sars cov 2  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD Catalog 40589 V08B1 YP 009724390 1 Val16 Pro1213 The specific IgG was purified by SARS CoV 2 2019 nCoV Spike affinity chromatography
    Catalog Number:
    40589-T62
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    2019 nCoV
    Applications:
    WB,ELISA
    Immunogen:
    Recombinant SARS-CoV-2 / 2019-nCoV Spike Protein (Catalog#40589-V08B1)
    Product Aliases:
    Anti-coronavirus spike Antibody, Anti-cov spike Antibody, Anti-ncov RBD Antibody, Anti-ncov s1 Antibody, Anti-ncov s2 Antibody, Anti-ncov spike Antibody, Anti-NCP-CoV RBD Antibody, Anti-NCP-CoV s1 Antibody, Anti-NCP-CoV s2 Antibody, Anti-NCP-CoV Spike Antibody, Anti-novel coronavirus RBD Antibody, Anti-novel coronavirus s1 Antibody, Anti-novel coronavirus s2 Antibody, Anti-novel coronavirus spike Antibody, Anti-RBD Antibody, Anti-S1 Antibody, Anti-S2 Antibody, Anti-Spike RBD Antibody
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological sars cov 2
    BLI-ISA evaluation of <t>SARS-CoV-2</t> spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD Catalog 40589 V08B1 YP 009724390 1 Val16 Pro1213 The specific IgG was purified by SARS CoV 2 2019 nCoV Spike affinity chromatography
    https://www.bioz.com/result/sars cov 2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry"

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78895-x

    BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
    Figure Legend Snippet: BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Techniques Used: Standard Deviation, Binding Assay

    ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).
    Figure Legend Snippet: ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.
    Figure Legend Snippet: BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Techniques Used: Binding Assay, Standard Deviation

    2) 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

    3) Product Images from "Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike"

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    Journal: bioRxiv

    doi: 10.1101/2021.03.10.434840

    The C93D9 class of antibodies. (A) Two views of 20 Fab structures, listed in (C), bound with SARS-CoV-2 RBD. Structures all superposed on the RBD; heavy-and light-chains of each Fab in a distinct color. The figure includes only the RBD from 6YZ5 (not one of the 20), with the RBM in light orange and the rest of the chain in gray. (B) View as in the right-hand panel in (A), but showing only the FAB from 7B3O (the closest in sequence to C93D9), with CDRs labeled. The most intimate contacts with RBM residues are from CDRH1, CDRH2 and CDRL1, many with residues constrained in potential variability by ACE2 interaction. (C) Maps of pairwise distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the 21 C93D9 class antibodies in (A) and (B). Pairwise distances analyzed by Mega X. Intensity of color shows the distance, from 0 (identical) to 1 (no identity). The VH and VL genes encoding the antibodies are shown in the indicated groups. Differences in CDR3s from the reference sequences (bold) are in red; dashes indicate missing amino acids; dots represent identical amino acids. IGHV3-66 and IGHV3-53 are very similar VH gene segments, differing by only one encoded amino-acid residue.
    Figure Legend Snippet: The C93D9 class of antibodies. (A) Two views of 20 Fab structures, listed in (C), bound with SARS-CoV-2 RBD. Structures all superposed on the RBD; heavy-and light-chains of each Fab in a distinct color. The figure includes only the RBD from 6YZ5 (not one of the 20), with the RBM in light orange and the rest of the chain in gray. (B) View as in the right-hand panel in (A), but showing only the FAB from 7B3O (the closest in sequence to C93D9), with CDRs labeled. The most intimate contacts with RBM residues are from CDRH1, CDRH2 and CDRL1, many with residues constrained in potential variability by ACE2 interaction. (C) Maps of pairwise distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the 21 C93D9 class antibodies in (A) and (B). Pairwise distances analyzed by Mega X. Intensity of color shows the distance, from 0 (identical) to 1 (no identity). The VH and VL genes encoding the antibodies are shown in the indicated groups. Differences in CDR3s from the reference sequences (bold) are in red; dashes indicate missing amino acids; dots represent identical amino acids. IGHV3-66 and IGHV3-53 are very similar VH gene segments, differing by only one encoded amino-acid residue.

    Techniques Used: Sequencing, Labeling

    Antibody sequence analyses. (A) Heavy-chain variable-domain genes of the 167 mAbs characterized by binding SARS-CoV-2 spike in either ELISA or cell-surface expression format. The inner ring of each pie chart shows the VH family and the outer ring, the gene. PBMC repertoire is from 350 million reads of deep sequencing ( 37 ). S binders include 167 clones in Table S2. *P
    Figure Legend Snippet: Antibody sequence analyses. (A) Heavy-chain variable-domain genes of the 167 mAbs characterized by binding SARS-CoV-2 spike in either ELISA or cell-surface expression format. The inner ring of each pie chart shows the VH family and the outer ring, the gene. PBMC repertoire is from 350 million reads of deep sequencing ( 37 ). S binders include 167 clones in Table S2. *P

    Techniques Used: Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Clone Assay

    Sorting strategy for SARS-CoV-2 specific memory B cells. ( A ) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 spike-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD - IgM-IgG + CD27 + and finally on spike ( B ) Representative flow cytometry plots showing spike-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the spike gate.
    Figure Legend Snippet: Sorting strategy for SARS-CoV-2 specific memory B cells. ( A ) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 spike-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD - IgM-IgG + CD27 + and finally on spike ( B ) Representative flow cytometry plots showing spike-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the spike gate.

    Techniques Used: Flow Cytometry, Binding Assay, Magnetic Beads

    Schemes followed for three-dimensional image reconstructions of C12C9 and G32R7 Fabs bound with SARS-CoV-2 spike ectodomain. See Methods for description of the procedures.
    Figure Legend Snippet: Schemes followed for three-dimensional image reconstructions of C12C9 and G32R7 Fabs bound with SARS-CoV-2 spike ectodomain. See Methods for description of the procedures.

    Techniques Used:

    mAb binding to SARS-CoV-2 S, RBD and NTD in cell-surface assay and EC 50 from ELISA-based and cell-based assay. (A) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK 293T cells. Cells were gated on DAPI-GFP + population. (B) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Fig. 1C for the screening color scheme. (D) Bar graph of EC 50 of antibodies targeting RBD, NTD and S2 using ELISA-based and cell-based assay. RBD (n=23), NTD clusters (n=15) and S2 (n=15). ***P
    Figure Legend Snippet: mAb binding to SARS-CoV-2 S, RBD and NTD in cell-surface assay and EC 50 from ELISA-based and cell-based assay. (A) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK 293T cells. Cells were gated on DAPI-GFP + population. (B) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Fig. 1C for the screening color scheme. (D) Bar graph of EC 50 of antibodies targeting RBD, NTD and S2 using ELISA-based and cell-based assay. RBD (n=23), NTD clusters (n=15) and S2 (n=15). ***P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Based Assay

    SARS-CoV-2 surface glycoprotein (spike) specificities of memory B cells from convalescent subjects. (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors (one dot per donor) and the subset of those that expressed spike-binding BCRs. Right: cells from 3 donors expressing spikebinding BCRs and sorted to recover principally those that did not bind recombinant receptor-binding domain (RBD). Sorting protocols as described in Methods and shown in Fig. S1 . (B) Summary of all antibodies (expressed as recombinant IgG1) screened by ELISA (with recombinant spike ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown to next to each chart. To the right of the charts for the two alternative sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding antibodies for those subjects from whom at least 10 paired-chain BCR sequences were recovered. (C) Binding to a panel of spike proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA (with recombinant spike ectodomain) and by association with spike expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for cells sorted just for spike binding (left) and for those sorted for positive spike binding but no RBD binding (right). The rows with pink highlighting are from the ELISA screen; those with blue highlighting, from the cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heat maps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of heavy-and light-chain somatic mutation counts in antibodies that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. The significantly higher numbers of mutations in the last group suggest recalled, affinity matured memory from previous exposures to seasonal coronaviruses. ****P
    Figure Legend Snippet: SARS-CoV-2 surface glycoprotein (spike) specificities of memory B cells from convalescent subjects. (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors (one dot per donor) and the subset of those that expressed spike-binding BCRs. Right: cells from 3 donors expressing spikebinding BCRs and sorted to recover principally those that did not bind recombinant receptor-binding domain (RBD). Sorting protocols as described in Methods and shown in Fig. S1 . (B) Summary of all antibodies (expressed as recombinant IgG1) screened by ELISA (with recombinant spike ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown to next to each chart. To the right of the charts for the two alternative sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding antibodies for those subjects from whom at least 10 paired-chain BCR sequences were recovered. (C) Binding to a panel of spike proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA (with recombinant spike ectodomain) and by association with spike expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for cells sorted just for spike binding (left) and for those sorted for positive spike binding but no RBD binding (right). The rows with pink highlighting are from the ELISA screen; those with blue highlighting, from the cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heat maps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of heavy-and light-chain somatic mutation counts in antibodies that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. The significantly higher numbers of mutations in the last group suggest recalled, affinity matured memory from previous exposures to seasonal coronaviruses. ****P

    Techniques Used: Binding Assay, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Mutagenesis

    Ab contact regions. Surface regions of the SARS-CoV-2 spike protein trimer contacted by antibodies in four of the seven principal clusters, according to the color scheme shown (taken from the color scheme in Fig. 2 ), with a representative Fab for all except RBD-3. The C81C10 Fab defines an epitope just outside the margin of NTD-1, but it does not compete with any antibodies in RBD-2. The RBD-2 Fv shown is that of C121 (PDB ID: 7K8X: Barnes et al, 2020), which fits most closely, of the many published RBD-2 antibodies, into our low-resolution map for C12A2. Left: views normal to and along threefold axis of the closed, all-RBD-down conformation; right: similar views of the one-RBD-up conformation. C121 (RBD-2) can bind both RBD down and RBD up; G32R7 (RBD-1) binds only the “up” conformation of the RBD. The epitopes of the several published RBD-3 antibodies are partly occluded in both closed and open conformations of the RBD; none are shown here as cartoons. A cartoon of the polypeptide chain of a single subunit (dark red) is shown within the surface contour for a spike trimer (gray).
    Figure Legend Snippet: Ab contact regions. Surface regions of the SARS-CoV-2 spike protein trimer contacted by antibodies in four of the seven principal clusters, according to the color scheme shown (taken from the color scheme in Fig. 2 ), with a representative Fab for all except RBD-3. The C81C10 Fab defines an epitope just outside the margin of NTD-1, but it does not compete with any antibodies in RBD-2. The RBD-2 Fv shown is that of C121 (PDB ID: 7K8X: Barnes et al, 2020), which fits most closely, of the many published RBD-2 antibodies, into our low-resolution map for C12A2. Left: views normal to and along threefold axis of the closed, all-RBD-down conformation; right: similar views of the one-RBD-up conformation. C121 (RBD-2) can bind both RBD down and RBD up; G32R7 (RBD-1) binds only the “up” conformation of the RBD. The epitopes of the several published RBD-3 antibodies are partly occluded in both closed and open conformations of the RBD; none are shown here as cartoons. A cartoon of the polypeptide chain of a single subunit (dark red) is shown within the surface contour for a spike trimer (gray).

