protein Data Bank; RBD, receptor-binding domain; RBM, receptor-binding motif; S protein, spike protein; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Binding Assay

    Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Cryo-EM structure of RdRp of SARS-CoV-2. (A) The domain architecture of RdRp or nsp12 of SARS-CoV-2 is subdivided into NiRAN, interface, fingers, palm, and thumb subdomains; A–G indicate conserved motifs. (B) The cryo-EM structure of apo-RdRp complex (shown as front view, PDB: 7BV1) consists of nsp12, nsp7 (brown), and 2 chains of nsp8 (nsp8.1 and nsp8.2, both in gray). The nsp8.1 interacts directly with nsp12, whereas the nsp8.2 binds to nsp7, which in turn interacts with nsp12. The RNA template is expected to enter the active site, which is formed by motifs A and C through a groove clamped by motifs F and G. Motif E and the thumb subdomain support the primer strand. The RdRp subdomain color scheme is according to Fig 4A. (C) The cryo-EM structure (in top view) of the RdRp complex bound to RNA (PDB: 6YYT) shows 2 chains of nsp8 stabilizing the extending RNA with their alpha helices. The apo-RdRp complex structure (PDB: 7BV1) is shown for comparison. The active site is expanded to show the RNA molecules coming out of the groove formed by the finger and the thumb subdomains. The figures were prepared using Pymol. cryo-EM, cryo-electron microscopy; NiRAN, nidovirus RdRp-associated nucleotidyltransferase; PDB, Protein Data Bank; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Electron Microscopy

    Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Phylogenetic relationships in the Coronavirinae subfamily. The subfamily is formed by 4 genera: Alphacoronavirus , Betacoronavirus (lineages A, B, C, and D), Gammacoronavirus , and Deltacoronavirus . We randomly picked 62 SARS-CoV-2 genome sequences, representing 15 different countries, together with other Coronavirinae subfamily members. The phylogenetic tree was created using NgPhylogeny.fr tool. The analysis indicates that SARS-CoV-2 has a close relationship with bat coronavirus RaTG13 and SARS-CoV; therefore, it is classified as a new member of the lineage B Betacoronavirus . SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used:

    Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.
    Figure Legend Snippet: Origin and transmission of pathogenic HCoVs. Yellow and red arrows indicate mild and severe infections in humans, respectively. The figure is inspired from Jie Cui and colleagues [ 46 ], and the illustrations of coronaviruses (left) are adapted from “Desiree Ho, Innovative Genomics Institute,” available at https://innovativegenomics.org/free-covid-19-illustrations/ . HCoV, Human Coronavirus; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2, SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus-2.

    Techniques Used: Transmission Assay

    13) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    14) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    15) Product Images from "A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS"

    Article Title: A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS

    Journal: Cell

    doi: 10.1016/j.cell.2020.06.035

    Pilot Scale Production of RBD-SC-Dimers of MERS-CoV and SARS-CoV-2 (A) RBD-sc-dimers were produced in industry-standard CHO cell system in GMP grade manufacturing. The immunogen yields and purities for vaccine stock solution are shown. (B) Non-reduced SDS-PAGE migration profile of increasing amounts of GMP grade RBD-sc-dimers are shown.
    Figure Legend Snippet: Pilot Scale Production of RBD-SC-Dimers of MERS-CoV and SARS-CoV-2 (A) RBD-sc-dimers were produced in industry-standard CHO cell system in GMP grade manufacturing. The immunogen yields and purities for vaccine stock solution are shown. (B) Non-reduced SDS-PAGE migration profile of increasing amounts of GMP grade RBD-sc-dimers are shown.

    Techniques Used: Produced, SDS Page, Migration

    Design and Assessment of RBD-SC-Dimer as a Vaccine against SARS-CoV-2 (A) A schematic diagram of SARS-CoV-2 RBD-sc-dimer. Two SARS-CoV-2 RBD (R319-K537) were dimerized as tandem repeat (SP, signal peptide). Analytical gel filtration of SARS-CoV-2 RBD-sc-dimer protein was performed with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curve is shown. Non-reduced and reduced SDS-PAGE migration profiles of the pooled samples are shown. (B) Ultracentrifugation sedimentation profiles of SARS-CoV-2 RBD-sc-dimer. (C) Representative BIAcore diagrams of SARS-CoV-2 RBD-sc-dimer and monomer bound to hACE2 protein. The K D value was calculated by the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. (D and E) Groups of BALB/c mice were immunized with a 10-μg dose of SARS-CoV-2 RBD-sc-dimer and conventional RBD-monomer, respectively, with AddaVax as adjuvant. PBS formulated with adjuvant was given as control. A three-dose vaccination regimen was performed. Serum samples were collected after each immunization (19 days after 1 st immunization, 14 days after 2 nd immunization, and 14 days after 3 rd immunization) to evaluate the humoral response dynamics. ELISA assay shows the SARS-CoV-2 RBD specific IgG titers in (D) and SARS-CoV-2 pseudovirus neutralization assay shows the NT 90 in (E). The values shown in (D) and (E) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P-values were analyzed with one-way ANOVA (ns, p > 0.05; ∗ p
    Figure Legend Snippet: Design and Assessment of RBD-SC-Dimer as a Vaccine against SARS-CoV-2 (A) A schematic diagram of SARS-CoV-2 RBD-sc-dimer. Two SARS-CoV-2 RBD (R319-K537) were dimerized as tandem repeat (SP, signal peptide). Analytical gel filtration of SARS-CoV-2 RBD-sc-dimer protein was performed with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curve is shown. Non-reduced and reduced SDS-PAGE migration profiles of the pooled samples are shown. (B) Ultracentrifugation sedimentation profiles of SARS-CoV-2 RBD-sc-dimer. (C) Representative BIAcore diagrams of SARS-CoV-2 RBD-sc-dimer and monomer bound to hACE2 protein. The K D value was calculated by the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. (D and E) Groups of BALB/c mice were immunized with a 10-μg dose of SARS-CoV-2 RBD-sc-dimer and conventional RBD-monomer, respectively, with AddaVax as adjuvant. PBS formulated with adjuvant was given as control. A three-dose vaccination regimen was performed. Serum samples were collected after each immunization (19 days after 1 st immunization, 14 days after 2 nd immunization, and 14 days after 3 rd immunization) to evaluate the humoral response dynamics. ELISA assay shows the SARS-CoV-2 RBD specific IgG titers in (D) and SARS-CoV-2 pseudovirus neutralization assay shows the NT 90 in (E). The values shown in (D) and (E) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P-values were analyzed with one-way ANOVA (ns, p > 0.05; ∗ p

