sars cov 2 s protein  (Sino Biological)


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    Structured Review

    Sino Biological sars cov 2 s protein
    <t>SARS-CoV-2</t> 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).
    Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    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

    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

    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

    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

    2) Product Images from "Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat"

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.217174

    pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.
    Figure Legend Snippet: pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP plasmid map.

    Techniques Used: Plasmid Preparation

    Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.
    Figure Legend Snippet: Micrographs of 293T cells transfected with pSARS-CoV-2-S (X 100). (A1, A2) 293T cells transfected with pSARS-CoV-2-S (pcDNA3.1(+)-CMV-SARS-CoV-2-S-GFP) at 48 hours after transfection. A1 fluorescence micrograph with GFP expression in cells and light micrograph with the same visual field as A2. (B) 293T cells transfected with pSARS-CoV-2-S at 48 hours after transfection. The SARS-CoV-2-S protein showed about 141 kDa.

    Techniques Used: Transfection, Fluorescence, Expressing

    Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2-S protein antigen in the rat after immunizationon day 0, day 14, and day 28 with Salmonella carrying the control vector or pSARS-CoV-2-S (as described in the methods). (A) After immunization with the control vector, test SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent test mean OD450 nm values (mean±SD) for each of 9 rats, or (B) After immunization with the pSARS-CoV-2-S vector, SARS-CoV-2-S protein antigen binding of IgG in serial serum dilutions from a rat at day (0, 14, 28). Data shown represent mean OD450 nm values (3 times measurement, mean±SD) for each of 9 rats.

    Techniques Used: Plasmid Preparation, Binding Assay

    3) Product Images from "D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity"

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.161323

    The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P
    Figure Legend Snippet: The S-G614 protein pseudotyped virus showed increased infectivity. a HEK 293T and 293T-ACE2 (human angiotensin-converting enzyme 2) cells were infected with lentiviruses pseudotyped with VSV-G and SARS-CoV-2 S protein variants. Virus titers were quantified by RT-qPCR and adjusted to 3.8 × 10 4 copies in 50 μL to normalize input virus doses. The relative luminescence units (RLU) detected 72 h post-infection (hpi). b Inhibition of pseudoviral entry by ACE2-Ig. Pseudoviruses were pre-incubated with ACE2-Ig and added to 293T-ACE2 cells, then RLU was measured at 72 hpi. c Viral entry efficiency meditated by S variants. The RLU was measured at 24-72 hpi. d-e D614G mutation facilitates elastase-2 induced pseudoviral entry. 293T-ACE2 cells were treated with elastase for 5 min and then infected with pseudotyped viruses containing the S-D614 or S-G614 mutant in the presence of various concentrations of sivelestat sodium. RLU was measured at 72 hpi. n = 3, ±SD. * P

    Techniques Used: Infection, Quantitative RT-PCR, Inhibition, Incubation, Mutagenesis

    4) Product Images from "Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2"

    Article Title: Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2

    Journal: Genes & Diseases

    doi: 10.1016/j.gendis.2020.07.006

    Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 spike (S) protein expression and localization. (A) Schematic illustration of the SARS-CoV-2 full-length spike (S-FL) and mutant S variants. The RBD (receptor binding domain) is in subunit S1; the FP (fusion peptide), HR1 (heptad repeat 1), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail) are in subunit S2. The endoplasmic reticulum retrieval signals (“KxHxx” motif) in the CT domain of S-FL were destroyed in S-Mut protein. The C-terminal 19 amino acids were lacking in S-C19del. (B) Detection of SARS-CoV-2 S expression in HKE293T cells by Western blot using the anti-RBD monoclonal antibody. Cells were transfected with pS-FL, pS-Mut, and pS-C19del plasmids or with an empty vector. (C) Detection of SARS-CoV-2 S subcellular localization in HKE293T cells by confocal microscopy. Cells were grown on glass coverslips for 24 h preceding transfection of plasmids encoding S protein variants. The cells were harvested and labeled with the corresponding antibodies. Calreticulin, ER marker. Nuclei were counterstained with DAPI. Bar = 20 μm.

    Techniques Used: Expressing, Mutagenesis, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Labeling, Marker

    Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.
    Figure Legend Snippet: Detection of SARS-CoV-2 S pseudotyped virus infectivity. (A) Schematic representation of the pseudovirus production and neutralization assay and the applications of the pseudovirus. (B) HEK293T and 293T-ACE2 cells were infected with lentiviruses pseudotyped with vesicular stomatitis virus G (VSV-G) and SARS-CoV-2 S protein variants. The y -axis shows the relative luminescence units (RLU) detected at 48 h post-pseudovirus inoculation. (C) Optimization of the incubation time for pseudovirus luciferase assay. Luciferase activities were measured 24–72 h post-virus infection. For this purpose, 72 h was chosen as the optimized incubation time. The data are presented as the means ± standard deviations (SDs) of three independent biological replicates.

    Techniques Used: Infection, Neutralization, Incubation, Luciferase

    5) 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

    6) Product Images from "3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor"

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.603830

    Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Activity Assay, Over Expression, Fluorescence, Microscopy, Inhibition

    Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Inhibition, Infection, Activity Assay

    HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p
    Figure Legend Snippet: HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Techniques Used: Binding Assay, Expressing, Western Blot, Flow Cytometry

    The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
    Figure Legend Snippet: The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Inhibition

    Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.
    Figure Legend Snippet: Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Techniques Used: Infection, Functional Assay, Activity Assay

    7) Product Images from "SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site"

    Article Title: SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

    Journal: eLife

    doi: 10.7554/eLife.64508

    A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p
    Figure Legend Snippet: A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection

    8) Product Images from "Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction"

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction

    Journal: International Dairy Journal

    doi: 10.1016/j.idairyj.2021.105002

    Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.
    Figure Legend Snippet: Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

    Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.
    Figure Legend Snippet: Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA

    Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .
    Figure Legend Snippet: Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .

    Techniques Used: Direct ELISA, Recombinant

    Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.
    Figure Legend Snippet: Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.

    Techniques Used: Recombinant, Binding Assay

    9) Product Images from "Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain"

    Article Title: Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

    Journal: bioRxiv

    doi: 10.1101/2021.02.03.429601

    Activity assessment of purified recombinant RBD-MBP. A) SPR binding assay of RBD-MBP to hACE2. B) Microsphere immunoassay of RBD-MBP binding to IgG antibodies in patient sera [(+) = SARS-CoV-2 IgG positive, (-) = SARS-CoV-2 IgG negative]. MFI: mean fluorescence intensity.
    Figure Legend Snippet: Activity assessment of purified recombinant RBD-MBP. A) SPR binding assay of RBD-MBP to hACE2. B) Microsphere immunoassay of RBD-MBP binding to IgG antibodies in patient sera [(+) = SARS-CoV-2 IgG positive, (-) = SARS-CoV-2 IgG negative]. MFI: mean fluorescence intensity.

    Techniques Used: Activity Assay, Purification, Recombinant, SPR Assay, Binding Assay, Fluorescence

    Ribbon diagram of SARS-CoV-2 spike RBD showing intramolecular disulfide bonds in yellow. As the structures solved for the RBD had missing gaps in the loops, we fed our sequence into I-Tasser, which generated the complete model ( 14 - 16 ). The ribbon was drawn using UCSF Chimera ( 17 ), is colored from blue (N-terminus) to red (C-terminus), and cysteines forming disulfide bonds are labeled..
    Figure Legend Snippet: Ribbon diagram of SARS-CoV-2 spike RBD showing intramolecular disulfide bonds in yellow. As the structures solved for the RBD had missing gaps in the loops, we fed our sequence into I-Tasser, which generated the complete model ( 14 - 16 ). The ribbon was drawn using UCSF Chimera ( 17 ), is colored from blue (N-terminus) to red (C-terminus), and cysteines forming disulfide bonds are labeled..

    Techniques Used: Sequencing, Generated, Labeling

    10) Product Images from "Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections"

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20465-w

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with the 3C1 Fab. a , b S-3C1-F3b cryo-EM map ( a ) and pseudo atomic model ( b ). All the three RBDs are up and each of them binds with a 3C1 Fab. The heavy chain of the 3C1 Fab in medium blue and light chain in violet red. c , d S-3C1-F3a cryo-EM map ( c ) and pseudo atomic model ( d ). There are two up RBDs and one down RBD, with each bound with a 3C1 Fab. e Structural alignment of the three up RBDs of S-3C1-F3b (in color) and the only up RBD from S-open (gray), suggesting 3C1 induced outward tilt of the RBDs within the S trimer. f , g Conformational comparation between S-3C1-F1 and S-open ( f ), as well as between S-3C1-F3a and S-3C1-F2 ( g ). h RBD/3C1 interaction interface (take RBD-3/3C1 of S-3C1-F3b as an example), with major involved structural elements labeled. i ACE2 (coral, PDB: 6M0J) would clash with the heavy chain of 3C1 Fab (blue). They share overlapping epitopes on the RBM (dotted black circle); additionally, the framework of 3C1-VH would clash with ACE2 (dotted black frame), which could be enhanced by the presence of an N-linked glycan at site N322 of ACE2. j 3C1 showed two distinct orientations to bind RBD within S trimer, i.e., adopting orientation 1 to associate with up RBD while orientation 2 with down RBD. k Contact footprint variations of 3C1 on up RBD (left) compared with that on down RBD (right), with unique epitopes indicated by dotted black frame. l – m Potential simultaneous binding of RBD by 2H2 and 3C1 cocktail. In 3C1 orientation 1, 3C1 and 2H2 could have minor clash (indicated by black frame, l ); while in origination 2, there is no clash between 3C1 and 2H2 Fabs ( m ).

    Techniques Used: Labeling, Binding Assay

    A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.
    Figure Legend Snippet: A proposed model of stepwise binding of 2H2/3C1 Fabs to the RBD of SARS-CoV-2 S trimer. a 2H2 and 3C1 Fabs appear to follow similar pathway to induce generally comparable conformational transitions of the S trimer to neutralize the virus. RBD-1, RBD-2, and RBD-3 are colored in light green, light blue, and gold, respectively; 2H2 and 3C1 Fab in violent red and medium blue, respectively. Red ellipsoid and black ellipsoid indicate Fab bound to up RBD and down RBD, respectively. The maps of S-2H2 and S-3C1 complexes shown here were generated by lowpass filtering of the corresponding models to 10 Å resolution. b Population distribution for the S-2H2 and S-3C1 dataset.

    Techniques Used: Binding Assay, Generated

    Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both
    Figure Legend Snippet: Cryo-EM structures of the SARS-CoV-2 S trimer in complex with 2H2 Fab. a , b Side and top views of the S-2H2-F3a cryo-EM map ( a ) and pseudo atomic model ( b ). RBD-1 and RBD-2 are in up configuration, while RBD-3 is down, with each of the RBDs bound with a 2H2 Fab. Protomer 1, 2, and 3 are shown in light green, powder blue, and gold, respectively. This color scheme is followed throughout. Heavy chain and light chain of 2H2 Fab in royal blue and violet red, respectively. c , d Side and top views of the S-2H2-F2 cryo-EM map ( c ) and pseudo atomic model ( d ), with two up RBDs (RBD-1 and RBD-2) each bound with a 2H2 Fab. e , f 2H2 Fab-induced conformational changes of the S trimer. Shown is the structural comparation of RBDs between S-2H2-F1 (in color) and S-open (dim gray) ( e ), and between S-2H2-F3a (in color) and S-2H2-F2 (dim gray) ( f ). g 2H2 Fab mainly binds to the RBM (light sea green surface) of RBD, with major involved structural elements labeled. RBD core is rendered as light green surface. h 2H2 Fab (left) and ACE2 (right, gold, PDB: 6M0J) share overlapping epitopes on RBM (second row) and would clash upon binding to the S trimer. i , j The involved regions/residues forming potential contacts between the light chain (in violent red, i ) or heavy chain (in royal blue, j ) of 2H2 and the RBD-1 of S-2H2-F3a. Asterisks highlight residues also involved in the interactions with ACE2. Note that considering the local resolution limitation in the RBD-2H2 portion of the map due to intrinsic dynamic nature in these regions, we analyzed the potential interactions that fulfill criteria of both

    Techniques Used: Labeling, Binding Assay

    Related Articles

    Clone Assay:

    Article Title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein
    Article Snippet: .. To express the SRBD subdomain of SARS-CoV-2 S protein, residues 319-541 were cloned into a mammalian expression vector downstream of an IL-2 signal peptide and upstream of a thrombin cleavage site, an AviTag, and a 6x-His tag. .. RBD protein fused to mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-V05H).

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
    Article Snippet: .. To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+. .. A C-terminal T4 fibritin trimerization motif, a TEV protease cleavage site, a FLAG tag and a His tag were cloned downstream of the SARS-CoV-2 S glycoprotein ectodomain.

    Mouse Assay:

    Article Title: Adopting STING agonist cyclic dinucleotides as a potential adjuvant for SARS-CoV-2 vaccine
    Article Snippet: .. SARS-CoV-2 vaccines immunization 6-8 week old Babl/c mice (4-5 mice per group, female) were separately subcutaneously vaccinated with SARS-CoV-2 S protein 5 μg/mouse, CDGSF 20 μg/mouse, Alhydrogel® adjuvant 2% 100 μg/mouse. .. SARS-CoV-2 Spike protein (S1+S2 ECD, gene: YP_009724390.1) was purchased from Sino Biological Inc. Alhydrogel® adjuvant 2% was purchased from InvivoGen.

    Concentration Assay:

    Article Title: The seroprevalence of SARS-CoV-2 IgG antibodies among asymptomatic blood donors in Saudi Arabia
    Article Snippet: .. The SARS-CoV-2 S protein was reconstituted in phosphate-buffered saline (PBS; pH 7.2), and the plates were coated with 100 µL/well of SARS-CoV-2 S protein, at an optimal concentration of 2 µg/mL. ..

    other:

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
    Article Snippet: Antibodies and inhibitorsThe anti-RBD monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University.

    Expressing:

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat
    Article Snippet: .. Detection of SARS-CoV-2-S protein expression in vitroFor the western blot detection of SARS-CoV-2-S protein, the cells were washed twice with ice-cold PBS and harvested from the dishes with a cell scraper by adding a WIP lysis buffer. ..

    Article Title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein
    Article Snippet: .. To express the SRBD subdomain of SARS-CoV-2 S protein, residues 319-541 were cloned into a mammalian expression vector downstream of an IL-2 signal peptide and upstream of a thrombin cleavage site, an AviTag, and a 6x-His tag. .. RBD protein fused to mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-V05H).

    Western Blot:

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat
    Article Snippet: .. Detection of SARS-CoV-2-S protein expression in vitroFor the western blot detection of SARS-CoV-2-S protein, the cells were washed twice with ice-cold PBS and harvested from the dishes with a cell scraper by adding a WIP lysis buffer. ..

    Article Title: Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain
    Article Snippet: .. RBD-MBP identity after each column was confirmed by Peggy Sue (Protein Simple) western analysis using Sino Biological Anti-Coronavirus spike antibody (Cat # 40591-T62). .. The concentrations were determined using a Fisher NanoDrop1000 using a molecular weight of 74 kDa for the fusion protein, and calculated ε280 =101.19 M−1 cm−1.

    Lysis:

    Article Title: Oral delivery of SARS-CoV-2 DNA vaccines using attenuated Salmonella typhimurium as a carrier in rat
    Article Snippet: .. Detection of SARS-CoV-2-S protein expression in vitroFor the western blot detection of SARS-CoV-2-S protein, the cells were washed twice with ice-cold PBS and harvested from the dishes with a cell scraper by adding a WIP lysis buffer. ..

    Plasmid Preparation:

    Article Title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein
    Article Snippet: .. To express the SRBD subdomain of SARS-CoV-2 S protein, residues 319-541 were cloned into a mammalian expression vector downstream of an IL-2 signal peptide and upstream of a thrombin cleavage site, an AviTag, and a 6x-His tag. .. RBD protein fused to mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-V05H).

    Article Title: Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
    Article Snippet: .. To prepare SARS-CoV-2 S protein, mammalian codon-optimized gene coding S ectodomain (residues 1–1208) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682–685) was cloned into vector pcDNA3.1+. .. A C-terminal T4 fibritin trimerization motif, a TEV protease cleavage site, a FLAG tag and a His tag were cloned downstream of the SARS-CoV-2 S glycoprotein ectodomain.

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    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and <t>VSV-SARS-CoV-2</t> replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 s protein
    <t>SARS-CoV-2</t> 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).
    Sars Cov 2 S Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 antigen
    Gross pathological examination of the lungs of <t>SARS-CoV-2</t> infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.
    Sars Cov 2 Antigen, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: JIB-04 exhibits distinct post-entry antiviral mechanisms (A) Drug combination dose-response matrix and VSV-SARS-CoV-2 replication. MA104 cells were treated with JIB-04 and chloroquine or JIB-04 and NTZ for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. (B) Time of compound addition and VSV-SARS-CoV-2 replication. MA104 cells were treated with NTZ or JIB-04 (10 μM) at indicated time points relative to VSV-SARS-CoV-2 infection (MOI=3, 0 hpi). GFP signals at 8 hpi were quantified to calculate the percentage of inhibition. (C) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. S RNA levels were measured by RT-qPCR. (D) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. MA104 cells were treated with JIB-04 (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1) for 1, 3, 5, and 7 hr. FL: full-length. S2: cleaved S2 fragment. (* non-specific band) (E) Histone demethylase siRNA knockdown and RV replication. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01). Viral RNA copy numbers at 12 hpi were quantified by RT-qPCR. (F) Volcano plot of differentially expressed transcripts with JIB-04 treatment and RV infection. HEK293 cells were treated with DMSO or JIB-04 (10 μM) for 12 hr, and mock-infected (left panel) or infected with porcine RV (MOI=0.01, right panel) for another 12 hr. Red dots represent upregulated genes and green dots represent downregulated genes in JIB-04 treated cells. (G) Expression of three top genes in (F) with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 12 hr and mock-infected or infected porcine RV (MOI=0.01) for 12 hr. mRNA levels of CYP1A1, CYP1B1, and AHRR at 12 hpi were measured by RT-qPCR. (H) Dose-response analysis of VSV-SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. (I) Dose-response analysis of wild-type SARS-CoV-2 replication with fluoxetine or fluvoxamine treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels at 24 hpi were quantified based on immunofluorescence. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 300 μM for 25 hr. For all panels except A and I, experiments were repeated at least three times with similar results. Fig. 3A was performed twice. Inhibition assay in Fig. 3I was performed once and cytotoxicity assay was performed in triplicates. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Inhibition, Quantitative RT-PCR, Western Blot, Transfection, Expressing, Immunofluorescence, Cytotoxicity Assay

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Chemical structures of NTZ and JIB-04 E-isomer from ChemSpider database. (B) Representative images of Vero E6 cells infected by SARS-CoV-2-mNeonGreen (MOI=0.5) at 24 hpi in Fig. 1A . Experiments were repeated at least three times with similar results.

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection

    Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit the replication of multiple viruses (A) Mean fluorescence intensity of GFP positive cells in Fig. 2B was quantified by flow cytometry. (B) Dose-response analysis of VSV-SARS-CoV-2 replication with NTZ or JIB-04 treatment. MA104 cells were treated with compounds at indicated concentrations for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). At 24 hpi, images of GFP positive infected cells were acquired by the ECHO fluorescence microscope. (C) Same as (B) except that cells were infected with an MOI of 0.1. (D) Dose-response analysis of intracellular viral RNA levels with NTZ, JIB-04, or chloroquine treatment. MA104 cells were treated with compounds at 0.1 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (E) Western blot analysis of RV antigen VP6 levels with JIB-04 treatment. HEK293 cells were treated with JIB-04 at 1, 5, or 10 μM for 6 hr and infected with porcine RV (MOI=0.01) for 12 hr. GAPDH was used as a loading control. All experiments were repeated at least three times with similar results. Data are represented as mean ± SEM.

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Fluorescence, Flow Cytometry, Infection, Microscopy, Quantitative RT-PCR, Western Blot

    Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 broadly inhibit DNA and RNA viruses in different cell types (A) Dose-response analysis of VSV and VSV-SARS-CoV-2 replication with 15 compounds. MA104 cells were treated with indicated compounds at 0.01 to 30 μM for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). GFP signals at 24 hpi were quantified to calculate the percentage of inhibition. EC 50 values for VSV and VSV-SARS-CoV-2 are shown in each graph in red and blue, respectively. (B) Virus infectivity with NTZ or JIB-04 treatment. Vero E6-TMPRSS2 cells were treated with compounds (10 μM) for 1 hr and infected with VSV or VSV-SARS-CoV-2 (MOI=3). At 6 hpi, percentages of GFP positive cells were quantified by flow cytometry. (C) Dose-response analysis of VSV-SARS-CoV-2 replication and cytotoxicity with NTZ or JIB-04 treatment. For EC 50 measurement, MA104 cells were treated with compounds at 0.01 to 30 μM for 1 hr and infected with VSV-SARS-CoV-2 (MOI=3) for 24 hr. For CC 50 measurement, cells were treated with compounds at 0.1 μM to 3 mM for 25 hr. SI: selectivity index. (D) Intracellular viral RNA levels with NTZ or JIB-04 treatment. MA104 cells were treated with compounds (10 μM) for 1 hr and infected with vaccinia virus (VACV), herpes simplex virus-1 (HSV-1), or rotavirus (RV, RRV and UK strains) (MOI=1). Viral RNA levels at 24 hpi were measured by RT-qPCR for VACV B10R, HSV-1 ICP-27, and RV NSP5, respectively. (E) Viral RNA copy numbers with JIB-04 treatment. HEK293 cells were treated with JIB-04 (10 μM) for 6 hr and infected with porcine rotavirus (MOI=0.01) for 6 hr. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with transmissible gastroenteritis virus (TGEV) (MOI=0.01) for 12 hr. Viral RNA copy numbers were measured by RT-qPCR. (F) TGEV titers in the cell supernatant with JIB-04 treatment. ST cells were treated with JIB-04 (10 μM) for 12 hr and infected with TGEV (MOI=0.01). Virus titers at 6 and 12 hpi were measured by plaque assays. (G) Intracellular viral RNA levels with NTZ or JIB-04 treatment in different cell types. HEK293-hACE2, HEK293-hACE2-TMPRSS2, and Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV-2 (MOI=1). VSV RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 2A was performed once. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Inhibition, Flow Cytometry, Quantitative RT-PCR

    Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Nitazoxanide and JIB-04 inhibit SARS-CoV-2 replication (A) Small molecule inhibitor screen. Vero E6 cells were treated with individual compounds (listed in Table S1) at 10 μM for 1 hour (hr) and infected with SARS-CoV-2-mNeonGreen (MOI=0.5). At 24 hr post infection (hpi), cells were fixed and nuclei were stained by Hoechst 33342. The intensities of mNeonGreen and Hoechst were quantified by the Typhoon biomolecular imager and Cytation plate reader, respectively. The ratio of mNeonGreen and Hoechst is plotted as percentage of inhibition. (B) Dose-response analysis of wild-type SARS-CoV-2 replication with NTZ or JIB-04 treatment. Vero E6 cells were treated with compounds for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). S protein levels were quantified at 24 hpi based on immunofluorescence. For CC 50 measurement, cells were treated with inhibitors at 0.3 μM to 1 mM for 25 hr. SI: selectivity index. (C) Dose-response analysis of intracellular viral RNA levels with compounds. Vero E6 cells were treated with NTZ (10 μM), JIB-04 (10 μM), chloroquine (10 μM), remdesivir (3 μM), or camostat (10 μM) for 1 hr and infected with a clinical isolate of SARS-CoV-2 (MOI=0.5). SARS-CoV-2 RNA levels at 24 hpi were measured by RT-qPCR. For all panels except A, experiments were repeated at least three times with similar results. Fig. 1A was performed once with raw data included in Dataset S1. Data are represented as mean ± SEM. Statistical significance is from pooled data of the multiple independent experiments (*p≤0.05).

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Infection, Staining, Inhibition, Immunofluorescence, Quantitative RT-PCR

    Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Journal: bioRxiv

    Article Title: Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model

    doi: 10.1101/2020.09.24.312165

    Figure Lengend Snippet: Inhibition or knockdown of specific KDM histone demethylases inhibits virus replication (A) Expression of IFN and IFN-stimulated genes with NTZ or JIB-04 treatment. HEK293 cells were treated with NTZ (10 μM), JIB-04 (3 μM), or transfected with low-molecular-weight poly(I:C) (100 ng/ml) for 24 hr. mRNA levels of IFNL3 and CXCL10 were measured by RT-qPCR. (B) Autophagy formation with compound treatment. HEK293 cells were transfected with EGFP-LC3 plasmid for 24 hr and treated with rapamycin (100 nM), NTZ (10 μM), or JIB-04 (3 μM) for another 18 hr. GFP positive punctate structures indicate autophagy activation. Scale bar, 20 μm. (C) Intracellular viral RNA levels with JIB-04 and camostat treatment. Calu-3 cells were treated with compounds (10 μM) for 1 hr and infected with VSV-SARS-CoV (MOI=3). VSV RNA levels at 24 hpi were measured by RT-qPCR. (D) Intracellular SARS-CoV-2 S RNA levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. S RNA levels were measured by RT-qPCR. (E) Western blot analysis of SARS-CoV-2 S protein levels with JIB-04 treatment. HEK293 cells were transfected with SARS-CoV-2 S plasmid for 2 hr and treated with JIB-04 (3 μM) for 24 hr. FL: full-length. S2: cleaved S2 fragment. (F) siRNA-mediated knockdown of JIB-04 target histone demethylases. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr. mRNA levels of indicated histone demethylases were measured by RT-qPCR. (G) Western blot analysis of RV antigen VP6 levels in cells with histone demethylase siRNA knockdown. HEK293 cells were transfected with scrambled siRNA or siRNA targeting indicated histone demethylases for 48 hr and infected with porcine RV (MOI=0.01) for 12 hr. (H) Pathway enrichment analysis of gene expression regulated by JIB-04 treatment. Downregulated genes in Fig. 3F with p values

    Article Snippet: Proteins were resolved in SDS-PAGE and detected as describedusing the following antibodies: GAPDH (631402, Biolegend), rotavirus VP6 (rabbit polyclonal, ABclonal technology), and SARS-CoV-2 S2 (40592-T62, Sino Biological).

    Techniques: Inhibition, Expressing, Transfection, Molecular Weight, Quantitative RT-PCR, Plasmid Preparation, Activation Assay, Infection, Western Blot

    Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Journal: eLife

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

    doi: 10.7554/eLife.61552

    Figure Lengend Snippet: Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

    Techniques: Binding Assay, Expressing, Generated

    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p

    Journal: eLife

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

    doi: 10.7554/eLife.61552

    Figure Lengend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

    Techniques: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Journal: eLife

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

    doi: 10.7554/eLife.61552

    Figure Lengend Snippet: Glycan coverage of Spike-ACE2 co-complex. SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

    Article Snippet: Rabbit anti-SARS-CoV-2 RBD (40592-T62) and anti-S2 (40590-T62) pAbs were from Sino Biologicals (Beijing, China).

    Techniques: Liquid Chromatography with Mass Spectroscopy

    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 SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

    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 SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

    Techniques: Binding Assay, Flow Cytometry

    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 SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

    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 SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

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

    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 SARS-CoV-2 Spike Protein Binds Heparin through the RBD To test experimentally if the SARS-CoV-2 S protein interacts with heparin/HS, recombinant ectodomain and RBD proteins were prepared and characterized.

    Techniques: Binding Assay, Sequencing

    Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Infection

    Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing, Immunohistochemistry

    Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Infection

    Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing

    Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    doi: 10.1101/2020.08.24.264630

    Figure Lengend Snippet: Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Article Snippet: Quantitative assessment of virus antigen expression in the lung was performed according to the same method, but using lung sections stained by immunohistochemistry for SARS-CoV-2 antigen.

    Techniques: Expressing