    Techniques Used:

    4) Product Images from "Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry"

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78895-x

    BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
    Figure Legend Snippet: BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Techniques Used: Standard Deviation, Binding Assay

    ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).
    Figure Legend Snippet: ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.
    Figure Legend Snippet: BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Techniques Used: Binding Assay, Standard Deviation

    5) 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

    6) Product Images from "Distinct conformational states of SARS-CoV-2 spike protein"

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.05.16.099317

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.
    Figure Legend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Techniques Used: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Techniques Used:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.
    Figure Legend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Techniques Used: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.
    Figure Legend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Techniques Used: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Techniques Used: Binding Assay

    7) Product Images from "Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease"

    Article Title: Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142032

    Infection of the nasal cavity and olfactory bulb in K18-hACE2 mice. ( A ) ISH labeling for SARS-CoV-2 RNA in a coronal section of the head, including the caudal aspect of the nasal cavity and olfactory bulb. Within the olfactory bulb, a strong positive signal is present in the glomerular layer, external plexiform layer, mitral cell layer, and internal plexiform layer, with multifocal positivity in the granular cell layer in the olfactory bulb hemisphere at right. Low numbers of cells within the olfactory epithelium lining the dorsal nasal meatus have a positive ISH signal (arrows). Cells were counterstained with hematoxylin (blue). ( B ) ISH labeling for SARS-CoV-2 RNA in the olfactory epithelium. Cells were counterstained with hematoxylin (blue). ( C ) IFA of olfactory epithelium stained for SARS-CoV-2 spike (green) and Pan-cytokeratin (red). Nuclei were stained with DAPI (blue). ( D ) Representative H E staining of the nasal cavity, including olfactory epithelium and olfactory bulb from uninfected or infected mice. In the infected mouse, there is a focal area of olfactory epithelium atrophy on a nasal turbinate located in the lateral nasal meatus. Inset, indicated area of detail under increased magnification.
    Figure Legend Snippet: Infection of the nasal cavity and olfactory bulb in K18-hACE2 mice. ( A ) ISH labeling for SARS-CoV-2 RNA in a coronal section of the head, including the caudal aspect of the nasal cavity and olfactory bulb. Within the olfactory bulb, a strong positive signal is present in the glomerular layer, external plexiform layer, mitral cell layer, and internal plexiform layer, with multifocal positivity in the granular cell layer in the olfactory bulb hemisphere at right. Low numbers of cells within the olfactory epithelium lining the dorsal nasal meatus have a positive ISH signal (arrows). Cells were counterstained with hematoxylin (blue). ( B ) ISH labeling for SARS-CoV-2 RNA in the olfactory epithelium. Cells were counterstained with hematoxylin (blue). ( C ) IFA of olfactory epithelium stained for SARS-CoV-2 spike (green) and Pan-cytokeratin (red). Nuclei were stained with DAPI (blue). ( D ) Representative H E staining of the nasal cavity, including olfactory epithelium and olfactory bulb from uninfected or infected mice. In the infected mouse, there is a focal area of olfactory epithelium atrophy on a nasal turbinate located in the lateral nasal meatus. Inset, indicated area of detail under increased magnification.

    Techniques Used: Infection, Mouse Assay, In Situ Hybridization, Labeling, Immunofluorescence, Staining

    Infection of SARS-CoV-2 in the lung of K18-hACE2 transgenic mice. ( A ) Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected mice at low and high magnification or uninfected mice. Cells were counterstained with hematoxylin (blue). ( B ) Costaining for viral spike protein (green) and E-cadherin (red) in infected lung tissues using IFA. Arrows point to double-positive cells. Nuclei are stained with DAPI (blue). ( C ) Costaining of viral nucleoprotein and the macrophage marker CD68 (red) in infected lungs using IFA. Arrows denote double-positive cells. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Infection of SARS-CoV-2 in the lung of K18-hACE2 transgenic mice. ( A ) Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected mice at low and high magnification or uninfected mice. Cells were counterstained with hematoxylin (blue). ( B ) Costaining for viral spike protein (green) and E-cadherin (red) in infected lung tissues using IFA. Arrows point to double-positive cells. Nuclei are stained with DAPI (blue). ( C ) Costaining of viral nucleoprotein and the macrophage marker CD68 (red) in infected lungs using IFA. Arrows denote double-positive cells. Nuclei are stained with DAPI (blue).

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, In Situ Hybridization, Immunofluorescence, Staining, Marker

    SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. ( A – D ) Representative H E staining of lungs in infected C57BL/6 mice ( A ) or K18-hACE2-infected mice ( B – D ). Numerous fibrin thrombi (black arrows) filling the lumen of small-to-intermediate size vessels ( B ) adjacent to a normal bronchus and surrounded by minimally inflamed, congested, and collapsed alveolar septa. ( C ) Extensive area of lung consolidation with inflammation and expansion of alveolar septa, exudation of fibrin and edema into alveolar lumina, and infiltration of vessel walls and perivascular area by numerous mononuclear inflammatory cells (arrows), disrupting/obscuring vessel architecture (vasculitis). ( D ) Extensive area of consolidated lung showing type II pneumocyte hyperplasia (arrowhead) and rare multinucleate cells (black arrow). ( E ) H E and ISH staining of infected mouse lung showing vasculitis with absence of viral RNA in the affected vessel walls; note there is viral RNA (red) in the adjacent alveolar septa. Highlighted vessels (broken black circles). ( F ) TUNEL staining of infected and uninfected K18-hACE2 mouse lungs. TUNEL (green) was performed as indicted in Methods. Cell nuclei stained with DAPI (blue). ( G ) Ki-67 staining (red) in infected and uninfected K18-hACE2 mouse lungs. Nuclei stained with DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. ( A – D ) Representative H E staining of lungs in infected C57BL/6 mice ( A ) or K18-hACE2-infected mice ( B – D ). Numerous fibrin thrombi (black arrows) filling the lumen of small-to-intermediate size vessels ( B ) adjacent to a normal bronchus and surrounded by minimally inflamed, congested, and collapsed alveolar septa. ( C ) Extensive area of lung consolidation with inflammation and expansion of alveolar septa, exudation of fibrin and edema into alveolar lumina, and infiltration of vessel walls and perivascular area by numerous mononuclear inflammatory cells (arrows), disrupting/obscuring vessel architecture (vasculitis). ( D ) Extensive area of consolidated lung showing type II pneumocyte hyperplasia (arrowhead) and rare multinucleate cells (black arrow). ( E ) H E and ISH staining of infected mouse lung showing vasculitis with absence of viral RNA in the affected vessel walls; note there is viral RNA (red) in the adjacent alveolar septa. Highlighted vessels (broken black circles). ( F ) TUNEL staining of infected and uninfected K18-hACE2 mouse lungs. TUNEL (green) was performed as indicted in Methods. Cell nuclei stained with DAPI (blue). ( G ) Ki-67 staining (red) in infected and uninfected K18-hACE2 mouse lungs. Nuclei stained with DAPI (blue).

    Techniques Used: Infection, Mouse Assay, Staining, In Situ Hybridization, TUNEL Assay

    Transcriptional activation in SARS-CoV-2–infected lungs. Transcriptional activation in lung homogenates from infected K18-hACE2 (male and female) and C57BL/6 (female) mice were examined by NanoString. ( A ) Log 2 fold changes in gene expression levels of selected genes categorized by group vs. infected C57BL/6 mice were graphed with SD. All graphed transcripts had a P value of less than 0.05. ( B ) Differential gene expression (log 2 changes) between infected male and female K18-hACE2 mice. All graphed transcripts had a P value of less than 0.05.
    Figure Legend Snippet: Transcriptional activation in SARS-CoV-2–infected lungs. Transcriptional activation in lung homogenates from infected K18-hACE2 (male and female) and C57BL/6 (female) mice were examined by NanoString. ( A ) Log 2 fold changes in gene expression levels of selected genes categorized by group vs. infected C57BL/6 mice were graphed with SD. All graphed transcripts had a P value of less than 0.05. ( B ) Differential gene expression (log 2 changes) between infected male and female K18-hACE2 mice. All graphed transcripts had a P value of less than 0.05.

    Techniques Used: Activation Assay, Infection, Mouse Assay, Expressing

    Neuropathogenesis of SARS-CoV-2 in K18-hACE2 transgenic and nontransgenic mice. ISH detection of for SARS-CoV-2 RNA in uninfected ( A ) and infected mice ( B and C ) in a coronal section of brain demonstrating a strong positive signal within neurons of thalamic nuclei. The boxed area is shown at increased magnification (right) ( C ). Note the absence of a positive signal from the vessel at center right ( C ) where the vessel wall and perivascular space are infiltrated by mononuclear inflammatory cells. ( D – G ) Representative H E staining of uninfected ( D ) or infected ( E – G ) mice. Perivascular hemorrhage extending into the adjacent neuropil ( E ), in the region of the rostral cerebral cortex. Two small caliber vessels ( F ) in the thalamus with fibrin thrombi (arrows), with mild microgliosis and some perivascular hemorrhage (arrowhead). The walls of small-to-intermediate size vessels and perivascular spaces ( G ) are multifocally expanded/obscured by mononuclear inflammatory cells and increased numbers of glial cells. ( H ) Detection of Iba-1 and GFAP, markers for microgliosis and astrogliosis, respectively, in uninfected or infected brain sections using IFA. Nuclei were stained with DAPI (blue). ( I ) Costaining for astrocyte marker GFAP (red) or neuron marker NeuN (red) with SARS-CoV-2 spike protein (green). Spike protein was predominantly detected in NeuN + neurons. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Neuropathogenesis of SARS-CoV-2 in K18-hACE2 transgenic and nontransgenic mice. ISH detection of for SARS-CoV-2 RNA in uninfected ( A ) and infected mice ( B and C ) in a coronal section of brain demonstrating a strong positive signal within neurons of thalamic nuclei. The boxed area is shown at increased magnification (right) ( C ). Note the absence of a positive signal from the vessel at center right ( C ) where the vessel wall and perivascular space are infiltrated by mononuclear inflammatory cells. ( D – G ) Representative H E staining of uninfected ( D ) or infected ( E – G ) mice. Perivascular hemorrhage extending into the adjacent neuropil ( E ), in the region of the rostral cerebral cortex. Two small caliber vessels ( F ) in the thalamus with fibrin thrombi (arrows), with mild microgliosis and some perivascular hemorrhage (arrowhead). The walls of small-to-intermediate size vessels and perivascular spaces ( G ) are multifocally expanded/obscured by mononuclear inflammatory cells and increased numbers of glial cells. ( H ) Detection of Iba-1 and GFAP, markers for microgliosis and astrogliosis, respectively, in uninfected or infected brain sections using IFA. Nuclei were stained with DAPI (blue). ( I ) Costaining for astrocyte marker GFAP (red) or neuron marker NeuN (red) with SARS-CoV-2 spike protein (green). Spike protein was predominantly detected in NeuN + neurons. Nuclei were stained with DAPI (blue).

    Techniques Used: Transgenic Assay, Mouse Assay, In Situ Hybridization, Infection, Staining, Immunofluorescence, Marker

    SARS-CoV-2 infection in K18-hACE2 transgenic mice. ( A ) Male and female K18-hACE2 transgenic mice (day 0–3, n = 7/group; day 3+, n = 5/group) were infected with 2 × 10 4 PFU or 2 × 10 3 PFU of SARS-CoV-2 by the IN route. C57BL/6 and RAG2 KO mice (day 0–3, n = 8/ group; day 3+, n = 5/group) were infected with 2 × 10 4 PFU by the IN route. Survival and weight loss (± SEM) were monitored and plotted using Prism software. ( B ) Titers in lung ( n = 2 mice/group) were examined on day 3 by qRT-PCR. Mean titers ± SEM of the genome molecules of viral RNA/mL were graphed. ( C ) Titers in lungs of individual K18-hACE2 mice. Numbers above bars denote day of death. Colors represent the 4 groups. ( D ) Monocyte chemoattractants and inflammatory cytokines were measured from the serum of SARS-CoV-3– infected mice on day 3 or at the time of euthanasia using a multiplex system. Mice from each group are aggregated from samples taken on day 3 (blue symbols) and when mice were euthanized (black symbols).
    Figure Legend Snippet: SARS-CoV-2 infection in K18-hACE2 transgenic mice. ( A ) Male and female K18-hACE2 transgenic mice (day 0–3, n = 7/group; day 3+, n = 5/group) were infected with 2 × 10 4 PFU or 2 × 10 3 PFU of SARS-CoV-2 by the IN route. C57BL/6 and RAG2 KO mice (day 0–3, n = 8/ group; day 3+, n = 5/group) were infected with 2 × 10 4 PFU by the IN route. Survival and weight loss (± SEM) were monitored and plotted using Prism software. ( B ) Titers in lung ( n = 2 mice/group) were examined on day 3 by qRT-PCR. Mean titers ± SEM of the genome molecules of viral RNA/mL were graphed. ( C ) Titers in lungs of individual K18-hACE2 mice. Numbers above bars denote day of death. Colors represent the 4 groups. ( D ) Monocyte chemoattractants and inflammatory cytokines were measured from the serum of SARS-CoV-3– infected mice on day 3 or at the time of euthanasia using a multiplex system. Mice from each group are aggregated from samples taken on day 3 (blue symbols) and when mice were euthanized (black symbols).