    Techniques Used: Filtration, SDS Page, Migration, Sedimentation, Software, Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    Characterization of the Cellular Immune Response for COVID-19 Vaccine, Related to Figure 3 Fourty-five after the last vaccination, the splenocytes were isolated from mice vaccinated with SARS-CoV-2 RBD-sc-dimer (plus AddaVax TM adjuvant) and PBS (plus AddaVax TM adjuvant), respectively. (A) ELISPOT assay was performed to evaluate the ability of splenocytes to secrete IFN-γ following stimulation with different concentrations of peptide pool of SARS-CoV-2 RBD (2 μg/mL, 10 μg/mL and 50 μg/mL). Spot-forming cells (SFCs) per million cells are shown. (B) An ICS assay was conducted to quantify the proportion of CD8+ and CD4+ T cells producing key cytokines (IFN-γ, IL-2, TNF-α and IL-4) following stimulation with 10 μg/mL peptide pool (SARS-CoV-2 RBD). Shown are the frequencies of respective cytokine-producing cells. The values are the mean ± SEM. P-values were analyzed with unpaired t test (ns, p > 0.05).
    Figure Legend Snippet: Characterization of the Cellular Immune Response for COVID-19 Vaccine, Related to Figure 3 Fourty-five after the last vaccination, the splenocytes were isolated from mice vaccinated with SARS-CoV-2 RBD-sc-dimer (plus AddaVax TM adjuvant) and PBS (plus AddaVax TM adjuvant), respectively. (A) ELISPOT assay was performed to evaluate the ability of splenocytes to secrete IFN-γ following stimulation with different concentrations of peptide pool of SARS-CoV-2 RBD (2 μg/mL, 10 μg/mL and 50 μg/mL). Spot-forming cells (SFCs) per million cells are shown. (B) An ICS assay was conducted to quantify the proportion of CD8+ and CD4+ T cells producing key cytokines (IFN-γ, IL-2, TNF-α and IL-4) following stimulation with 10 μg/mL peptide pool (SARS-CoV-2 RBD). Shown are the frequencies of respective cytokine-producing cells. The values are the mean ± SEM. P-values were analyzed with unpaired t test (ns, p > 0.05).

    Techniques Used: Isolation, Mouse Assay, Enzyme-linked Immunospot

    16) Product Images from "Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection"

    Article Title: Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.431177

    Viral RNA levels in monocultures. A) qPCR analysis shows presence of viral RNA (MRPO) in SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 represented as fold change relative to mock infection at 48h after infection with 6 × 10 4 PFU. n=4 independent experiments. B) qPCR shows presence of viral RNA (MPRO) in SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 represented as fold change relative to mock infection at 72h after infection with 2 × 10 6 PFU. n=3 independent experiments. C) Quantification of % NP positive cells that are extruded upon SARS-CoV-2 infection of HMVEC-Ls. n=15 images from 3 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between Calu-3 and all others (A, B). *P
    Figure Legend Snippet: Viral RNA levels in monocultures. A) qPCR analysis shows presence of viral RNA (MRPO) in SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 represented as fold change relative to mock infection at 48h after infection with 6 × 10 4 PFU. n=4 independent experiments. B) qPCR shows presence of viral RNA (MPRO) in SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 represented as fold change relative to mock infection at 72h after infection with 2 × 10 6 PFU. n=3 independent experiments. C) Quantification of % NP positive cells that are extruded upon SARS-CoV-2 infection of HMVEC-Ls. n=15 images from 3 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between Calu-3 and all others (A, B). *P

    Techniques Used: Real-time Polymerase Chain Reaction, Infection

    Endothelial cells mount an inflammatory response upon infection. Representative immunofluorescent images of HMVEC-L stained for ICAM-1 (shown as single channel in top panel) (green), NP (magenta), phalloidin (grey) and DAPI (blue) with A) mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection or with B) control and TNFα treatment for 16h. Scalebar 50 µm. C) Quantification of ICAM-1 staining intensity in HMVEC-L. n=15 images from 3 independent experiments. D) Western blot analysis showing ICAM-1 and NP protein levels in HMVEC-L and Calu-3 cells after 72h of infection. E) Quantification of ICAM-1 protein levels in HMVEC-L and Calu-3. n=3 independent experiments. Measurement of cytokines with AlphaLISA Immunoassay kit for F) CXCL10 and G) IL6 in the supernatant of HMVEC-L with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection. n=4 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Mann-Whitney test between mock and + SARS-CoV-2 (C, E, F, G). *P
    Figure Legend Snippet: Endothelial cells mount an inflammatory response upon infection. Representative immunofluorescent images of HMVEC-L stained for ICAM-1 (shown as single channel in top panel) (green), NP (magenta), phalloidin (grey) and DAPI (blue) with A) mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection or with B) control and TNFα treatment for 16h. Scalebar 50 µm. C) Quantification of ICAM-1 staining intensity in HMVEC-L. n=15 images from 3 independent experiments. D) Western blot analysis showing ICAM-1 and NP protein levels in HMVEC-L and Calu-3 cells after 72h of infection. E) Quantification of ICAM-1 protein levels in HMVEC-L and Calu-3. n=3 independent experiments. Measurement of cytokines with AlphaLISA Immunoassay kit for F) CXCL10 and G) IL6 in the supernatant of HMVEC-L with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection. n=4 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Mann-Whitney test between mock and + SARS-CoV-2 (C, E, F, G). *P

    Techniques Used: Infection, Staining, Western Blot, MANN-WHITNEY

    Endothelial cells are not productively infected with 6 × 10 4 PFU of SARS-CoV-2. Schematic of A) apical and B) basolateral infection of cells cultured on transwell membranes. C) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 cells at 24h, 48h and 72h after infection. n=2 (HUVEC and HMVEC-L), n=3 (Calu-3) independent experiments. Representative immunofluorescent images of D) HUVEC, E) Calu-3 or F) HMVEC-L stained for NP (shown as single channel in top panel) (green), phalloidin (magenta) and DAPI (blue) with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 48h after infection. Scalebar 50 µm. G) Quantification of NP staining intensity in HUVEC, HMVEC-L and Calu-3. n=9 images from 3 independent experiments. H) Western blot analysis showing NP protein levels in HUVEC, HMVEC-L and Calu-3 cells after 48h of infection. I) Quantification of protein levels for NP in HUVEC, HMVEC-L and Calu-3. n=2 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between 24h and other time points (C) or Mann-Whitney test between mock and + SARS-CoV-2 (G, I). *P
    Figure Legend Snippet: Endothelial cells are not productively infected with 6 × 10 4 PFU of SARS-CoV-2. Schematic of A) apical and B) basolateral infection of cells cultured on transwell membranes. C) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 cells at 24h, 48h and 72h after infection. n=2 (HUVEC and HMVEC-L), n=3 (Calu-3) independent experiments. Representative immunofluorescent images of D) HUVEC, E) Calu-3 or F) HMVEC-L stained for NP (shown as single channel in top panel) (green), phalloidin (magenta) and DAPI (blue) with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 48h after infection. Scalebar 50 µm. G) Quantification of NP staining intensity in HUVEC, HMVEC-L and Calu-3. n=9 images from 3 independent experiments. H) Western blot analysis showing NP protein levels in HUVEC, HMVEC-L and Calu-3 cells after 48h of infection. I) Quantification of protein levels for NP in HUVEC, HMVEC-L and Calu-3. n=2 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between 24h and other time points (C) or Mann-Whitney test between mock and + SARS-CoV-2 (G, I). *P

