mers cov spike antibody rabbit pab  (Sino Biological)


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    Name:
    MERS CoV Spike Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Spike Protein Catalog 40069 V08B AFS88936 1 Met1 Trp1297 Total IgG was purified by Protein A affinity chromatography
    Catalog Number:
    40069-RP01
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    MERS CoV
    Applications:
    ELISA
    Immunogen:
    Recombinant MERS-CoV (NCoV / Novel coronavirus) Spike Protein (ECD, aa 1-1297) (Catalog#40069-V08B)
    Product Aliases:
    Anti-coronavirus s1 Antibody, Anti-coronavirus s2 Antibody, 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-RBD Antibody, Anti-S Antibody, Anti-s1 Antibody, Anti-Spike RBD Antibody
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
    Buy from Supplier


    Structured Review

    Sino Biological mers cov spike antibody rabbit pab
    Pseudo-particle assays of <t>MERS-CoV</t> S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Spike Protein Catalog 40069 V08B AFS88936 1 Met1 Trp1297 Total IgG was purified by Protein A affinity chromatography
    https://www.bioz.com/result/mers cov spike antibody rabbit pab/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov spike antibody rabbit pab - by Bioz Stars, 2021-07
    95/100 stars

    Images

    1) Product Images from "Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity"

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    Journal: bioRxiv

    doi: 10.1101/2019.12.18.881391

    Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.
    Figure Legend Snippet: Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Techniques Used: Infection, Luciferase, Activity Assay, Standard Deviation

    ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.
    Figure Legend Snippet: ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Techniques Used: Binding Assay, Concentration Assay, Standard Deviation

    Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.
    Figure Legend Snippet: Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Techniques Used: Sequencing, Mutagenesis, Labeling

    Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.
    Figure Legend Snippet: Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.

    Techniques Used: Western Blot, SDS Page

    Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .
    Figure Legend Snippet: Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .

    Techniques Used: Infection, Standard Deviation, Mutagenesis

    Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.
    Figure Legend Snippet: Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Techniques Used: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Standard Deviation, Mutagenesis

    Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.
    Figure Legend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

    2) Product Images from "Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity"

    Article Title: Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.00426-20

    Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.
    Figure Legend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

    Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations ( n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.
    Figure Legend Snippet: Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations ( n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Techniques Used: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Mutagenesis

    Western blot analysis of S proteins incorporated into PPs. A total of 1 ml of DMEM containing PPs per each tested S protein was ultracentrifuged, washed in PBS, and resuspended in SDS Laemmli buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S protein antibodies.
    Figure Legend Snippet: Western blot analysis of S proteins incorporated into PPs. A total of 1 ml of DMEM containing PPs per each tested S protein was ultracentrifuged, washed in PBS, and resuspended in SDS Laemmli buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S protein antibodies.

    Techniques Used: Western Blot, SDS Page

    Sequence and model of MERS-CoV S protein fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FPs). FP1 and FP2 designate the two different domains in the FP. Sequences illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E; in green are the A substitutions. (B) Modeling of the MERS-CoV S protein monomer with an emphasis on the FP. Negatively charges D and E are depicted as atomic bonds in red. The S2’ site is orange, and the FP1 and FP2 domains are labeled blue and pink, respectively.
    Figure Legend Snippet: Sequence and model of MERS-CoV S protein fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FPs). FP1 and FP2 designate the two different domains in the FP. Sequences illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E; in green are the A substitutions. (B) Modeling of the MERS-CoV S protein monomer with an emphasis on the FP. Negatively charges D and E are depicted as atomic bonds in red. The S2’ site is orange, and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Techniques Used: Sequencing, Mutagenesis, Labeling

    ESR and ITC analysis of the MERS-CoV FP. (A and B) Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5; and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of the difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1 × 10 −3 to 5 × 10 −3 , while the uncertainties from repeated experiments were 5 × 10 −3 to 8 × 10 −3 or less than ±0.01. We show the standard deviation bars in A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.
    Figure Legend Snippet: ESR and ITC analysis of the MERS-CoV FP. (A and B) Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5; and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of the difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol of 3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1 × 10 −3 to 5 × 10 −3 , while the uncertainties from repeated experiments were 5 × 10 −3 to 8 × 10 −3 or less than ±0.01. We show the standard deviation bars in A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Techniques Used: Binding Assay, Concentration Assay, Standard Deviation

    Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations ( n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.
    Figure Legend Snippet: Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations ( n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.

    Techniques Used: Infection, Luciferase, Activity Assay

    Related Articles

    Expressing:

    Article Title: Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity
    Article Snippet: For immunoblotting, the lysates were separated by sodium dodecyl sulfate gel electrophoresis and transferred onto nitrocellulose membranes (Hartenstein). .. MERS-S expression was detected using a monoclonal antibody directed against the V5 tag (Invitrogen) or a polyclonal antibody directed against the S2 subunit of the MERS-S protein (Sino Biological). .. For detection of MERS-S protein in infected cells, Caco-2 and Vero B4 cells were infected with MERS-CoV (human betacoronavirus 2c EMC/2012) at a multiplicity of infection (MOI) of 0.01 and 5, respectively.

    Article Title: Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity
    Article Snippet: At 24 hours after infection, the cells were washed and harvested, and the pellet was lysed with RIPA lysis buffer, supplemented with 4xNuPAGE (Invitrogen) and boiled for 20 minutes at 95°C. .. S protein expression in lysates was detected by Western blot, using a polyclonal antibody directed against the S2 subunit of MERS-S (Sino Biological). .. In parallel, for quantification of viral RNA, 50 µL of the cell supernatant was dissolved in RAV1 buffer (Macherey-Nagel) for RNA extraction, followed by quantitative reverse-transcription PCR analysis, using the upE assay as previously described [ ].

    Western Blot:

    Article Title: Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity
    Article Snippet: At 24 hours after infection, the cells were washed and harvested, and the pellet was lysed with RIPA lysis buffer, supplemented with 4xNuPAGE (Invitrogen) and boiled for 20 minutes at 95°C. .. S protein expression in lysates was detected by Western blot, using a polyclonal antibody directed against the S2 subunit of MERS-S (Sino Biological). .. In parallel, for quantification of viral RNA, 50 µL of the cell supernatant was dissolved in RAV1 buffer (Macherey-Nagel) for RNA extraction, followed by quantitative reverse-transcription PCR analysis, using the upE assay as previously described [ ].

    Enzyme-linked Immunosorbent Assay:

    Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva
    Article Snippet: For all optical measurements, the secondary antibody labelled with alkaline phosphate enzyme was added subsequently using 4-nitrophenyl phosphate as enzymatic substrate and monitoring the binding through the formation of enzymatic by-product 4-nitrophenol at 415 nm. .. In A the response of ELISA was reported testing the two PAb and two S proteins and demonstrating the better affinity between PAb belonging from Sinobiological, Germany and Recombinant Spike protein SARS-CoV, S1 subunit from Sinobiological, Germany, thus these reagents were selected for further studies. .. Subsequently, MAb was tested towards this S protein in ELISA and the binding curve was reported in B, demonstrating high affinity.

    Article Title: Comparative Serological Study for the Prevalence of Anti-MERS Coronavirus Antibodies in High- and Low-Risk Groups in Qatar
    Article Snippet: Since samples from CC were collected within the first week of primary case identification, these samples (n = 135) were also tested for the presence of IgM antibodies using whole-virus anti-MERS-CoV IgM IIFT kit (IgM-IIFT) (Euroimmun, cat no. FI 2604-1010 M). .. The anti-MERS-CoV (IgM/IgG) IIFT is based on MERS-CoV-infected eukaryotic cells and the anti-MERS-CoV ELISA (IgG) on purified S1 antigens of MERS-CoV. .. As recommended by the WHO, all borderline and reactive samples were then tested for the presence of anti-MERS-CoV antibodies using whole-virus indirect immunofluorescence assay (IgM- and IgG-IIFT) (Euroimmun, cat no. FI 2604-1010).

    Recombinant:

    Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva
    Article Snippet: For all optical measurements, the secondary antibody labelled with alkaline phosphate enzyme was added subsequently using 4-nitrophenyl phosphate as enzymatic substrate and monitoring the binding through the formation of enzymatic by-product 4-nitrophenol at 415 nm. .. In A the response of ELISA was reported testing the two PAb and two S proteins and demonstrating the better affinity between PAb belonging from Sinobiological, Germany and Recombinant Spike protein SARS-CoV, S1 subunit from Sinobiological, Germany, thus these reagents were selected for further studies. .. Subsequently, MAb was tested towards this S protein in ELISA and the binding curve was reported in B, demonstrating high affinity.

    Purification:

    Article Title: Comparative Serological Study for the Prevalence of Anti-MERS Coronavirus Antibodies in High- and Low-Risk Groups in Qatar
    Article Snippet: Since samples from CC were collected within the first week of primary case identification, these samples (n = 135) were also tested for the presence of IgM antibodies using whole-virus anti-MERS-CoV IgM IIFT kit (IgM-IIFT) (Euroimmun, cat no. FI 2604-1010 M). .. The anti-MERS-CoV (IgM/IgG) IIFT is based on MERS-CoV-infected eukaryotic cells and the anti-MERS-CoV ELISA (IgG) on purified S1 antigens of MERS-CoV. .. As recommended by the WHO, all borderline and reactive samples were then tested for the presence of anti-MERS-CoV antibodies using whole-virus indirect immunofluorescence assay (IgM- and IgG-IIFT) (Euroimmun, cat no. FI 2604-1010).

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    Sino Biological mers cov s rabbit polyclonal antibodies
    Two Selected G2-escape Mutations and Their Impact on G2 Binding and Neutralization. (A-D) Binding of G2 Fab to immobilized (A) <t>MERS-CoV</t> S1-NTD, (B) MERS-CoV S-2P, (C) MERS-CoV S-2P-S28F and (D) MERS-CoV S-2P-G198D measured by surface plasmon resonance (SPR). The same concentration series of G2 Fab was used in A-D. Best global fit of the data to a 1:1 binding model is shown as colored lines. (E) Neutralization activity of G2 IgG was measured against pseudotyped lentivirus bearing MERS-CoV S (WT) and two variants (S28F and G198D). Percent neutralization of WT (red), S28F (blue) and G198D (black) S pseudovirions at the different antibody concentrations is shown. Data points represent the mean of three technical replicates with standard errors. (F) Neutralization activity of G2 IgG was measured against authentic MERS-CoV (WT) and the G198D variant. Percent neutralization of WT (red) and G198D (black) MERS-CoV at the different antibody concentrations is shown. Data points for the wild-type virus represent the mean of two technical replicates.
    Mers Cov S Rabbit Polyclonal Antibodies, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Sino Biological pab
    A) <t>ELISA</t> response for two different <t>PAb</t> anti-SARS-CoV-2 1 μg/mL (Sinobiological and ProSci) towards two different Spike proteins coated at 2 ng/mL. B) Binding curve of colorimetric ELISA for MAb anti-SARS-CoV-2 ranging from 0.12 – 2 μg/mL. Coating of Spike protein: 2 ng/mL. C) Electrochemical response using the MBs-based assay using CB-based modified electrode (blue line) and bare electrode (black line). The mean value (n = 3) with the corresponding standard deviation was reported for each measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological mers cov antigen
    <t>MERS-CoV</t> viral RNA in respiratory tissues of llamas (A) and pigs (B). Viral RNA was determined in tissue homogenates at postinoculation days 4 and 24. Error bars indicate SDs when results were positive in > 1 animal. Dashed lines depict the detection limit of the assays (C t ≤40). C t , cycle threshold; MERS-CoV, Middle East respiratory syndrome coronavirus; PI, postinoculation.
    Mers Cov Antigen, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Two Selected G2-escape Mutations and Their Impact on G2 Binding and Neutralization. (A-D) Binding of G2 Fab to immobilized (A) MERS-CoV S1-NTD, (B) MERS-CoV S-2P, (C) MERS-CoV S-2P-S28F and (D) MERS-CoV S-2P-G198D measured by surface plasmon resonance (SPR). The same concentration series of G2 Fab was used in A-D. Best global fit of the data to a 1:1 binding model is shown as colored lines. (E) Neutralization activity of G2 IgG was measured against pseudotyped lentivirus bearing MERS-CoV S (WT) and two variants (S28F and G198D). Percent neutralization of WT (red), S28F (blue) and G198D (black) S pseudovirions at the different antibody concentrations is shown. Data points represent the mean of three technical replicates with standard errors. (F) Neutralization activity of G2 IgG was measured against authentic MERS-CoV (WT) and the G198D variant. Percent neutralization of WT (red) and G198D (black) MERS-CoV at the different antibody concentrations is shown. Data points for the wild-type virus represent the mean of two technical replicates.

    Journal: Cell reports

    Article Title: Structural Definition of a Neutralization-sensitive Epitope on the MERS-CoV S1-NTD

    doi: 10.1016/j.celrep.2019.08.052

    Figure Lengend Snippet: Two Selected G2-escape Mutations and Their Impact on G2 Binding and Neutralization. (A-D) Binding of G2 Fab to immobilized (A) MERS-CoV S1-NTD, (B) MERS-CoV S-2P, (C) MERS-CoV S-2P-S28F and (D) MERS-CoV S-2P-G198D measured by surface plasmon resonance (SPR). The same concentration series of G2 Fab was used in A-D. Best global fit of the data to a 1:1 binding model is shown as colored lines. (E) Neutralization activity of G2 IgG was measured against pseudotyped lentivirus bearing MERS-CoV S (WT) and two variants (S28F and G198D). Percent neutralization of WT (red), S28F (blue) and G198D (black) S pseudovirions at the different antibody concentrations is shown. Data points represent the mean of three technical replicates with standard errors. (F) Neutralization activity of G2 IgG was measured against authentic MERS-CoV (WT) and the G198D variant. Percent neutralization of WT (red) and G198D (black) MERS-CoV at the different antibody concentrations is shown. Data points for the wild-type virus represent the mean of two technical replicates.

    Article Snippet: Subsequently, cells were stained with MERS-CoV S rabbit polyclonal antibodies (Sino Biological, Beijing, China) and then secondary goat anti-rabbit IgG H & L labeled with Alexa Fluor® 488 (AF488) was added.

    Techniques: Binding Assay, Neutralization, SPR Assay, Concentration Assay, Activity Assay, Variant Assay

    G2 IgG Prevents the Binding of MERS-CoV S Protein to DDP4-Expressing Cells. Normalized binding efficiency of GFP-tagged MERS-CoV S-2P proteins to DPP4-expressing FreeStyle 293F cells in the presence or absence of IgGs was calculated from median fluorescence intensity (MFI) values. FreeStyle 293-F cells were transfected with a plasmid encoding full-length DPP4 60 h before the experiment. Non-transfected cells (NT) incubated with MERS-CoV S-2P, as well as transfected cells incubated with PBS, were used as negative controls. AM14 is an irrelevant RSV F-specific neutralizing antibody used as another negative control. Bar graph shows the mean and error bars indicate the standard deviation (n = 3 biologically independent experiments with two technical replicates).

    Journal: Cell reports

    Article Title: Structural Definition of a Neutralization-sensitive Epitope on the MERS-CoV S1-NTD

    doi: 10.1016/j.celrep.2019.08.052

    Figure Lengend Snippet: G2 IgG Prevents the Binding of MERS-CoV S Protein to DDP4-Expressing Cells. Normalized binding efficiency of GFP-tagged MERS-CoV S-2P proteins to DPP4-expressing FreeStyle 293F cells in the presence or absence of IgGs was calculated from median fluorescence intensity (MFI) values. FreeStyle 293-F cells were transfected with a plasmid encoding full-length DPP4 60 h before the experiment. Non-transfected cells (NT) incubated with MERS-CoV S-2P, as well as transfected cells incubated with PBS, were used as negative controls. AM14 is an irrelevant RSV F-specific neutralizing antibody used as another negative control. Bar graph shows the mean and error bars indicate the standard deviation (n = 3 biologically independent experiments with two technical replicates).

    Article Snippet: Subsequently, cells were stained with MERS-CoV S rabbit polyclonal antibodies (Sino Biological, Beijing, China) and then secondary goat anti-rabbit IgG H & L labeled with Alexa Fluor® 488 (AF488) was added.

    Techniques: Binding Assay, Expressing, Fluorescence, Transfection, Plasmid Preparation, Incubation, Negative Control, Standard Deviation

    G2-mediated Inhibition of DPP4 binding to MERS-CoV S Depends on Oligomeric State of the Spike (A–C) Normalized binding efficiency of DPP4, in the presence or absence of Fab, to cells transfected with plasmids encoding (A) membrane-anchored S1 (S1-TM), (B) full-length S-WT (S-WT-FL) and (C) full-length S-2P (S-2P-FL). Cells incubated with PBS were used as negative controls. Bar graph shows the mean and error bars indicate the standard deviation (n = 3 biologically independent experiments with two technical replicates). (D) Surface plasmon resonance competition assay. Response curves for MERS-CoV S-2P, alone or in the presence of a 5-fold molar excess of indicated Fabs or IgGs, passed over immobilized DPP4 ectodomain. The curves for S-2P or S-2P supplemented with Fab or IgG are shown with a solid line, whereas control curves for samples without S-2P are shown with a dotted line.

    Journal: Cell reports

    Article Title: Structural Definition of a Neutralization-sensitive Epitope on the MERS-CoV S1-NTD

    doi: 10.1016/j.celrep.2019.08.052

    Figure Lengend Snippet: G2-mediated Inhibition of DPP4 binding to MERS-CoV S Depends on Oligomeric State of the Spike (A–C) Normalized binding efficiency of DPP4, in the presence or absence of Fab, to cells transfected with plasmids encoding (A) membrane-anchored S1 (S1-TM), (B) full-length S-WT (S-WT-FL) and (C) full-length S-2P (S-2P-FL). Cells incubated with PBS were used as negative controls. Bar graph shows the mean and error bars indicate the standard deviation (n = 3 biologically independent experiments with two technical replicates). (D) Surface plasmon resonance competition assay. Response curves for MERS-CoV S-2P, alone or in the presence of a 5-fold molar excess of indicated Fabs or IgGs, passed over immobilized DPP4 ectodomain. The curves for S-2P or S-2P supplemented with Fab or IgG are shown with a solid line, whereas control curves for samples without S-2P are shown with a dotted line.

    Article Snippet: Subsequently, cells were stained with MERS-CoV S rabbit polyclonal antibodies (Sino Biological, Beijing, China) and then secondary goat anti-rabbit IgG H & L labeled with Alexa Fluor® 488 (AF488) was added.

    Techniques: Inhibition, Binding Assay, Transfection, Incubation, Standard Deviation, SPR Assay, Competitive Binding Assay

    A) ELISA response for two different PAb anti-SARS-CoV-2 1 μg/mL (Sinobiological and ProSci) towards two different Spike proteins coated at 2 ng/mL. B) Binding curve of colorimetric ELISA for MAb anti-SARS-CoV-2 ranging from 0.12 – 2 μg/mL. Coating of Spike protein: 2 ng/mL. C) Electrochemical response using the MBs-based assay using CB-based modified electrode (blue line) and bare electrode (black line). The mean value (n = 3) with the corresponding standard deviation was reported for each measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biosensors & Bioelectronics

    Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

    doi: 10.1016/j.bios.2020.112686

    Figure Lengend Snippet: A) ELISA response for two different PAb anti-SARS-CoV-2 1 μg/mL (Sinobiological and ProSci) towards two different Spike proteins coated at 2 ng/mL. B) Binding curve of colorimetric ELISA for MAb anti-SARS-CoV-2 ranging from 0.12 – 2 μg/mL. Coating of Spike protein: 2 ng/mL. C) Electrochemical response using the MBs-based assay using CB-based modified electrode (blue line) and bare electrode (black line). The mean value (n = 3) with the corresponding standard deviation was reported for each measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: In A the response of ELISA was reported testing the two PAb and two S proteins and demonstrating the better affinity between PAb belonging from Sinobiological, Germany and Recombinant Spike protein SARS-CoV, S1 subunit from Sinobiological, Germany, thus these reagents were selected for further studies.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Modification, Standard Deviation

    Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Infection, Luciferase, Activity Assay, Standard Deviation

    ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Binding Assay, Concentration Assay, Standard Deviation

    Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Sequencing, Mutagenesis, Labeling

    Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Western Blot, SDS Page

    Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Infection, Standard Deviation, Mutagenesis

    Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Standard Deviation, Mutagenesis

    Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

    MERS-CoV viral RNA in respiratory tissues of llamas (A) and pigs (B). Viral RNA was determined in tissue homogenates at postinoculation days 4 and 24. Error bars indicate SDs when results were positive in > 1 animal. Dashed lines depict the detection limit of the assays (C t ≤40). C t , cycle threshold; MERS-CoV, Middle East respiratory syndrome coronavirus; PI, postinoculation.

    Journal: Emerging Infectious Diseases

    Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

    doi: 10.3201/eid2302.161239

    Figure Lengend Snippet: MERS-CoV viral RNA in respiratory tissues of llamas (A) and pigs (B). Viral RNA was determined in tissue homogenates at postinoculation days 4 and 24. Error bars indicate SDs when results were positive in > 1 animal. Dashed lines depict the detection limit of the assays (C t ≤40). C t , cycle threshold; MERS-CoV, Middle East respiratory syndrome coronavirus; PI, postinoculation.

    Article Snippet: Sequential slides were either stained with hematoxylin and eosin or used to detect the DPP4 receptor and MERS-CoV antigen by IHC and viral genome by ISH ( , ).

    Techniques:

    Antibody responses after experimental inoculation of MERS-CoV into llamas and pigs. A) MERS-CoV S1 antibody responses were analyzed in serum from all animals at postinoculation days 0, 14, and 24. An ELISA with recombinant MERS-CoV S1 protein was used, and results are represented individually. B) Individual MERS-CoV neutralization titers from llamas and pigs as determined from serum. Dashed lines depict the detection limit of the assays. MERS-CoV, Middle East respiratory syndrome coronavirus; OD, optical density; PRNT 90 , 90% plaque reduction neutralization test.

    Journal: Emerging Infectious Diseases

    Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

    doi: 10.3201/eid2302.161239

    Figure Lengend Snippet: Antibody responses after experimental inoculation of MERS-CoV into llamas and pigs. A) MERS-CoV S1 antibody responses were analyzed in serum from all animals at postinoculation days 0, 14, and 24. An ELISA with recombinant MERS-CoV S1 protein was used, and results are represented individually. B) Individual MERS-CoV neutralization titers from llamas and pigs as determined from serum. Dashed lines depict the detection limit of the assays. MERS-CoV, Middle East respiratory syndrome coronavirus; OD, optical density; PRNT 90 , 90% plaque reduction neutralization test.

    Article Snippet: Sequential slides were either stained with hematoxylin and eosin or used to detect the DPP4 receptor and MERS-CoV antigen by IHC and viral genome by ISH ( , ).

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Neutralization, Plaque Reduction Neutralization Test

    Viral shedding of llamas and pigs after experimental inoculation with MERS-CoV. A) Viral RNA and B) infectious MERS-CoV from nasal swab samples collected from llamas (top) and pigs (bottom) at different times after challenge. Each bar represents an individual animal. Dashed lines depict the detection limit of the assays. C t , cycle threshold; MERS-CoV, Middle East respiratory syndrome coronavirus; TCID 50 , 50% tissue culture infective dose.

    Journal: Emerging Infectious Diseases

    Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

    doi: 10.3201/eid2302.161239

    Figure Lengend Snippet: Viral shedding of llamas and pigs after experimental inoculation with MERS-CoV. A) Viral RNA and B) infectious MERS-CoV from nasal swab samples collected from llamas (top) and pigs (bottom) at different times after challenge. Each bar represents an individual animal. Dashed lines depict the detection limit of the assays. C t , cycle threshold; MERS-CoV, Middle East respiratory syndrome coronavirus; TCID 50 , 50% tissue culture infective dose.

    Article Snippet: Sequential slides were either stained with hematoxylin and eosin or used to detect the DPP4 receptor and MERS-CoV antigen by IHC and viral genome by ISH ( , ).

    Techniques:

    Histology and expression of viral antigen (IHC) and viral RNA (ISH) at postinoculation day 4 in the nasal respiratory epithelium of sheep, pigs, llamas, and horses inoculated with MERS-CoV. A mild to severe rhinitis with epithelial necrosis and hypertrophy and inflammation of the epithelium and lamina propria was observed in the nasal respiratory tissue of pigs and llamas. Associated with these was presence of virus antigen (IHC) and RNA (ISH). No substantial lesions, virus antigen, or virus RNA were detected in the nasal respiratory tissues of sheep and horses (HE, IHC, ISH). Original magnification ×200 for all images. HE, hematoxylin and eosin; IHC, immunohistochemistry; ISH, in situ hybridization; MERS-CoV, Middle East respiratory syndrome coronavirus.

    Journal: Emerging Infectious Diseases

    Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

    doi: 10.3201/eid2302.161239

    Figure Lengend Snippet: Histology and expression of viral antigen (IHC) and viral RNA (ISH) at postinoculation day 4 in the nasal respiratory epithelium of sheep, pigs, llamas, and horses inoculated with MERS-CoV. A mild to severe rhinitis with epithelial necrosis and hypertrophy and inflammation of the epithelium and lamina propria was observed in the nasal respiratory tissue of pigs and llamas. Associated with these was presence of virus antigen (IHC) and RNA (ISH). No substantial lesions, virus antigen, or virus RNA were detected in the nasal respiratory tissues of sheep and horses (HE, IHC, ISH). Original magnification ×200 for all images. HE, hematoxylin and eosin; IHC, immunohistochemistry; ISH, in situ hybridization; MERS-CoV, Middle East respiratory syndrome coronavirus.

    Article Snippet: Sequential slides were either stained with hematoxylin and eosin or used to detect the DPP4 receptor and MERS-CoV antigen by IHC and viral genome by ISH ( , ).

    Techniques: Expressing, Immunohistochemistry, In Situ Hybridization

    Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

    Journal: Emerging Infectious Diseases

    Article Title: Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

    doi: 10.3201/eid2302.161239

    Figure Lengend Snippet: Presence of MERS-CoV receptor DPP4 (IHC) and of mucosubstances (PAS) in upper and lower respiratory tract tissues from sheep, pigs, llamas, and horses. A) In the nose, DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of pigs, llamas, and horses but not sheep. PAS staining (magenta) demonstrated more mucous cells in the lining epithelium of sheep and horses and a layer of mucus on the lining epithelium of the horses. B) DPP4 (red cytoplasmic or membrane staining) was present on the lining epithelium of the trachea, bronchus/bronchioles, and alveoli in the pigs, llamas and horses but not in the sheep. Original magnification ×400 for all images. DPP4, dipeptidyl peptidase-4; IHC, immunohistochemistry; MERS-CoV, Middle East respiratory syndrome coronavirus; PAS, periodic acid–Schiff; term., terminal.

    Article Snippet: Sequential slides were either stained with hematoxylin and eosin or used to detect the DPP4 receptor and MERS-CoV antigen by IHC and viral genome by ISH ( , ).

    Techniques: Immunohistochemistry, Staining