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Software, Quantitative RT-PCR, Multiplex Assay

    8) Product Images from "Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease"

    Article Title: Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease

    Journal: bioRxiv

    doi: 10.1101/2020.07.09.195230

    Brain lesions in SARS-CoV-2 infected mice. Representative H E staining of lungs in infected K18-hACE2 mice. Multifocal areas of gliosis within the amygdala, predominantly characterized by increased numbers of microglia, and there are individual shrunken, angular cells with hypereosinophilic cytoplasm, pyknotic nuclei and surrounded by a clear halo, consistent with necrosis. While the morphology and location of individual necrotic cells is suggestive of neuronal necrosis, additional diagnostics are necessary to confirm the cell of origin. The right panel is a enhanced magnification of the boxed area
    Figure Legend Snippet: Brain lesions in SARS-CoV-2 infected mice. Representative H E staining of lungs in infected K18-hACE2 mice. Multifocal areas of gliosis within the amygdala, predominantly characterized by increased numbers of microglia, and there are individual shrunken, angular cells with hypereosinophilic cytoplasm, pyknotic nuclei and surrounded by a clear halo, consistent with necrosis. While the morphology and location of individual necrotic cells is suggestive of neuronal necrosis, additional diagnostics are necessary to confirm the cell of origin. The right panel is a enhanced magnification of the boxed area

    Techniques Used: Infection, Mouse Assay, Staining

    Infection of SARS-CoV-2 in the neurons of the olfactory bulb. Detection of viral spike protein (green) and the neuron marker NeuN (red) in infected olfactory bulb. Arrows denote co-stained cells. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Infection of SARS-CoV-2 in the neurons of the olfactory bulb. Detection of viral spike protein (green) and the neuron marker NeuN (red) in infected olfactory bulb. Arrows denote co-stained cells. Nuclei are stained with DAPI (blue).

    Techniques Used: Infection, Marker, Staining

    Infection of SARS-CoV-2 in the lung of K18-ACE2 transgenic mice. A. Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected at low and high magnification (i iii) or uninfected (ii) K18-hACE2 mice. Cells were counterstained with hematoxylin (blue). B. Co-staining for viral spike protein (green) and E-cadherin (red) in infected lung tissues using IFA. Arrows point to double positive cells. Nuclei are stained with DAPI (blue). C. Co-staining of viral nucleoprotein and the macrophage marker CD68 (red) in infected lungs using IFA. Arrows denote double positive cells. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Infection of SARS-CoV-2 in the lung of K18-ACE2 transgenic mice. A. Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected at low and high magnification (i iii) or uninfected (ii) K18-hACE2 mice. Cells were counterstained with hematoxylin (blue). B. Co-staining for viral spike protein (green) and E-cadherin (red) in infected lung tissues using IFA. Arrows point to double positive cells. Nuclei are stained with DAPI (blue). C. Co-staining of viral nucleoprotein and the macrophage marker CD68 (red) in infected lungs using IFA. Arrows denote double positive cells. Nuclei are stained with DAPI (blue).

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, In Situ Hybridization, Staining, Immunofluorescence, Marker

    SARS-CoV-2 infection of eyes and nasal turbinates. Representative ISH staining showing the presence of SARS-CoV-2 RNA (red) in the eyes of infected K18-hACE2 mice with staining in the retina (arrows) (i ii). Viral RNA was detected in the nasal turbiantes (iii iv) with sloughing of infected cells (iv). Cells were counterstained with hematoxylin (blue).
    Figure Legend Snippet: SARS-CoV-2 infection of eyes and nasal turbinates. Representative ISH staining showing the presence of SARS-CoV-2 RNA (red) in the eyes of infected K18-hACE2 mice with staining in the retina (arrows) (i ii). Viral RNA was detected in the nasal turbiantes (iii iv) with sloughing of infected cells (iv). Cells were counterstained with hematoxylin (blue).

    Techniques Used: Infection, In Situ Hybridization, Staining, Mouse Assay

    hACE2 transgene expression in neurons of SARS-CoV-2 infected mice. Representative ISH staining showing the presence of the hACE2 transgene (red) in infected K18-hACE2 mice. Duplex ISH staining further shows hACE2-exressping neurons are also positive to SARS-CoV-2 genomic RNA (green). Cells were counterstained with hematoxylin. C57BL/6 mice do not express the transgene.
    Figure Legend Snippet: hACE2 transgene expression in neurons of SARS-CoV-2 infected mice. Representative ISH staining showing the presence of the hACE2 transgene (red) in infected K18-hACE2 mice. Duplex ISH staining further shows hACE2-exressping neurons are also positive to SARS-CoV-2 genomic RNA (green). Cells were counterstained with hematoxylin. C57BL/6 mice do not express the transgene.

    Techniques Used: Expressing, Infection, Mouse Assay, In Situ Hybridization, Staining

    Infiltrating cells in the lungs of SARS-CoV-2 infected mice. A–C. IFA demonstrates increased number of myeloperoxidase (MPO)+ polymorphonuclear cells (neutrophils, eosinophils, and basophils) (A, red), CD68+ macrophages (B, red) CD45+ leukocytes (C, red) including CD3+ T cells (C, green) infiltrates in the lung of infected mice in comparison with the lung of uninfected mice. MPO positive cells (red) were devoid of viral NP protein (A, green). Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: Infiltrating cells in the lungs of SARS-CoV-2 infected mice. A–C. IFA demonstrates increased number of myeloperoxidase (MPO)+ polymorphonuclear cells (neutrophils, eosinophils, and basophils) (A, red), CD68+ macrophages (B, red) CD45+ leukocytes (C, red) including CD3+ T cells (C, green) infiltrates in the lung of infected mice in comparison with the lung of uninfected mice. MPO positive cells (red) were devoid of viral NP protein (A, green). Nuclei are stained with DAPI (blue).

    Techniques Used: Infection, Mouse Assay, Immunofluorescence, Staining

    SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. A. Representative H E staining of lungs in infected C57BL/6 mice (i) or K18-hACE2 infected mice (ii-iv). Numerous fibrin thrombi (black arrows) filling the lumen of small to intermediate sized vessels (ii) adjacent to a normal bronchus and surrounded by minimally inflamed, congested and collapsed alveolar septa. Extensive area of lung consolidation (iii) with inflammation/expansion of alveolar septa, exudation of fibrin and edema into alveolar lumina and infiltration of vessel walls and perivascular area by numerous mononuclear inflammatory cells (arrows), disrupting/obscuring vessel architecture (vasculitis). Extensive area of consolidated lung (iv) showing type II pneumocyte hyperplasia (arrowhead) and rare multinucleate cells (black arrow), B . H E and ISH staining of infected mouse lung showing vasculitis with absence of viral RNA in the affected vessel walls; note there is viral RNA (red) in the adjacent alveolar septa. Vessels are highlighted by the broken black circles. C . TUNEL staining of infected and uninfected K18-hACE2 mouse lungs. TUNEL (green) was performed as indicted in the materials and methods. Cell nuclei are stained with DAPI (blue). D . Ki-67 staining (red) in infected and uninfected K18-hACE2 mouse lungs. Nuclei are stained with DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. A. Representative H E staining of lungs in infected C57BL/6 mice (i) or K18-hACE2 infected mice (ii-iv). Numerous fibrin thrombi (black arrows) filling the lumen of small to intermediate sized vessels (ii) adjacent to a normal bronchus and surrounded by minimally inflamed, congested and collapsed alveolar septa. Extensive area of lung consolidation (iii) with inflammation/expansion of alveolar septa, exudation of fibrin and edema into alveolar lumina and infiltration of vessel walls and perivascular area by numerous mononuclear inflammatory cells (arrows), disrupting/obscuring vessel architecture (vasculitis). Extensive area of consolidated lung (iv) showing type II pneumocyte hyperplasia (arrowhead) and rare multinucleate cells (black arrow), B . H E and ISH staining of infected mouse lung showing vasculitis with absence of viral RNA in the affected vessel walls; note there is viral RNA (red) in the adjacent alveolar septa. Vessels are highlighted by the broken black circles. C . TUNEL staining of infected and uninfected K18-hACE2 mouse lungs. TUNEL (green) was performed as indicted in the materials and methods. Cell nuclei are stained with DAPI (blue). D . Ki-67 staining (red) in infected and uninfected K18-hACE2 mouse lungs. Nuclei are stained with DAPI (blue).

    Techniques Used: Infection, Mouse Assay, Staining, In Situ Hybridization, TUNEL Assay

    Infection of SARS-CoV-2 in the lungs in K18-Ace2 transgenic mice. Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected K18-hACE2 mice. ISH was performed in a different mouse than that in Fig. 2 . Cells were counterstained with hematoxylin.
    Figure Legend Snippet: Infection of SARS-CoV-2 in the lungs in K18-Ace2 transgenic mice. Representative ISH images showing the presence of SARS-CoV-2 RNA (red) in the lungs of infected K18-hACE2 mice. ISH was performed in a different mouse than that in Fig. 2 . Cells were counterstained with hematoxylin.

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, In Situ Hybridization

    Transcriptional activation in SARS-CoV-2 infected lungs. Transcriptional activation in lung homogenates from infected K18-hACE2 (male and female) and C57BL/6 (female) mice were examined by NanoString. A. Log 2 fold changes in gene expression levels of selected genes categorized by group versus infected C57BL/6 mice were graphed with SD. All graphed transcripts had a p value of
    Figure Legend Snippet: Transcriptional activation in SARS-CoV-2 infected lungs. Transcriptional activation in lung homogenates from infected K18-hACE2 (male and female) and C57BL/6 (female) mice were examined by NanoString. A. Log 2 fold changes in gene expression levels of selected genes categorized by group versus infected C57BL/6 mice were graphed with SD. All graphed transcripts had a p value of

    Techniques Used: Activation Assay, Infection, Mouse Assay, Expressing

    SARS-CoV-2 infection in K18-hACE2 transgenic mice. A. Male and Female K18-hACE2 transgenic mice (Day 0-3 n=7/group; Day 3+, n=5/group) were infected with 2×10 4 PFU or 2 × 10 3 PFU of SARS-CoV-2 by the IN route. C57BL/6 and RAG2 KO mice (Day 0-3 n=8/ group; Day 3+, n=5/group) were infected with 2×10 4 pfu by the IN route. Survival and weight loss (+/− SEM) were monitored and plotted using Prism software. B . Titers in lung (n=2 mice/group) were examined on day 3 by qRT-PCR. Mean titers +/− SEM of the genome molecules of viral RNA/ml were graphed. C. Titers in lungs of individual K18-hACE2 mice. Numbers above bars denote day of death. Colors represent the four groups. D. Monocyte chemoattractants and inflammatory cytokines were measured from the serum of SARS-CoV-3 infected mice on day 3 or at the time of euthanasia using a multiplex system. Mice from each group are aggregated from samples taken on day 3 (blue symbols) and when mice were euthanized (black symbols).
    Figure Legend Snippet: SARS-CoV-2 infection in K18-hACE2 transgenic mice. A. Male and Female K18-hACE2 transgenic mice (Day 0-3 n=7/group; Day 3+, n=5/group) were infected with 2×10 4 PFU or 2 × 10 3 PFU of SARS-CoV-2 by the IN route. C57BL/6 and RAG2 KO mice (Day 0-3 n=8/ group; Day 3+, n=5/group) were infected with 2×10 4 pfu by the IN route. Survival and weight loss (+/− SEM) were monitored and plotted using Prism software. B . Titers in lung (n=2 mice/group) were examined on day 3 by qRT-PCR. Mean titers +/− SEM of the genome molecules of viral RNA/ml were graphed. C. Titers in lungs of individual K18-hACE2 mice. Numbers above bars denote day of death. Colors represent the four groups. D. Monocyte chemoattractants and inflammatory cytokines were measured from the serum of SARS-CoV-3 infected mice on day 3 or at the time of euthanasia using a multiplex system. Mice from each group are aggregated from samples taken on day 3 (blue symbols) and when mice were euthanized (black symbols).

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Software, Quantitative RT-PCR, Multiplex Assay

    SARS-CoV-2 infection in K18-hAce2 transgenic mice. A . Individual weight loss in each challenge group is shown up to day 15. Red indicates an animal that died or was euthanized. B . Maximal weight loss over 15 day in infected mice. Red indicates mice that succumbed to disease. C . Titers in liver and spleen (n=2 mice/group) were examined on day 3 by qRT-PCR. Mean titers +/− SEM of the genome copies/ml were graphed.
    Figure Legend Snippet: SARS-CoV-2 infection in K18-hAce2 transgenic mice. A . Individual weight loss in each challenge group is shown up to day 15. Red indicates an animal that died or was euthanized. B . Maximal weight loss over 15 day in infected mice. Red indicates mice that succumbed to disease. C . Titers in liver and spleen (n=2 mice/group) were examined on day 3 by qRT-PCR. Mean titers +/− SEM of the genome copies/ml were graphed.

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Quantitative RT-PCR

    Infection of the nasal cavity and olfactory bulb. A. ISH labeling for SARS-CoV-2 RNA in a coronal section of the head including the caudal aspect of the nasal cavity and olfactory bulb. Within the olfactory bulb, a strong positive signal is present in the glomerular layer, external plexiform layer, mitral cell layer and internal plexiform layer, with multifocal positivity in the granular cell layer in the olfactory bulb hemisphere at right. Low numbers of cells within the olfactory epithelium lining the dorsal nasal meatus have a positive ISH signal (arrows). Cells were counterstained with hematoxylin (blue). B . ISH labeling for SARS-CoV-2 RNA in the olfactory epithelium. Cells were counterstained with hematoxylin (blue). C . IFA of olfactory epithelium stained for SARS-CoV-2 spike (green) and Pan-cytokeratin (red). Nuclei were stained with DAPI (blue). D. Representative H E staining of the nasal cavity including olfactory epithelium and olfactory bulb from uninfected or infected K18-hACE2 mice. In the infected mouse there is a focal area of olfactory epithelium atrophy on a nasal turbinate located in the lateral nasal meatus. Inset image shows the indicated area of detail under increased magnification.
    Figure Legend Snippet: Infection of the nasal cavity and olfactory bulb. A. ISH labeling for SARS-CoV-2 RNA in a coronal section of the head including the caudal aspect of the nasal cavity and olfactory bulb. Within the olfactory bulb, a strong positive signal is present in the glomerular layer, external plexiform layer, mitral cell layer and internal plexiform layer, with multifocal positivity in the granular cell layer in the olfactory bulb hemisphere at right. Low numbers of cells within the olfactory epithelium lining the dorsal nasal meatus have a positive ISH signal (arrows). Cells were counterstained with hematoxylin (blue). B . ISH labeling for SARS-CoV-2 RNA in the olfactory epithelium. Cells were counterstained with hematoxylin (blue). C . IFA of olfactory epithelium stained for SARS-CoV-2 spike (green) and Pan-cytokeratin (red). Nuclei were stained with DAPI (blue). D. Representative H E staining of the nasal cavity including olfactory epithelium and olfactory bulb from uninfected or infected K18-hACE2 mice. In the infected mouse there is a focal area of olfactory epithelium atrophy on a nasal turbinate located in the lateral nasal meatus. Inset image shows the indicated area of detail under increased magnification.

    Techniques Used: Infection, In Situ Hybridization, Labeling, Immunofluorescence, Staining, Mouse Assay

    SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. Representative H E staining of lungs in infected K18-ACE2 mice. Edema, moderate numbers of mononuclear inflammatory cells, and fewer neutrophils expand the perivascular space surrounding an intermediate sized artery in the lung (i). Area of lung consolidation with inflammation/expansion of alveolar septa by fibrin, edema and mononuclear inflammatory cells; adjacent alveolar lumina are correspondingly filled with fibrin, edema and increased numbers of alveolar macrophages (ii).
    Figure Legend Snippet: SARS-CoV-2 infection causes respiratory damage in K18-hACE2 mice. Representative H E staining of lungs in infected K18-ACE2 mice. Edema, moderate numbers of mononuclear inflammatory cells, and fewer neutrophils expand the perivascular space surrounding an intermediate sized artery in the lung (i). Area of lung consolidation with inflammation/expansion of alveolar septa by fibrin, edema and mononuclear inflammatory cells; adjacent alveolar lumina are correspondingly filled with fibrin, edema and increased numbers of alveolar macrophages (ii).

    Techniques Used: Infection, Mouse Assay, Staining

    Neuropathogenesis of SARS-CoV-2 in K18-ACE2 transgenic and non-transgenic mice. A. ISH detection of for SARS-CoV-2 RNA in uninfected (i) and infected mice (ii and iii) in a coronal section of brain demonstrating a strong positive signal within neurons of thalamic nuclei. The boxed (ii) area is shown at increased magnification in the right panel (iii). Note the absence of a positive signal from the vessel at center right (iii) where the vessel wall and perivascular space are infiltrated by mononuclear inflammatory cells. B. Representative H E staining of uninfected (i) or infected (ii-iv) K18-hACE2 mice. Perivascular hemorrhage extending into the adjacent neuropil (ii), in the region of the rostral cerebral cortex. Two small caliber vessels (iii) in the thalamus with fibrin thrombi (arrows), with mild microgliosis and some perivascular hemorrhage (arrowhead). The walls of small to intermediate size vessels and perivascular spaces (iv) are multifocally expanded/obscured by mononuclear inflammatory cells and increased numbers of glial cells C. Detection of Iba-1 and GFAP, markers for microgliosis and astrogliosis, respectively, in uninfected or infected brain sections using IFA. Nuclei were stained with DAPI (blue). D. Co-staining for astrocyte marker GFAP (red) or neuron marker NeuN (red) with SARS-CoV-2 spike protein (green). Spike protein was predominantly detected in NeuN + neurons. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Neuropathogenesis of SARS-CoV-2 in K18-ACE2 transgenic and non-transgenic mice. A. ISH detection of for SARS-CoV-2 RNA in uninfected (i) and infected mice (ii and iii) in a coronal section of brain demonstrating a strong positive signal within neurons of thalamic nuclei. The boxed (ii) area is shown at increased magnification in the right panel (iii). Note the absence of a positive signal from the vessel at center right (iii) where the vessel wall and perivascular space are infiltrated by mononuclear inflammatory cells. B. Representative H E staining of uninfected (i) or infected (ii-iv) K18-hACE2 mice. Perivascular hemorrhage extending into the adjacent neuropil (ii), in the region of the rostral cerebral cortex. Two small caliber vessels (iii) in the thalamus with fibrin thrombi (arrows), with mild microgliosis and some perivascular hemorrhage (arrowhead). The walls of small to intermediate size vessels and perivascular spaces (iv) are multifocally expanded/obscured by mononuclear inflammatory cells and increased numbers of glial cells C. Detection of Iba-1 and GFAP, markers for microgliosis and astrogliosis, respectively, in uninfected or infected brain sections using IFA. Nuclei were stained with DAPI (blue). D. Co-staining for astrocyte marker GFAP (red) or neuron marker NeuN (red) with SARS-CoV-2 spike protein (green). Spike protein was predominantly detected in NeuN + neurons. Nuclei were stained with DAPI (blue).

    Techniques Used: Transgenic Assay, Mouse Assay, In Situ Hybridization, Infection, Staining, Immunofluorescence, Marker

    9) Product Images from "Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein"

    Article Title: Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein

    Journal: Stem Cell Reviews and Reports

    doi: 10.1007/s12015-020-10056-z

    Cord blood HSCs/HPCs exhibit reduced colony forming capacity in the presence of SARS-CoV-2 S protein. ( a ) CD34+ enriched cells were plated at 100,000 cells/mL in media with stimulating growth factors (rhuTPO/rhuSCF/rhuFLT3L) and with 1 μg/mL recombinant S protein or PBS control and grown for 4 days in 5% O 2 and 5% CO 2 at 37 °C. Viable cells were counted using a hemocytometer and Trypan Blue viability stain. n = 5, stats: paired t-test, different symbols indicate the same cord blood unit in different treatment conditions. ( b-c ) CD34+ enriched cells were either taken for direct plating (unstimulated) or were grown for 24 h with stimulating growth factors (stimulated). 300 CD34+ enriched cells were plated in triplicate with the indicated doses of recombinant S protein or PBS control in 1% v /v methylcellulose with growth factors and serum and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. CFU/1x10 6 cells were calculated. Stats: general linearized modeling including stimulated and unstimulated cells at varying doses in the same model followed by ANOVA with TukeyHSD post hoc tests. Significance codes indicate comparison of sample to 0 ng/mL (PBS) control in unstimulated cells. ( d-e ) 350 freshly harvested CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and with PBS control, 250 ng/mL recombinant S protein alone, 250 ng/mL S protein pre-incubated with 250 ng/mL SARS-CoV-2 neutralizing antibody (Antibody), or 250 ng/mL S protein with 250 ng/mL Angiotensin1–7 (Ang1–7) and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU/1x10 6 cells were calculated. Stats: general linearized modeling followed by ANOVA with TukeyHSD post hoc tests, matched symbols indicate the same cord blood unit in different treatment conditions. Shown are significance codes comparing all treatment levels to PBS control. * P
    Figure Legend Snippet: Cord blood HSCs/HPCs exhibit reduced colony forming capacity in the presence of SARS-CoV-2 S protein. ( a ) CD34+ enriched cells were plated at 100,000 cells/mL in media with stimulating growth factors (rhuTPO/rhuSCF/rhuFLT3L) and with 1 μg/mL recombinant S protein or PBS control and grown for 4 days in 5% O 2 and 5% CO 2 at 37 °C. Viable cells were counted using a hemocytometer and Trypan Blue viability stain. n = 5, stats: paired t-test, different symbols indicate the same cord blood unit in different treatment conditions. ( b-c ) CD34+ enriched cells were either taken for direct plating (unstimulated) or were grown for 24 h with stimulating growth factors (stimulated). 300 CD34+ enriched cells were plated in triplicate with the indicated doses of recombinant S protein or PBS control in 1% v /v methylcellulose with growth factors and serum and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. CFU/1x10 6 cells were calculated. Stats: general linearized modeling including stimulated and unstimulated cells at varying doses in the same model followed by ANOVA with TukeyHSD post hoc tests. Significance codes indicate comparison of sample to 0 ng/mL (PBS) control in unstimulated cells. ( d-e ) 350 freshly harvested CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and with PBS control, 250 ng/mL recombinant S protein alone, 250 ng/mL S protein pre-incubated with 250 ng/mL SARS-CoV-2 neutralizing antibody (Antibody), or 250 ng/mL S protein with 250 ng/mL Angiotensin1–7 (Ang1–7) and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU/1x10 6 cells were calculated. Stats: general linearized modeling followed by ANOVA with TukeyHSD post hoc tests, matched symbols indicate the same cord blood unit in different treatment conditions. Shown are significance codes comparing all treatment levels to PBS control. * P

    Techniques Used: Recombinant, Staining, Incubation

    Cord blood HSCs/HPCs exhibit reduced expansion in the presence of SARS-CoV-2 S protein. ( a-h ) CD34+ enriched cells were plated at 100,000–200,000 cells/mL in media with stimulating growth factors and with PBS control, 1 μg/mL recombinant S protein alone, 1 μg/mL S protein pre-incubated with 1 μg/mL SARS-CoV-2 neutralizing antibody (Antibody), 1 μg/mL S protein pre-incubated with 1 μg/mL rhu ACE2, or 1 μg/mL S protein with 1 μg/mL Angiotensin1–7 (Ang1–7) and grown for 7 days in 5% O 2 and 5% CO 2 at 37 °C. ( a-d ). Cells were then analyzed by flow cytometry for the indicated cell populations and total cell numbers were calculated or ( e-h ) 350–500 CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU were calculated. n = 4/2 for ( a-e )/( f-h ), stats: generalized linear modelling followed by ANOVA with TukeyHSD post hoc tests, matched colors of points for 3a-e and matched symbols for points for 3f-h indicate the same cord blood unit in different treatment conditions. For ( f-h ) significance codes shown are for the comparison of the indicated treatment PBS control. * P
    Figure Legend Snippet: Cord blood HSCs/HPCs exhibit reduced expansion in the presence of SARS-CoV-2 S protein. ( a-h ) CD34+ enriched cells were plated at 100,000–200,000 cells/mL in media with stimulating growth factors and with PBS control, 1 μg/mL recombinant S protein alone, 1 μg/mL S protein pre-incubated with 1 μg/mL SARS-CoV-2 neutralizing antibody (Antibody), 1 μg/mL S protein pre-incubated with 1 μg/mL rhu ACE2, or 1 μg/mL S protein with 1 μg/mL Angiotensin1–7 (Ang1–7) and grown for 7 days in 5% O 2 and 5% CO 2 at 37 °C. ( a-d ). Cells were then analyzed by flow cytometry for the indicated cell populations and total cell numbers were calculated or ( e-h ) 350–500 CD34+ cells were plated in triplicate in 1% v/v methylcellulose with serum and growth factors and grown for 12 days in 5% O 2 and 5% CO 2 at 37 °C. Total CFU were calculated. n = 4/2 for ( a-e )/( f-h ), stats: generalized linear modelling followed by ANOVA with TukeyHSD post hoc tests, matched colors of points for 3a-e and matched symbols for points for 3f-h indicate the same cord blood unit in different treatment conditions. For ( f-h ) significance codes shown are for the comparison of the indicated treatment PBS control. * P

    Techniques Used: Recombinant, Incubation, Flow Cytometry

    Peripheral blood cells respond ex vivo to exposure to SARS-CoV-2 S protein. ( a-d ) Low density PB was incubated for 2 h with 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( a ) Representative histogram from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes and ( b ) mean fluorescence intensities for CD14 staining. n = 4, stats: paired t-test, different colors of points indicate PBs from the same donor. ( c ) Representative contour plot from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes, the gating strategy for CD14hi cells and ( d ) difference in CD14hi monocytes represented as a fold-change of S protein treated cells relative to PBS treated cells. n = 4, stats: paired t-test performed on total numbers of CD14hi monocytes. ( e-g ) Low density PB was incubated for 18 h with serum in the presence of 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( e ) Representative contour plot from 1 PB sample showing all cells excluding debris and the monocyte gate used. ( f ) Mean values for forward scatter (FSC) and ( g ) side scatter (SSC) of monocytes from PB samples after incubation with S protein or PBS. n = 5, stats = paired t-test, different colors of points indicate PBs from the same donor. FACS = fluorescence activated flow cytometry. *P
    Figure Legend Snippet: Peripheral blood cells respond ex vivo to exposure to SARS-CoV-2 S protein. ( a-d ) Low density PB was incubated for 2 h with 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( a ) Representative histogram from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes and ( b ) mean fluorescence intensities for CD14 staining. n = 4, stats: paired t-test, different colors of points indicate PBs from the same donor. ( c ) Representative contour plot from 1 PB sample treated with S protein or PBS showing CD14 expression of forward and side scatter gated monocytes, the gating strategy for CD14hi cells and ( d ) difference in CD14hi monocytes represented as a fold-change of S protein treated cells relative to PBS treated cells. n = 4, stats: paired t-test performed on total numbers of CD14hi monocytes. ( e-g ) Low density PB was incubated for 18 h with serum in the presence of 1 μg/mL SARS-CoV-2 S recombinant S protein or PBS control. ( e ) Representative contour plot from 1 PB sample showing all cells excluding debris and the monocyte gate used. ( f ) Mean values for forward scatter (FSC) and ( g ) side scatter (SSC) of monocytes from PB samples after incubation with S protein or PBS. n = 5, stats = paired t-test, different colors of points indicate PBs from the same donor. FACS = fluorescence activated flow cytometry. *P

    Techniques Used: Ex Vivo, Incubation, Recombinant, Expressing, Fluorescence, Staining, FACS, Flow Cytometry

    10) Product Images from "A novel cell culture system modeling the SARS-CoV-2 life cycle"

    Article Title: A novel cell culture system modeling the SARS-CoV-2 life cycle

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009439

    Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n = 6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n = 3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d. = not detected; n.a. = not applicable.
    Figure Legend Snippet: Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n = 6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n = 3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d. = not detected; n.a. = not applicable.

    Techniques Used: Inhibition, Recombinant, Infection, Flow Cytometry, Fluorescence, Viability Assay, Concentration Assay, Software, Activity Assay

    High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d. = not detected; n.a. = not applicable.
    Figure Legend Snippet: High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d. = not detected; n.a. = not applicable.

    Techniques Used: High Throughput Screening Assay, Infection, Selection, Activity Assay, Generated, Software

    Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of virus derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.
    Figure Legend Snippet: Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of virus derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, RNA Sequencing Assay, Derivative Assay, Expressing

    Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.
    Figure Legend Snippet: Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.

    Techniques Used: Polymerase Chain Reaction, Purification, In Vitro, Western Blot, Expressing

    Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P
    Figure Legend Snippet: Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P

    Techniques Used: Transduction, Cell Culture, Expressing, Flow Cytometry, Microscopy, Quantitative RT-PCR, Western Blot, Infection, Glo Assay

    The recombinant SARS-CoV-2-GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n = 6). ***, P
    Figure Legend Snippet: The recombinant SARS-CoV-2-GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n = 6). ***, P

    Techniques Used: Recombinant, Infection, Incubation, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Expressing, SDS Page, Reverse Transcription Polymerase Chain Reaction, Marker

    Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n = 4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P
    Figure Legend Snippet: Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n = 4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P

    Techniques Used: Functional Assay, Western Blot, Transduction, Fluorescence, Infection, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR

    11) Product Images from "Distinct conformational states of SARS-CoV-2 spike protein"

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.05.16.099317

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.
    Figure Legend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Techniques Used: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Techniques Used:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.
    Figure Legend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Techniques Used: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.
    Figure Legend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Techniques Used: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.
    Figure Legend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Techniques Used: Binding Assay

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008762

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

    Techniques Used: Binding Assay

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

    Techniques Used: Binding Assay

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

    Techniques Used: Electron Microscopy

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

    Techniques Used:

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

    Techniques Used: Transmission Assay

    13) Product Images from "A novel cell culture system modeling the SARS-CoV-2 life cycle"

    Article Title: A novel cell culture system modeling the SARS-CoV-2 life cycle

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422469

    Generation of Caco-2 cell expressing SARS-CoV-2 N by lentiviral transduction. (A) Scheme depicting the bicistronic lentiviral constructs for expressing SARS-CoV-2 N protein with C-terminal Flag tag. (B) Representative flow cytometry plots demonstrating efficient lentivirus transduction. Caco-2 cells were transduced with pLVX-N-Flag-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of N-expressing cells. The flow cytometry result was representative of one of three independent experiments.
    Figure Legend Snippet: Generation of Caco-2 cell expressing SARS-CoV-2 N by lentiviral transduction. (A) Scheme depicting the bicistronic lentiviral constructs for expressing SARS-CoV-2 N protein with C-terminal Flag tag. (B) Representative flow cytometry plots demonstrating efficient lentivirus transduction. Caco-2 cells were transduced with pLVX-N-Flag-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of N-expressing cells. The flow cytometry result was representative of one of three independent experiments.

    Techniques Used: Expressing, Transduction, Construct, FLAG-tag, Flow Cytometry

    The recombinant SARS-CoV-2 GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n=6). ***, P
    Figure Legend Snippet: The recombinant SARS-CoV-2 GFP/ΔN trVLP can propagate with the help of viral N protein. (A) Experimental scheme. Caco-2 or Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN for 3h (MOI 0.05), washed, and incubated for an additional 72 h. GFP fluorescence were observed or quantified by microscopy or flow cytometry analysis. Viral RNA was determined by RT-qPCR assay; (B) GFP expression was observed in Caco-2 or Caco-2-N cells using microscopy at indicated time point after inoculation; Representative images from one of three independent experiments. (C) Cell lysates were resolved by SDS-PAGE and probed with anti-Spike and anti-Tubulin antibodies. Representative images from two independent experiments; (D) The total RNAs were extracted and RT-qPCR assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate; (E) RT-PCR analysis of the SARS-CoV-2 GFP/ΔN genome in Caco-2-N cells infected with recombinant virus using a primer set flanking the N region. The expected DNA sized were indicated in each genome, and DNA marker is shown on the left. Representative images from one of two independent experiments; (F-G) Recombinant SARS-CoV-2 GFP/ΔN virus was incubated with indicated doses of neutralizing mAbs against SARS-CoV-2 (1F11 and 2F6) or HIV (VCR01), as well as soluble human ACE2-Fc or F10sFV for 1 h prior to inoculation. The infection was analyzed by GFP expression 2 days later, and the number of positive cells was expressed as a percentage of that for the VRC01 or F10sFV treatment control. Error bars represent the standard deviations from three independent experiments (n=6). ***, P

    Techniques Used: Recombinant, Infection, Incubation, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Expressing, SDS Page, Reverse Transcription Polymerase Chain Reaction, Marker

    High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.
    Figure Legend Snippet: High throughput screening of antivirals against SARS-CoV-2 infection using trVLP system. (A) Screening of 377 compounds from Topscience Natural Product Library and hits selection. The purple dot line represents the threshold (40%) for positive hit compounds. DMSO (blue) and remdesivir (red) are used as the control for the screening. Each dot represents a single compound, and the green dots represent the promising candidates which exhibited potent antiviral activity without dramatic cytotoxic effect. (B-F) Dose response curves of selected hit compounds. Compounds concentrations are presented in log scale for logarithmic interpolation. Dose response curves were generated using GraphPad Prism software version 7.0. IC 50 values were calculated using Prism software and is representative of one of three independent experiments. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. (G) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.

    Techniques Used: High Throughput Screening Assay, Infection, Selection, Activity Assay, Generated, Software

    Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of viruse derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.
    Figure Legend Snippet: Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN trVLP. (A) Detection of the GFP reporter gene during viral passage. RNAs were extracted from the VLP infected cells of P0 to P10 passage, respectively. (B) RT-PCR was performed with a primer pair flanking the N region of ORF8 and 3’UTR. The PCR products were resolved on an agarose gel using electrophoresis. The numbers of time points-samples-passage were denoted on the top of each lane. Representative images from one of three independent experiments; (C-D) RNA-seq coverage of viruse derived reads aligned to SARS-CoV-2 (C) or SARS-CoV-2 GFP/ΔN (D) genome, respectively. (E) Heatmap shows the expression levels of each subgenomic RNA of P1 or P10 trVLP.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, RNA Sequencing Assay, Derivative Assay, Expressing

    Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P
    Figure Legend Snippet: Site-specific phosphorylation of N is required to support virus life cycle. (A) Schematics and alignments of N proteins from MERS-CoV, SARS-CoV and SARS-CoV-2. The phosphorylation sites in SARS-CoV-2 N protein were highlighted. (B) Schematic presentation of assessment of N variants function. The trVLP inoculated with Caco-2 cells transduced with N variants, and the cell culture medium were collected to infect the Caco-2-N cells, and GFP expression analyzed by flow cytometry/microscopy or viral subgenomic RNA abundance were determined by RT-qPCR. (C) Western blotting assay was performed to detect the N proteins expression in Caco-2 cells transduced with distinct N genes from SARS-CoV-2, SARS-CoV or MERS-CoV. (D-E) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing N from SARS-CoV-2, SARS-CoV or MERS-CoV to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (F) Western blotting assay detected the expression of SARS-CoV-2 N WT or mutants in Caco-2 cells. (G-H) The cell culture medium was collected from SARS-CoV-2 GFP/ΔN trVLP infected Caco-2 cells expressing SARS-CoV-2 N mutants to infect the naïve Caco-2-N cells. GFP were observed using microscopy and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. (I) GSK-3 inhibitors LiCl or SB216763 treated Caco-2-N cells inoculated with SARS-CoV-2 GFP/ΔN trVLP, the cell culture medium was then inoculated with Caco-2-N cells. RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Cell viability was evaluated by CellTiter-Glo assay. Error bars (E, H and I) represent the standard deviations from one of three independent experiments performed in triplicate. n.s. no significance; *, P

    Techniques Used: Transduction, Cell Culture, Expressing, Flow Cytometry, Microscopy, Quantitative RT-PCR, Western Blot, Infection, Glo Assay

    GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. (A) GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. Caco-2-N cells were electroporated with 20 μg of SARS-CoV-2 GFP/ΔN RNA. From 21h-96h p.t., GFP expression in the cells was observed with microscopy. (B) GFP expression was quantified by flowcytometry at 96h post transfection of the RNA. This experiment was representative of three independent experiments.
    Figure Legend Snippet: GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. (A) GFP expression in Caco-2-N cells electroporated with SARS-CoV-2 GFP/ΔN RNA. Caco-2-N cells were electroporated with 20 μg of SARS-CoV-2 GFP/ΔN RNA. From 21h-96h p.t., GFP expression in the cells was observed with microscopy. (B) GFP expression was quantified by flowcytometry at 96h post transfection of the RNA. This experiment was representative of three independent experiments.

    Techniques Used: Expressing, Microscopy, Transfection

    Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n=4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P
    Figure Legend Snippet: Reconstitution of functional N protein by intein-mediated protein splicing. (A) Scheme depicting of intein-mediated protein trans-splicing to reconstitute full length N protein. (B) Western blot (WB) analysis of lysates from Caco-2 cells transduced with either full-length N or intein-N lentiviruses. The star indicates the full-length N protein. The WB is representative of three independent experiments. (C) GFP fluorescence in Caco-2-N cells infected cell culture medium (containing SARS-CoV-2 GFP/ΔN progeny) collected from each Caco-2-N int cells which was inoculated with SARS-CoV-2 GFP/ΔN trVLP at 2 days of culture. The image is representative of n=4. (D) Cells were harvested to quantify GFP expression by flow cytometry analysis, and (E) Subgenomic RNA of E were determined by RT-qPCR assay. Error bars represent the standard deviations from one of three independent experiments performed in triplicate. *, P

    Techniques Used: Functional Assay, Western Blot, Transduction, Fluorescence, Infection, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR

    Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.
    Figure Legend Snippet: Production of SARS-CoV-2 GFP/ΔN trVLP. (A) The top rows show genetic organizations of the SARS-CoV-2 and SARS-CoV-2 GFP/ΔN genomes. The ORF of N is replaced with reporter gene (GFP here). The cDNA of SARS-CoV-2 GFP/ΔN genome was divided into five fragments designated as Fragment A, B, C, D and E, which could be obtained by PCR (B). Each cDNA fragment was flanked by a class IIS restriction endonuclease site (BsaI or BsmBI) and the nucleotide sequences and locations of the cohesive overhangs are indicated. The fragment cDNA were digested and purified for directed assembly of SARS-CoV-2 GFP/ΔN cDNA (see C panel, and the star indicates the genome-length cDNA), which served as the template for in vitro transcription to generate viral RNA genome (see D panel, and the star indicates the genome-length RNA transcript). The viral genomic RNAs were electroporated into Caco-2-N cells. After 3 days, the supernatant was collected and inoculated with Caco-2 or Caco-2-N cells. (E). Western blotting assay was performed to detect the expression of N proteins and ACE2 in Caco-2-N cells, Vero E6 and Vero E6-N cells.

    Techniques Used: Polymerase Chain Reaction, Purification, In Vitro, Western Blot, Expressing

    Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN virus. (A) RT-PCR products from P10 virus infected cell passage were cloned into pEASY-Blunt vector, and 12 colonies were randomly chosen for DNA sequences analysis. Multiple deletions were detected in the amplicon. (B) Categories of mapped reads from P1 and P10 virus infected Caco-2-N cells. (C) Canonical discontinuous transcription (top) that is mediated by TRS-L (TRS in the leader) and TRS-B (TRS in the body). Quantification of junction-reads from canonical discontinuous transcripts post P1 and P10 virus infection.
    Figure Legend Snippet: Characterization of the genetic stability of SARS-CoV-2 GFP/ΔN virus. (A) RT-PCR products from P10 virus infected cell passage were cloned into pEASY-Blunt vector, and 12 colonies were randomly chosen for DNA sequences analysis. Multiple deletions were detected in the amplicon. (B) Categories of mapped reads from P1 and P10 virus infected Caco-2-N cells. (C) Canonical discontinuous transcription (top) that is mediated by TRS-L (TRS in the leader) and TRS-B (TRS in the body). Quantification of junction-reads from canonical discontinuous transcripts post P1 and P10 virus infection.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Clone Assay, Plasmid Preparation, Amplification

    Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n=6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n=3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.
    Figure Legend Snippet: Inhibition of recombinant SARS-CoV-2 GFP/ΔN trVLP infection by IFN and antivirals. (A) IFN-β pretreated Caco-2-N int cells were subsequently infected with trVLP and cells were subjected to flow cytometry analysis for quantify the GFP fluorescence at 2 days post-infection. Error bars represent the standard deviations from three independent experiments (n=6). (B-E) Antiviral effect of remdesivir, GC376, lopinavir and ritonavir. The drug treated cells were infected with trVLP and GFP fluorescence was quantified at 48h after infection. The cytotoxic effect of each drug at indicated concentrations were determined by CellTiter-Glo cell viability assay. The virus infection or cytotoxicity is plotted versus compound concentration (n=3 biological replicates for all compounds). The black dots indicate replicate measurements, and the black lines indicate dose-response curve fits. The red dots indicate cytotoxicity. IC 50 values were calculated using Prism software and is representative of one of three independent experiments performed in triplicate. Three independent experiments had similar results. (F) Comparison of antiviral activity and cytotoxicity of each compound. Selectivity Index (SI), a ratio that compares a drug’s cytotoxicity and antiviral activity was also calculated. n.d.=not detected; n.a.=not applicable.

    Techniques Used: Inhibition, Recombinant, Infection, Flow Cytometry, Fluorescence, Viability Assay, Concentration Assay, Software, Activity Assay

    Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.
    Figure Legend Snippet: Identification of host factors associated with N protein and phosphorylation on N protein by mass spectrometry. (A) Flag tagged N protein was immunoprecipitated from Caco-2-N cells infected with recombinant SARS-CoV-2 GFP/ΔN trVLP using Flag antibody, and the proteins were analyzed on SDS-PAGE gel. The proteins were visualized by Coomassie blue staining. N, G3BP1 and G3BP2 were labelled. (B) Phosphorylated peptides of N protein derived from Caco-2-N cells. Caco-2-N cells in which N was C-terminal Flag-tagged were collected and cell lysates were immunoprecipitated with anti-Flag coupled beads. Phosphorylated peptides of the immunoprecipitates were analyzed by mass spectrometry.

    Techniques Used: Mass Spectrometry, Immunoprecipitation, Infection, Recombinant, SDS Page, Staining, Derivative Assay

    14) Product Images from "Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody"

    Article Title: Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody

    Journal: bioRxiv

    doi: 10.1101/2020.12.21.423787

    (a) The FTIR scan of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle), exclusively associated with the proteins, but not found in the PBS solvent, attributed to the possible Van der Waals bonding between the large pendant group such as phenyl. (b) Similar FTIR result of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (c) Similar FTIR result of SARS-CoV-2 spike protein alone, obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (d) The infrared absorption of 750cm -1 under variable temperature of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1), and the spike protein alone, obtained from the difference between the peak near 750cm -1 and the trough near 725cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation.
    Figure Legend Snippet: (a) The FTIR scan of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle), exclusively associated with the proteins, but not found in the PBS solvent, attributed to the possible Van der Waals bonding between the large pendant group such as phenyl. (b) Similar FTIR result of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (c) Similar FTIR result of SARS-CoV-2 spike protein alone, obtained from more concentrated samples, showing a visible 750 cm -1 bands at 27-29°C (red circle). (d) The infrared absorption of 750cm -1 under variable temperature of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2), SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1), and the spike protein alone, obtained from the difference between the peak near 750cm -1 and the trough near 725cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation.

    Techniques Used:

    (a) The FTIR scan of SARS-CoV-2 spike protein (S protein) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (b) The FTIR scan of SARS-CoV-2 antibody (antibody 2) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (c) The FTIR scan of SARS-CoV-1 antibody (antibody 1) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (d) The infrared absorption of 1550cm -1 under variable temperature, of SARS-CoV-2 spike protein, SARS-CoV-2 antibody, and SARS-CoV-1 antibody, respectively, given by the difference between the crest near 1550cm -1 , and the trough near 1480cm -1 . The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. All 3 proteins followed a similar trend in temperature, which is attributed to the thermal agitation, when the likelihood increases for such binding sites to pair, either between the neighbouring proteins, or amongst various strands within a protein, or even along a protein strand. (e) Schematic illustration of the hydrogen bonding between carboxyl and amino groups within a protein strand, showing the intra-molecular binding of the quaternary structure of proteins, when the Van der Waals bonds between large pendant groups, such as phenyl, were disrupted, which causes unfolding and exposing more bonding sites.
    Figure Legend Snippet: (a) The FTIR scan of SARS-CoV-2 spike protein (S protein) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (b) The FTIR scan of SARS-CoV-2 antibody (antibody 2) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (c) The FTIR scan of SARS-CoV-1 antibody (antibody 1) in PBS solution, for the wavenumber range of 1400-1800cm -1 . (d) The infrared absorption of 1550cm -1 under variable temperature, of SARS-CoV-2 spike protein, SARS-CoV-2 antibody, and SARS-CoV-1 antibody, respectively, given by the difference between the crest near 1550cm -1 , and the trough near 1480cm -1 . The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. All 3 proteins followed a similar trend in temperature, which is attributed to the thermal agitation, when the likelihood increases for such binding sites to pair, either between the neighbouring proteins, or amongst various strands within a protein, or even along a protein strand. (e) Schematic illustration of the hydrogen bonding between carboxyl and amino groups within a protein strand, showing the intra-molecular binding of the quaternary structure of proteins, when the Van der Waals bonds between large pendant groups, such as phenyl, were disrupted, which causes unfolding and exposing more bonding sites.

    Techniques Used: Binding Assay

    (a) Schematic illustration of the hydrogen bonding between carboxyl and amino groups of the protein strands, representing a typical intermolecular binding for the secondary structure of proteins. (b) The FTIR apparatus, the ATR model has been adopted to improve the signal noise ratio, which offers 0.1% error margin in the absorption measurement, and 0.5cm -1 along the wavenumber scan. To facilitate the possible temperature variation, an electric heating element, and a thermometer were attached to the metallic plate of the ATR assembly, which also acts as an ideal heat sink. Samples of antibodies and spike proteins were mixed together, and undergone FTIR spectroscopic examination conditioned by variable temperatures. (c) The FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution. Comparing the FTIR signal of the PBS solvent, it indicates that 1550 cm -1 was the only absorption band exclusively associated with the proteins, but not overshadowed by the PBS solvent. (d) The same FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution.
    Figure Legend Snippet: (a) Schematic illustration of the hydrogen bonding between carboxyl and amino groups of the protein strands, representing a typical intermolecular binding for the secondary structure of proteins. (b) The FTIR apparatus, the ATR model has been adopted to improve the signal noise ratio, which offers 0.1% error margin in the absorption measurement, and 0.5cm -1 along the wavenumber scan. To facilitate the possible temperature variation, an electric heating element, and a thermometer were attached to the metallic plate of the ATR assembly, which also acts as an ideal heat sink. Samples of antibodies and spike proteins were mixed together, and undergone FTIR spectroscopic examination conditioned by variable temperatures. (c) The FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution. Comparing the FTIR signal of the PBS solvent, it indicates that 1550 cm -1 was the only absorption band exclusively associated with the proteins, but not overshadowed by the PBS solvent. (d) The same FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution.

    Techniques Used: Binding Assay

    (a) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution, within the wavenumber range of 1400-1800cm -1 . (b) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution, in the wavenumber range of 1400-1800cm -1 . (c) The infrared absorptions of 1550cm -1 under variable temperature of the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody, versus the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody, obtained from the difference between the peak near 1550cm -1 and the trough near 1480cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. A strong temperature dependence is observed, where the bonding number of the antibody is enhanced sharply beyond 31°C, rather than at the usual room temperature. (d) The possible temperature influence on the structure of spike protein and antibodies, where only the top part of the IgM, similar to IgG, was depicted: a higher temperature increases the probability of protein quaternary structure unfolding, and exposes more binding sites, hence more bonds.
    Figure Legend Snippet: (a) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody (S + Antibody 2) in PBS solution, within the wavenumber range of 1400-1800cm -1 . (b) Detailed FTIR scan of the sample mixture of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody (S + Antibody 1) in PBS solution, in the wavenumber range of 1400-1800cm -1 . (c) The infrared absorptions of 1550cm -1 under variable temperature of the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-2 antibody, versus the sample combination of SARS-CoV-2 spike protein/ SARS-CoV-1 antibody, obtained from the difference between the peak near 1550cm -1 and the trough near 1480cm -1 , after subtracting that of the buffer solution. The associated error bars were estimated by combining the instrumental error, and the possible deviation involved in the computation. A strong temperature dependence is observed, where the bonding number of the antibody is enhanced sharply beyond 31°C, rather than at the usual room temperature. (d) The possible temperature influence on the structure of spike protein and antibodies, where only the top part of the IgM, similar to IgG, was depicted: a higher temperature increases the probability of protein quaternary structure unfolding, and exposes more binding sites, hence more bonds.

