nucleoprotein  (Sino Biological)


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    • 96
    Name:
    MERS CoV Nucleoprotein NP protein
    Description:
    A DNA sequence encoding the Nucleoprotein MERS CoV AFS88943 1 Met1 Asp413 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    40068-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus NP Protein MERS-CoV, coronavirus Nucleocapsid Protein MERS-CoV, coronavirus Nucleoprotein Protein MERS-CoV, cov np Protein MERS-CoV, ncov NP Protein MERS-CoV, novel coronavirus Nucleoprotein Protein MERS-CoV, NP Protein MERS-CoV, Nucleocapsid Protein MERS-CoV, Nucleoprotein Protein MERS-CoV
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological nucleoprotein
    A DNA sequence encoding the Nucleoprotein MERS CoV AFS88943 1 Met1 Asp413 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/nucleoprotein/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nucleoprotein - by Bioz Stars, 2021-04
    96/100 stars

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    Related Articles

    Expressing:

    Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus
    Article Snippet: Briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. .. Expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) (Sino Biological Inc., Beijing, China), neutrophil marker NIMP-14, (Santa Cruz Biotechnology, Paso Robles, CA, USA) and CD68 (Abcam, Cambridge, MA, USA). .. The reaction was detected using a standard streptavidin-biotin detection system (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) and visualized using DAB with hematoxylin counterstaining.

    Article Title: Species-Specific Colocalization of Middle East Respiratory Syndrome Coronavirus Attachment and Entry Receptors
    Article Snippet: The bat tissues included in this study were histologically normal as determined by using hematoxylin-eosin staining prior to our experiment. .. MERS-CoV nucleoprotein was detected with 5 μg/ml mouse anti-MERS nucleoprotein (Sino-Biological, Beijing, China), while DPP4 expression was detected with either 5 μg/ml goat anti-human DPP4 (R & D, Minneapolis, MN, USA) or 10 μg/ml mouse anti-human DPP4 (clone 11D7; Origene, Rockville, MD, USA). .. Briefly, the paraffin-embedded tissues were deparaffinized using xylene, hydrated using graded concentrations of alcohol, boiled in 10 mM citric acid buffer (pH 6) for 15 min, and subsequently incubated in 3% H2 O2 for 10 min and in 5% normal goat serum for 30 min before staining with antibodies.

    Marker:

    Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus
    Article Snippet: Briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. .. Expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) (Sino Biological Inc., Beijing, China), neutrophil marker NIMP-14, (Santa Cruz Biotechnology, Paso Robles, CA, USA) and CD68 (Abcam, Cambridge, MA, USA). .. The reaction was detected using a standard streptavidin-biotin detection system (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) and visualized using DAB with hematoxylin counterstaining.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins
    Article Snippet: 2.2 Development of phage display-derived SARS-CoV-2-specific antibodiesThe scFv clones were isolated from a chicken naïve phage library (YntoAb, South Korea) by biopanning using purified recombinant SARS-CoV-2 NP. .. Monoclonal binders were selected using an ELISA based on SARS-CoV-2 NP (40588-V08B; Sino Biological, Inc., China); clones that were cross-reactive for SARS-CoV NP (40143-V08B, Sino Biological, Inc.) and MERS-CoV NP (40068-V08B, Sino Biological, Inc.) were eliminated. ..

    Clone Assay:

    Article Title: Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins
    Article Snippet: 2.2 Development of phage display-derived SARS-CoV-2-specific antibodiesThe scFv clones were isolated from a chicken naïve phage library (YntoAb, South Korea) by biopanning using purified recombinant SARS-CoV-2 NP. .. Monoclonal binders were selected using an ELISA based on SARS-CoV-2 NP (40588-V08B; Sino Biological, Inc., China); clones that were cross-reactive for SARS-CoV NP (40143-V08B, Sino Biological, Inc.) and MERS-CoV NP (40068-V08B, Sino Biological, Inc.) were eliminated. ..

    Immunohistochemistry:

    Article Title: Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits
    Article Snippet: .. Samples were collected in a standard manner from the cranial and caudal parts of the lung, embedded in paraffin, sectioned at 4 μm, and used for immunohistochemistry (IHC) with sera from human MERS patients, a monoclonal antibody to the MERS-CoV nucleocapsid protein (Sino Biological, Beijing), for in situ hybridization (ISH), or for histopathology after staining with hematoxylin and eosin (HE). ..

    In Situ Hybridization:

    Article Title: Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits
    Article Snippet: .. Samples were collected in a standard manner from the cranial and caudal parts of the lung, embedded in paraffin, sectioned at 4 μm, and used for immunohistochemistry (IHC) with sera from human MERS patients, a monoclonal antibody to the MERS-CoV nucleocapsid protein (Sino Biological, Beijing), for in situ hybridization (ISH), or for histopathology after staining with hematoxylin and eosin (HE). ..

    Histopathology:

    Article Title: Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits
    Article Snippet: .. Samples were collected in a standard manner from the cranial and caudal parts of the lung, embedded in paraffin, sectioned at 4 μm, and used for immunohistochemistry (IHC) with sera from human MERS patients, a monoclonal antibody to the MERS-CoV nucleocapsid protein (Sino Biological, Beijing), for in situ hybridization (ISH), or for histopathology after staining with hematoxylin and eosin (HE). ..

    Staining:

    Article Title: Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits
    Article Snippet: .. Samples were collected in a standard manner from the cranial and caudal parts of the lung, embedded in paraffin, sectioned at 4 μm, and used for immunohistochemistry (IHC) with sera from human MERS patients, a monoclonal antibody to the MERS-CoV nucleocapsid protein (Sino Biological, Beijing), for in situ hybridization (ISH), or for histopathology after staining with hematoxylin and eosin (HE). ..

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus
    Article Snippet: Cells were incubated in a blocking solution [PBS, 10% donor calf serum (Sigma, Cat #C9676) and 0.1% Tween 20 (Fisher Bioreagents, Cat #BP337–500)]. .. Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302). .. Secondary staining was performed using 4 μg/ml goat anti-mouse Alexa 488 (Molecular Probes, Cat #A-11001), 0.1 μg/ml goat anti-rabbit Cy5 (GE Healthcare, Cat #PA45012) and 0.2 μg/ml Hoechst 33342 (Molecular Probes, Cat #H3570) in blocking solution.

    Recombinant:

    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
    Article Snippet: Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1). .. Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1 ). ..

    Sequencing:

    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
    Article Snippet: Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1). .. Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1 ). ..

    Derivative Assay:

    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
    Article Snippet: Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1). .. Recombinant influenza A virus nucleoprotein (rNP) was purchased from Sino Biological Inc. (Beijing, China) (DNA sequence derived from A/Anhui/1-BALF_RG6/2013, H7N9; accession number: AHZ59787.1 ). ..

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    • monoclonal mouse anti mers cov nucleocapsid antibody  (Sino Biological)
    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, 2021-04
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    polyclonal rabbit anti mers cov nucleocapsid antibodies  (Sino Biological)
    94
    Sino Biological polyclonal rabbit anti mers cov nucleocapsid antibodies
    Representative HE findings in respiratory epithelium of the nasal turbinates of <t>MERS-CoV-infected</t> alpacas and immunohistochemical reactions of the commercially available positive controls for comparison. (A) Multifocal infiltration of lamina propria and submucosa by moderate numbers of lymphocytes, macrophages, and single neutrophilic granulocytes (asterisk). (B) Exocytosis of single neutrophilic granulocytes (grey arrow). (C, D) Abundant viral antigen was detected multifocally in the cytoplasm and along the apical membranous region of epithelial cells using a monoclonal mouse (C, Sino Biological Inc.) and <t>polyclonal</t> rabbit anti-MERS-CoV nucleocapsid antibody (D, Sino Biological Inc.) with citrate pretreatment, in a dilution of 1:70 and 1:2,000, respectively. Both antibodies exhibited a similar staining intensity. (A, B) HE staining; 400x, (C, D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.
    Polyclonal Rabbit Anti Mers Cov Nucleocapsid Antibodies, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit anti mers cov nucleocapsid antibodies/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit anti mers cov nucleocapsid antibodies - by Bioz Stars, 2021-04
    94/100 stars
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    mers cov nucleoprotein  (Sino Biological)
    96
    Sino Biological mers cov nucleoprotein
    ΔORF5 <t>MERS-CoV</t> infection induces higher levels of IFNβ and ISGs in bat cells. ( a–e ) Transcript levels of IFNβ and interferon stimulated genes (ISGs), IFI6, GBP1, Mx1 and MDA5 in Efk and MRC5 cells infected with W+ or ΔORF5 MERS-CoV. (f) DPP4 transcript levels in W+ or ΔORF5 MERS-CoV infected Efk cells (n = 4; Mean ± SD). Bars represent average fold changes (2 −ΔΔCT ) in transcript levels compared to mock infected cells and normalized to GAPDH levels in each sample (n = 4; Mean ± SD). *P
    Mers Cov Nucleoprotein, 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 nucleoprotein/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov nucleoprotein - by Bioz Stars, 2021-04
    96/100 stars
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    Image Search Results


    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

    Representative HE findings in respiratory epithelium of the nasal turbinates of MERS-CoV-infected alpacas and immunohistochemical reactions of the commercially available positive controls for comparison. (A) Multifocal infiltration of lamina propria and submucosa by moderate numbers of lymphocytes, macrophages, and single neutrophilic granulocytes (asterisk). (B) Exocytosis of single neutrophilic granulocytes (grey arrow). (C, D) Abundant viral antigen was detected multifocally in the cytoplasm and along the apical membranous region of epithelial cells using a monoclonal mouse (C, Sino Biological Inc.) and polyclonal rabbit anti-MERS-CoV nucleocapsid antibody (D, Sino Biological Inc.) with citrate pretreatment, in a dilution of 1:70 and 1:2,000, respectively. Both antibodies exhibited a similar staining intensity. (A, B) HE staining; 400x, (C, D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Journal: Veterinary Immunology and Immunopathology

    Article Title: Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

    doi: 10.1016/j.vetimm.2019.109939

    Figure Lengend Snippet: Representative HE findings in respiratory epithelium of the nasal turbinates of MERS-CoV-infected alpacas and immunohistochemical reactions of the commercially available positive controls for comparison. (A) Multifocal infiltration of lamina propria and submucosa by moderate numbers of lymphocytes, macrophages, and single neutrophilic granulocytes (asterisk). (B) Exocytosis of single neutrophilic granulocytes (grey arrow). (C, D) Abundant viral antigen was detected multifocally in the cytoplasm and along the apical membranous region of epithelial cells using a monoclonal mouse (C, Sino Biological Inc.) and polyclonal rabbit anti-MERS-CoV nucleocapsid antibody (D, Sino Biological Inc.) with citrate pretreatment, in a dilution of 1:70 and 1:2,000, respectively. Both antibodies exhibited a similar staining intensity. (A, B) HE staining; 400x, (C, D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Article Snippet: For positive control and elucidation of virus distribution, additional sections were stained with two different commercially available, previously published monoclonal mouse and polyclonal rabbit anti-MERS-CoV nucleocapsid antibodies (Sino Biological Inc.; ; ), respectively.

    Techniques: Infection, Immunohistochemistry, Staining, Avidin-Biotin Assay

    Representative positive immunohistochemical reactions for human monoclonal antibodies. (A) Antibody 1.2g5 with citrate pretreatment exhibited a strong multifocal MERS-CoV antigen specific signal in the cytoplasm of ciliated respiratory epithelial cells (arrow) and segmentally along the apical membranous region (arrow heads). (B) The same protocol without pretreatment revealed a much fainter but similarly distributed staining of cytoplasm (arrow) and ciliary base (arrow heads). (C) Antibody 1.10f3 with citrate pretreatment displayed a strong diffuse background staining and only few positively stained cilia (arrowhead). Single intraepithelial plasma cells displayed a strong false positive intracytoplasmic staining (white arrow). (D) Antibody 1.6c7 with citrate pretreatment exhibited also a strong diffuse background staining and only few positive staining cilia (arrow heads). (A–D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Journal: Veterinary Immunology and Immunopathology

    Article Title: Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

    doi: 10.1016/j.vetimm.2019.109939

    Figure Lengend Snippet: Representative positive immunohistochemical reactions for human monoclonal antibodies. (A) Antibody 1.2g5 with citrate pretreatment exhibited a strong multifocal MERS-CoV antigen specific signal in the cytoplasm of ciliated respiratory epithelial cells (arrow) and segmentally along the apical membranous region (arrow heads). (B) The same protocol without pretreatment revealed a much fainter but similarly distributed staining of cytoplasm (arrow) and ciliary base (arrow heads). (C) Antibody 1.10f3 with citrate pretreatment displayed a strong diffuse background staining and only few positively stained cilia (arrowhead). Single intraepithelial plasma cells displayed a strong false positive intracytoplasmic staining (white arrow). (D) Antibody 1.6c7 with citrate pretreatment exhibited also a strong diffuse background staining and only few positive staining cilia (arrow heads). (A–D) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Article Snippet: For positive control and elucidation of virus distribution, additional sections were stained with two different commercially available, previously published monoclonal mouse and polyclonal rabbit anti-MERS-CoV nucleocapsid antibodies (Sino Biological Inc.; ; ), respectively.

    Techniques: Immunohistochemistry, Staining, Avidin-Biotin Assay

    Representative negative immunohistochemical reactions for human monoclonal antibodies. (A–E) Specific staining for MERS-CoV antigen was absent for the antibodies 1.6f9 (A), 4.6e10 (B), 7.7g6 (C), 1.8e5 (D), and 3.5g6 (E), respectively. Reactions were accompanied by mild to severe non-specific, intracytoplasmic background staining. (A–E) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Journal: Veterinary Immunology and Immunopathology

    Article Title: Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein

    doi: 10.1016/j.vetimm.2019.109939

    Figure Lengend Snippet: Representative negative immunohistochemical reactions for human monoclonal antibodies. (A–E) Specific staining for MERS-CoV antigen was absent for the antibodies 1.6f9 (A), 4.6e10 (B), 7.7g6 (C), 1.8e5 (D), and 3.5g6 (E), respectively. Reactions were accompanied by mild to severe non-specific, intracytoplasmic background staining. (A–E) Avidin-biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x.

    Article Snippet: For positive control and elucidation of virus distribution, additional sections were stained with two different commercially available, previously published monoclonal mouse and polyclonal rabbit anti-MERS-CoV nucleocapsid antibodies (Sino Biological Inc.; ; ), respectively.

    Techniques: Immunohistochemistry, Staining, Avidin-Biotin Assay

    ΔORF5 MERS-CoV infection induces higher levels of IFNβ and ISGs in bat cells. ( a–e ) Transcript levels of IFNβ and interferon stimulated genes (ISGs), IFI6, GBP1, Mx1 and MDA5 in Efk and MRC5 cells infected with W+ or ΔORF5 MERS-CoV. (f) DPP4 transcript levels in W+ or ΔORF5 MERS-CoV infected Efk cells (n = 4; Mean ± SD). Bars represent average fold changes (2 −ΔΔCT ) in transcript levels compared to mock infected cells and normalized to GAPDH levels in each sample (n = 4; Mean ± SD). *P

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: ΔORF5 MERS-CoV infection induces higher levels of IFNβ and ISGs in bat cells. ( a–e ) Transcript levels of IFNβ and interferon stimulated genes (ISGs), IFI6, GBP1, Mx1 and MDA5 in Efk and MRC5 cells infected with W+ or ΔORF5 MERS-CoV. (f) DPP4 transcript levels in W+ or ΔORF5 MERS-CoV infected Efk cells (n = 4; Mean ± SD). Bars represent average fold changes (2 −ΔΔCT ) in transcript levels compared to mock infected cells and normalized to GAPDH levels in each sample (n = 4; Mean ± SD). *P

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Infection

    MERS-CoV gene expression varies between acute and persistently infected bat cells. RNA from persistently infected Efk cells and acutely infected Efk cells were harvested at several time points and MERS-CoV genome quantities (upE levels) and gene expression levels were analyzed by real time quantitative PCR. ( a–i) Genome and gene expression (−(ΔCT gene − ΔCT GAPDH )) levels for MERS-CoV upE, S, ORF3, ORF4a, ORF4b, ORF5, E, M and N genes in acute (green) vs. persistent (purple) infections at 0, 12, 24 and 48 hours post-infection or seeding, respectively (n = 4; Mean ± SD). ***P

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: MERS-CoV gene expression varies between acute and persistently infected bat cells. RNA from persistently infected Efk cells and acutely infected Efk cells were harvested at several time points and MERS-CoV genome quantities (upE levels) and gene expression levels were analyzed by real time quantitative PCR. ( a–i) Genome and gene expression (−(ΔCT gene − ΔCT GAPDH )) levels for MERS-CoV upE, S, ORF3, ORF4a, ORF4b, ORF5, E, M and N genes in acute (green) vs. persistent (purple) infections at 0, 12, 24 and 48 hours post-infection or seeding, respectively (n = 4; Mean ± SD). ***P

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction

    Proposed model for establishment of persistent MERS-CoV infection in bat cells. As with the stocks of most RNA viruses, the MERS-CoV inoculum is made up of the dominant W + virus as well as smaller numbers of variants, including variants with inactivating mutations in ORF5 (ΔORF5). The cells infected with the more cytolytic W + virus die, while the small number of cells infected with ΔORF5 MERS-CoV survive because of an ensuing antiviral response and the induction of anti-apoptotic processes. ΔORF5 MERS-CoV infected cells are resistant to infection with the W + virus and the W + virus is soon diluted out. After a process of cell death and recovery, ΔORF5 MERS-CoV infected cells survive and take over, leading to a culture of persistently infected cells that produce small, but consistent amounts of virus over time.

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: Proposed model for establishment of persistent MERS-CoV infection in bat cells. As with the stocks of most RNA viruses, the MERS-CoV inoculum is made up of the dominant W + virus as well as smaller numbers of variants, including variants with inactivating mutations in ORF5 (ΔORF5). The cells infected with the more cytolytic W + virus die, while the small number of cells infected with ΔORF5 MERS-CoV survive because of an ensuing antiviral response and the induction of anti-apoptotic processes. ΔORF5 MERS-CoV infected cells are resistant to infection with the W + virus and the W + virus is soon diluted out. After a process of cell death and recovery, ΔORF5 MERS-CoV infected cells survive and take over, leading to a culture of persistently infected cells that produce small, but consistent amounts of virus over time.

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Infection

    MERS-CoV ΔORF5 mutant persistently infects bat (Efk) cells. ( a ) Schematic highlighting mutations (red arrows) in the MERS-CoV genome that were identified by sequencing the dominant virus strain in persistently infected bat (Efk) cells (passage 15) are shown. ( b ) Levels of W+ and ΔORF5 MERS-CoV replication in bat (Efk) and human (MRC5) cells. Expression levels of MERS-CoV upE gene (−(ΔCT gene − ΔCT GAPDH )), normalized to mock infected cells are shown (n = 4; Mean ± SD). *P = 0.015 and ***P = 0.0003 (Holm-Sidak t test with α = 0.05).

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: MERS-CoV ΔORF5 mutant persistently infects bat (Efk) cells. ( a ) Schematic highlighting mutations (red arrows) in the MERS-CoV genome that were identified by sequencing the dominant virus strain in persistently infected bat (Efk) cells (passage 15) are shown. ( b ) Levels of W+ and ΔORF5 MERS-CoV replication in bat (Efk) and human (MRC5) cells. Expression levels of MERS-CoV upE gene (−(ΔCT gene − ΔCT GAPDH )), normalized to mock infected cells are shown (n = 4; Mean ± SD). *P = 0.015 and ***P = 0.0003 (Holm-Sidak t test with α = 0.05).

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Mutagenesis, Sequencing, Infection, Expressing

    Persistently infected bat cells are resistant to super-infection with wildtype or ΔORF5 MERS-CoV. ( a–c ) Efk cells persistently infected with MERS-CoV were superinfected with W + MERS-CoV (blue) and transcript levels for ( a ) upE, ( b ) ORF5 or ( c ) E. fuscus dipeptidyl peptidase 4 (DPP4) were measured. The expression levels of upE, ORF5 and DPP4 transcripts (−(ΔCT gene − ΔCT GAPDH )) were also measured in Efk cells that were persistently infected with MERS-CoV in the absence of additional virus (red) and naïve Efk cells infected with W + MERS-CoV (green), with respect to time 0 for input W + virus (n = 3; Mean ± SD). DPP4 qRT-PCR amplicons were analyzed on an agarose gel (gel inset) and a ratio of basal DPP4 transcript levels (−(ΔCT DPP4 − ΔCT GAPDH )) in naïve, uninfected Efk and MRC5 cells is shown (right panel). ***P

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: Persistently infected bat cells are resistant to super-infection with wildtype or ΔORF5 MERS-CoV. ( a–c ) Efk cells persistently infected with MERS-CoV were superinfected with W + MERS-CoV (blue) and transcript levels for ( a ) upE, ( b ) ORF5 or ( c ) E. fuscus dipeptidyl peptidase 4 (DPP4) were measured. The expression levels of upE, ORF5 and DPP4 transcripts (−(ΔCT gene − ΔCT GAPDH )) were also measured in Efk cells that were persistently infected with MERS-CoV in the absence of additional virus (red) and naïve Efk cells infected with W + MERS-CoV (green), with respect to time 0 for input W + virus (n = 3; Mean ± SD). DPP4 qRT-PCR amplicons were analyzed on an agarose gel (gel inset) and a ratio of basal DPP4 transcript levels (−(ΔCT DPP4 − ΔCT GAPDH )) in naïve, uninfected Efk and MRC5 cells is shown (right panel). ***P

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Infection, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis

    Bat cells can be persistently infected with MERS-CoV. ( a ) Big brown bat kidney cells (Efk) were infected with MERS-CoV (MOI = 0.01 TCID 50 /cell) for 12 days and then passaged weekly. Supernatant was collected during each passage to determine the presence of virus by titration on Vero cells, along with immunofluorescent and electron microscopic studies of infected cells. ( b ) Levels of MERS-CoV at different times following initial infection. ( c ) Phase contrast micrographs showing cytopathic effects on MERS-CoV infection and subsequent recovery of Efk cells at various time points. ( d ) Immunofluorescent images showing MERS-CoV nucleocapsid (N) protein in persistently infected Efk cells (bottom row; red arrows). The contrast for persistently infected Efk cells (inset) was adjusted to visualize low levels of protein. High MOI acute infection (middle row) and mock infection of Efk cells (top row) were used as positive and negative controls, respectively. Images were processed using ImageJ. ( e ) In-situ hybridization to detect the presence of MERS-CoV nucleoprotein RNA in persistently infected Efk cells. High, intermediate and low levels of MERS-CoV nucleoprotein RNA have been shown in the insets. Acutely infected (right) and mock infected (left) cells were used as positive and negative controls, respectively.

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: Bat cells can be persistently infected with MERS-CoV. ( a ) Big brown bat kidney cells (Efk) were infected with MERS-CoV (MOI = 0.01 TCID 50 /cell) for 12 days and then passaged weekly. Supernatant was collected during each passage to determine the presence of virus by titration on Vero cells, along with immunofluorescent and electron microscopic studies of infected cells. ( b ) Levels of MERS-CoV at different times following initial infection. ( c ) Phase contrast micrographs showing cytopathic effects on MERS-CoV infection and subsequent recovery of Efk cells at various time points. ( d ) Immunofluorescent images showing MERS-CoV nucleocapsid (N) protein in persistently infected Efk cells (bottom row; red arrows). The contrast for persistently infected Efk cells (inset) was adjusted to visualize low levels of protein. High MOI acute infection (middle row) and mock infection of Efk cells (top row) were used as positive and negative controls, respectively. Images were processed using ImageJ. ( e ) In-situ hybridization to detect the presence of MERS-CoV nucleoprotein RNA in persistently infected Efk cells. High, intermediate and low levels of MERS-CoV nucleoprotein RNA have been shown in the insets. Acutely infected (right) and mock infected (left) cells were used as positive and negative controls, respectively.

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Infection, Titration, In Situ Hybridization

    IRF3 and MAP kinase-mediated signaling regulate persistent infection in Efk cells. Persistently infected Efk cells were transfected with siRNA targeting IRF3 mRNA and the subsequent effect on virus replication was measured. ( a) MERS-CoV titres in persistently infected bat cells 24 hours post treatment with IRF3-siRNA (red bar; n = 4; Mean ± SD). Scrambled siRNA (blue bar; control-siRNA) was used as a negative control. **P = 0.0049 (Unpaired t test with α = 0.05). ( b) Western blot for IRF3 and GAPDH in Efk cells treated or mock treated with IRF3 siRNA. (c) MERS-CoV upE transcript levels in Efk cells after treatment or mock treatment with MAPK inhibitor, URMC-99 for 24 and 48 hours. * P = 0.0053 ( Holm-Sidak t test with α=0.05). ( d) RERG transcript levels 48-hours post treatment with URMC-99 (n = 4; Mean ± SD). * P = 0.017 ( Holm-Sidak t test with α = 0.05). For full size gel images in (b) , see supplementary Fig. S3 .

    Journal: Scientific Reports

    Article Title: Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

    doi: 10.1038/s41598-020-64264-1

    Figure Lengend Snippet: IRF3 and MAP kinase-mediated signaling regulate persistent infection in Efk cells. Persistently infected Efk cells were transfected with siRNA targeting IRF3 mRNA and the subsequent effect on virus replication was measured. ( a) MERS-CoV titres in persistently infected bat cells 24 hours post treatment with IRF3-siRNA (red bar; n = 4; Mean ± SD). Scrambled siRNA (blue bar; control-siRNA) was used as a negative control. **P = 0.0049 (Unpaired t test with α = 0.05). ( b) Western blot for IRF3 and GAPDH in Efk cells treated or mock treated with IRF3 siRNA. (c) MERS-CoV upE transcript levels in Efk cells after treatment or mock treatment with MAPK inhibitor, URMC-99 for 24 and 48 hours. * P = 0.0053 ( Holm-Sidak t test with α=0.05). ( d) RERG transcript levels 48-hours post treatment with URMC-99 (n = 4; Mean ± SD). * P = 0.017 ( Holm-Sidak t test with α = 0.05). For full size gel images in (b) , see supplementary Fig. S3 .

    Article Snippet: Primary staining for MERS-CoV nucleoprotein (N) and GAPDH was performed using 1:100 dilution of rabbit anti-MERS-CoV N (Sino Biological, Cat #40068-RP01) and mouse anti-GAPDH (EMD Milipore, Cat #AB2302).

    Techniques: Infection, Transfection, Negative Control, Western Blot