    Techniques Used: Infection, Cell Culture, Staining, Western Blot, MANN-WHITNEY

    Endothelial cells can be infected with 2× 10 6 PFU of SARS-CoV-2, but infection is abortive. A) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 cells at 24h, 48h and 72h after infection. n=2 (HUVEC and HMVEC-L), n=3 (Calu-3) independent experiments. B) Representative immunofluorescent images of HMVEC-L stained for NP (shown as single channel in top panel) (green), phalloidin (magenta) and DAPI (blue) with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection. Scalebar 50 µm. B’) Enlargement of white box in B shows Phalloidin and DAPI single channels and merge. Scalebar 10 µm. B’’) XZ projection of merged image in B’, with apical side on top of image and basolateral side on the bottom. Scalebar 10 µm. C) Quantification of NP staining intensity in HMVEC-L. n=15 images from 3 independent experiments. D) Western blot analysis showing NP and ORF7a protein levels in HUVEC, HMVEC-L and Calu-3 cells after 72h of infection. Quantification of E) NP protein levels and F) ORF7a protein levels in HUVEC, HMVEC-L and Calu-3. n=3 (NP), n=2(ORF7a) independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between 24h and other time points (A) or Mann-Whitney test between mock and + SARS-CoV-2 (C, E, F). *P
    Figure Legend Snippet: Endothelial cells can be infected with 2× 10 6 PFU of SARS-CoV-2, but infection is abortive. A) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected HUVEC, HMVEC-L and Calu-3 cells at 24h, 48h and 72h after infection. n=2 (HUVEC and HMVEC-L), n=3 (Calu-3) independent experiments. B) Representative immunofluorescent images of HMVEC-L stained for NP (shown as single channel in top panel) (green), phalloidin (magenta) and DAPI (blue) with mock or SARS-CoV-2 infection from either apical or basolateral side of the cells at 72h after infection. Scalebar 50 µm. B’) Enlargement of white box in B shows Phalloidin and DAPI single channels and merge. Scalebar 10 µm. B’’) XZ projection of merged image in B’, with apical side on top of image and basolateral side on the bottom. Scalebar 10 µm. C) Quantification of NP staining intensity in HMVEC-L. n=15 images from 3 independent experiments. D) Western blot analysis showing NP and ORF7a protein levels in HUVEC, HMVEC-L and Calu-3 cells after 72h of infection. Quantification of E) NP protein levels and F) ORF7a protein levels in HUVEC, HMVEC-L and Calu-3. n=3 (NP), n=2(ORF7a) independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Kruskal Wallis test between 24h and other time points (A) or Mann-Whitney test between mock and + SARS-CoV-2 (C, E, F). *P

    Techniques Used: Infection, Staining, Western Blot, MANN-WHITNEY

    In vivo sections of lung show endothelial cells are not infected. A) Lung tissue from a deceased COVID-19 patient was sectioned and stained for Hematoxylin and Eosin (H E). Consecutive section was stained using RNAscope probes targeting SARS-CoV-2 spike mRNA and Syto13 for DNA. Enlargement of numbered insets show artery (1), vein (2) and venule (3) outlined with red dashed line are negative for SARS-CoV-2 spike mRNA in the endothelial layer of the vessels.
    Figure Legend Snippet: In vivo sections of lung show endothelial cells are not infected. A) Lung tissue from a deceased COVID-19 patient was sectioned and stained for Hematoxylin and Eosin (H E). Consecutive section was stained using RNAscope probes targeting SARS-CoV-2 spike mRNA and Syto13 for DNA. Enlargement of numbered insets show artery (1), vein (2) and venule (3) outlined with red dashed line are negative for SARS-CoV-2 spike mRNA in the endothelial layer of the vessels.

    Techniques Used: In Vivo, Infection, Staining

    Co-culture shows infection of Calu-3 and inflammation of HMVEC-L. A) Representative immunofluorescent images of co-cultured Calu-3 and HMVEC-L stained for NP (shown as single channel in top panel) (magenta), ICAM-1 (shown as single channel in middle panel) (green), phalloidin (grey) and DAPI (blue) with mock or SARS-CoV-2 infection at 72h after infection. Scalebar 50 µm. B) Quantification of NP staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. C) Schematic of Calu-3 and HMVEC-L co-cultures on transwell membranes. D) Quantification of ICAM-1 staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. E) Representative immunofluorescent images of co-cultured Calu-3 and HMVEC-L stained for dsRNA (shown as single channel in top panel) (magenta), phalloidin (grey) and DAPI (blue) with mock or SARS-CoV-2 infection at 72h after infection. Scalebar 50 µm. F) Quantification of dsRNA staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. G) qPCR shows presence of viral RNA (MPRO) in SARS-CoV-2 infected Calu-3 and HMVEC-L co-cultured cells represented as fold change relative to mock infection at 72h after infection. n=3 independent experiments. H) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected Calu-3 and HMVEC-L co-cultured cells at 72h after infection. n=3 independent experiments. Measurement of cytokines with AlphaLISA Immunoassay kit for I) CXCL10 and J) IL6 in the supernatant of Calu-3 and HMVEC-L co-cultured cells with mock or SARS-CoV-2 infection at 72h after infection. n=4 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Mann-Whitney test between mock and + SARS-CoV-2 (B, D, F, G, H, I, J). *P
    Figure Legend Snippet: Co-culture shows infection of Calu-3 and inflammation of HMVEC-L. A) Representative immunofluorescent images of co-cultured Calu-3 and HMVEC-L stained for NP (shown as single channel in top panel) (magenta), ICAM-1 (shown as single channel in middle panel) (green), phalloidin (grey) and DAPI (blue) with mock or SARS-CoV-2 infection at 72h after infection. Scalebar 50 µm. B) Quantification of NP staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. C) Schematic of Calu-3 and HMVEC-L co-cultures on transwell membranes. D) Quantification of ICAM-1 staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. E) Representative immunofluorescent images of co-cultured Calu-3 and HMVEC-L stained for dsRNA (shown as single channel in top panel) (magenta), phalloidin (grey) and DAPI (blue) with mock or SARS-CoV-2 infection at 72h after infection. Scalebar 50 µm. F) Quantification of dsRNA staining intensity in Calu-3 and HMVEC-L. n=9 images from 3 independent experiments. G) qPCR shows presence of viral RNA (MPRO) in SARS-CoV-2 infected Calu-3 and HMVEC-L co-cultured cells represented as fold change relative to mock infection at 72h after infection. n=3 independent experiments. H) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected Calu-3 and HMVEC-L co-cultured cells at 72h after infection. n=3 independent experiments. Measurement of cytokines with AlphaLISA Immunoassay kit for I) CXCL10 and J) IL6 in the supernatant of Calu-3 and HMVEC-L co-cultured cells with mock or SARS-CoV-2 infection at 72h after infection. n=4 independent experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using Mann-Whitney test between mock and + SARS-CoV-2 (B, D, F, G, H, I, J). *P

    Techniques Used: Co-Culture Assay, Infection, Cell Culture, Staining, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    17) Product Images from "Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV"

    Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15562-9

    Characterization of polyclonal rabbit anti-SARS S1 antibodies T62. a Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to SARS-CoV-2, SARS-CoV S, and chimeric S proteins. HEK293T cells transiently expressing either SARS-CoV-2 S, SARS-CoV S, SARS-CoV S/nRBD, or SARS-CoV-2 S/sRBD proteins were incubated with polyclonal rabbit anti-SARS-CoV S1 antibody T62 for 1 h on ice, followed by a FITC-conjugated secondary antibody, then cells were analyzed by flow cytometry. Experiments were done three times and one representative is shown. b Expression of SARS-CoV-2 S, SARS-CoV S, or chimeric S proteins on 293T cells. Cells from panel A were lyzed and blotted with anti-FLAG M2 antibody and polyclonal anti-SARS S1 antibody T62. c Amino acid sequence alignment of SARS-CoV and SARS-CoV-2 S RBDs. Stars (*) indicate the seven critical residues different between SARS-CoV-2 and SARS-CoV RBDs. d Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to mutant SARS-CoV S proteins. e Neutralization of SARS-CoV-2 S and SARS-CoV S pseudovirions by polyclonal rabbit anti-SARS S1 antibody T62. Pseudovirons were pre-incubated with serially diluted polyclonal rabbit anti-SARS S1 antibodies T62 on ice, then virus-antibody mixture was added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.
    Figure Legend Snippet: Characterization of polyclonal rabbit anti-SARS S1 antibodies T62. a Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to SARS-CoV-2, SARS-CoV S, and chimeric S proteins. HEK293T cells transiently expressing either SARS-CoV-2 S, SARS-CoV S, SARS-CoV S/nRBD, or SARS-CoV-2 S/sRBD proteins were incubated with polyclonal rabbit anti-SARS-CoV S1 antibody T62 for 1 h on ice, followed by a FITC-conjugated secondary antibody, then cells were analyzed by flow cytometry. Experiments were done three times and one representative is shown. b Expression of SARS-CoV-2 S, SARS-CoV S, or chimeric S proteins on 293T cells. Cells from panel A were lyzed and blotted with anti-FLAG M2 antibody and polyclonal anti-SARS S1 antibody T62. c Amino acid sequence alignment of SARS-CoV and SARS-CoV-2 S RBDs. Stars (*) indicate the seven critical residues different between SARS-CoV-2 and SARS-CoV RBDs. d Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to mutant SARS-CoV S proteins. e Neutralization of SARS-CoV-2 S and SARS-CoV S pseudovirions by polyclonal rabbit anti-SARS S1 antibody T62. Pseudovirons were pre-incubated with serially diluted polyclonal rabbit anti-SARS S1 antibodies T62 on ice, then virus-antibody mixture was added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Expressing, Incubation, Flow Cytometry, Sequencing, Mutagenesis, Neutralization, Transduction

    Limited cross-neutralization of SARS and COVID-19 sera. All sera were incubated on 56 °C for 30 min to eliminate complement. SARS-CoV S and SARS-CoV-2 S pseudovirons were pre-incubated with serially diluted SARS patient serum ( a ) or COVID-19 patient sera ( b ) for 1 h on ice and then added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.
    Figure Legend Snippet: Limited cross-neutralization of SARS and COVID-19 sera. All sera were incubated on 56 °C for 30 min to eliminate complement. SARS-CoV S and SARS-CoV-2 S pseudovirons were pre-incubated with serially diluted SARS patient serum ( a ) or COVID-19 patient sera ( b ) for 1 h on ice and then added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.

    Techniques Used: Neutralization, Incubation, Transduction

    Incorporation of SARS-CoV-2 S protein into pseudovirions. a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b – f Detection of CoVs S protein in cells lysate by western blot. Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g – j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa. Experiments were done twice and one is shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: Incorporation of SARS-CoV-2 S protein into pseudovirions. a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b – f Detection of CoVs S protein in cells lysate by western blot. Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g – j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa. Experiments were done twice and one is shown. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Western Blot, Transfection, Plasmid Preparation

    Entry and receptor of SARS-CoV-2 S pseudovirons. a , b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.
    Figure Legend Snippet: Entry and receptor of SARS-CoV-2 S pseudovirons. a , b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.

    Techniques Used: Transduction, Luciferase, Stable Transfection, Expressing, Binding Assay, Incubation, Flow Cytometry, Inhibition

    Activation of SARS-CoV-2 S protein by cathepsin and trypsin. a Effects of cathepsin inhibitors on entry of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. HEK 293/hACE2 cells were pretreated with broad-spectrum cathepsin inhibitor E64D, cathepsin L-specific inhibitor (SID 26681509), or cathepsin B-specific inhibitor (CA-074) and then transduced with SARS-CoV-2 S and VSV-G pseudovirions. Pseudoviral transduction was measured at 40 h post inoculation. Experiments were done in triplicates and repeated at least three times. One representative is shown. Error bars indicate SEM of technical triplicates. b Cell–cell fusion mediated by SARS-CoV-2 S protein. HEK 293T cells were transiently expressing eGFP and either SARS-CoV-2 or SARS-CoV S protein were detached with either trypsin or EDTA, and co-cultured with 293/hACE2 or 293 cells for 4 h at 37 °C. The scale bar indicates 250 µm. c Quantitative analysis of syncytia in panel b . d , e Thermostability analysis of SARS-CoV-2 S protein. d SARS-CoV and SARS-CoV-2 S pseudovirons were incubated at 37 °C for the specified times (0 to 4 h) in the absence of serum, and then assayed on 293/hACE2 cells. The results from infection at 0 h were set as 100%, and the experiments were repeated four times, and means with standard deviations are shown. e SARS-CoV and SARS-CoV-2 S pseudovirions without serum were incubated at the indicated temperature (37 to 51 °C) for 2 h and then assayed on 293/hACE2 cells. The results are reported as the percentage of transduction at 37 °C. The experiments were repeated four times, and means with standard deviations are shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: Activation of SARS-CoV-2 S protein by cathepsin and trypsin. a Effects of cathepsin inhibitors on entry of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. HEK 293/hACE2 cells were pretreated with broad-spectrum cathepsin inhibitor E64D, cathepsin L-specific inhibitor (SID 26681509), or cathepsin B-specific inhibitor (CA-074) and then transduced with SARS-CoV-2 S and VSV-G pseudovirions. Pseudoviral transduction was measured at 40 h post inoculation. Experiments were done in triplicates and repeated at least three times. One representative is shown. Error bars indicate SEM of technical triplicates. b Cell–cell fusion mediated by SARS-CoV-2 S protein. HEK 293T cells were transiently expressing eGFP and either SARS-CoV-2 or SARS-CoV S protein were detached with either trypsin or EDTA, and co-cultured with 293/hACE2 or 293 cells for 4 h at 37 °C. The scale bar indicates 250 µm. c Quantitative analysis of syncytia in panel b . d , e Thermostability analysis of SARS-CoV-2 S protein. d SARS-CoV and SARS-CoV-2 S pseudovirons were incubated at 37 °C for the specified times (0 to 4 h) in the absence of serum, and then assayed on 293/hACE2 cells. The results from infection at 0 h were set as 100%, and the experiments were repeated four times, and means with standard deviations are shown. e SARS-CoV and SARS-CoV-2 S pseudovirions without serum were incubated at the indicated temperature (37 to 51 °C) for 2 h and then assayed on 293/hACE2 cells. The results are reported as the percentage of transduction at 37 °C. The experiments were repeated four times, and means with standard deviations are shown. Source data are provided as a Source Data file.

    Techniques Used: Activation Assay, Transduction, Expressing, Cell Culture, Incubation, Infection

    Endocytosis of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d , e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod ( d ), YM201636 ( e ), or tetrandrine ( f ), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file.
    Figure Legend Snippet: Endocytosis of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d , e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod ( d ), YM201636 ( e ), or tetrandrine ( f ), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file.

    Techniques Used: Inhibition, Transduction, Luciferase, Activity Assay, Infection, Real-time Polymerase Chain Reaction, MTT Assay

    18) Product Images from "Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development"

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    Journal: Biomedical Journal

    doi: 10.1016/j.bj.2020.06.003

    Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.
    Figure Legend Snippet: Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.

    Techniques Used: Transduction, Generated

    Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.
    Figure Legend Snippet: Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.

    Techniques Used: Expressing, Plasmid Preparation, Derivative Assay, Transfection, Generated

    Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.
    Figure Legend Snippet: Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.

    Techniques Used:

    Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.
    Figure Legend Snippet: Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.

    Techniques Used: Transduction, Generated

    Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.
    Figure Legend Snippet: Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.

    Techniques Used: Transduction, Generated, Fluorescence, Expressing

    19) Product Images from "Human Mesenchymal Stromal Cells Are Resistant to SARS-CoV-2 Infection under Steady-State, Inflammatory Conditions and in the Presence of SARS-CoV-2-Infected Cells"

    Article Title: Human Mesenchymal Stromal Cells Are Resistant to SARS-CoV-2 Infection under Steady-State, Inflammatory Conditions and in the Presence of SARS-CoV-2-Infected Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2020.09.003

    Evaluation of SARS-CoV-2 Infection of MSCs Evaluation of SARS-CoV-2 infection of MSCs under steady-state and inflammatory conditions and in the presence of SARS-CoV-2-infected Caco-2 cells. SARS-CoV-2 infection is identified by SARS-CoV-2 S protein staining (red). All MSCs and Caco-2 cells experiments were repeated in three independent settings from three BM-MSC donors and three ASC donors, and were performed in three biological replicates each. One representative picture is shown for each condition. (A) Caco-2 cells without SARS-CoV-2; (B) Caco-2 cells with SARS-CoV-2 MOI1; (C) SARS-CoV-2 replication quantified by qPCR detecting high copy numbers in Caco-2 cells infected by SARS-CoV-2; error bars: SD; (D) BM-MSC steady state with SARS-CoV-2 MOI1; (E) ASC steady state with SARS-CoV-2 MOI1; (F) BM-MSC inflammatory conditions with SARS-CoV-2 MOI1; (G) Co-culture BM-MSC:Caco-2 cells (10:1) with SARS-CoV-2 MOI1; BM-MSCs (black star) + Caco-2 cells (white star). Scale bars, 100 μm and 20 μm (inset in G).
    Figure Legend Snippet: Evaluation of SARS-CoV-2 Infection of MSCs Evaluation of SARS-CoV-2 infection of MSCs under steady-state and inflammatory conditions and in the presence of SARS-CoV-2-infected Caco-2 cells. SARS-CoV-2 infection is identified by SARS-CoV-2 S protein staining (red). All MSCs and Caco-2 cells experiments were repeated in three independent settings from three BM-MSC donors and three ASC donors, and were performed in three biological replicates each. One representative picture is shown for each condition. (A) Caco-2 cells without SARS-CoV-2; (B) Caco-2 cells with SARS-CoV-2 MOI1; (C) SARS-CoV-2 replication quantified by qPCR detecting high copy numbers in Caco-2 cells infected by SARS-CoV-2; error bars: SD; (D) BM-MSC steady state with SARS-CoV-2 MOI1; (E) ASC steady state with SARS-CoV-2 MOI1; (F) BM-MSC inflammatory conditions with SARS-CoV-2 MOI1; (G) Co-culture BM-MSC:Caco-2 cells (10:1) with SARS-CoV-2 MOI1; BM-MSCs (black star) + Caco-2 cells (white star). Scale bars, 100 μm and 20 μm (inset in G).

    Techniques Used: Infection, Staining, Real-time Polymerase Chain Reaction, Co-Culture Assay

    20) Product Images from "Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2"

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2

    Journal: Viruses

    doi: 10.3390/v12070699

    Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.
    Figure Legend Snippet: Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.

    Techniques Used: Cell Culture, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker

    21) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    22) Product Images from "Engineered Trimeric ACE2 Binds and Locks “Three-up” Spike Protein to Potently Inhibit SARS-CoVs and Mutants"

    Article Title: Engineered Trimeric ACE2 Binds and Locks “Three-up” Spike Protein to Potently Inhibit SARS-CoVs and Mutants

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.274704

    Binding affinities measurement between ACE2 proteins and SARS-CoV-2 spike protein ectodomain (S-ECD). Low loading means S-ECD was loaded at thickness signal of 0.3 nm, whereas normal loading is thickness signal of 0.6 nm.
    Figure Legend Snippet: Binding affinities measurement between ACE2 proteins and SARS-CoV-2 spike protein ectodomain (S-ECD). Low loading means S-ECD was loaded at thickness signal of 0.3 nm, whereas normal loading is thickness signal of 0.6 nm.

    Techniques Used: Binding Assay

    Short-linker ACE2 proteins inhibition of SARS-CoV-2 pseudotyped virus.
    Figure Legend Snippet: Short-linker ACE2 proteins inhibition of SARS-CoV-2 pseudotyped virus.

    Techniques Used: Inhibition

    Cryo-EM analysis of S-ECD in complex with ACE2. (A) Representative SEC purification profile of the S-ECD in complex with T-ACE2. (B) Euler angle distribution in the final 3D reconstruction of S-ECD in the SARS-CoV-2/T-ACE2 complex. (C) Representative cryo-EM micrograph and 2D class averages of cryo-EM particle images. The scale bar in 2D class averages is 10 nm. (D) and (E) Local resolution maps for the 3D reconstruction of the RBD-ACE2 subcomplex and overall structure, respectively. (F) FSC curve of the overall structure (blue) and RBD-ACE2 subcomplex (orange). (G) FSC curve of the refined model of S-ECD of SARS-CoV-2 bound with ACE2 complex versus the overall structure against which it is refined (black), the refined model against the first half of the map versus the same map (red); and the refined model against the first half of the map versus the second half map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not allow enough for overfitting. (H) FSC curve of the refined model of RBD-ACE2 subcomplex is the same as (G) .
    Figure Legend Snippet: Cryo-EM analysis of S-ECD in complex with ACE2. (A) Representative SEC purification profile of the S-ECD in complex with T-ACE2. (B) Euler angle distribution in the final 3D reconstruction of S-ECD in the SARS-CoV-2/T-ACE2 complex. (C) Representative cryo-EM micrograph and 2D class averages of cryo-EM particle images. The scale bar in 2D class averages is 10 nm. (D) and (E) Local resolution maps for the 3D reconstruction of the RBD-ACE2 subcomplex and overall structure, respectively. (F) FSC curve of the overall structure (blue) and RBD-ACE2 subcomplex (orange). (G) FSC curve of the refined model of S-ECD of SARS-CoV-2 bound with ACE2 complex versus the overall structure against which it is refined (black), the refined model against the first half of the map versus the same map (red); and the refined model against the first half of the map versus the second half map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not allow enough for overfitting. (H) FSC curve of the refined model of RBD-ACE2 subcomplex is the same as (G) .

    Techniques Used: Purification

    ACE2 proteins inhibition of SARS-CoVs pseudotyped viruses (n=3). (A) ACE2 proteins inhibition of SARS-CoV-2. (B) ACE2 proteins inhibition of SARS-CoV. (C-J) ACE2-rigid-foldon (T-ACE2) inhibition of SARS-CoV-2 mutants. (K-L) ACE2-rigid-foldon (T-ACE2) inhibition of SARSr-CoVs WIV1 and Rs3367.
    Figure Legend Snippet: ACE2 proteins inhibition of SARS-CoVs pseudotyped viruses (n=3). (A) ACE2 proteins inhibition of SARS-CoV-2. (B) ACE2 proteins inhibition of SARS-CoV. (C-J) ACE2-rigid-foldon (T-ACE2) inhibition of SARS-CoV-2 mutants. (K-L) ACE2-rigid-foldon (T-ACE2) inhibition of SARSr-CoVs WIV1 and Rs3367.

    Techniques Used: Inhibition

    Binding affinities measurements between ACE2 proteins and SARS-CoV-2 spike protein ectodomain (S-ECD). (A) Binding affinities measured using ELISA assay. (B-F) Binding affinities measured using biolayer interferometry.
    Figure Legend Snippet: Binding affinities measurements between ACE2 proteins and SARS-CoV-2 spike protein ectodomain (S-ECD). (A) Binding affinities measured using ELISA assay. (B-F) Binding affinities measured using biolayer interferometry.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    ACE2-rigid-foldon (T-ACE2) inhibition of authentic SARS-CoV-2 virus (n=3). (A) Vero E6 cells were infected with authentic SARS-CoV-2, and inhibitory effects were evaluated using quantitative real-time (qPCR). (B) Immunofluorescence microscopy of virus infection upon treatment of ACE2-rigid-foldon (T-ACE2) or ACE2 monomer.
    Figure Legend Snippet: ACE2-rigid-foldon (T-ACE2) inhibition of authentic SARS-CoV-2 virus (n=3). (A) Vero E6 cells were infected with authentic SARS-CoV-2, and inhibitory effects were evaluated using quantitative real-time (qPCR). (B) Immunofluorescence microscopy of virus infection upon treatment of ACE2-rigid-foldon (T-ACE2) or ACE2 monomer.

    Techniques Used: Inhibition, Infection, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy

    23) Product Images from "Collapsing Glomerulopathy in a Patient With Coronavirus Disease 2019 (COVID-19)"

    Article Title: Collapsing Glomerulopathy in a Patient With Coronavirus Disease 2019 (COVID-19)

    Journal: Kidney International Reports

    doi: 10.1016/j.ekir.2020.04.002

    In situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Tissue quality was evaluated by performing RNAscope analysis for mRNA of the housekeeping gene peptidylprolyl isomerase B ( PPIB ). Positive cytoplasmic staining confirms adequate quality. Signal was detected using 3,3′-diaminobenzidine (DAB) (brown) chromogen. (periodic acid–Schiff counter stain; original magnification ×400). (b) RNAscope using probes directed against SARS-CoV-2 shows absence of signal in the patient's kidney parenchyma (periodic acid–Schiff counter stain; original magnification ×400).
    Figure Legend Snippet: In situ hybridization for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Tissue quality was evaluated by performing RNAscope analysis for mRNA of the housekeeping gene peptidylprolyl isomerase B ( PPIB ). Positive cytoplasmic staining confirms adequate quality. Signal was detected using 3,3′-diaminobenzidine (DAB) (brown) chromogen. (periodic acid–Schiff counter stain; original magnification ×400). (b) RNAscope using probes directed against SARS-CoV-2 shows absence of signal in the patient's kidney parenchyma (periodic acid–Schiff counter stain; original magnification ×400).

    Techniques Used: In Situ Hybridization, Staining

    Related Articles

    Neutralization:

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection
    Article Snippet: .. Virus neutralization assay Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications . .. In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 μl was mixed with 50 μl (500 TCID50 ) SARS-CoV or SARS-CoV-2 for 1 hour.

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2
    Article Snippet: .. SARS-CoV-2 exhibits distinct epitope features on the RBD from SARS-CoVNumerous binding and neutralization epitopes have been identified on the S protein of coronaviruses, making the S protein an essential target for vaccine design [ , , ]. ..

    Incubation:

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2
    Article Snippet: .. Fr 1C was incubated with cell supernatant containing SARS-CoV-2. .. The concentration of Fr 1C was 250 µg/mL in the mixture containing SARS-CoV-2 (4.75 log10 TCID50 /mL).

    other:

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection
    Article Snippet: In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 μl was mixed with 50 μl (500 TCID50 ) SARS-CoV or SARS-CoV-2 for 1 hour.

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2
    Article Snippet: Their analysis identified 5 shared epitopes, along with 40 and 29 unique epitopes, on the S proteins of SARS-CoV and SARS-CoV-2, respectively.

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2
    Article Snippet: Therefore, it was expected that SARS-CoV–specific antibody/antibodies alone or in combination can interfere or even inhibit SARS-CoV-2 and hACE2 receptor interactions.

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2
    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Plasmid Preparation:

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development
    Article Snippet: .. Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively. .. For SARS-CoV or SARS-CoV-2 pseudovirus, the sequences of S protein were de novo synthesized.

    Expressing:

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development
    Article Snippet: .. Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively. .. For SARS-CoV or SARS-CoV-2 pseudovirus, the sequences of S protein were de novo synthesized.

    Binding Assay:

    Article Title: COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2
    Article Snippet: .. SARS-CoV-2 exhibits distinct epitope features on the RBD from SARS-CoVNumerous binding and neutralization epitopes have been identified on the S protein of coronaviruses, making the S protein an essential target for vaccine design [ , , ]. ..

    Plaque Reduction Neutralization Test:

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection
    Article Snippet: .. Virus neutralization assay Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications . .. In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 μl was mixed with 50 μl (500 TCID50 ) SARS-CoV or SARS-CoV-2 for 1 hour.

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    Sino Biological sars cov 2
    The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and <t>SARS-CoV-2</t> to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.
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    The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    doi: 10.1038/s41467-020-16256-y

    Figure Lengend Snippet: The neutralizing 47D11 mAb binds SARS1-S and SARS2-S RBD without eliminating receptor interaction. a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analyzed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Supplementary Fig. 3 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analyzed for (ACE2-)GFP expression ( x axis) and S1 B binding ( y axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. Experiment was done twice, a representative experiment is shown. c Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 24 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white. Source data are provided as a Source Data file.

    Article Snippet: In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 μl was mixed with 50 μl (500 TCID50 ) SARS-CoV or SARS-CoV-2 for 1 hour.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Expressing

    47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    doi: 10.1038/s41467-020-16256-y

    Figure Lengend Snippet: 47D11 neutralizes SARS-CoV and SARS-CoV-2. a Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see Methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least three independent experiments with technical triplicates is shown. Iso-CTRL: an anti-Strep-tag human monoclonal antibody 11 was used as an antibody isotype control. c Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown. Source data are provided as a Source Data file.

    Article Snippet: In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 μl was mixed with 50 μl (500 TCID50 ) SARS-CoV or SARS-CoV-2 for 1 hour.

    Techniques: Binding Assay, Expressing, Immunofluorescence, Negative Control, Neutralization, Infection, Luciferase, Incubation, Transduction, Strep-tag

    Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.

    Journal: Biomedical Journal

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    doi: 10.1016/j.bj.2020.06.003

    Figure Lengend Snippet: Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.33 × 10 5 transduction unit.

    Article Snippet: Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively.

    Techniques: Transduction, Generated

    Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.

    Journal: Biomedical Journal

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    doi: 10.1016/j.bj.2020.06.003

    Figure Lengend Snippet: Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.

    Article Snippet: Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively.

    Techniques: Expressing, Plasmid Preparation, Derivative Assay, Transfection, Generated

    Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.

    Journal: Biomedical Journal

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    doi: 10.1016/j.bj.2020.06.003

    Figure Lengend Snippet: Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.

    Article Snippet: Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively.

    Techniques:

    Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.

    Journal: Biomedical Journal

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    doi: 10.1016/j.bj.2020.06.003

    Figure Lengend Snippet: Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.

    Article Snippet: Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively.

    Techniques: Transduction, Generated

    Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.

    Journal: Biomedical Journal

    Article Title: Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

    doi: 10.1016/j.bj.2020.06.003

    Figure Lengend Snippet: Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.

    Article Snippet: Production of SARS-CoV/SARS-CoV-2 and avian influenza viruses H5Nx pseudoviruses To generate SARS-CoV or SARS-CoV-2 and avian influenza virus pseudovirus, we applied the lentiviral vector system provided by National RNAi Core of Academic Sinica Taiwan to produce the pseudoviruses expressing full-length S protein and HA/NA proteins, respectively.

    Techniques: Transduction, Generated, Fluorescence, Expressing

    SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: SARS-CoV-2 Spike Ectodomain Protein Binding to Cells Is Differentially Affected by HS from Different Organs and Potently Inhibited by Heparinoids (A) LC-MS/MS disaccharide analysis of HS isolated from human kidney, liver, tonsil, and lung tissue. (B) Inhibition of binding of recombinant SARS-CoV-2 S RBD protein to H1299 cells, using tissue HS. Analysis by flow cytometry. (C) Inhibition of recombinant trimeric SARS-CoV-2 protein (20 μg/mL) binding to H1299 cells, using CHO HS, heparin, MST heparin, and split-glycol heparin. Analysis by flow cytometry. (D) Similar analysis of A549 cells. Curve fitting was performed using non-linear regression and the inhibitor versus response least-squares fit algorithm. IC 50 values are listed in Table 1 . Graphs show representative experiments performed in technical duplicates or triplicates. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Isolation, Inhibition, Binding Assay, Recombinant, Flow Cytometry

    ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: ACE2 and Cellular Heparan Sulfate Are Both Necessary for Binding of SARS-CoV-2 Spike Ectodomain (A) Western blot shows overexpression of ACE2 in the A375 and A375 B4GALT7 −/− cells. A representative blot is shown. (B) Binding of SARS-CoV-2 spike protein to cells with and without ACE2 overexpression. Note that binding is reduced in the cells deficient in HS. (C) Gene targeting of ACE2 in A549 using CRISPR-Cas9. The bars show spike binding to two independent ACE2 CRISPR-Cas9 knockout clones with and without HSase treatment. Note that binding depends on ACE2 expression and that residual binding depends in part on HS. All analyses were done by flow cytometry. The graphs show representative experiments performed in triplicate technical replicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S5 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay, Western Blot, Over Expression, CRISPR, Knock-Out, Clone Assay, Expressing, Flow Cytometry

    SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: SARS-CoV-2 Spike Binds Heparin through the RBD (A) Recombinant trimeric SARS-CoV-2 spike and RBD proteins were bound to heparin-Sepharose and eluted with a gradient of sodium chloride. (B) RBD protein from SARS-CoV-1 and SARS-CoV-2 binding to immobilized heparin-BSA. (C) Binding of spike protein or ACE2 to heparin-BSA. Insert shows SARS-CoV-2 spike protein binding to heparin-BSA in the presence of ACE2. (D) Binding of spike protein in the active RBD open (Mut7) and inactive RBD closed (Mut2) conformation to heparin-BSA. (E) Binding of ACE2 to spike protein in active RBD open (Mut7) and inactive RBD closed (Mut 2) conformation. (F) ACE2 binding to spike protein immobilized on heparin-BSA. The broken line represents background binding. Statistical analysis was by one-way ANOVA. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). (G and H) Negative stain-electron microscopy analysis of binding of heparin and ACE2 to spike protein. ACE2 binding to spike protein increases in the presence of heparin. 3D class averages of SARS-CoV-2 spike bound to zero, one, two, or three ACE2 (white, orange, blue, or gray) when complexed with and without a heparin dp20. The incubation was done for 15 min (G) or 60 min (H). The percentage of particles belonging to each class is shown in pie charts. See also Figure S3 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Recombinant, Binding Assay, Protein Binding, Staining, Electron Microscopy, Incubation

    Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Binding of RBD Protein to Hep3B Mutants, Related to Figure 3 Binding of SARS-CoV-2 S RBD protein (20 μg/mL) to Hep3B mutants. Binding was measured by flow cytometry. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay, Flow Cytometry

    Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Infection of Hep3B by MERS Pseudovirus, Related to Figure 6 Infection of Hep3B cells with SARS-CoV-2 and MERS-CoV S protein pseudotyped viruses carrying luciferase with and without treatment with heparin lyases.

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Infection, Luciferase

    Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Infection, Flow Cytometry, Staining, Incubation

    SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: SARS-CoV-2 Spike Ectodomain Binding to Cells Is Dependent on Cellular HS (A) Titration of recombinant SARS-CoV-2 spike protein binding to human H1299 cells with and without treatment with a mix of heparin lyases I, II, and III (HSase). (B) Recombinant SARS-CoV-2 spike protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (C) SARS-CoV-2 S RBD protein binding (20 μg/mL) to H1299, A549, and Hep3B cells with and without HSase treatment. (D) SARS-CoV-2 spike protein binding (20 μg/mL) to H1299 and A375 cells with and without HSase treatment. (E) Anti-HS (F58-10E4) staining of H1299, A549, Hep3B, and A375 cells with and without HSase treatment. (F) Binding of recombinant SARS-CoV-2 spike protein (20 μg/mL) to Hep3B mutants altered in HS biosynthesis enzymes. Specific enzymes that were lacking in the mutants are listed along the x axis. All values were obtained by flow cytometry. Graphs shows representative experiments performed in technical triplicate. The experiments were repeated at least three times. Statistical analysis by unpaired t test (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S4 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay, Titration, Recombinant, Protein Binding, Staining, Flow Cytometry

    Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Analysis of Recombinant Spike Proteins and Receptor-Binding Domain, Related to Star Methods, Protein Production (A) SDS-PAGE gel of recombinant SARS-CoV-2 spike ectodomain protein produced in ExpiCho cells and commercial recombinant SARS-CoV-2 RBD. (B) Transmission electron micrographs of recombinant SARS-CoV-2 spike ectodomain protein. (C) Size exclusion chromatography of recombinant SARS-CoV-2 spike ectodomain protein on a Superose 6 column. (D) SDS-PAGE gel of recombinant SARS-CoV-2 RBD produced in ExpiHEK cells. (E) Size exclusion chromatography of recombinant SARS-CoV-2 RBD on a Superdex200 column.

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Recombinant, Binding Assay, SDS Page, Produced, Transmission Assay, Size-exclusion Chromatography

    SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Infection, Binding Assay, Flow Cytometry, Western Blot, Expressing, Imaging, Luciferase

    Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Location of the Putative Heparin/HS Binding Site in the Spike Protein RBD from SARS-CoV-2, Related to Figure 1 PDB files 6VSB and 6M0J were used to model the spike protein. The residues colored pink on the three RBDs (444+509+346+354+356+357+355+466+ 347+348+349+353+450+448+451+352) make up a potential binding site for heparin and heparan sulfate.

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay

    Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Molecular Modeling of the SARS-Cov-2 Spike RBD Interaction with Heparin (A) A molecular model of SARS CoV-2 S protein trimer (PDB: 6VSB and 6M0J ) rendered with Pymol. ACE2 is shown in blue and the RBD open conformation in green. A set of positively charged residues lies distal to the ACE2 binding site. (B) Electrostatic surface rendering of the SARS-CoV-2 RBD (PDB: 6M17 ) docked with dp4 heparin oligosaccharides. Blue and red surfaces indicate electropositive and electronegative surfaces, respectively. Oligosaccharides are represented using standard CPK format. (C) Mesh surface rendering of the RBD (green) docked with dp4 heparin oligosaccharides (red). (D) Number of contacts between the RBD amino acids and a set of docked heparin dp4 oligosaccharides from (A and B). (E) Calculated energy contributions of each amino acid residue in the RBD that can interact with heparin. (F) Amino acid sequence alignment of the SARS-CoV-1 and SARS-Cov-2 RBD. Red boxes indicate amino acid residues contributing to the electropositive patch in (A and B). Identical residues are shaded dark gray. Conservative substitutions have backgrounds in blue. Non-conserved residues have a white background (G) Structural alignment of SARS-CoV-1 (cyan; PDB: 3BGF ) and SARS-CoV-2 (red; PDB: 6M17 ) RBD. (H) Electrostatic surface rendering of the SARS-CoV-1 and SAR-CoV-2 RBDs. See also Figure S1 .

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay, Sequencing

    Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.

    Journal: Cell

    Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

    doi: 10.1016/j.cell.2020.09.033

    Figure Lengend Snippet: Binding of Spike Protein to Heparin and ACE2 and Electron Micrographs of the Spike-ACE2 Complexes, Related to Figure 2 (A) SARS-CoV-2 spike binding to immobilized heparin or BSA. (B) ACE2 binding to immobilized spike protein. (C) Transmission electron micrographs of stabilized spike protein treated with ACE2 and with or without dp20 for 15 min or 1 h. (D) 2D classes averages for each condition.

    Article Snippet: The RBD protein from SARS-CoV-2 also bound in a saturable manner to heparin-BSA immobilized on a plate ( B).

    Techniques: Binding Assay, Transmission Assay

    Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.

    Journal: Viruses

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2

    doi: 10.3390/v12070699

    Figure Lengend Snippet: Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.

    Article Snippet: Fr 1C was incubated with cell supernatant containing SARS-CoV-2.

    Techniques: Cell Culture, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker