mers cov spike protein  (Sino Biological)


Bioz Verified Symbol Sino Biological is a verified supplier
Bioz Manufacturer Symbol Sino Biological manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    MERS CoV Spike Protein
    Description:
    A DNA sequence encoding the extracellular domain of spike protein MERS CoV AFS88936 1 Met1 Trp1297 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    40069-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus s1 Protein MERS-CoV, coronavirus s2 Protein MERS-CoV, coronavirus spike Protein MERS-CoV, cov spike Protein MERS-CoV, ncov RBD Protein MERS-CoV, ncov s1 Protein MERS-CoV, ncov s2 Protein MERS-CoV, ncov spike Protein MERS-CoV, RBD Protein MERS-CoV, S Protein MERS-CoV, s1 Protein MERS-CoV, Spike RBD Protein MERS-CoV
    Host:
    Baculovirus-Insect Cells
    Buy from Supplier


    Structured Review

    Sino Biological mers cov spike protein
    Immunohistochemistry of <t>MERS</t> spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and <t>MERS-CoV</t> on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.
    A DNA sequence encoding the extracellular domain of spike protein MERS CoV AFS88936 1 Met1 Trp1297 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/mers cov spike protein/product/Sino Biological
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov spike protein - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques"

    Article Title: A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28900-1

    Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.
    Figure Legend Snippet: Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.

    Techniques Used: Immunohistochemistry

    2) Product Images from "A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis"

    Article Title: A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000715

    DMVs are sites of vRNA synthesis. Analysis of the association of autoradiography signal with DMVs in MERS-CoV-infected Huh7 cells (MOI 5). The cells were pretreated with actinomycin D at 10 hpi and labeled with tritiated uridine for 30 minutes immediately before fixation (12 hpi). (A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM), and orange (DMSs). Several densely labeled regions containing DMVs (but not the other virus-induced structures) are apparent. A close-up of one of these regions (boxed area) is shown in (B), with DMVs highlighted by yellow asterisks. (C, D) Distribution of the autoradiography signal around DMVs ( n DMVs = 36, see Materials and methods for selection criteria and details, and S4 Data for the underlying numerical data). The data are plotted (C) as a histogram or (D) normalized by the radius to the DMV center to account for the increase in the perimeter of the screened area with the distance. Scale bars, (A) 5 μm, (B) 500 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; N, nucleus; vRNA, viral RNA.
    Figure Legend Snippet: DMVs are sites of vRNA synthesis. Analysis of the association of autoradiography signal with DMVs in MERS-CoV-infected Huh7 cells (MOI 5). The cells were pretreated with actinomycin D at 10 hpi and labeled with tritiated uridine for 30 minutes immediately before fixation (12 hpi). (A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM), and orange (DMSs). Several densely labeled regions containing DMVs (but not the other virus-induced structures) are apparent. A close-up of one of these regions (boxed area) is shown in (B), with DMVs highlighted by yellow asterisks. (C, D) Distribution of the autoradiography signal around DMVs ( n DMVs = 36, see Materials and methods for selection criteria and details, and S4 Data for the underlying numerical data). The data are plotted (C) as a histogram or (D) normalized by the radius to the DMV center to account for the increase in the perimeter of the screened area with the distance. Scale bars, (A) 5 μm, (B) 500 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; N, nucleus; vRNA, viral RNA.

    Techniques Used: Autoradiography, Infection, Labeling, Selection

    CoV RNA synthesis is confined to RO regions. Newly synthesized vRNA was metabolically labeled by providing tritiated uridine to CoV-infected cells pretreated with actinomycin D to limit host transcription. (A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labeling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells (underlying numerical data in S2 Data ). The label was provided simultaneously to all the samples at 6 hpi. (B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10, 7 hpi, labeled for 20 minutes). Autoradiography grains accumulate in the RO regions. Scale bar, 1 μm. (C, D) Quantification of the autoradiography signal per subcellular structure (see also S3 Data ). Labeling densities and RLIs in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively labeled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. These time points represent, respectively, the middle (SARS-CoV) or late (MERS-CoV) exponential phase of viral replication [ 21 , 34 ]. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions. CM, convoluted membranes; CoV, coronavirus; cpm, counts per minute; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; HCoV-229E, human coronavirus 229E; hpi, hours postinfection; IBV, infectious bronchitis virus; LD, lipid droplet; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; MHV, murine hepatitis virus; MOI, multiplicity of infection; N, nucleus; RLI, relative labeling index;; RO, replication organelle; SARS-CoV, severe acute respiratory syndrome-CoV; VCR, virion-containing region; vRNA, viral RNA.
    Figure Legend Snippet: CoV RNA synthesis is confined to RO regions. Newly synthesized vRNA was metabolically labeled by providing tritiated uridine to CoV-infected cells pretreated with actinomycin D to limit host transcription. (A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labeling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells (underlying numerical data in S2 Data ). The label was provided simultaneously to all the samples at 6 hpi. (B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10, 7 hpi, labeled for 20 minutes). Autoradiography grains accumulate in the RO regions. Scale bar, 1 μm. (C, D) Quantification of the autoradiography signal per subcellular structure (see also S3 Data ). Labeling densities and RLIs in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively labeled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. These time points represent, respectively, the middle (SARS-CoV) or late (MERS-CoV) exponential phase of viral replication [ 21 , 34 ]. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions. CM, convoluted membranes; CoV, coronavirus; cpm, counts per minute; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; HCoV-229E, human coronavirus 229E; hpi, hours postinfection; IBV, infectious bronchitis virus; LD, lipid droplet; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; MHV, murine hepatitis virus; MOI, multiplicity of infection; N, nucleus; RLI, relative labeling index;; RO, replication organelle; SARS-CoV, severe acute respiratory syndrome-CoV; VCR, virion-containing region; vRNA, viral RNA.

    Techniques Used: Synthesized, Metabolic Labelling, Labeling, Infection, Isolation, Autoradiography, Electron Microscopy

    Membrane structures induced by MERS-CoV infection. Electron microscopy analysis of Huh7 cells infected with MERS-CoV (MOI 5, 12 hpi). (A) Electron micrograph of an area with abundant DMSs. DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: CM (blue), DMSs (orange), and DMVs (yellow and lilac, outer and inner membranes, respectively). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video .) (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs ( n = 58), virions ( n = 28), and DMVs ( n = 109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), whereas the median diameter of the DMVs is 247 nm ( S1 Data ). (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two slices approximately 8 nm apart in the reconstruction are shown. For closed DMSs, only the outer membrane is connected to CM, whereas the inner membrane seems to define a closed compartment. (F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (blue arrowheads), and CM-DMS (orange arrowhead) connections. Constrictions in the DMVs are indicated by arrows. Scale bars, 250 nm (A, B), and 100 nm (C-F). CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; ER, endoplasmic reticulum; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; RO, replication organelle.
    Figure Legend Snippet: Membrane structures induced by MERS-CoV infection. Electron microscopy analysis of Huh7 cells infected with MERS-CoV (MOI 5, 12 hpi). (A) Electron micrograph of an area with abundant DMSs. DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: CM (blue), DMSs (orange), and DMVs (yellow and lilac, outer and inner membranes, respectively). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video .) (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs ( n = 58), virions ( n = 28), and DMVs ( n = 109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), whereas the median diameter of the DMVs is 247 nm ( S1 Data ). (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two slices approximately 8 nm apart in the reconstruction are shown. For closed DMSs, only the outer membrane is connected to CM, whereas the inner membrane seems to define a closed compartment. (F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (blue arrowheads), and CM-DMS (orange arrowhead) connections. Constrictions in the DMVs are indicated by arrows. Scale bars, 250 nm (A, B), and 100 nm (C-F). CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; ER, endoplasmic reticulum; hpi, hours postinfection; MERS-CoV, Middle East respiratory syndrome-coronavirus; MOI, multiplicity of infection; RO, replication organelle.

    Techniques Used: Infection, Electron Microscopy, Whisker Assay

    IEM detection of viral markers in MERS-CoV-infected cells. (A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labeled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (black arrows), whereas the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, were devoid of dsRNA signal. Scale bars, 250 nm. CM, convoluted membranes; DMS, double-membrane spherule; dsRNA, double-stranded RNA; G, Golgi complex; HPF-FS, high-pressure freezing, freeze-substitution; hpi, hours postinfection; IEM, immunoelectron microscopy; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; nsp3, nonstructural protein 3.
    Figure Legend Snippet: IEM detection of viral markers in MERS-CoV-infected cells. (A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labeled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (black arrows), whereas the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, were devoid of dsRNA signal. Scale bars, 250 nm. CM, convoluted membranes; DMS, double-membrane spherule; dsRNA, double-stranded RNA; G, Golgi complex; HPF-FS, high-pressure freezing, freeze-substitution; hpi, hours postinfection; IEM, immunoelectron microscopy; m, mitochondrion; MERS-CoV, Middle East respiratory syndrome-CoV; nsp3, nonstructural protein 3.

    Techniques Used: Infection, Labeling, Immuno-Electron Microscopy

    Newly synthesized vRNA signal does not clearly associate with CM or DMSs. (A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig 5 . Some DMSs are boxed in orange, and regions with CM are encircled in blue. In comparison with the densely labeled surrounding DMVs, these regions are relatively devoid of autoradiography signal. (B) The distribution of autoradiography grains on CM was not homogeneous ( n CM = 9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs. (C-E) Analysis of the label around/on the DMSs (see Materials and methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A). Most DMSs were devoid of signal, and those who contained label were close to labeled DMVs (D) ( n DMS = 127). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS center, as expected from a random distribution ( n DMSs = 58). The underlying numerical data for the plots are in S5 Data . Scale bars, (A) 500 nm, (C) 100 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; vRNA, viral RNA.
    Figure Legend Snippet: Newly synthesized vRNA signal does not clearly associate with CM or DMSs. (A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig 5 . Some DMSs are boxed in orange, and regions with CM are encircled in blue. In comparison with the densely labeled surrounding DMVs, these regions are relatively devoid of autoradiography signal. (B) The distribution of autoradiography grains on CM was not homogeneous ( n CM = 9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs. (C-E) Analysis of the label around/on the DMSs (see Materials and methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A). Most DMSs were devoid of signal, and those who contained label were close to labeled DMVs (D) ( n DMS = 127). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS center, as expected from a random distribution ( n DMSs = 58). The underlying numerical data for the plots are in S5 Data . Scale bars, (A) 500 nm, (C) 100 nm. CM, convoluted membranes; DMS, double-membrane spherule; DMV, double-membrane vesicle; vRNA, viral RNA.

    Techniques Used: Synthesized, Labeling, Autoradiography, Selection

    Membrane structures induced by gamma-CoV infections. Tomography of Vero cells infected with IBV, fixed at 16 hpi, and processed for EM following the same protocol as for MERS-CoV-infected cells ( Fig 1 ). Tomographic slices through 2 regions containing IBV-induced membrane modifications. These include DMVs (asterisks), DMSs (white arrowheads), and zippered ER (white arrows). Most zippered ER consists of long stretches of ER-derived paired membranes (A), though branching zippered ER, closer to the CM described for beta-CoV, was also present. (B) Virus particles (black arrowheads) budding into the ER membranes were often observed. Scale bars, 250 nm. CM, convoluted membranes; CoV, coronavirus; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; hpi, hours postinfection; IBV, infectious bronchitis virus; MERS-CoV, Middle East respiratory syndrome-CoV.
    Figure Legend Snippet: Membrane structures induced by gamma-CoV infections. Tomography of Vero cells infected with IBV, fixed at 16 hpi, and processed for EM following the same protocol as for MERS-CoV-infected cells ( Fig 1 ). Tomographic slices through 2 regions containing IBV-induced membrane modifications. These include DMVs (asterisks), DMSs (white arrowheads), and zippered ER (white arrows). Most zippered ER consists of long stretches of ER-derived paired membranes (A), though branching zippered ER, closer to the CM described for beta-CoV, was also present. (B) Virus particles (black arrowheads) budding into the ER membranes were often observed. Scale bars, 250 nm. CM, convoluted membranes; CoV, coronavirus; DMS, double-membrane spherule; DMV, double-membrane vesicle; EM, electron microscopy; ER, endoplasmic reticulum; hpi, hours postinfection; IBV, infectious bronchitis virus; MERS-CoV, Middle East respiratory syndrome-CoV.

    Techniques Used: Infection, Derivative Assay, Electron Microscopy

    3) Product Images from "High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan"

    Article Title: High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan

    Journal: Vector Borne and Zoonotic Diseases

    doi: 10.1089/vbz.2016.2062

    Phylogenetic analysis of a partial spike S2 domain. A maximum likelihood tree based on the GTR+G+I model using 1000 bootstraps was generated from a spike S2 domain genome fragment corresponding to nucleotides 23781–24395 of HCoV-EMC/2012. The newly identified MERS-CoV sequences are depicted in bold , recent MERS-CoV sequences associated with an outbreak in Jordan in 2015 are depicted in bold . Bootstrap values of
    Figure Legend Snippet: Phylogenetic analysis of a partial spike S2 domain. A maximum likelihood tree based on the GTR+G+I model using 1000 bootstraps was generated from a spike S2 domain genome fragment corresponding to nucleotides 23781–24395 of HCoV-EMC/2012. The newly identified MERS-CoV sequences are depicted in bold , recent MERS-CoV sequences associated with an outbreak in Jordan in 2015 are depicted in bold . Bootstrap values of

    Techniques Used: Generated

    4) Product Images from "A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques"

    Article Title: A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28900-1

    Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.
    Figure Legend Snippet: Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.

    Techniques Used: Immunohistochemistry

    5) Product Images from "Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection"

    Article Title: Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2019.1620083

    Intranasal immunization with AdC68-S induces a robust antibody and T cell response in BALB/c mice. (A) Timeline for vaccination and characterization of virologic and immunologic responses in two batches of animals. In batch I, a total of 12 groups of mice were immunized and monitored for serum binding and neutralizing activities. Group 1–6 (G1-G6) were negative controls whereas group 7–12 (G7–G12) were vaccinated with a single and varying dose of AdC68-S. In batch II, G8 and G11 mice were immunized along with G1 and G4* controls mice. These mice were examined for serum IgG subtypes, saliva IgA, and cytokine release up to 14-weeks post-immunization. The specific dose and route of immunization are indicated. i.n.: intranasal. i.m.: intramuscular. The open and solid drops indicate the blood collection for animals in the batch I and II, respectively. The temporal changes in serum binding activity to MERS-S1 (B) and neutralizing activity against autologous (C) and heterologous (D) MERS-CoV variants up to 40-weeks post-immunization are shown. Live virus neutralization by immune sera from G4, G8 (E) and G6, G11 (F) at 40-weeks post-immunization. The mean ID50 for G8 and G11 immune sera are indicated. The temporal changes in IgA in saliva (G), serum IgG subtypes (H), and cytokine release (I) in G8 and G11 animals were studied for up to 14-weeks post-immunization. Red symbols represent sera from i.n. vaccination groups and blue colour indicates sera from i.m. immunized animals. The grey colour is indicative of the control groups. ED50 means dilutions of serum at which half of the binding to antigen was identified. ID50 means dilutions of serum at which half of the viruses are neutralized. G4* indicates 10 10 vp used in batch I and 10 9 in batch II.
    Figure Legend Snippet: Intranasal immunization with AdC68-S induces a robust antibody and T cell response in BALB/c mice. (A) Timeline for vaccination and characterization of virologic and immunologic responses in two batches of animals. In batch I, a total of 12 groups of mice were immunized and monitored for serum binding and neutralizing activities. Group 1–6 (G1-G6) were negative controls whereas group 7–12 (G7–G12) were vaccinated with a single and varying dose of AdC68-S. In batch II, G8 and G11 mice were immunized along with G1 and G4* controls mice. These mice were examined for serum IgG subtypes, saliva IgA, and cytokine release up to 14-weeks post-immunization. The specific dose and route of immunization are indicated. i.n.: intranasal. i.m.: intramuscular. The open and solid drops indicate the blood collection for animals in the batch I and II, respectively. The temporal changes in serum binding activity to MERS-S1 (B) and neutralizing activity against autologous (C) and heterologous (D) MERS-CoV variants up to 40-weeks post-immunization are shown. Live virus neutralization by immune sera from G4, G8 (E) and G6, G11 (F) at 40-weeks post-immunization. The mean ID50 for G8 and G11 immune sera are indicated. The temporal changes in IgA in saliva (G), serum IgG subtypes (H), and cytokine release (I) in G8 and G11 animals were studied for up to 14-weeks post-immunization. Red symbols represent sera from i.n. vaccination groups and blue colour indicates sera from i.m. immunized animals. The grey colour is indicative of the control groups. ED50 means dilutions of serum at which half of the binding to antigen was identified. ID50 means dilutions of serum at which half of the viruses are neutralized. G4* indicates 10 10 vp used in batch I and 10 9 in batch II.

    Techniques Used: Mouse Assay, Binding Assay, Activity Assay, Neutralization

    Intranasal immunization with AdC68-S provides complete protection against lethal MERS-CoV challenge in human DPP4 knock-in mice. (A) Timeline for immunization, challenge and evaluation of protective efficacy. Human DPP4 knock-in (KI) mice were immunized with either 2 × 10 9 vp AdC68-S or empty AdC68 via i.n. route. Ten weeks later, the same set of animals were challenged intranasally with 2000 PFU of mouse-adapted MERS-CoV strain MERS-CoV-MA and monitored daily for (B) survival and (C) weight loss. On day-4 post-infection, lung virus titres (D) were examined. Data are shown as mean ± SEM. p -values were analysed with Student’s t-test (**** P
    Figure Legend Snippet: Intranasal immunization with AdC68-S provides complete protection against lethal MERS-CoV challenge in human DPP4 knock-in mice. (A) Timeline for immunization, challenge and evaluation of protective efficacy. Human DPP4 knock-in (KI) mice were immunized with either 2 × 10 9 vp AdC68-S or empty AdC68 via i.n. route. Ten weeks later, the same set of animals were challenged intranasally with 2000 PFU of mouse-adapted MERS-CoV strain MERS-CoV-MA and monitored daily for (B) survival and (C) weight loss. On day-4 post-infection, lung virus titres (D) were examined. Data are shown as mean ± SEM. p -values were analysed with Student’s t-test (**** P

    Techniques Used: Knock-In, Mouse Assay, Infection

    Generation and evaluation of recombinant AdC68 expressing full-length MERS-CoV spike protein. (A) Schematic representation of the recombinant AdC68 expressing the full-length MERS-CoV spike gene (AdC68-S) (Genbank accession number: JX869059). Spike gene was inserted into the E1 region of AdC68 under the control of the CMV promoter and terminated by bovine growth hormone (BGH) polyadenylation signal sequence. (B) Western blot analysis of MERS-CoV S protein expression in 293 T cells after infection with AdC68-S (10 8 , 10 9 and 10 10 vp). MERS-S protein in cell lysates was probed by Rabbit anti-MERS-S1 polyclonal antibody (Sino biological). The solid arrow points to the band of S protein while the hollow arrow points to the band of S1 protein cleaved from S by the protease. (C) Cell surface expression of MERS-CoV S protein analysed by MERS-CoV-specific antibodies MERS-4, MERS-27, MERS-GD27. Cell lysates or cells infected by empty AdC68 (10 10 vp) were used as negative controls. 17b, an antibody against HIV-1, was used as a negative control antibody.
    Figure Legend Snippet: Generation and evaluation of recombinant AdC68 expressing full-length MERS-CoV spike protein. (A) Schematic representation of the recombinant AdC68 expressing the full-length MERS-CoV spike gene (AdC68-S) (Genbank accession number: JX869059). Spike gene was inserted into the E1 region of AdC68 under the control of the CMV promoter and terminated by bovine growth hormone (BGH) polyadenylation signal sequence. (B) Western blot analysis of MERS-CoV S protein expression in 293 T cells after infection with AdC68-S (10 8 , 10 9 and 10 10 vp). MERS-S protein in cell lysates was probed by Rabbit anti-MERS-S1 polyclonal antibody (Sino biological). The solid arrow points to the band of S protein while the hollow arrow points to the band of S1 protein cleaved from S by the protease. (C) Cell surface expression of MERS-CoV S protein analysed by MERS-CoV-specific antibodies MERS-4, MERS-27, MERS-GD27. Cell lysates or cells infected by empty AdC68 (10 10 vp) were used as negative controls. 17b, an antibody against HIV-1, was used as a negative control antibody.

    Techniques Used: Recombinant, Expressing, Sequencing, Western Blot, Infection, Negative Control

    Protective efficacy of passive immunization with AdC68-S immune sera. (A) Timeline of immunization, serum transfer, challenge, and monitoring for various biological and clinical outcomes. BALB/c and human DPP4 KI mice were immunized with either 2 × 10 9 vp AdC68-S or empty vector AdC68 via i.n. route. 175 µl of immune sera collected from these BALB/c and hDPP4-KI mice were transferred to hDPP4-KI mice via intraperitoneal route one day before lethal 2000 PFU MERS-CoV-MA infection. (B) Neutralizing activity of immune sera from immunized BALB/c (B) and hDPP4-KI (F) mice. (C-E) Survival (C), weight loss (D) and lung viral titres (E) in hDPP4-KI mice receiving sera from immunized BALB/c mice. (G-I) Survival (G), weight loss (H) and lung viral titres (I) in hDPP4-KI mice receiving sera from immunized hDPP4-KI mice. Data are mean ± SEM. p -values were analysed with Student’s t-test (** P
    Figure Legend Snippet: Protective efficacy of passive immunization with AdC68-S immune sera. (A) Timeline of immunization, serum transfer, challenge, and monitoring for various biological and clinical outcomes. BALB/c and human DPP4 KI mice were immunized with either 2 × 10 9 vp AdC68-S or empty vector AdC68 via i.n. route. 175 µl of immune sera collected from these BALB/c and hDPP4-KI mice were transferred to hDPP4-KI mice via intraperitoneal route one day before lethal 2000 PFU MERS-CoV-MA infection. (B) Neutralizing activity of immune sera from immunized BALB/c (B) and hDPP4-KI (F) mice. (C-E) Survival (C), weight loss (D) and lung viral titres (E) in hDPP4-KI mice receiving sera from immunized BALB/c mice. (G-I) Survival (G), weight loss (H) and lung viral titres (I) in hDPP4-KI mice receiving sera from immunized hDPP4-KI mice. Data are mean ± SEM. p -values were analysed with Student’s t-test (** P

    Techniques Used: Mouse Assay, Plasmid Preparation, Infection, Activity Assay

    Characterization of AdC68-S-elicited monoclonal antibodies. (A) Summary of isolated 14 mouse mAbs on their family designations and degree of similarity compared to their germline sequences, together with their sequences of complementarity-determining region 3 (CDR3) for both VH and VL. (B) Unrooted neighbor-joining tree depicting the relationship of isolated mAbs, left panel for the heavy chain and right panel for the light chain variable region. The branch length is drawn to scale so that the relatedness between different amino acid sequences can be readily assessed. Individual sequences are named at the tip of the branches. Binding (C) and neutralizing (D) activities of 14 mAbs measured by ELISA and pseudovirus bearing naturally occurring MERS-CoV mutant strains. (E) Epitope specificity analysed by competitive ELISA. HIV-1-specific mAb 17b was used as a negative control whereas previously isolated MERS-CoV-specific human mAb MERS-4, MERS-27 and MERS-GD27 as positive controls. Data are presented as mean ± SEM. The four antibodies with potent neutralizing activities are colored in light orange in (A) and (B).
    Figure Legend Snippet: Characterization of AdC68-S-elicited monoclonal antibodies. (A) Summary of isolated 14 mouse mAbs on their family designations and degree of similarity compared to their germline sequences, together with their sequences of complementarity-determining region 3 (CDR3) for both VH and VL. (B) Unrooted neighbor-joining tree depicting the relationship of isolated mAbs, left panel for the heavy chain and right panel for the light chain variable region. The branch length is drawn to scale so that the relatedness between different amino acid sequences can be readily assessed. Individual sequences are named at the tip of the branches. Binding (C) and neutralizing (D) activities of 14 mAbs measured by ELISA and pseudovirus bearing naturally occurring MERS-CoV mutant strains. (E) Epitope specificity analysed by competitive ELISA. HIV-1-specific mAb 17b was used as a negative control whereas previously isolated MERS-CoV-specific human mAb MERS-4, MERS-27 and MERS-GD27 as positive controls. Data are presented as mean ± SEM. The four antibodies with potent neutralizing activities are colored in light orange in (A) and (B).

    Techniques Used: Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Competitive ELISA, Negative Control

    6) Product Images from "Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice"

    Article Title: Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0223684

    Immunofluorescence staining of RV-N and MERS-CoV S1 protein expression. BHK-P cells were inoculated with either RVΔP-MERS/S1 or RVΔP at an MOI of 0.1/cell and incubated at 33 °C for 48 h. Cells were stained with the monoclonal antibody against RV-N (green) or the monoclonal antibody MERS-CoV S1 protein (red), respectively.
    Figure Legend Snippet: Immunofluorescence staining of RV-N and MERS-CoV S1 protein expression. BHK-P cells were inoculated with either RVΔP-MERS/S1 or RVΔP at an MOI of 0.1/cell and incubated at 33 °C for 48 h. Cells were stained with the monoclonal antibody against RV-N (green) or the monoclonal antibody MERS-CoV S1 protein (red), respectively.

    Techniques Used: Immunofluorescence, Staining, Expressing, Incubation

    Schematic illustration of recombinant RV genome constructs used in this study. Recombinant HEP-Flury (rHEP) has a complete genome of RV HEP-Flury strain (upper). RVΔP lacks the RV-P gene (middle). RVΔP-MERS/S1 harbors the MERS-CoV S1 gene fused with the C-terminal region of RV G protein (amino acids 446 to 524), which includes transmembrane, cytoplasmic domain, and stem domains of RV-G gene between RV-N and RV-M genes of the genome (lower).
    Figure Legend Snippet: Schematic illustration of recombinant RV genome constructs used in this study. Recombinant HEP-Flury (rHEP) has a complete genome of RV HEP-Flury strain (upper). RVΔP lacks the RV-P gene (middle). RVΔP-MERS/S1 harbors the MERS-CoV S1 gene fused with the C-terminal region of RV G protein (amino acids 446 to 524), which includes transmembrane, cytoplasmic domain, and stem domains of RV-G gene between RV-N and RV-M genes of the genome (lower).

    Techniques Used: Recombinant, Construct

    Western blotting analysis of RV-G and MERS-S1 protein expression. BHK-P cells were inoculated with RVΔP-MERS/S1 or RVΔP and incubated at 33 °C for 48 h. RV-G protein and MERS-S1 protein expression were confirmed with western blotting using monoclonal antibody against RV-G and polyclonal antibody against MERS-CoV S1 protein, respectively, a) in cell lysate preparations and b) in polyethylene glycol (PEG)-precipitated or sucrose-purified viruses. Recombinant MERS-CoV S1 protein were used as a positive control.
    Figure Legend Snippet: Western blotting analysis of RV-G and MERS-S1 protein expression. BHK-P cells were inoculated with RVΔP-MERS/S1 or RVΔP and incubated at 33 °C for 48 h. RV-G protein and MERS-S1 protein expression were confirmed with western blotting using monoclonal antibody against RV-G and polyclonal antibody against MERS-CoV S1 protein, respectively, a) in cell lysate preparations and b) in polyethylene glycol (PEG)-precipitated or sucrose-purified viruses. Recombinant MERS-CoV S1 protein were used as a positive control.

    Techniques Used: Western Blot, Expressing, Incubation, Purification, Recombinant, Positive Control

    Analysis of VNAs against MERS-CoV and RV in mice inoculated with RVΔP-MERS/S1 or RVΔP. a) Immunization and whole-blood collection schedules of 4-week-old female BALB/c mice. Mice were inoculated with RVΔP-MERS/S1, RVΔP, or PBS as control. All mice were inoculated intraperitoneally with 100 μL of virus solution containing 10 7 FFU/mL of each virus. Titers of VNAs against MERS-CoV and RV were determined using the serum of mice 14 days after last inoculation. b) Titers of VNAs against MERS-CoV detected in the serum of mice inoculated with RVΔP-MERS/S1 (n = 12 in mice inoculated once or twice, respectively), RVΔP (n = 10 in mice inoculated once or twice, respectively), or PBS (n = 10 in mice inoculated twice). c) Titers of VNAs against RV were also detected in the same samples. The Steel-Dwass nonparametric test was used, and asterisks indicate a significant difference (p
    Figure Legend Snippet: Analysis of VNAs against MERS-CoV and RV in mice inoculated with RVΔP-MERS/S1 or RVΔP. a) Immunization and whole-blood collection schedules of 4-week-old female BALB/c mice. Mice were inoculated with RVΔP-MERS/S1, RVΔP, or PBS as control. All mice were inoculated intraperitoneally with 100 μL of virus solution containing 10 7 FFU/mL of each virus. Titers of VNAs against MERS-CoV and RV were determined using the serum of mice 14 days after last inoculation. b) Titers of VNAs against MERS-CoV detected in the serum of mice inoculated with RVΔP-MERS/S1 (n = 12 in mice inoculated once or twice, respectively), RVΔP (n = 10 in mice inoculated once or twice, respectively), or PBS (n = 10 in mice inoculated twice). c) Titers of VNAs against RV were also detected in the same samples. The Steel-Dwass nonparametric test was used, and asterisks indicate a significant difference (p

    Techniques Used: Mouse Assay

    7) Product Images from "Proof of Concept for a Quick and Highly Sensitive On-Site Detection of SARS-CoV-2 by Plasmonic Optical Fibers and Molecularly Imprinted Polymers"

    Article Title: Proof of Concept for a Quick and Highly Sensitive On-Site Detection of SARS-CoV-2 by Plasmonic Optical Fibers and Molecularly Imprinted Polymers

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s21051681

    ( a ) Blue resonance peak: bare gold surface before the functionalization. Red resonance peak: red shifted resonance due to the MIP layer before template extraction with trypsin enzyme 4.2 × 10 −8 M in buffer phosphate pH 7.4 and sodium dodecyl sulfate (SDS) 5%. Magenta resonance peak: Blue shift due to the freeing of sites from the template protein. ( b ) Response curves of Sars-Cov-2 Spike S1 subunit-MIP at different concentrations of protein. ( c ) SARS-CoV-2 Spike protein dose-response curve with the Hill fitting of the data. ( d ) Specificity test: sensor’s responses for MERS-CoV Spike protein and SARS-CoV-2 Spike protein, both with a concentration of 1 µM in UTM buffer.
    Figure Legend Snippet: ( a ) Blue resonance peak: bare gold surface before the functionalization. Red resonance peak: red shifted resonance due to the MIP layer before template extraction with trypsin enzyme 4.2 × 10 −8 M in buffer phosphate pH 7.4 and sodium dodecyl sulfate (SDS) 5%. Magenta resonance peak: Blue shift due to the freeing of sites from the template protein. ( b ) Response curves of Sars-Cov-2 Spike S1 subunit-MIP at different concentrations of protein. ( c ) SARS-CoV-2 Spike protein dose-response curve with the Hill fitting of the data. ( d ) Specificity test: sensor’s responses for MERS-CoV Spike protein and SARS-CoV-2 Spike protein, both with a concentration of 1 µM in UTM buffer.

    Techniques Used: Concentration Assay

    8) Product Images from "Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome"

    Article Title: Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome

    Journal: Acta Naturae

    doi: 10.32607/actanaturae.11042

    Survival of vaccinated (n = 10) and non-vaccinated (control group, n = 10) animals after a lethal infection of MERS-CoV. The ordinate axis shows the survival rate of animals (%). The abscissa axis represents time after immunization (days)
    Figure Legend Snippet: Survival of vaccinated (n = 10) and non-vaccinated (control group, n = 10) animals after a lethal infection of MERS-CoV. The ordinate axis shows the survival rate of animals (%). The abscissa axis represents time after immunization (days)

    Techniques Used: Infection

    Study of the lymphoproliferative activity of splenocytes in mice immunized with the vaccine or placebo. The levels (in %) of proliferating CD4 + and CD8 + T cells re-stimulated with recombinant MERS-CoV S glycoprotein on the 18th day after vaccination are presented. Medians of the percentage of proliferating cells after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p
    Figure Legend Snippet: Study of the lymphoproliferative activity of splenocytes in mice immunized with the vaccine or placebo. The levels (in %) of proliferating CD4 + and CD8 + T cells re-stimulated with recombinant MERS-CoV S glycoprotein on the 18th day after vaccination are presented. Medians of the percentage of proliferating cells after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p

    Techniques Used: Activity Assay, Mouse Assay, Recombinant

    Increase in the concentration of IFN-gamma in the splenocyte media of immunized and non-immunized mice after re-stimulation with recombinant MERS-CoV S glycoprotein. Median increase in the concentration of IFN-gamma after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p
    Figure Legend Snippet: Increase in the concentration of IFN-gamma in the splenocyte media of immunized and non-immunized mice after re-stimulation with recombinant MERS-CoV S glycoprotein. Median increase in the concentration of IFN-gamma after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p

    Techniques Used: Concentration Assay, Mouse Assay, Recombinant

    9) Product Images from "Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection"

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    Journal: mBio

    doi: 10.1128/mBio.00554-20

    Single-dose intranasal immunization with PIV5-MERS-S induced robust MERS-CoV-specific CD8 T cell response in human DPP4 knockin (hDPP4 KI) mice. (A) Schematic diagram showing the experimental plan to examine CD8 T cell response after immunization and challenge. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. At 4 weeks or 4 days following immunization, mice were challenged with MERS MA 6.1.2, single-cell suspensions from the lungs of immunized mice were stimulated with MERS-CoV spike peptides (S343 and S1165), and specific CD8 T cells were determined by IFN-γ intracellular staining. (B to D) Representative FACS plots (B), percentage (C), and total number (D) of MERS-CoV-specific CD8 T cells in the lungs at 4 weeks after immunization. (E to G) Representative FACS plots (E), percentage (F), and total number (G) of MERS-CoV-specific CD8 T cells in the lungs at 4 days after MERS MA 6.1.2 challenge ( n = 6 mice per group). Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P
    Figure Legend Snippet: Single-dose intranasal immunization with PIV5-MERS-S induced robust MERS-CoV-specific CD8 T cell response in human DPP4 knockin (hDPP4 KI) mice. (A) Schematic diagram showing the experimental plan to examine CD8 T cell response after immunization and challenge. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. At 4 weeks or 4 days following immunization, mice were challenged with MERS MA 6.1.2, single-cell suspensions from the lungs of immunized mice were stimulated with MERS-CoV spike peptides (S343 and S1165), and specific CD8 T cells were determined by IFN-γ intracellular staining. (B to D) Representative FACS plots (B), percentage (C), and total number (D) of MERS-CoV-specific CD8 T cells in the lungs at 4 weeks after immunization. (E to G) Representative FACS plots (E), percentage (F), and total number (G) of MERS-CoV-specific CD8 T cells in the lungs at 4 days after MERS MA 6.1.2 challenge ( n = 6 mice per group). Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Techniques Used: Knock-In, Mouse Assay, Staining, FACS

    Serum neutralizing antibodies produced in mice 4 weeks after single-dose intranasal immunization with PIV5-MERS-S. Naive mice were intranasally immunized with PIV5-GFP or PIV5-MERS-S. Sera were collected at 4 weeks postimmunization. Neutralization assay against MERS-CoV spike pseudovirions was performed as described in Materials and Methods. The neutralization results were measured in luciferase activity and plotted relative to mock-treatment value. (A and B) Neutralization assay results from C57BL/6 mice immunized with 10 4 PFU (A) and 10 6 PFU (B) PIV5-MERS-S or PIV5-GFP. (C) Neutralization assay results from BALB/c mice immunized with 10 6 PFU PIV5-MERS-S or PIV5-GFP. Data presented represent means ± SEs.
    Figure Legend Snippet: Serum neutralizing antibodies produced in mice 4 weeks after single-dose intranasal immunization with PIV5-MERS-S. Naive mice were intranasally immunized with PIV5-GFP or PIV5-MERS-S. Sera were collected at 4 weeks postimmunization. Neutralization assay against MERS-CoV spike pseudovirions was performed as described in Materials and Methods. The neutralization results were measured in luciferase activity and plotted relative to mock-treatment value. (A and B) Neutralization assay results from C57BL/6 mice immunized with 10 4 PFU (A) and 10 6 PFU (B) PIV5-MERS-S or PIV5-GFP. (C) Neutralization assay results from BALB/c mice immunized with 10 6 PFU PIV5-MERS-S or PIV5-GFP. Data presented represent means ± SEs.

    Techniques Used: Produced, Mouse Assay, Neutralization, Luciferase, Activity Assay

    Representative images of lung tissues from mice receiving PBS (A), UV-inactivated MERS-CoV (B), or PIV5-MERS-S (C) treatment, followed by infection with MERS MA 6.1.2. Images obtained from tissues at 3 days after MERS MA 6.1.2 infection. Compared to PBS or PIV5-MERS, perivascular eosinophilic infiltration (arrows) in UV-MERS-treated mice was greatly increased. n = 3 to 4 mice/group. (D) Graph representing eosinophil infiltration in the lung tissues of mice from groups shown in panels A to . * denotes P
    Figure Legend Snippet: Representative images of lung tissues from mice receiving PBS (A), UV-inactivated MERS-CoV (B), or PIV5-MERS-S (C) treatment, followed by infection with MERS MA 6.1.2. Images obtained from tissues at 3 days after MERS MA 6.1.2 infection. Compared to PBS or PIV5-MERS, perivascular eosinophilic infiltration (arrows) in UV-MERS-treated mice was greatly increased. n = 3 to 4 mice/group. (D) Graph representing eosinophil infiltration in the lung tissues of mice from groups shown in panels A to . * denotes P

    Techniques Used: Mouse Assay, Infection

    Histopathology in immunized mice challenged with MERS-CoV. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (A) Representative images of H E-stained lung sections from PIV5-MERS-S- or PIV5-GFP-immunized hDPP4 KI mice at indicated days after MERS MA 6.1.2 challenge. Note the cellular infiltration (black arrows) and the hyaline membranes (red arrows). (B) Summary scores for disease in the lung sections. n = 3 to 5 mice/group. * denotes P
    Figure Legend Snippet: Histopathology in immunized mice challenged with MERS-CoV. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (A) Representative images of H E-stained lung sections from PIV5-MERS-S- or PIV5-GFP-immunized hDPP4 KI mice at indicated days after MERS MA 6.1.2 challenge. Note the cellular infiltration (black arrows) and the hyaline membranes (red arrows). (B) Summary scores for disease in the lung sections. n = 3 to 5 mice/group. * denotes P

    Techniques Used: Histopathology, Mouse Assay, Infection, Staining

    Comparison of the protective efficacy between single-dose immunization with UV light-inactivated MERS-CoV and PIV5-MERS-S. hDPP4 KI mice were immunized with 10 4 PFU PIV5-MERS-S via intranasal route; 10 6 PFU UV-inactivated MERS MA 6.1.2, mixed with Imject alum; or PBS via intramuscular route. Four weeks after immunization, immunized mice were infected with 10 5 PFU MERS-CoV. (A) Schematic timeline outlining experimental plan. (B and C) Survival (B) and weight loss (C) were monitored daily until 10 days postinfection. PBS, n = 9; UV MERS-CoV, n = 12; PIV5-MERS-S, n = 8. Data represent mean ± SE.
    Figure Legend Snippet: Comparison of the protective efficacy between single-dose immunization with UV light-inactivated MERS-CoV and PIV5-MERS-S. hDPP4 KI mice were immunized with 10 4 PFU PIV5-MERS-S via intranasal route; 10 6 PFU UV-inactivated MERS MA 6.1.2, mixed with Imject alum; or PBS via intramuscular route. Four weeks after immunization, immunized mice were infected with 10 5 PFU MERS-CoV. (A) Schematic timeline outlining experimental plan. (B and C) Survival (B) and weight loss (C) were monitored daily until 10 days postinfection. PBS, n = 9; UV MERS-CoV, n = 12; PIV5-MERS-S, n = 8. Data represent mean ± SE.

    Techniques Used: Mouse Assay, Infection

    Generation and characterization of recombinant PIV5 expressing MERS-CoV spike protein. (A) Schematic of PIV5-MERS-S. NP, nucleoprotein; V, V protein; P, phosphoprotein; M, matrix protein; F, fusion protein; SH, small hydrophobic protein; HN, hemagglutinin-neuraminidase protein; L, RNA-dependent RNA polymerase. (B) Confirmation of MERS-CoV spike protein expression by Western blotting. Vero 81 cells were infected with PIV5-MERS-S at MOIs of 0.01, 0.1, and 1.0 or mock infected. At 2 days postinfection, MERS-CoV spike was detected with anti-MERS-S antibody by Western blotting. (C) Immunofluorescence of Vero cells infected with PIV5 and PIV5-MERS-S. Vero cells were infected with PIV5 and PIV5-MERS-S (MOI = 0.1) or mock infected. At 2 days postinfection, cells were fixed, permeabilized, and stained with anti-PIV5 V/P or anti-MERS-spike antibodies. Scale bar = 200 μm. (D) Growth rate of PIV5-MERS-S. MDBK cells were infected with PIV5 or PIV5-MERS-S at an MOI of 0.1. Media were collected daily for 5 days, and titers of viruses in the media were determined using plaque assay.
    Figure Legend Snippet: Generation and characterization of recombinant PIV5 expressing MERS-CoV spike protein. (A) Schematic of PIV5-MERS-S. NP, nucleoprotein; V, V protein; P, phosphoprotein; M, matrix protein; F, fusion protein; SH, small hydrophobic protein; HN, hemagglutinin-neuraminidase protein; L, RNA-dependent RNA polymerase. (B) Confirmation of MERS-CoV spike protein expression by Western blotting. Vero 81 cells were infected with PIV5-MERS-S at MOIs of 0.01, 0.1, and 1.0 or mock infected. At 2 days postinfection, MERS-CoV spike was detected with anti-MERS-S antibody by Western blotting. (C) Immunofluorescence of Vero cells infected with PIV5 and PIV5-MERS-S. Vero cells were infected with PIV5 and PIV5-MERS-S (MOI = 0.1) or mock infected. At 2 days postinfection, cells were fixed, permeabilized, and stained with anti-PIV5 V/P or anti-MERS-spike antibodies. Scale bar = 200 μm. (D) Growth rate of PIV5-MERS-S. MDBK cells were infected with PIV5 or PIV5-MERS-S at an MOI of 0.1. Media were collected daily for 5 days, and titers of viruses in the media were determined using plaque assay.

    Techniques Used: Recombinant, Expressing, Western Blot, Infection, Immunofluorescence, Staining, Plaque Assay

    Single-dose intranasal immunization with PIV5-MERS-S completely protects hDPP4 KI mice from lethal MERS-CoV challenge. (A) Schematic timeline showing immunization, challenge, and the evaluation of protection. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S, PIV5-GFP, or PBS. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (B and C) Survival (B) and weight loss (C) were monitored daily for 12 days. PIV5-MERS-S or PIV5-GFP, n = 10; PBS, n = 5. (D) At indicated days postinfection, virus lung titers were quantified by plaque assay. Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P
    Figure Legend Snippet: Single-dose intranasal immunization with PIV5-MERS-S completely protects hDPP4 KI mice from lethal MERS-CoV challenge. (A) Schematic timeline showing immunization, challenge, and the evaluation of protection. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S, PIV5-GFP, or PBS. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (B and C) Survival (B) and weight loss (C) were monitored daily for 12 days. PIV5-MERS-S or PIV5-GFP, n = 10; PBS, n = 5. (D) At indicated days postinfection, virus lung titers were quantified by plaque assay. Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Techniques Used: Mouse Assay, Infection, Plaque Assay

    10) Product Images from "A Human DPP4-Knockin Mouse’s Susceptibility to Infection by Authentic and Pseudotyped MERS-CoV"

    Article Title: A Human DPP4-Knockin Mouse’s Susceptibility to Infection by Authentic and Pseudotyped MERS-CoV

    Journal: Viruses

    doi: 10.3390/v10090448

    R26-hDPP4-knockin mice were susceptible to infection by authentic MERS-CoV at low dose. ( A ) Weight loss in R26-hDPP4 mice challenged with hCoV-EMC at a dose of 1.5 × 10 5 PFUs. ( B ) Viral titers in lungs of challenged R26-hDPP4 mice on day 5 p.i. LOD (limitation of detection): 0.85 PFU. ( C ) Quantitative analysis of pathological lesions in lungs. W. A. S. = widened alveolar septa; I. S. F. = inflammatory cells, serous and fibrinous exudation; D. N. B. = degeneration and necrosis of bronchial epithelial cells; P. I. I. = perivascular inflammatory cell infiltration; V. H. = vasodilator hyperemia. ( D – O ) Histopathological changes and viral loads in the lungs, brains, and kidneys of mice. R26-hDDP4 mice exhibited disease symptoms similar to those of MERS-CoV-infected human patients ( D ), while no or mild symptoms were observed in wild-type mice. ( J ). Perivascular gliosis in the cerebellum was observed in R26-hDPP4 mice ( F ) but not in wild-type mice ( L ). No pathological lesions were identified in the kidneys of either R26-hDPP4 or wild-type mice ( I , O ). IHC assays confirmed viral loads in lungs ( E ) and cerebella ( G ) of R26-hDPP4 mice; little or no virus was detected in the lungs ( K ) and cerebella ( M ) of wild-type mice. Four mice in each group were infected, and samples from all mice were subjected to tittering and histopathological analysis (* p
    Figure Legend Snippet: R26-hDPP4-knockin mice were susceptible to infection by authentic MERS-CoV at low dose. ( A ) Weight loss in R26-hDPP4 mice challenged with hCoV-EMC at a dose of 1.5 × 10 5 PFUs. ( B ) Viral titers in lungs of challenged R26-hDPP4 mice on day 5 p.i. LOD (limitation of detection): 0.85 PFU. ( C ) Quantitative analysis of pathological lesions in lungs. W. A. S. = widened alveolar septa; I. S. F. = inflammatory cells, serous and fibrinous exudation; D. N. B. = degeneration and necrosis of bronchial epithelial cells; P. I. I. = perivascular inflammatory cell infiltration; V. H. = vasodilator hyperemia. ( D – O ) Histopathological changes and viral loads in the lungs, brains, and kidneys of mice. R26-hDDP4 mice exhibited disease symptoms similar to those of MERS-CoV-infected human patients ( D ), while no or mild symptoms were observed in wild-type mice. ( J ). Perivascular gliosis in the cerebellum was observed in R26-hDPP4 mice ( F ) but not in wild-type mice ( L ). No pathological lesions were identified in the kidneys of either R26-hDPP4 or wild-type mice ( I , O ). IHC assays confirmed viral loads in lungs ( E ) and cerebella ( G ) of R26-hDPP4 mice; little or no virus was detected in the lungs ( K ) and cerebella ( M ) of wild-type mice. Four mice in each group were infected, and samples from all mice were subjected to tittering and histopathological analysis (* p

    Techniques Used: Knock-In, Mouse Assay, Infection, Immunohistochemistry

    Establishment of the R26-hDDP4 knockin model of infection with MERS-CoV pseudovirus. ( A – C ) Four-week-old R26-hDPP4 mice were inoculated with 1.27 × 10 7.5 TCID 50 (I.P. route) or 3.8 × 10 6.5 TCID 50 (I.T. route) of pHIV/MERSS/Fluc per animal. ( B ) Bioluminescent images (BLI) are shown at different days p.i. Relative bioluminescence intensity was shown in pseudocolor, with red representing the strongest and blue representing the weakest photon fluxes. Data are shown as means ± deviation ( C ). Organs were examined for Fluc expression using BLI: 1 = thymus; 2 = heart; 3 = liver; 4 = spleen; 5 = kidney; 6 = lung; 7 = lymph node; 8 = muscle; 9 = skin; 10 = ovary or testis; 11 = brain; 12 = intestine. ( D ) The copy number of pHIV/MERSS/Flucmeasured by RT-qPCR (I.P. challenge route). ( E , F ) Susceptibility tests for mice at different ages. Four- to 9- week-old mice could be infected, but younger mice were more susceptible, * p
    Figure Legend Snippet: Establishment of the R26-hDDP4 knockin model of infection with MERS-CoV pseudovirus. ( A – C ) Four-week-old R26-hDPP4 mice were inoculated with 1.27 × 10 7.5 TCID 50 (I.P. route) or 3.8 × 10 6.5 TCID 50 (I.T. route) of pHIV/MERSS/Fluc per animal. ( B ) Bioluminescent images (BLI) are shown at different days p.i. Relative bioluminescence intensity was shown in pseudocolor, with red representing the strongest and blue representing the weakest photon fluxes. Data are shown as means ± deviation ( C ). Organs were examined for Fluc expression using BLI: 1 = thymus; 2 = heart; 3 = liver; 4 = spleen; 5 = kidney; 6 = lung; 7 = lymph node; 8 = muscle; 9 = skin; 10 = ovary or testis; 11 = brain; 12 = intestine. ( D ) The copy number of pHIV/MERSS/Flucmeasured by RT-qPCR (I.P. challenge route). ( E , F ) Susceptibility tests for mice at different ages. Four- to 9- week-old mice could be infected, but younger mice were more susceptible, * p

    Techniques Used: Knock-In, Infection, Mouse Assay, Expressing, Quantitative RT-PCR

    Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4 mice by the novel mAb H111-1 and the well-characterized mAb m336. For evaluation of H111-1, mice were administered 1 mg/kg of mAb either I.P. ( A ) or I.T. ( B ) and 6 h later, challenged with pseudovirus I.T. at a dose of 3.8 × 10 6.5 TCID 50 . On day 11 p.i., BLI of the whole body or specific organs was conducted ( D ). To evaluate the efficacy of m336 ( C ), mice were administered mAb and challenged using the same doses as for H111-1, and typical images ( E ) are shown. ( F ) Bar of photo flux; for details see Figure 2 . N = 4 mice in each group, * means p
    Figure Legend Snippet: Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4 mice by the novel mAb H111-1 and the well-characterized mAb m336. For evaluation of H111-1, mice were administered 1 mg/kg of mAb either I.P. ( A ) or I.T. ( B ) and 6 h later, challenged with pseudovirus I.T. at a dose of 3.8 × 10 6.5 TCID 50 . On day 11 p.i., BLI of the whole body or specific organs was conducted ( D ). To evaluate the efficacy of m336 ( C ), mice were administered mAb and challenged using the same doses as for H111-1, and typical images ( E ) are shown. ( F ) Bar of photo flux; for details see Figure 2 . N = 4 mice in each group, * means p

    Techniques Used: Inhibition, Infection, Mouse Assay

    Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4-knockin mice by peptide HR2P-M2. Mice were administered HR2P-M2 peptide or phosphate-buffered saline (PBS) I.T., respectively. Thirty min later, mice were infected I.T. with pseudotyped MERS-CoV (3.8 × 10 6.5 TCID 50 ). BLI images were taken on day 11 p.i., and pseudovirus signals were recorded for the whole body or specific organs. ( A ) Flux value of pseudovirus for assessment of the protective efficacy of HR2P-M2. ( B ) BLI images of whole mice or their organs. ( C ) Bar of photo flux; for details see Figure 2 . Four mice were used for each group, and representative images are shown.
    Figure Legend Snippet: Inhibition of pseudotyped MERS-CoV infection in R26-hDPP4-knockin mice by peptide HR2P-M2. Mice were administered HR2P-M2 peptide or phosphate-buffered saline (PBS) I.T., respectively. Thirty min later, mice were infected I.T. with pseudotyped MERS-CoV (3.8 × 10 6.5 TCID 50 ). BLI images were taken on day 11 p.i., and pseudovirus signals were recorded for the whole body or specific organs. ( A ) Flux value of pseudovirus for assessment of the protective efficacy of HR2P-M2. ( B ) BLI images of whole mice or their organs. ( C ) Bar of photo flux; for details see Figure 2 . Four mice were used for each group, and representative images are shown.

    Techniques Used: Inhibition, Infection, Knock-In, Mouse Assay

    MERS-CoV S-RBD-specific humanized neutralizing antibody H111-1 protected R26-hDPP4 mice from challenge with authentic MERS-CoV. Four-week-old mice were administered either PBS (control), 1 mg/kg mAb H111-1 or 5 mg/kg mAb H111-1 via the I.P. route, and 6 h later they were challenged I.N. with hCoV-EMC (1.5 × 10 5 PFUs). On day 5 p.i., mice were sacrificed for virus titering and pathological analysis. ( A ) Treatment with mAb H111-1 significantly decreased viral titers in lungs. The dashed line indicates the LOD. ( B ) Efficacy of mAbH111-1 in abating pathological lesions caused by infection with authentic MERS-CoV. Explanation of pathological changes is given in Figure 2 . ( C ➊– C ➇) Histopathological changes and viral loads in lungs and brains of R26-hDPP4 mice administered 1 mg/kg mAb H111-1 ( C ➊, C ➌, C ➎, C ➆) or 5 mg/kg mAb H111-1 ( C ➋, C ➍, C ➏, C ➇), * p
    Figure Legend Snippet: MERS-CoV S-RBD-specific humanized neutralizing antibody H111-1 protected R26-hDPP4 mice from challenge with authentic MERS-CoV. Four-week-old mice were administered either PBS (control), 1 mg/kg mAb H111-1 or 5 mg/kg mAb H111-1 via the I.P. route, and 6 h later they were challenged I.N. with hCoV-EMC (1.5 × 10 5 PFUs). On day 5 p.i., mice were sacrificed for virus titering and pathological analysis. ( A ) Treatment with mAb H111-1 significantly decreased viral titers in lungs. The dashed line indicates the LOD. ( B ) Efficacy of mAbH111-1 in abating pathological lesions caused by infection with authentic MERS-CoV. Explanation of pathological changes is given in Figure 2 . ( C ➊– C ➇) Histopathological changes and viral loads in lungs and brains of R26-hDPP4 mice administered 1 mg/kg mAb H111-1 ( C ➊, C ➌, C ➎, C ➆) or 5 mg/kg mAb H111-1 ( C ➋, C ➍, C ➏, C ➇), * p

    Techniques Used: Mouse Assay, Infection

    Relationship between pseudovirus and authentic virus models. ( A – D ) Pseudovirus and authentic virus infection in the lungs of R26-hDPP4 mice showed a similar pattern, as shown by IHC using mAb R723 against the RBD of the MERS-CoV S protein. ( C , D ) Both pseudovirus and authentic virus infected ciliated columnar epithelium of bronchi. ( E ) Dose conversion of pseudovirus and authentic virus. The full black line represented the inhibition rate (◆) of humanized mAb H111-1 (1 mg/kg) in vivo using different pseudovirus doses. The blue dashed line represents inhibition rate of H111-1 against authentic virus. When the dose of authentic virus was 1.5 × 10 5 PFUs, we calculated that the inhibition rate of H111-1 at a dose of 1 mg/kg would be 60% (see main text). From the inhibition rate curve of pseudovirus, the corresponding pseudovirus dose was 3.25 × 10 7 TCID 50 . That is, 1 TCID 50 of pseudovirus corresponded to 0.0046 PFU of authentic virus (1.5 × 10 5 /3.25 × 10 7 ). n = 4–6 mice per group.
    Figure Legend Snippet: Relationship between pseudovirus and authentic virus models. ( A – D ) Pseudovirus and authentic virus infection in the lungs of R26-hDPP4 mice showed a similar pattern, as shown by IHC using mAb R723 against the RBD of the MERS-CoV S protein. ( C , D ) Both pseudovirus and authentic virus infected ciliated columnar epithelium of bronchi. ( E ) Dose conversion of pseudovirus and authentic virus. The full black line represented the inhibition rate (◆) of humanized mAb H111-1 (1 mg/kg) in vivo using different pseudovirus doses. The blue dashed line represents inhibition rate of H111-1 against authentic virus. When the dose of authentic virus was 1.5 × 10 5 PFUs, we calculated that the inhibition rate of H111-1 at a dose of 1 mg/kg would be 60% (see main text). From the inhibition rate curve of pseudovirus, the corresponding pseudovirus dose was 3.25 × 10 7 TCID 50 . That is, 1 TCID 50 of pseudovirus corresponded to 0.0046 PFU of authentic virus (1.5 × 10 5 /3.25 × 10 7 ). n = 4–6 mice per group.

    Techniques Used: Infection, Mouse Assay, Immunohistochemistry, Inhibition, In Vivo

    11) Product Images from "A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques"

    Article Title: A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28900-1

    Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.
    Figure Legend Snippet: Immunohistochemistry of MERS spike protein in the lung. ( a ) Rare MERS spike antigen positive pneumocytes (arrows) of NHP6 at day 5 pi. ( b ) Rare MERS spike antigen positive cells in the submucosal glands (arrow) and lymphoid aggregates (^) of NHP6 at day 5 pi; ( c ) Many epithelial cells (arrow) of submucosal glands in the bronchi and fewer cells in the BALTs (^) were positive for MERS spike antigen of NHP2 at day 30 pi. ( d ) Increased numbers of alveolar macrophages that are positive for CD26 and MERS-CoV on day 5 pi. ( e ) Reduced hyperplasia and fewer CD26+ cells and alveolar macrophages were present on day 30 pi.

    Techniques Used: Immunohistochemistry

    12) Product Images from "A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis"

    Article Title: A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

    Journal: bioRxiv

    doi: 10.1101/2020.03.24.005298

    CoV RNA synthesis is confined to RO regions. Newly-synthesized vRNA was metabolically labelled by providing tritiated uridine to CoV-infected cells pre-treated with actinomycin D to limit host transcription. (A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi. (B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm. (C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.
    Figure Legend Snippet: CoV RNA synthesis is confined to RO regions. Newly-synthesized vRNA was metabolically labelled by providing tritiated uridine to CoV-infected cells pre-treated with actinomycin D to limit host transcription. (A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi. (B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm. (C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.

    Techniques Used: Synthesized, Metabolic Labelling, Infection, Isolation, Autoradiography

    DMVs are sites of vRNA synthesis. Analysis of the association of autoradiography signal with DMVs in MERS-CoV-infected Huh7 cells (MOI 5). The cells were pre-treated with actinomycin D at 10 hpi, and labelled with tritiated uridine for 30 min immediately before fixation (12 hpi). (A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B). (C, D) Distribution of the autoradiography signal around DMVs (n DMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.
    Figure Legend Snippet: DMVs are sites of vRNA synthesis. Analysis of the association of autoradiography signal with DMVs in MERS-CoV-infected Huh7 cells (MOI 5). The cells were pre-treated with actinomycin D at 10 hpi, and labelled with tritiated uridine for 30 min immediately before fixation (12 hpi). (A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B). (C, D) Distribution of the autoradiography signal around DMVs (n DMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.

    Techniques Used: Autoradiography, Infection, Selection

    Detection of DMSs in cryo-fixed and FS samples of CoV-infected cells. Analysis of previously described samples of CoV-infected cells, prepared for EM either by HPF(A) or cryo-plunging (B). A targeted search revealed the presence of DMSs (white arrowheads) in close association with CM. In comparison with the chemically fixed samples used in this study, the superior ultrastructural preservation of cryo-fixation results in less distorted membranes, but also in a denser cytoplasm and darker CM that makes DMS less apparent. (A) Example from a MERS-CoV-infected Huh7 cell (16 hpi) in a sample used in [ 6 ]. (B) Region in a SARS-CoV-infected cell (8 hpi), adapted from [ 7 ]. Scale bars, 250 nm.
    Figure Legend Snippet: Detection of DMSs in cryo-fixed and FS samples of CoV-infected cells. Analysis of previously described samples of CoV-infected cells, prepared for EM either by HPF(A) or cryo-plunging (B). A targeted search revealed the presence of DMSs (white arrowheads) in close association with CM. In comparison with the chemically fixed samples used in this study, the superior ultrastructural preservation of cryo-fixation results in less distorted membranes, but also in a denser cytoplasm and darker CM that makes DMS less apparent. (A) Example from a MERS-CoV-infected Huh7 cell (16 hpi) in a sample used in [ 6 ]. (B) Region in a SARS-CoV-infected cell (8 hpi), adapted from [ 7 ]. Scale bars, 250 nm.

    Techniques Used: Infection, Preserving

    Newly-synthesized vRNA signal does not clearly associate with CM or DMSs. (A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal. (B) The distribution of autoradiography grains on CM was not homogeneous (n CM =9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs. (C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (n DMS = 105). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (n DMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.
    Figure Legend Snippet: Newly-synthesized vRNA signal does not clearly associate with CM or DMSs. (A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal. (B) The distribution of autoradiography grains on CM was not homogeneous (n CM =9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs. (C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (n DMS = 105). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (n DMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.

    Techniques Used: Synthesized, Autoradiography, Selection

    IEM detection of viral markers in MERS-CoV-infected cells. (A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.
    Figure Legend Snippet: IEM detection of viral markers in MERS-CoV-infected cells. (A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.

    Techniques Used: Infection, Labeling

    Membrane structures induced by MERS-CoV infection. Electron microscopy analysis of Huh7 cells infected with MERS-CoV (MOI 5, 12 hpi). (A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment. (F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).
    Figure Legend Snippet: Membrane structures induced by MERS-CoV infection. Electron microscopy analysis of Huh7 cells infected with MERS-CoV (MOI 5, 12 hpi). (A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment. (F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).

    Techniques Used: Infection, Electron Microscopy, Whisker Assay

    13) Product Images from "Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial"

    Article Title: Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial

    Journal: The Lancet. Infectious Diseases

    doi: 10.1016/S1473-3099(20)30160-2

    MERS-CoV spike-pseudotyped neutralisation p values were calculated using Kruskall-Wallis with Dunn's multiple comparison post test. The dashed lines represent lower limit of detection under our experimental condition. Data points represent geometric means, and error bars represent 95% CIs. MERS-CoV=Middle East respiratory syndrome coronavirus.
    Figure Legend Snippet: MERS-CoV spike-pseudotyped neutralisation p values were calculated using Kruskall-Wallis with Dunn's multiple comparison post test. The dashed lines represent lower limit of detection under our experimental condition. Data points represent geometric means, and error bars represent 95% CIs. MERS-CoV=Middle East respiratory syndrome coronavirus.

    Techniques Used:

    14) Product Images from "Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome"

    Article Title: Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome

    Journal: Acta Naturae

    doi: 10.32607/actanaturae.11042

    Survival of vaccinated (n = 10) and non-vaccinated (control group, n = 10) animals after a lethal infection of MERS-CoV. The ordinate axis shows the survival rate of animals (%). The abscissa axis represents time after immunization (days)
    Figure Legend Snippet: Survival of vaccinated (n = 10) and non-vaccinated (control group, n = 10) animals after a lethal infection of MERS-CoV. The ordinate axis shows the survival rate of animals (%). The abscissa axis represents time after immunization (days)

    Techniques Used: Infection

    Study of the lymphoproliferative activity of splenocytes in mice immunized with the vaccine or placebo. The levels (in %) of proliferating CD4 + and CD8 + T cells re-stimulated with recombinant MERS-CoV S glycoprotein on the 18th day after vaccination are presented. Medians of the percentage of proliferating cells after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p
    Figure Legend Snippet: Study of the lymphoproliferative activity of splenocytes in mice immunized with the vaccine or placebo. The levels (in %) of proliferating CD4 + and CD8 + T cells re-stimulated with recombinant MERS-CoV S glycoprotein on the 18th day after vaccination are presented. Medians of the percentage of proliferating cells after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p

    Techniques Used: Activity Assay, Mouse Assay, Recombinant

    Increase in the concentration of IFN-gamma in the splenocyte media of immunized and non-immunized mice after re-stimulation with recombinant MERS-CoV S glycoprotein. Median increase in the concentration of IFN-gamma after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p
    Figure Legend Snippet: Increase in the concentration of IFN-gamma in the splenocyte media of immunized and non-immunized mice after re-stimulation with recombinant MERS-CoV S glycoprotein. Median increase in the concentration of IFN-gamma after re-stimulation and 95% CI for the median for each group (n = 6) are indicated. * – p

    Techniques Used: Concentration Assay, Mouse Assay, Recombinant

    Related Articles

    Infection:

    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: 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. .. At 24 hours after infection, the cells were harvested and treated with NuPAGE LDS sample buffer (Invitrogen), boiled for 20 minutes at 95°C, and analyzed by Western blot as described above.

    other:

    Article Title: Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial
    Article Snippet: Peptides were pooled into 13 pools for the MERS-CoV spike protein containing 18 or 21 peptides, plus a single pool of five peptides for the tissue plasminogen activator leader.

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: MERS-GD27 and MERS-GD33 showed subnanomolar affinity for the MERS-CoV S protein (K D equivalent to 0.775 and 0.575 nM, respectively), which was consistent with MERS-4 (K D equivalent to 0.978 nM) [ ].

    Article Title: A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis
    Article Snippet: The human monoclonal antibody used against MERS-CoV spike protein was kindly provided by Dr. Berend Jan Bosch (Utrecht University) and has been described elsewhere as 1.6f9 [ ].

    Incubation:

    Article Title: HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV
    Article Snippet: Immunostaining and confocal imaging.Fixed cells were permeabilized with 0.1% Triton X-100–1× PBS and incubated overnight at 4°C in 1× PBS supplemented with 5% bovine serum albumin (BSA) and 0.5% Tween 20. .. To visualize MERS-CoV particles, cells were incubated for 2 h at room temperature with mouse anti-MERS-CoV N IgGs (Sino Biological, China) (1:1,000 dilution), followed by 1 h of incubation with Alexa Fluor 488-labeled goat anti-mouse IgG (Thermo Fisher Scientific, Poland) (2.5 μg/ml). .. Actin filaments were stained using phalloidin coupled with Alexa Fluor 633 (Thermo Fisher Scientific, Poland) (0.2 U/ml).

    Enzyme-linked Immunosorbent Assay:

    Article Title: High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan
    Article Snippet: Phylogenetic trees of the S2 domain were generated using Mega 6.0.6 with the maximum likelihood statistical method based on the GTR+G+I model with 1000 bootstraps replicates. .. Sera were analyzed by MERS-CoV spike protein (S) enzyme-linked immunosorbent assay (ELISA); Maxisorp (Nunc) plates were coated overnight with S1 protein (Sino Biological) and blocked with 1% milk. ..

    Functional Assay:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Activity Assay:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Produced:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Expressing:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Plasmid Preparation:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Transfection:

    Article Title: Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient
    Article Snippet: .. Functional Characterization of Antibodies With Potent Neutralizing Activity Against Middle East Respiratory Syndrome Coronavirus In VitroTo characterize the function of human mAbs targeting the MERS-CoV S protein, we produced 13 mAbs from 11 cell cultures in large quantities from different combinations of VH and VL/VK genes in the same well by a double gene expression vector transiently transfected into human embryonic kidney (HEK) 293FS cells. .. All of the Abs target the S protein from the endoplasmic reticulum membrane protein complex (EMC) strain, as determined by ELISA, whereas the H7 (a mAb against hemagglutination of the influenza virus) was used as an irrelevant Ab control and phosphate-buffered saline was used as a blank control ( ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    Sino Biological mers cov spike protein
    Single-dose intranasal immunization with <t>PIV5-MERS-S</t> induced robust <t>MERS-CoV-specific</t> CD8 T cell response in human DPP4 knockin (hDPP4 KI) mice. (A) Schematic diagram showing the experimental plan to examine CD8 T cell response after immunization and challenge. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. At 4 weeks or 4 days following immunization, mice were challenged with MERS MA 6.1.2, single-cell suspensions from the lungs of immunized mice were stimulated with MERS-CoV spike peptides (S343 and S1165), and specific CD8 T cells were determined by IFN-γ intracellular staining. (B to D) Representative FACS plots (B), percentage (C), and total number (D) of MERS-CoV-specific CD8 T cells in the lungs at 4 weeks after immunization. (E to G) Representative FACS plots (E), percentage (F), and total number (G) of MERS-CoV-specific CD8 T cells in the lungs at 4 days after MERS MA 6.1.2 challenge ( n = 6 mice per group). Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P
    Mers Cov Spike Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov spike protein/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov spike protein - by Bioz Stars, 2021-05
    96/100 stars
      Buy from Supplier

    97
    Sino Biological mouse anti mers cov n iggs
    Coronavirus internalization into susceptible cells is hampered by HTCC. (A and B) Precooled HAE cultures were incubated with an ice-cold <t>MERS-CoV</t> suspension in the presence or absence of HTCC-63 (200 μg/ml) for 2 h at 37°C. Next, cells were fixed in paraformaldehyde (PFA) and immunostained for MERS-CoV N protein and actin. Virus entry was analyzed with confocal microscopy. The data shown are representative of results from three independent experiments, each performed in triplicate. Mann-Whitney test, *** * , P
    Mouse Anti Mers Cov N Iggs, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti mers cov n iggs/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti mers cov n iggs - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    96
    Sino Biological rabbit anti mers cov antibodies
    Characterization of <t>anti-MERS-S2P</t> IgGs. ( A ) Immunofluorescence assay of anti-MERS-S2P IgGs on cell lines infected with various human coronaviruses (hCoVs) to determine their <t>MERS-CoV</t> specificity. Scale bar, 200 μm. ( B ) Size-exclusion chromatography analysis of S2A3 (IgG) and S2D5 (IgG). The molecular weights (kDa) of the molecular mass markers are shown above the corresponding peaks at the top chromatogram. ( C ) Surface plasmon resonance (SPR) analysis of S2A3 (IgG) and S2D5 (IgG) on a MERS-S2P-immobilized sensor chip to determine their apparent binding affinities. The fitted-lines are marked by red.
    Rabbit Anti Mers Cov Antibodies, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mers cov antibodies/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mers cov antibodies - by Bioz Stars, 2021-05
    96/100 stars
      Buy from Supplier

    93
    Sino Biological monoclonal mouse anti mers cov nucleocapsid antibody
    Mapping of H2-d restricted T cell epitopes in <t>MERS-CoV</t> N protein; ( a–b ) BALB/c mice (n = 2 to 4) were immunized twice (21-day interval) i.p. or i.m. with 10 8 PFU of recombinant MVA-MERS-N (MVA-N), non-recombinant MVA (MVA) or PBS. Splenocytes from vaccinated mice were incubated in the presence of subpools (V8.1, V8.2, H8.1, H8.2) from positive matrix pools ( a ) or individual 15-mers peptides #89 or #90 ( b ). IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were quantified by ELISPOT. The lines represent means.
    Monoclonal Mouse Anti Mers Cov Nucleocapsid Antibody, 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
    https://www.bioz.com/result/monoclonal mouse anti mers cov nucleocapsid antibody/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal mouse anti mers cov nucleocapsid antibody - by Bioz Stars, 2021-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    Single-dose intranasal immunization with PIV5-MERS-S induced robust MERS-CoV-specific CD8 T cell response in human DPP4 knockin (hDPP4 KI) mice. (A) Schematic diagram showing the experimental plan to examine CD8 T cell response after immunization and challenge. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. At 4 weeks or 4 days following immunization, mice were challenged with MERS MA 6.1.2, single-cell suspensions from the lungs of immunized mice were stimulated with MERS-CoV spike peptides (S343 and S1165), and specific CD8 T cells were determined by IFN-γ intracellular staining. (B to D) Representative FACS plots (B), percentage (C), and total number (D) of MERS-CoV-specific CD8 T cells in the lungs at 4 weeks after immunization. (E to G) Representative FACS plots (E), percentage (F), and total number (G) of MERS-CoV-specific CD8 T cells in the lungs at 4 days after MERS MA 6.1.2 challenge ( n = 6 mice per group). Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Single-dose intranasal immunization with PIV5-MERS-S induced robust MERS-CoV-specific CD8 T cell response in human DPP4 knockin (hDPP4 KI) mice. (A) Schematic diagram showing the experimental plan to examine CD8 T cell response after immunization and challenge. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. At 4 weeks or 4 days following immunization, mice were challenged with MERS MA 6.1.2, single-cell suspensions from the lungs of immunized mice were stimulated with MERS-CoV spike peptides (S343 and S1165), and specific CD8 T cells were determined by IFN-γ intracellular staining. (B to D) Representative FACS plots (B), percentage (C), and total number (D) of MERS-CoV-specific CD8 T cells in the lungs at 4 weeks after immunization. (E to G) Representative FACS plots (E), percentage (F), and total number (G) of MERS-CoV-specific CD8 T cells in the lungs at 4 days after MERS MA 6.1.2 challenge ( n = 6 mice per group). Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Knock-In, Mouse Assay, Staining, FACS

    Serum neutralizing antibodies produced in mice 4 weeks after single-dose intranasal immunization with PIV5-MERS-S. Naive mice were intranasally immunized with PIV5-GFP or PIV5-MERS-S. Sera were collected at 4 weeks postimmunization. Neutralization assay against MERS-CoV spike pseudovirions was performed as described in Materials and Methods. The neutralization results were measured in luciferase activity and plotted relative to mock-treatment value. (A and B) Neutralization assay results from C57BL/6 mice immunized with 10 4 PFU (A) and 10 6 PFU (B) PIV5-MERS-S or PIV5-GFP. (C) Neutralization assay results from BALB/c mice immunized with 10 6 PFU PIV5-MERS-S or PIV5-GFP. Data presented represent means ± SEs.

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Serum neutralizing antibodies produced in mice 4 weeks after single-dose intranasal immunization with PIV5-MERS-S. Naive mice were intranasally immunized with PIV5-GFP or PIV5-MERS-S. Sera were collected at 4 weeks postimmunization. Neutralization assay against MERS-CoV spike pseudovirions was performed as described in Materials and Methods. The neutralization results were measured in luciferase activity and plotted relative to mock-treatment value. (A and B) Neutralization assay results from C57BL/6 mice immunized with 10 4 PFU (A) and 10 6 PFU (B) PIV5-MERS-S or PIV5-GFP. (C) Neutralization assay results from BALB/c mice immunized with 10 6 PFU PIV5-MERS-S or PIV5-GFP. Data presented represent means ± SEs.

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Produced, Mouse Assay, Neutralization, Luciferase, Activity Assay

    Representative images of lung tissues from mice receiving PBS (A), UV-inactivated MERS-CoV (B), or PIV5-MERS-S (C) treatment, followed by infection with MERS MA 6.1.2. Images obtained from tissues at 3 days after MERS MA 6.1.2 infection. Compared to PBS or PIV5-MERS, perivascular eosinophilic infiltration (arrows) in UV-MERS-treated mice was greatly increased. n = 3 to 4 mice/group. (D) Graph representing eosinophil infiltration in the lung tissues of mice from groups shown in panels A to . * denotes P

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Representative images of lung tissues from mice receiving PBS (A), UV-inactivated MERS-CoV (B), or PIV5-MERS-S (C) treatment, followed by infection with MERS MA 6.1.2. Images obtained from tissues at 3 days after MERS MA 6.1.2 infection. Compared to PBS or PIV5-MERS, perivascular eosinophilic infiltration (arrows) in UV-MERS-treated mice was greatly increased. n = 3 to 4 mice/group. (D) Graph representing eosinophil infiltration in the lung tissues of mice from groups shown in panels A to . * denotes P

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Mouse Assay, Infection

    Histopathology in immunized mice challenged with MERS-CoV. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (A) Representative images of H E-stained lung sections from PIV5-MERS-S- or PIV5-GFP-immunized hDPP4 KI mice at indicated days after MERS MA 6.1.2 challenge. Note the cellular infiltration (black arrows) and the hyaline membranes (red arrows). (B) Summary scores for disease in the lung sections. n = 3 to 5 mice/group. * denotes P

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Histopathology in immunized mice challenged with MERS-CoV. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S or PIV5-GFP. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (A) Representative images of H E-stained lung sections from PIV5-MERS-S- or PIV5-GFP-immunized hDPP4 KI mice at indicated days after MERS MA 6.1.2 challenge. Note the cellular infiltration (black arrows) and the hyaline membranes (red arrows). (B) Summary scores for disease in the lung sections. n = 3 to 5 mice/group. * denotes P

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Histopathology, Mouse Assay, Infection, Staining

    Comparison of the protective efficacy between single-dose immunization with UV light-inactivated MERS-CoV and PIV5-MERS-S. hDPP4 KI mice were immunized with 10 4 PFU PIV5-MERS-S via intranasal route; 10 6 PFU UV-inactivated MERS MA 6.1.2, mixed with Imject alum; or PBS via intramuscular route. Four weeks after immunization, immunized mice were infected with 10 5 PFU MERS-CoV. (A) Schematic timeline outlining experimental plan. (B and C) Survival (B) and weight loss (C) were monitored daily until 10 days postinfection. PBS, n = 9; UV MERS-CoV, n = 12; PIV5-MERS-S, n = 8. Data represent mean ± SE.

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Comparison of the protective efficacy between single-dose immunization with UV light-inactivated MERS-CoV and PIV5-MERS-S. hDPP4 KI mice were immunized with 10 4 PFU PIV5-MERS-S via intranasal route; 10 6 PFU UV-inactivated MERS MA 6.1.2, mixed with Imject alum; or PBS via intramuscular route. Four weeks after immunization, immunized mice were infected with 10 5 PFU MERS-CoV. (A) Schematic timeline outlining experimental plan. (B and C) Survival (B) and weight loss (C) were monitored daily until 10 days postinfection. PBS, n = 9; UV MERS-CoV, n = 12; PIV5-MERS-S, n = 8. Data represent mean ± SE.

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Mouse Assay, Infection

    Generation and characterization of recombinant PIV5 expressing MERS-CoV spike protein. (A) Schematic of PIV5-MERS-S. NP, nucleoprotein; V, V protein; P, phosphoprotein; M, matrix protein; F, fusion protein; SH, small hydrophobic protein; HN, hemagglutinin-neuraminidase protein; L, RNA-dependent RNA polymerase. (B) Confirmation of MERS-CoV spike protein expression by Western blotting. Vero 81 cells were infected with PIV5-MERS-S at MOIs of 0.01, 0.1, and 1.0 or mock infected. At 2 days postinfection, MERS-CoV spike was detected with anti-MERS-S antibody by Western blotting. (C) Immunofluorescence of Vero cells infected with PIV5 and PIV5-MERS-S. Vero cells were infected with PIV5 and PIV5-MERS-S (MOI = 0.1) or mock infected. At 2 days postinfection, cells were fixed, permeabilized, and stained with anti-PIV5 V/P or anti-MERS-spike antibodies. Scale bar = 200 μm. (D) Growth rate of PIV5-MERS-S. MDBK cells were infected with PIV5 or PIV5-MERS-S at an MOI of 0.1. Media were collected daily for 5 days, and titers of viruses in the media were determined using plaque assay.

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Generation and characterization of recombinant PIV5 expressing MERS-CoV spike protein. (A) Schematic of PIV5-MERS-S. NP, nucleoprotein; V, V protein; P, phosphoprotein; M, matrix protein; F, fusion protein; SH, small hydrophobic protein; HN, hemagglutinin-neuraminidase protein; L, RNA-dependent RNA polymerase. (B) Confirmation of MERS-CoV spike protein expression by Western blotting. Vero 81 cells were infected with PIV5-MERS-S at MOIs of 0.01, 0.1, and 1.0 or mock infected. At 2 days postinfection, MERS-CoV spike was detected with anti-MERS-S antibody by Western blotting. (C) Immunofluorescence of Vero cells infected with PIV5 and PIV5-MERS-S. Vero cells were infected with PIV5 and PIV5-MERS-S (MOI = 0.1) or mock infected. At 2 days postinfection, cells were fixed, permeabilized, and stained with anti-PIV5 V/P or anti-MERS-spike antibodies. Scale bar = 200 μm. (D) Growth rate of PIV5-MERS-S. MDBK cells were infected with PIV5 or PIV5-MERS-S at an MOI of 0.1. Media were collected daily for 5 days, and titers of viruses in the media were determined using plaque assay.

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Recombinant, Expressing, Western Blot, Infection, Immunofluorescence, Staining, Plaque Assay

    Single-dose intranasal immunization with PIV5-MERS-S completely protects hDPP4 KI mice from lethal MERS-CoV challenge. (A) Schematic timeline showing immunization, challenge, and the evaluation of protection. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S, PIV5-GFP, or PBS. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (B and C) Survival (B) and weight loss (C) were monitored daily for 12 days. PIV5-MERS-S or PIV5-GFP, n = 10; PBS, n = 5. (D) At indicated days postinfection, virus lung titers were quantified by plaque assay. Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Journal: mBio

    Article Title: Single-Dose, Intranasal Immunization with Recombinant Parainfluenza Virus 5 Expressing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Spike Protein Protects Mice from Fatal MERS-CoV Infection

    doi: 10.1128/mBio.00554-20

    Figure Lengend Snippet: Single-dose intranasal immunization with PIV5-MERS-S completely protects hDPP4 KI mice from lethal MERS-CoV challenge. (A) Schematic timeline showing immunization, challenge, and the evaluation of protection. hDPP4 KI mice were intranasally immunized with 10 4 PFU PIV5-MERS-S, PIV5-GFP, or PBS. Four weeks after immunization, the mice were intranasally infected with 10 5 PFU MERS MA 6.1.2. (B and C) Survival (B) and weight loss (C) were monitored daily for 12 days. PIV5-MERS-S or PIV5-GFP, n = 10; PBS, n = 5. (D) At indicated days postinfection, virus lung titers were quantified by plaque assay. Data are representative of two independent experiments. Data presented represent mean ± SE; * denotes P

    Article Snippet: The expression of MERS-CoV spike protein was detected by a rabbit anti-MERS-CoV S2 antibody (Sino Biological, catalog no. 40070-T60) and colocalized using a mouse anti-α-tubulin antibody (Sigma, catalog no. T9026).

    Techniques: Mouse Assay, Infection, Plaque Assay

    Coronavirus internalization into susceptible cells is hampered by HTCC. (A and B) Precooled HAE cultures were incubated with an ice-cold MERS-CoV suspension in the presence or absence of HTCC-63 (200 μg/ml) for 2 h at 37°C. Next, cells were fixed in paraformaldehyde (PFA) and immunostained for MERS-CoV N protein and actin. Virus entry was analyzed with confocal microscopy. The data shown are representative of results from three independent experiments, each performed in triplicate. Mann-Whitney test, *** * , P

    Journal: Journal of Virology

    Article Title: HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV

    doi: 10.1128/JVI.01622-20

    Figure Lengend Snippet: Coronavirus internalization into susceptible cells is hampered by HTCC. (A and B) Precooled HAE cultures were incubated with an ice-cold MERS-CoV suspension in the presence or absence of HTCC-63 (200 μg/ml) for 2 h at 37°C. Next, cells were fixed in paraformaldehyde (PFA) and immunostained for MERS-CoV N protein and actin. Virus entry was analyzed with confocal microscopy. The data shown are representative of results from three independent experiments, each performed in triplicate. Mann-Whitney test, *** * , P

    Article Snippet: Mouse anti-MERS-CoV N IgGs (Sino Biological, China) (1:1,000 dilution) and horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (Dako, Denmark) (65 ng/ml) were used to detect MERS-CoV nucleocapsid protein.

    Techniques: Incubation, Confocal Microscopy, MANN-WHITNEY

    HTCC blocks the interaction between the virus and its entry receptor. Precooled Huh7 cells were incubated for 3 h at 4°C with ice-cold MERS-CoV or subjected to mock treatment in the presence or absence of HTCC-63 (100 μg/ml). Next, cells were fixed with PFA and immunostained for MERS-CoV-N (green), DPP4 (red), and nuclear DNA (blue). MERS-CoV interaction with the DPP4 protein was analyzed with confocal microscopy. Colocalization of DPP4 with MERS-CoV-N was determined by confocal microscopy, and the results are presented as Manders’ M2 coefficient values after excluding nonspecific nuclear signal. Colocalization analysis was carried out with ImageJ JACoP (Just Another Colocalization Plugin). The decrease in colocalization was statistically significant ( P

    Journal: Journal of Virology

    Article Title: HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV

    doi: 10.1128/JVI.01622-20

    Figure Lengend Snippet: HTCC blocks the interaction between the virus and its entry receptor. Precooled Huh7 cells were incubated for 3 h at 4°C with ice-cold MERS-CoV or subjected to mock treatment in the presence or absence of HTCC-63 (100 μg/ml). Next, cells were fixed with PFA and immunostained for MERS-CoV-N (green), DPP4 (red), and nuclear DNA (blue). MERS-CoV interaction with the DPP4 protein was analyzed with confocal microscopy. Colocalization of DPP4 with MERS-CoV-N was determined by confocal microscopy, and the results are presented as Manders’ M2 coefficient values after excluding nonspecific nuclear signal. Colocalization analysis was carried out with ImageJ JACoP (Just Another Colocalization Plugin). The decrease in colocalization was statistically significant ( P

    Article Snippet: Mouse anti-MERS-CoV N IgGs (Sino Biological, China) (1:1,000 dilution) and horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (Dako, Denmark) (65 ng/ml) were used to detect MERS-CoV nucleocapsid protein.

    Techniques: Incubation, Confocal Microscopy

    Characterization of anti-MERS-S2P IgGs. ( A ) Immunofluorescence assay of anti-MERS-S2P IgGs on cell lines infected with various human coronaviruses (hCoVs) to determine their MERS-CoV specificity. Scale bar, 200 μm. ( B ) Size-exclusion chromatography analysis of S2A3 (IgG) and S2D5 (IgG). The molecular weights (kDa) of the molecular mass markers are shown above the corresponding peaks at the top chromatogram. ( C ) Surface plasmon resonance (SPR) analysis of S2A3 (IgG) and S2D5 (IgG) on a MERS-S2P-immobilized sensor chip to determine their apparent binding affinities. The fitted-lines are marked by red.

    Journal: Antibodies

    Article Title: Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning

    doi: 10.3390/antib8030042

    Figure Lengend Snippet: Characterization of anti-MERS-S2P IgGs. ( A ) Immunofluorescence assay of anti-MERS-S2P IgGs on cell lines infected with various human coronaviruses (hCoVs) to determine their MERS-CoV specificity. Scale bar, 200 μm. ( B ) Size-exclusion chromatography analysis of S2A3 (IgG) and S2D5 (IgG). The molecular weights (kDa) of the molecular mass markers are shown above the corresponding peaks at the top chromatogram. ( C ) Surface plasmon resonance (SPR) analysis of S2A3 (IgG) and S2D5 (IgG) on a MERS-S2P-immobilized sensor chip to determine their apparent binding affinities. The fitted-lines are marked by red.

    Article Snippet: The rabbit anti-MERS-CoV antibodies (1:3000, Sino Biological), serially two-fold-diluted MERS-S2P, and HRP-conjugated goat anti-rabbit antibodies (1:6000, Sigma Aldrich) were added and incubated for 30 min at RT.

    Techniques: Immunofluorescence, Infection, Size-exclusion Chromatography, SPR Assay, Chromatin Immunoprecipitation, Binding Assay

    Output of the panning of the phage-displayed synthetic Fab library on MERS-S2P. ( A ) Monitoring of phage titers over three rounds (R1–R3) of panning. Black and gray bars indicate a ratio of phage output to input titers presented as a percentage (%) from panning on MERS-S2P-immobilized and -non-immobilized surfaces, respectively. The ratio of output to input (%) = (phage output titer ÷ phage input titer) × 100. ( B ) Phage ELISAs performed on MERS-S2P-, SARS-CoV spike protein-, a CoV spike protein-immobilized surfaces (blue, red, and green, respectively). ( C ) Amino acid sequences of three unique clones identified from panning (left) and their relative frequencies (%) (right). The sequences were aligned using the Kabat numbering system [ 38 ]. ELISA, enzyme-linked immunosorbent assay; MERS-S2P, Middle East respiratory syndrome-CoV S2 subunit protein; SARS-SP, severe acute respiratory syndrome-CoV S protein; HKU1-SP, hCoV HKU1 S protein; CoV, coronavirus; CDR, complementarity-determining region; FR, framework region.

    Journal: Antibodies

    Article Title: Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning

    doi: 10.3390/antib8030042

    Figure Lengend Snippet: Output of the panning of the phage-displayed synthetic Fab library on MERS-S2P. ( A ) Monitoring of phage titers over three rounds (R1–R3) of panning. Black and gray bars indicate a ratio of phage output to input titers presented as a percentage (%) from panning on MERS-S2P-immobilized and -non-immobilized surfaces, respectively. The ratio of output to input (%) = (phage output titer ÷ phage input titer) × 100. ( B ) Phage ELISAs performed on MERS-S2P-, SARS-CoV spike protein-, a CoV spike protein-immobilized surfaces (blue, red, and green, respectively). ( C ) Amino acid sequences of three unique clones identified from panning (left) and their relative frequencies (%) (right). The sequences were aligned using the Kabat numbering system [ 38 ]. ELISA, enzyme-linked immunosorbent assay; MERS-S2P, Middle East respiratory syndrome-CoV S2 subunit protein; SARS-SP, severe acute respiratory syndrome-CoV S protein; HKU1-SP, hCoV HKU1 S protein; CoV, coronavirus; CDR, complementarity-determining region; FR, framework region.

    Article Snippet: The rabbit anti-MERS-CoV antibodies (1:3000, Sino Biological), serially two-fold-diluted MERS-S2P, and HRP-conjugated goat anti-rabbit antibodies (1:6000, Sigma Aldrich) were added and incubated for 30 min at RT.

    Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay

    Detection of MERS-S2P using S2A3 (IgG) on ACCEL ELISA™ plates. ( A ) Schematic depicting the sandwich ELISA format to detect MERS-S2P using S2A3 (IgG) (capture antibody) and rabbit anti-MERS-CoV IgG (detection antibody) on ACCEL ELISA™ plates. ( B ) ELISA detection of MERS-S2P on a capture antibody (S2A3 (IgG)) immobilized using three different concentrations (3 µg/mL, 5 µg/mL, and 10 µg/mL) on ACCEL ELISA™ plates. The goodness of fit is indicated by the R 2 value. LOD, limit of detection.

    Journal: Antibodies

    Article Title: Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning

    doi: 10.3390/antib8030042

    Figure Lengend Snippet: Detection of MERS-S2P using S2A3 (IgG) on ACCEL ELISA™ plates. ( A ) Schematic depicting the sandwich ELISA format to detect MERS-S2P using S2A3 (IgG) (capture antibody) and rabbit anti-MERS-CoV IgG (detection antibody) on ACCEL ELISA™ plates. ( B ) ELISA detection of MERS-S2P on a capture antibody (S2A3 (IgG)) immobilized using three different concentrations (3 µg/mL, 5 µg/mL, and 10 µg/mL) on ACCEL ELISA™ plates. The goodness of fit is indicated by the R 2 value. LOD, limit of detection.

    Article Snippet: The rabbit anti-MERS-CoV antibodies (1:3000, Sino Biological), serially two-fold-diluted MERS-S2P, and HRP-conjugated goat anti-rabbit antibodies (1:6000, Sigma Aldrich) were added and incubated for 30 min at RT.

    Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

    Mapping of H2-d restricted T cell epitopes in MERS-CoV N protein; ( a–b ) BALB/c mice (n = 2 to 4) were immunized twice (21-day interval) i.p. or i.m. with 10 8 PFU of recombinant MVA-MERS-N (MVA-N), non-recombinant MVA (MVA) or PBS. Splenocytes from vaccinated mice were incubated in the presence of subpools (V8.1, V8.2, H8.1, H8.2) from positive matrix pools ( a ) or individual 15-mers peptides #89 or #90 ( b ). IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were quantified by ELISPOT. The lines represent means.

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Mapping of H2-d restricted T cell epitopes in MERS-CoV N protein; ( a–b ) BALB/c mice (n = 2 to 4) were immunized twice (21-day interval) i.p. or i.m. with 10 8 PFU of recombinant MVA-MERS-N (MVA-N), non-recombinant MVA (MVA) or PBS. Splenocytes from vaccinated mice were incubated in the presence of subpools (V8.1, V8.2, H8.1, H8.2) from positive matrix pools ( a ) or individual 15-mers peptides #89 or #90 ( b ). IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were quantified by ELISPOT. The lines represent means.

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Mouse Assay, Recombinant, Incubation, Enzyme-linked Immunospot

    Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; ( a–d ) Groups of BALB/c mice ( n = 3 to 8) were vaccinated in a prime-boost regime with 10 8 PFU of MVA-MERS-N via i.p. ( a ) or i.m. ( b–d ) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. ( a-b ) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. ( c–d ) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide ( c ) or F2L 26-34 peptide ( d ) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; ( a–d ) Groups of BALB/c mice ( n = 3 to 8) were vaccinated in a prime-boost regime with 10 8 PFU of MVA-MERS-N via i.p. ( a ) or i.m. ( b–d ) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. ( a-b ) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. ( c–d ) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide ( c ) or F2L 26-34 peptide ( d ) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Mouse Assay, Recombinant, Enzyme-linked Immunospot, Staining, FACS

    Analysis of recombinant MVA-MERS proteins; ( a ) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; ( b – d ) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody ( b ) or polyclonal sera from MERS-CoV infected rabbits ( c ) or cynomolgus macaques ( d ). Arrows indicate the N- or S-specific protein bands.

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Analysis of recombinant MVA-MERS proteins; ( a ) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; ( b – d ) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody ( b ) or polyclonal sera from MERS-CoV infected rabbits ( c ) or cynomolgus macaques ( d ). Arrows indicate the N- or S-specific protein bands.

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Recombinant, Western Blot, Produced, Infection

    Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; ( a – b ) groups of BALB/c mice ( n = 2 to 6) were vaccinated twice (21-day interval) by i.p. ( a ) or i.m. ( b ) application with 10 8 plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; ( a – b ) groups of BALB/c mice ( n = 2 to 6) were vaccinated twice (21-day interval) by i.p. ( a ) or i.m. ( b ) application with 10 8 plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Mouse Assay, Recombinant, In Vitro, Enzyme-linked Immunospot