    Techniques Used: Binding Assay

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry
    Article Snippet: Finally, we believe that BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens, for example to evaluate plasma antibody levels to inform patients on vaccinations, or to quickly identify and prioritize donors for convalescent plasma therapy donation , . .. Reagents and suppliesPhosphate buffered saline (PBS) tablets (Sigma P4417), Tween-20 (Fisher BP337), dry milk powder (RPI 50488786), ELISA plates (Corning 3590), Goat anti-Human IgG Fc HRP (Thermo Fisher A18817), OPD tablets (Pierce PI34006), bovine serum albumin (BSA) (Fisher BP1600), ChonBlock (Chondrex 9068), Biotinylated SARS-CoV-2 protein RBD His AviTag (Acro Biosystems SPD-C82E9), 4 nm Colloidal Gold-AffiPure Goat Anti-Human IgG Fcg fragment specific (Jackson ImmunoResearch 109-185-098) rehydrated in 1 mL deionized water per the manufacturer’s instructions, 4 nm Colloidal Gold-AffiPure Goat Anti-Human Serum IgA alpha chain specific (Jackson ImmunoResearch 109-185-011) rehydrated in 1 mL deionized water per the manufacturer’s instructions, Human coronavirus spike glycoprotein Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40021-T60), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40589-T62), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit MAb (40150-R007), Anti-SARS-CoV S Therapeutic Antibody (CR3022) (Creative Biolabs MRO-1214LC), Octet Anti-Penta-His (HIS1K) sensor tips (Sartorius ForteBio 18-5120), Octet Streptavidin (SA) sensor tips (Sartorius ForteBio 18-5019), tilted bottom (TW384) microplates (Sartorius ForteBio 18-5080), electroporation cuvettes (MaxCyte SOC4), suspension adapted CHO-S cells (Thermo Fisher R80007). .. CD-CHO medium (Thermo Fisher 10743029), CD OptiCHO medium (Thermo Fisher 12681011), HisTrap FF (GE Healthcare 17-5286-01), StrepTrap HP (GE Healthcare 28-9075-47), Superdex 200 Increase GL (GE Healthcare 28-9909-44).

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike
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    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
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    Affinity Purification:

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry
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    Electroporation:

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry
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    Purification:

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction
    Article Snippet: .. 2.1 Construction and purification of recombinant SARS-CoV-2 spike protein (S) and nucleocapsid protein (N) A partial-length of SARS-CoV-2 S gene (2055 bp) and the full-length of SARS-CoV-2 N gene (1260 bp) were synthesised based on SARS-CoV-2 isolate 2019-nCoV WHU01, complete genome (accession no. MN988668). .. Five sequences of SARS-CoV-2 S gene (529–1536 bp, 862–1536 bp, 1042–1734 bp, 1159–1,548 bp and 1222–1992 bp), corresponding to amino acid (aa) 177–512, 288–512, 348–578, 387–516 and 408–664, and five sequences of SARS-CoV-2 N gene (1–360 bp, 330–660 bp, 1–660 bp, 628–1260 bp and 1–1260 bp), corresponding to amino acid (aa) 1–120, 111–220, 1–220, 210–419 and 1–419, were cloned into pET28a expression vectors (Novagen, Inc., USA) using primers listed in .

    Recombinant:

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction
    Article Snippet: .. 2.1 Construction and purification of recombinant SARS-CoV-2 spike protein (S) and nucleocapsid protein (N) A partial-length of SARS-CoV-2 S gene (2055 bp) and the full-length of SARS-CoV-2 N gene (1260 bp) were synthesised based on SARS-CoV-2 isolate 2019-nCoV WHU01, complete genome (accession no. MN988668). .. Five sequences of SARS-CoV-2 S gene (529–1536 bp, 862–1536 bp, 1042–1734 bp, 1159–1,548 bp and 1222–1992 bp), corresponding to amino acid (aa) 177–512, 288–512, 348–578, 387–516 and 408–664, and five sequences of SARS-CoV-2 N gene (1–360 bp, 330–660 bp, 1–660 bp, 628–1260 bp and 1–1260 bp), corresponding to amino acid (aa) 1–120, 111–220, 1–220, 210–419 and 1–419, were cloned into pET28a expression vectors (Novagen, Inc., USA) using primers listed in .

    Incubation:

    Article Title: Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease
    Article Snippet: After rinses with PBS (pH 7.4), the section were blocked with PBT (PBS +0.1% Tween-20) containing 5% normal goat serum overnight at 4°C. .. Then the sections were incubated with primary antibodies: rabbit polyclonal anti-SARS-CoV Spike at a dilution of 1:200 (40150-T62-COV2, Sino Biological, Chesterbrook, PA, USA), mouse monoclonal anti-SARS-CoV NP at a dilution of 1:200 (40143-MM05, Sino Biological), mouse monoclonal anti-pan cytokeratin at a dilution of 1:100 (sc-8018, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-e-cadherin at a dilution of 1:100 (33-4000, Thermo Fisher Scientific, Waltham, MA, USA), rabbit polyclonal anti- myeloperoxidase (MPO) at a dilution of 1:200 (A039829-2, Dako Agilent Pathology Solutions, Carpinteria, CA, USA), rabbit polyclonal anti-CD3 antibody at a dilution of 1:200 (A045229-2, Dako Agilent Pathology Solutions), rat monoclonal anti-CD45 antibody at a dilution of 1:100 (05-1416, Millipore Sigma, Burlington, MA, USA), rabbit polyclonal anti-CD68 at a dilution of 1:200 (ab125212, Abcam, Cambridge, MA, USA), mouse monoclonal anti-NeuN at a dilution of1:200 (MAB377, Millipore Sigma), and/or chicken polyclonal anti-GFAP at a dilution of 1:200 (ab4674, Abcam) for 2 hours at room temperature. .. After rinses with PBT, the sections were incubated with secondary goat anti-rabbit or anti-chicken Alexa Fluor 488 at dilution of 1:500 (Thermo Fisher Scientific) and goat anti-mouse or anti-rat Alexa Fluor 568 at a dilution of 1:500 (Thermo Fisher Scientific) antibodies, for 1 hour at room temperature.

    Article Title: Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease
    Article Snippet: .. Then, the sections were incubated with primary antibodies: rabbit polyclonal anti-SARS-CoV Spike at a dilution of 1:200 (40150-T62-COV2, Sino Biological), mouse monoclonal anti-SARS-CoV NP at a dilution of 1:200 (40143-MM05, Sino Biological), mouse monoclonal anti-pan cytokeratin at a dilution of 1:100 (sc-8018, Santa Cruz Biotechnology), mouse monoclonal anti-e-cadherin at a dilution of 1:100 (33-4000, Thermo Fisher Scientific), rabbit polyclonal anti-MPO at a dilution of 1:200 (A039829-2, Dako Agilent Pathology Solutions), rabbit polyclonal anti-CD3 antibody at a dilution of 1:200 (A045229-2, Dako Agilent Pathology Solutions), rat monoclonal anti-CD45 antibody at a dilution of 1:100 (05-1416, MilliporeSigma), rabbit polyclonal anti-CD68 at a dilution of 1:200 (ab125212, Abcam), mouse monoclonal anti-NeuN at a dilution of 1:200 (MAB377, MilliporeSigma), and/or chicken polyclonal anti-GFAP at a dilution of 1:200 (ab4674, Abcam) for 2 hours at room temperature. .. After rinses with PBT, the sections were incubated with secondary goat anti-rabbit or anti-chicken Alexa Fluor 488 at a dilution of 1:500 (Thermo Fisher Scientific) and goat anti-mouse or anti-rat Alexa Fluor 568 at a dilution of 1:500 (Thermo Fisher Scientific) antibodies, for 1 hour at room temperature.

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    BLI-ISA evaluation of <t>SARS-CoV-2</t> spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.
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    BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Journal: Scientific Reports

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    doi: 10.1038/s41598-020-78895-x

    Figure Lengend Snippet: BLI-ISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a , b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:8 dilution. Bars and dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( a ) The Total Antibody Binding signal is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. ( b ) The Detection signal is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( c ) Dilution series BLI-ISA from representative strong (SP7) and moderate (SP8) seropositive samples. ( d ) Dilution series BLI-ISA from the weakest seropositive sample (SP3) compared to seronegative plasma samples.

    Article Snippet: Reagents and suppliesPhosphate buffered saline (PBS) tablets (Sigma P4417), Tween-20 (Fisher BP337), dry milk powder (RPI 50488786), ELISA plates (Corning 3590), Goat anti-Human IgG Fc HRP (Thermo Fisher A18817), OPD tablets (Pierce PI34006), bovine serum albumin (BSA) (Fisher BP1600), ChonBlock (Chondrex 9068), Biotinylated SARS-CoV-2 protein RBD His AviTag (Acro Biosystems SPD-C82E9), 4 nm Colloidal Gold-AffiPure Goat Anti-Human IgG Fcg fragment specific (Jackson ImmunoResearch 109-185-098) rehydrated in 1 mL deionized water per the manufacturer’s instructions, 4 nm Colloidal Gold-AffiPure Goat Anti-Human Serum IgA alpha chain specific (Jackson ImmunoResearch 109-185-011) rehydrated in 1 mL deionized water per the manufacturer’s instructions, Human coronavirus spike glycoprotein Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40021-T60), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40589-T62), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit MAb (40150-R007), Anti-SARS-CoV S Therapeutic Antibody (CR3022) (Creative Biolabs MRO-1214LC), Octet Anti-Penta-His (HIS1K) sensor tips (Sartorius ForteBio 18-5120), Octet Streptavidin (SA) sensor tips (Sartorius ForteBio 18-5019), tilted bottom (TW384) microplates (Sartorius ForteBio 18-5080), electroporation cuvettes (MaxCyte SOC4), suspension adapted CHO-S cells (Thermo Fisher R80007).

    Techniques: Standard Deviation, Binding Assay

    ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Journal: Scientific Reports

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    doi: 10.1038/s41598-020-78895-x

    Figure Lengend Snippet: ELISA evaluation of SARS-CoV-2 spike RBD reactivity of pre-pandemic and convalescent plasma. ( a ) Single-dilution ELISA to evaluate the presence of RBD-reactive human IgG in pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples compared to no-antigen controls (grey). The assays were performed with plasma at a 1:50 dilution. Samples were evaluated in biological duplicates and error bars represent one standard deviation from the mean. Blue and green dashed lines represent the mean of seronegative samples plus 3 and 5 standard deviations, respectively. ( b ) Dilution series ELISA was performed to quantitate RBD-reactive human IgG in plasma. Samples were evaluated in biological duplicates. Dashed curves represent fit lines from a four-parameter logistic regression applied over each series. ( c ) Data from ( b ) plotted as area-under-the-curve (AUC).

    Article Snippet: Reagents and suppliesPhosphate buffered saline (PBS) tablets (Sigma P4417), Tween-20 (Fisher BP337), dry milk powder (RPI 50488786), ELISA plates (Corning 3590), Goat anti-Human IgG Fc HRP (Thermo Fisher A18817), OPD tablets (Pierce PI34006), bovine serum albumin (BSA) (Fisher BP1600), ChonBlock (Chondrex 9068), Biotinylated SARS-CoV-2 protein RBD His AviTag (Acro Biosystems SPD-C82E9), 4 nm Colloidal Gold-AffiPure Goat Anti-Human IgG Fcg fragment specific (Jackson ImmunoResearch 109-185-098) rehydrated in 1 mL deionized water per the manufacturer’s instructions, 4 nm Colloidal Gold-AffiPure Goat Anti-Human Serum IgA alpha chain specific (Jackson ImmunoResearch 109-185-011) rehydrated in 1 mL deionized water per the manufacturer’s instructions, Human coronavirus spike glycoprotein Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40021-T60), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40589-T62), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit MAb (40150-R007), Anti-SARS-CoV S Therapeutic Antibody (CR3022) (Creative Biolabs MRO-1214LC), Octet Anti-Penta-His (HIS1K) sensor tips (Sartorius ForteBio 18-5120), Octet Streptavidin (SA) sensor tips (Sartorius ForteBio 18-5019), tilted bottom (TW384) microplates (Sartorius ForteBio 18-5080), electroporation cuvettes (MaxCyte SOC4), suspension adapted CHO-S cells (Thermo Fisher R80007).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Journal: Scientific Reports

    Article Title: Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry

    doi: 10.1038/s41598-020-78895-x

    Figure Lengend Snippet: BLI-ISA evaluation of plasma antibodies to SARS-CoV-2 prefusion Spike and plasma IgA to SARS-CoV-2 spike RBD. ( a ) Single-dilution BLI-ISA to evaluate the presence of prefusion Spike-reactive human antibodies in the pre-pandemic seronegative (SN, cyan) and convalescent seropositive (SP, red) samples. The Total Antibody Binding signal (left) is measured when prefusion Spike-His-loaded HIS1K biosensors are dipped into plasma samples. The Detection signal (right) is measured when prefusion Spike-His-loaded HIS1K biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgG. ( b ) Single-dilution BLI-ISA to evaluate the presence of RBD-reactive human antibodies in the samples. The Total Antibody Binding signal (left) is measured when RBD-biotin-loaded SA biosensors are dipped into plasma samples. The Detection signal (right) is measured when RBD-biotin-loaded SA biosensors that had been dipped into plasma are subsequently dipped into colloidal gold-conjugated anti-human IgA. The SP7 dot is colored pink to indicate that this sample had a negative signal (value in parentheses) in the Detection step. All assays were performed with plasma at a 1:8 dilution. Dots represent the mean of biological duplicates, and error bars represent one standard deviation from the mean.

    Article Snippet: Reagents and suppliesPhosphate buffered saline (PBS) tablets (Sigma P4417), Tween-20 (Fisher BP337), dry milk powder (RPI 50488786), ELISA plates (Corning 3590), Goat anti-Human IgG Fc HRP (Thermo Fisher A18817), OPD tablets (Pierce PI34006), bovine serum albumin (BSA) (Fisher BP1600), ChonBlock (Chondrex 9068), Biotinylated SARS-CoV-2 protein RBD His AviTag (Acro Biosystems SPD-C82E9), 4 nm Colloidal Gold-AffiPure Goat Anti-Human IgG Fcg fragment specific (Jackson ImmunoResearch 109-185-098) rehydrated in 1 mL deionized water per the manufacturer’s instructions, 4 nm Colloidal Gold-AffiPure Goat Anti-Human Serum IgA alpha chain specific (Jackson ImmunoResearch 109-185-011) rehydrated in 1 mL deionized water per the manufacturer’s instructions, Human coronavirus spike glycoprotein Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40021-T60), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit PAb, Antigen Affinity Purified (Sino Biological 40589-T62), SARS-CoV-2 (2019-nCoV) spike Antibody, Rabbit MAb (40150-R007), Anti-SARS-CoV S Therapeutic Antibody (CR3022) (Creative Biolabs MRO-1214LC), Octet Anti-Penta-His (HIS1K) sensor tips (Sartorius ForteBio 18-5120), Octet Streptavidin (SA) sensor tips (Sartorius ForteBio 18-5019), tilted bottom (TW384) microplates (Sartorius ForteBio 18-5080), electroporation cuvettes (MaxCyte SOC4), suspension adapted CHO-S cells (Thermo Fisher R80007).

    Techniques: Binding Assay, Standard Deviation

    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

    Journal: Frontiers in Pharmacology

    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

    doi: 10.3389/fphar.2020.603830

    Figure Lengend 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

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: 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

    Journal: Frontiers in Pharmacology

    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

    doi: 10.3389/fphar.2020.603830

    Figure Lengend 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

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: 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

    Journal: Frontiers in Pharmacology

    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

    doi: 10.3389/fphar.2020.603830

    Figure Lengend 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

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: 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

    Journal: Frontiers in Pharmacology

    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

    doi: 10.3389/fphar.2020.603830

    Figure Lengend 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

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: 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.

    Journal: Frontiers in Pharmacology

    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

    doi: 10.3389/fphar.2020.603830

    Figure Lengend 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.

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Infection, Functional Assay, Activity Assay

    The C93D9 class of antibodies. (A) Two views of 20 Fab structures, listed in (C), bound with SARS-CoV-2 RBD. Structures all superposed on the RBD; heavy-and light-chains of each Fab in a distinct color. The figure includes only the RBD from 6YZ5 (not one of the 20), with the RBM in light orange and the rest of the chain in gray. (B) View as in the right-hand panel in (A), but showing only the FAB from 7B3O (the closest in sequence to C93D9), with CDRs labeled. The most intimate contacts with RBM residues are from CDRH1, CDRH2 and CDRL1, many with residues constrained in potential variability by ACE2 interaction. (C) Maps of pairwise distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the 21 C93D9 class antibodies in (A) and (B). Pairwise distances analyzed by Mega X. Intensity of color shows the distance, from 0 (identical) to 1 (no identity). The VH and VL genes encoding the antibodies are shown in the indicated groups. Differences in CDR3s from the reference sequences (bold) are in red; dashes indicate missing amino acids; dots represent identical amino acids. IGHV3-66 and IGHV3-53 are very similar VH gene segments, differing by only one encoded amino-acid residue.

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: The C93D9 class of antibodies. (A) Two views of 20 Fab structures, listed in (C), bound with SARS-CoV-2 RBD. Structures all superposed on the RBD; heavy-and light-chains of each Fab in a distinct color. The figure includes only the RBD from 6YZ5 (not one of the 20), with the RBM in light orange and the rest of the chain in gray. (B) View as in the right-hand panel in (A), but showing only the FAB from 7B3O (the closest in sequence to C93D9), with CDRs labeled. The most intimate contacts with RBM residues are from CDRH1, CDRH2 and CDRL1, many with residues constrained in potential variability by ACE2 interaction. (C) Maps of pairwise distances of CDRH3 (lower left triangle) and CDRL3 (upper right triangle) for the 21 C93D9 class antibodies in (A) and (B). Pairwise distances analyzed by Mega X. Intensity of color shows the distance, from 0 (identical) to 1 (no identity). The VH and VL genes encoding the antibodies are shown in the indicated groups. Differences in CDR3s from the reference sequences (bold) are in red; dashes indicate missing amino acids; dots represent identical amino acids. IGHV3-66 and IGHV3-53 are very similar VH gene segments, differing by only one encoded amino-acid residue.

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: Sequencing, Labeling

    Antibody sequence analyses. (A) Heavy-chain variable-domain genes of the 167 mAbs characterized by binding SARS-CoV-2 spike in either ELISA or cell-surface expression format. The inner ring of each pie chart shows the VH family and the outer ring, the gene. PBMC repertoire is from 350 million reads of deep sequencing ( 37 ). S binders include 167 clones in Table S2. *P

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: Antibody sequence analyses. (A) Heavy-chain variable-domain genes of the 167 mAbs characterized by binding SARS-CoV-2 spike in either ELISA or cell-surface expression format. The inner ring of each pie chart shows the VH family and the outer ring, the gene. PBMC repertoire is from 350 million reads of deep sequencing ( 37 ). S binders include 167 clones in Table S2. *P

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Clone Assay

    Sorting strategy for SARS-CoV-2 specific memory B cells. ( A ) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 spike-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD - IgM-IgG + CD27 + and finally on spike ( B ) Representative flow cytometry plots showing spike-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the spike gate.

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: Sorting strategy for SARS-CoV-2 specific memory B cells. ( A ) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 spike-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD - IgM-IgG + CD27 + and finally on spike ( B ) Representative flow cytometry plots showing spike-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the spike gate.

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: Flow Cytometry, Binding Assay, Magnetic Beads

    Schemes followed for three-dimensional image reconstructions of C12C9 and G32R7 Fabs bound with SARS-CoV-2 spike ectodomain. See Methods for description of the procedures.

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: Schemes followed for three-dimensional image reconstructions of C12C9 and G32R7 Fabs bound with SARS-CoV-2 spike ectodomain. See Methods for description of the procedures.

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques:

    mAb binding to SARS-CoV-2 S, RBD and NTD in cell-surface assay and EC 50 from ELISA-based and cell-based assay. (A) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK 293T cells. Cells were gated on DAPI-GFP + population. (B) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Fig. 1C for the screening color scheme. (D) Bar graph of EC 50 of antibodies targeting RBD, NTD and S2 using ELISA-based and cell-based assay. RBD (n=23), NTD clusters (n=15) and S2 (n=15). ***P

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: mAb binding to SARS-CoV-2 S, RBD and NTD in cell-surface assay and EC 50 from ELISA-based and cell-based assay. (A) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK 293T cells. Cells were gated on DAPI-GFP + population. (B) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Fig. 1C for the screening color scheme. (D) Bar graph of EC 50 of antibodies targeting RBD, NTD and S2 using ELISA-based and cell-based assay. RBD (n=23), NTD clusters (n=15) and S2 (n=15). ***P

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Based Assay

    SARS-CoV-2 surface glycoprotein (spike) specificities of memory B cells from convalescent subjects. (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors (one dot per donor) and the subset of those that expressed spike-binding BCRs. Right: cells from 3 donors expressing spikebinding BCRs and sorted to recover principally those that did not bind recombinant receptor-binding domain (RBD). Sorting protocols as described in Methods and shown in Fig. S1 . (B) Summary of all antibodies (expressed as recombinant IgG1) screened by ELISA (with recombinant spike ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown to next to each chart. To the right of the charts for the two alternative sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding antibodies for those subjects from whom at least 10 paired-chain BCR sequences were recovered. (C) Binding to a panel of spike proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA (with recombinant spike ectodomain) and by association with spike expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for cells sorted just for spike binding (left) and for those sorted for positive spike binding but no RBD binding (right). The rows with pink highlighting are from the ELISA screen; those with blue highlighting, from the cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heat maps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of heavy-and light-chain somatic mutation counts in antibodies that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. The significantly higher numbers of mutations in the last group suggest recalled, affinity matured memory from previous exposures to seasonal coronaviruses. ****P

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: SARS-CoV-2 surface glycoprotein (spike) specificities of memory B cells from convalescent subjects. (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors (one dot per donor) and the subset of those that expressed spike-binding BCRs. Right: cells from 3 donors expressing spikebinding BCRs and sorted to recover principally those that did not bind recombinant receptor-binding domain (RBD). Sorting protocols as described in Methods and shown in Fig. S1 . (B) Summary of all antibodies (expressed as recombinant IgG1) screened by ELISA (with recombinant spike ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown to next to each chart. To the right of the charts for the two alternative sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding antibodies for those subjects from whom at least 10 paired-chain BCR sequences were recovered. (C) Binding to a panel of spike proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA (with recombinant spike ectodomain) and by association with spike expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for cells sorted just for spike binding (left) and for those sorted for positive spike binding but no RBD binding (right). The rows with pink highlighting are from the ELISA screen; those with blue highlighting, from the cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heat maps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of heavy-and light-chain somatic mutation counts in antibodies that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. The significantly higher numbers of mutations in the last group suggest recalled, affinity matured memory from previous exposures to seasonal coronaviruses. ****P

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: Binding Assay, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Mutagenesis

    Ab contact regions. Surface regions of the SARS-CoV-2 spike protein trimer contacted by antibodies in four of the seven principal clusters, according to the color scheme shown (taken from the color scheme in Fig. 2 ), with a representative Fab for all except RBD-3. The C81C10 Fab defines an epitope just outside the margin of NTD-1, but it does not compete with any antibodies in RBD-2. The RBD-2 Fv shown is that of C121 (PDB ID: 7K8X: Barnes et al, 2020), which fits most closely, of the many published RBD-2 antibodies, into our low-resolution map for C12A2. Left: views normal to and along threefold axis of the closed, all-RBD-down conformation; right: similar views of the one-RBD-up conformation. C121 (RBD-2) can bind both RBD down and RBD up; G32R7 (RBD-1) binds only the “up” conformation of the RBD. The epitopes of the several published RBD-3 antibodies are partly occluded in both closed and open conformations of the RBD; none are shown here as cartoons. A cartoon of the polypeptide chain of a single subunit (dark red) is shown within the surface contour for a spike trimer (gray).

    Journal: bioRxiv

    Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

    doi: 10.1101/2021.03.10.434840

    Figure Lengend Snippet: Ab contact regions. Surface regions of the SARS-CoV-2 spike protein trimer contacted by antibodies in four of the seven principal clusters, according to the color scheme shown (taken from the color scheme in Fig. 2 ), with a representative Fab for all except RBD-3. The C81C10 Fab defines an epitope just outside the margin of NTD-1, but it does not compete with any antibodies in RBD-2. The RBD-2 Fv shown is that of C121 (PDB ID: 7K8X: Barnes et al, 2020), which fits most closely, of the many published RBD-2 antibodies, into our low-resolution map for C12A2. Left: views normal to and along threefold axis of the closed, all-RBD-down conformation; right: similar views of the one-RBD-up conformation. C121 (RBD-2) can bind both RBD down and RBD up; G32R7 (RBD-1) binds only the “up” conformation of the RBD. The epitopes of the several published RBD-3 antibodies are partly occluded in both closed and open conformations of the RBD; none are shown here as cartoons. A cartoon of the polypeptide chain of a single subunit (dark red) is shown within the surface contour for a spike trimer (gray).

    Article Snippet: Monoclonal antibody screening with ELISA SARS-CoV-2 S protein and the RBD proteins of other coronaviruses were prepared as described ( ).

    Techniques: