polyclonal rabbit anti mers cov nucleocapsid antibodies  (Sino Biological)


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    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, 2021-06
    94/100 stars

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    1) Product Images from "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"

    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

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/j.vetimm.2019.109939

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Immunohistochemistry, Staining, Avidin-Biotin Assay

    Related Articles

    Western Blot:

    Article Title: High Expression of IL-36γ in Influenza Patients Regulates Interferon Signaling Pathway and Causes Programmed Cell Death During Influenza Virus Infection
    Article Snippet: Films were scanned and band volume intensity were measured using Image J software. .. The following reagents were used in western blotting: antibodies against human IL-36γ, LC-3B, p62, Mx1, PKR (Abcam), phosphorylated (p)-STAT1 (pY701), STAT1, (p)-STAT2 (pY141), STAT2, p-Akt, Akt, p-mTOR, mTOR, p-ULK1, ULK1, and GAPDH (Cell Signal Technology) and antibodies against IAV-Nucleocapsid (NP) and Matrix protein 1 (M1) (Sino Biological). .. Cell Apoptosis AnalysisA549 and 16HBE cells was pre-treated with IL-36γ or not and infected with IAV-Ca07 for 24 hours.

    Positive Control:

    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.
    Article Snippet: Sections were finally slightly counterstained with Mayer's hematoxylin (Roth C. GmbH Co KG). .. 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.; Haagmans et al., 2016; Haverkamp et al., 2018) , respectively. .. For negative controls, the primary antibody was replaced by ascites fluid from Balb/c mice (1:1,000) and normal rabbit serum (1:3,000), respectively.

    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
    Article Snippet: Sections were finally slightly counterstained with Mayer’s hematoxylin (Roth C. GmbH & Co KG). .. 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. .. For negative controls, the primary antibody was replaced by ascites fluid from Balb/c mice (1:1,000) and normal rabbit serum (1:3,000), respectively.

    Staining:

    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.
    Article Snippet: Sections were finally slightly counterstained with Mayer's hematoxylin (Roth C. GmbH Co KG). .. 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.; Haagmans et al., 2016; Haverkamp et al., 2018) , respectively. .. For negative controls, the primary antibody was replaced by ascites fluid from Balb/c mice (1:1,000) and normal rabbit serum (1:3,000), respectively.

    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
    Article Snippet: Sections were finally slightly counterstained with Mayer’s hematoxylin (Roth C. GmbH & Co KG). .. 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. .. For negative controls, the primary antibody was replaced by ascites fluid from Balb/c mice (1:1,000) and normal rabbit serum (1:3,000), respectively.

    other:

    Article Title: Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro
    Article Snippet: Rabbit anti-MERS-CoV nucleocapsid (N) antibody was purchased from Sino Biological Inc. (Cat. 100211-RP02, Beijing, China).

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    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, 2021-06
    94/100 stars
      Buy from Supplier

    95
    Sino Biological mers cov nucleocapsid protein antibody rabbit pab
    IHC detection of virus antigen expression in mouse tissue after challenge with <t>MERS-CoV.</t> Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.
    Mers Cov Nucleocapsid Protein Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov nucleocapsid protein antibody rabbit pab/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov nucleocapsid protein antibody rabbit pab - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

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    Sino Biological mers cov nucleoprotein np antibody rabbit pab
    Kinetics of serum and egg yolk <t>anti-MERS</t> <t>COV-S</t> IgY antibodies response of chickens after immunization with MERS COV-S recombinant protein compared with the adjuvant-immunized chicken (adjuvant control). Each week is represented by a pool of egg yolks of individual chicken in each group (S1-immunized and adjuvant-immunized).
    Mers Cov Nucleoprotein Np Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov nucleoprotein np antibody rabbit pab/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov nucleoprotein np antibody rabbit pab - by Bioz Stars, 2021-06
    94/100 stars
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    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

    IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Immunohistochemistry, Expressing

    rNTD or rRBD vaccination reduced respiratory tract pathology in mice after MERS-CoV challenge. Representative results of hematoxylin-eosin (HE) staining in the lung (A – C) and trachea (D – F) of mock-treated or immunized mice. Severe lesions including the loss of pulmonary alveolus (represented by the white vacuole) and diffuse inflammatory cell infiltration (represented by the dark purple point) are shown (figure A). In contrast, milder lesions were observed among mice immunized with rRBD (figure B) or NTD (figure C), as the pulmonary alveolus was highly visible with less inflammatory cell infiltration. Inflammatory cell infiltration and impaired epithelium of the tunica mucosa bronchiorum were seen in the mock group (D). rRBD (E) or rNTD (F) alleviated the pathologic damage in the trachea of immunized mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: rNTD or rRBD vaccination reduced respiratory tract pathology in mice after MERS-CoV challenge. Representative results of hematoxylin-eosin (HE) staining in the lung (A – C) and trachea (D – F) of mock-treated or immunized mice. Severe lesions including the loss of pulmonary alveolus (represented by the white vacuole) and diffuse inflammatory cell infiltration (represented by the dark purple point) are shown (figure A). In contrast, milder lesions were observed among mice immunized with rRBD (figure B) or NTD (figure C), as the pulmonary alveolus was highly visible with less inflammatory cell infiltration. Inflammatory cell infiltration and impaired epithelium of the tunica mucosa bronchiorum were seen in the mock group (D). rRBD (E) or rNTD (F) alleviated the pathologic damage in the trachea of immunized mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Mouse Assay, Staining

    Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Mouse Assay, Enzyme-linked Immunospot, Crocin Bleaching Assay

    Thapsigargin inhibits the replication of high- and low-pathogenic human coronaviruses in multiple cell types. (A-D) Human embryonic MRC-5 lung fibroblasts were infected with HCoV-229E according to the scheme shown in Fig. 1B . Viral titers (A, upper graph), and expression of viral S gene-containing RNAs (A, lower graph), viral and host cell proteins (B, C) and cell viability (D) were analyzed and quantified as described in the legend of Fig. 1 . (E-K) Similarly, HuH7 cells or Vero E6 African green monkey kidney epithelial cells were infected with MERS-CoV (MOI=0.5) or SARS-CoV-2 (MOI=0.5) for 12 h or 24 in the presence / absence of 0.4 μM or 1 μM thapsigargin. (E, F) show viral titers and (G, H) the corresponding expression of MERS-CoV / SARS-CoV-2 nucleocapsid (N) and host cell proteins, respectively. (I) Dose-dependent suppression of MERS-CoV-2 replication by thapsigargin (upper graph) and the estimated effective inhibitory concentration (EC 50 ) in HuH7 cells infected with an MOI of 0.5 (lower graph). (J) Dose-dependent suppression of SARS-CoV-2 replication by thapsigargin (left graph) and the calculated effective inhibitory concentration (EC 50 ) in Vero E6 cells infected with an MOI of 0.5 (right graph). (K) The CC 50 of thapsigargin in Vero E6 cells was calculated by MTS assays as described in the legend of Fig. 2G-H . Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests. See Fig. S2 for quantifications from replicates for MERS-CoV / SARS-CoV-2 immunoblot experiments.

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Thapsigargin inhibits the replication of high- and low-pathogenic human coronaviruses in multiple cell types. (A-D) Human embryonic MRC-5 lung fibroblasts were infected with HCoV-229E according to the scheme shown in Fig. 1B . Viral titers (A, upper graph), and expression of viral S gene-containing RNAs (A, lower graph), viral and host cell proteins (B, C) and cell viability (D) were analyzed and quantified as described in the legend of Fig. 1 . (E-K) Similarly, HuH7 cells or Vero E6 African green monkey kidney epithelial cells were infected with MERS-CoV (MOI=0.5) or SARS-CoV-2 (MOI=0.5) for 12 h or 24 in the presence / absence of 0.4 μM or 1 μM thapsigargin. (E, F) show viral titers and (G, H) the corresponding expression of MERS-CoV / SARS-CoV-2 nucleocapsid (N) and host cell proteins, respectively. (I) Dose-dependent suppression of MERS-CoV-2 replication by thapsigargin (upper graph) and the estimated effective inhibitory concentration (EC 50 ) in HuH7 cells infected with an MOI of 0.5 (lower graph). (J) Dose-dependent suppression of SARS-CoV-2 replication by thapsigargin (left graph) and the calculated effective inhibitory concentration (EC 50 ) in Vero E6 cells infected with an MOI of 0.5 (right graph). (K) The CC 50 of thapsigargin in Vero E6 cells was calculated by MTS assays as described in the legend of Fig. 2G-H . Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests. See Fig. S2 for quantifications from replicates for MERS-CoV / SARS-CoV-2 immunoblot experiments.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques: Infection, Expressing, Concentration Assay, Two Tailed Test

    Proteome profiling of thapsigargin effects on MERS-CoV or SARS-CoV-2-infected cells reveals multiple virus- and thapsigargin-specific protein and pathway patterns. (A-D) Total cell extracts from uninfected cells (-), HuH7 cells infected with MERS-CoV (M, MOI=0.5) for 12 h (A) or 24 h (B), or Vero E6 cells infected with SARS-CoV-2 (S, MOI=0.5) for 12 h (C) or 24 h (D), in the presence or absence of thapsigargin (T, 1 μM) were subjected to LC-MS/MS analysis. In total, 5,372 (from HuH7) or 5,072 (from Vero E6 cells) majority protein IDs were identified. Quantile-normalized log 2 -tranformed protein intensities were used for all further calculations. Volcano plots show the distribution of pairwise ratio comparisons and the corresponding p values which were obtained by pooling data from two independent experiments and from three technical replicates per sample. Blue and red colors indicate differentially expressed proteins (DEPs, ratio > 0, p ≥ −log 10 1.3). Purple and light red colors indicate individual viral proteins. (E) Scheme of multiple gene ID lists (corresponding to the DEPs shown in (A)-(D)) that were used for overrepresentation analyses to identify the top 100 enriched pathway categories per virus and time point using Metascape software ( Zhou et al ., 2019 ). Complete lists of pathways are shown in Fig. S3 and S4 as clustered heatmaps. The top 5 enriched pathway categories for up- or downregulated DEPs are shown in Fig. S5A . (F, G) The 400 enriched pathway categories were pooled and filtered for common and distinct pathways considering only terms with enrichment p values ≤ log 10 −3. (F) Venn diagram showing pathway terms specific to MERS-CoV (M), SARS-CoV-2 (S) or thapsigargin (T). The top 20 enriched pathway categories are shown in Fig. S5B . (G) Venn diagram showing pathway terms specific for virus, thapsigargin, or infection plus thapsigargin (virus + T) conditions. (H) The heatmap shows the top differentially enriched pathways corresponding to the Venn diagram shown in (G). Green colors refer to the pathways highlighted in (I). (I) Ten examples of differential and joint gene ID compositions of pathways enriched in HuH7 or Vero E6 cells.

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Proteome profiling of thapsigargin effects on MERS-CoV or SARS-CoV-2-infected cells reveals multiple virus- and thapsigargin-specific protein and pathway patterns. (A-D) Total cell extracts from uninfected cells (-), HuH7 cells infected with MERS-CoV (M, MOI=0.5) for 12 h (A) or 24 h (B), or Vero E6 cells infected with SARS-CoV-2 (S, MOI=0.5) for 12 h (C) or 24 h (D), in the presence or absence of thapsigargin (T, 1 μM) were subjected to LC-MS/MS analysis. In total, 5,372 (from HuH7) or 5,072 (from Vero E6 cells) majority protein IDs were identified. Quantile-normalized log 2 -tranformed protein intensities were used for all further calculations. Volcano plots show the distribution of pairwise ratio comparisons and the corresponding p values which were obtained by pooling data from two independent experiments and from three technical replicates per sample. Blue and red colors indicate differentially expressed proteins (DEPs, ratio > 0, p ≥ −log 10 1.3). Purple and light red colors indicate individual viral proteins. (E) Scheme of multiple gene ID lists (corresponding to the DEPs shown in (A)-(D)) that were used for overrepresentation analyses to identify the top 100 enriched pathway categories per virus and time point using Metascape software ( Zhou et al ., 2019 ). Complete lists of pathways are shown in Fig. S3 and S4 as clustered heatmaps. The top 5 enriched pathway categories for up- or downregulated DEPs are shown in Fig. S5A . (F, G) The 400 enriched pathway categories were pooled and filtered for common and distinct pathways considering only terms with enrichment p values ≤ log 10 −3. (F) Venn diagram showing pathway terms specific to MERS-CoV (M), SARS-CoV-2 (S) or thapsigargin (T). The top 20 enriched pathway categories are shown in Fig. S5B . (G) Venn diagram showing pathway terms specific for virus, thapsigargin, or infection plus thapsigargin (virus + T) conditions. (H) The heatmap shows the top differentially enriched pathways corresponding to the Venn diagram shown in (G). Green colors refer to the pathways highlighted in (I). (I) Ten examples of differential and joint gene ID compositions of pathways enriched in HuH7 or Vero E6 cells.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Software

    Top pathways regulated by MERS-CoV, SARS-CoV-2 or thapsigargin. (A) Top ten enriched pathways containing up- or downregulated DEPs extracted from the 100 enriched deregulated pathways shown in Fig. S3 / Fig. S4 . Colors indicate highly common categories. (B) Top 20 pathways enriched with thapsigargin alone or jointly by MERS-CoV, SARS-CoV-2 and thapsigargin according to the Venn diagram shown in Fig. 4F .

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Top pathways regulated by MERS-CoV, SARS-CoV-2 or thapsigargin. (A) Top ten enriched pathways containing up- or downregulated DEPs extracted from the 100 enriched deregulated pathways shown in Fig. S3 / Fig. S4 . Colors indicate highly common categories. (B) Top 20 pathways enriched with thapsigargin alone or jointly by MERS-CoV, SARS-CoV-2 and thapsigargin according to the Venn diagram shown in Fig. 4F .

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques:

    Opposing effects of HCoV-229E or MERS-CoV on ER stress genes at the mRNA and protein level. Projection of normalized ratio values for transcriptomic (by RNA-seq) and proteomic (by LC-MS/MS) data derived in parallel from HuH7 cells infected for 24 h with HCoV-229E or MERS-CoV with a MOI=1 on the components of the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. The left side of the boxes show mRNA values, right sides show protein values.

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Opposing effects of HCoV-229E or MERS-CoV on ER stress genes at the mRNA and protein level. Projection of normalized ratio values for transcriptomic (by RNA-seq) and proteomic (by LC-MS/MS) data derived in parallel from HuH7 cells infected for 24 h with HCoV-229E or MERS-CoV with a MOI=1 on the components of the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. The left side of the boxes show mRNA values, right sides show protein values.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Infection

    Thapsigargin effects on MERS-CoV or SARS-CoV-2-infected cells reveal the regulation of a specific network of proteins involved in transport, ERQC / ERAD and ER stress. (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Ve ro cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also Fig. S6 for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Thapsigargin effects on MERS-CoV or SARS-CoV-2-infected cells reveal the regulation of a specific network of proteins involved in transport, ERQC / ERAD and ER stress. (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Ve ro cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also Fig. S6 for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques: Infection, Expressing, Software

    Identification of deregulated cellular pathways in MERS-CoV-infected HuH7 cells. Top 100 overrepresented pathways containing up- or downregulated DEPs (ratio > 0, p ≥ log 10 1.3) for the 12 h p.i. and 24 h p.i. time points of MERS-CoV-infected cells based on gene IDs derived from protein IDs. Blue and red colors indicate differentially expressed proteins as shown in Fig. 4A-B .

    Journal: bioRxiv

    Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

    doi: 10.1101/2020.08.26.266304

    Figure Lengend Snippet: Identification of deregulated cellular pathways in MERS-CoV-infected HuH7 cells. Top 100 overrepresented pathways containing up- or downregulated DEPs (ratio > 0, p ≥ log 10 1.3) for the 12 h p.i. and 24 h p.i. time points of MERS-CoV-infected cells based on gene IDs derived from protein IDs. Blue and red colors indicate differentially expressed proteins as shown in Fig. 4A-B .

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

    Techniques: Infection, Derivative Assay

    Kinetics of serum and egg yolk anti-MERS COV-S IgY antibodies response of chickens after immunization with MERS COV-S recombinant protein compared with the adjuvant-immunized chicken (adjuvant control). Each week is represented by a pool of egg yolks of individual chicken in each group (S1-immunized and adjuvant-immunized).

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Kinetics of serum and egg yolk anti-MERS COV-S IgY antibodies response of chickens after immunization with MERS COV-S recombinant protein compared with the adjuvant-immunized chicken (adjuvant control). Each week is represented by a pool of egg yolks of individual chicken in each group (S1-immunized and adjuvant-immunized).

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Recombinant

    Recognition by anti-S1 IgY antibodies of viral antigen expressed in MERS-CoV-infected Vero E6 cells, using indirect immunofluorescence assay. ( A ) Vero E6 cells inoculated with MERS-CoV and stained with anti-S1 IgY antibodies and FITC-conjugated anti-chicken antibodies; and ( B ) control adjuvant IgY (Bright-field).

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Recognition by anti-S1 IgY antibodies of viral antigen expressed in MERS-CoV-infected Vero E6 cells, using indirect immunofluorescence assay. ( A ) Vero E6 cells inoculated with MERS-CoV and stained with anti-S1 IgY antibodies and FITC-conjugated anti-chicken antibodies; and ( B ) control adjuvant IgY (Bright-field).

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Infection, Immunofluorescence, Staining

    Dot blotting analysis. Purified anti-S1 IgY antibodies showed reactivity with different concentrations of the spike protein (S), S1, and receptor binding domain (RBD), but had no reactivity with nucleocapsid (NP) protein of MERS CoV.

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Dot blotting analysis. Purified anti-S1 IgY antibodies showed reactivity with different concentrations of the spike protein (S), S1, and receptor binding domain (RBD), but had no reactivity with nucleocapsid (NP) protein of MERS CoV.

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Purification, Binding Assay

    Examples of different concentrations of anti-S1 IgY antibodies tested against MERS-CoV on Vero-E6 cells examined by CPE. The IC100 neutralization of the antibody were determined as the reciprocal of the highest dilution at which no CPE was observed.

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Examples of different concentrations of anti-S1 IgY antibodies tested against MERS-CoV on Vero-E6 cells examined by CPE. The IC100 neutralization of the antibody were determined as the reciprocal of the highest dilution at which no CPE was observed.

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Neutralization

    ( A ) Viral titer in the lungs of MERS-CoV mice treated with anti-SI IgY antibodies and control IgY (adjuvant). ( B ) Body weight changes after MERS-CoV infection between anti-SI IgY antibodies and IgY of adjuvant control group. ( C – F ) Histopathology of the lungs from human dipeptidyl peptidase 4 (hDPP4)-transgenic mice on day 8 after inoculation with MERS-CoV. Representative histopathological findings of mice with the highest cellular infiltration in alveoli by H E staining ( C ) Massive mononuclear cell infiltrations including macrophages and lymphocytes with regenerated type II pneumocytes were seen in adjuvant control group (right column), but less in the anti-S1 IgY treated group (left column). Scale bars: 200 μm (upper row) and 20 μm (lower row). Al, alveoli; Br, bronchi; V, vessel. Detection of viral antigen in lung tissues of mice by immunohistochemistry ( D ) A few antigen positive cells were seen in the lungs of anti-S1 IgY treated group compared to adjuvant control group. Quantification of inflammation areas ( E ) The area of pulmonary lesion was determined based on the mean percentage of affected areas in each section of the collected lobes form each animal ( n = 8 or 6). Circles indicate averages from three observation lobes in each mouse. p = 0.1709 by Mann-Whitney test. Numbers of viral antigen positive cells in the alveoli ( F ) Data were obtained from 8 or 6 mice. Circles indicate averages of 5 observation fields in each mouse. * p = 0.0196 by Mann-Whitney test.

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: ( A ) Viral titer in the lungs of MERS-CoV mice treated with anti-SI IgY antibodies and control IgY (adjuvant). ( B ) Body weight changes after MERS-CoV infection between anti-SI IgY antibodies and IgY of adjuvant control group. ( C – F ) Histopathology of the lungs from human dipeptidyl peptidase 4 (hDPP4)-transgenic mice on day 8 after inoculation with MERS-CoV. Representative histopathological findings of mice with the highest cellular infiltration in alveoli by H E staining ( C ) Massive mononuclear cell infiltrations including macrophages and lymphocytes with regenerated type II pneumocytes were seen in adjuvant control group (right column), but less in the anti-S1 IgY treated group (left column). Scale bars: 200 μm (upper row) and 20 μm (lower row). Al, alveoli; Br, bronchi; V, vessel. Detection of viral antigen in lung tissues of mice by immunohistochemistry ( D ) A few antigen positive cells were seen in the lungs of anti-S1 IgY treated group compared to adjuvant control group. Quantification of inflammation areas ( E ) The area of pulmonary lesion was determined based on the mean percentage of affected areas in each section of the collected lobes form each animal ( n = 8 or 6). Circles indicate averages from three observation lobes in each mouse. p = 0.1709 by Mann-Whitney test. Numbers of viral antigen positive cells in the alveoli ( F ) Data were obtained from 8 or 6 mice. Circles indicate averages of 5 observation fields in each mouse. * p = 0.0196 by Mann-Whitney test.

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Mouse Assay, Infection, Histopathology, Transgenic Assay, Staining, Immunohistochemistry, MANN-WHITNEY

    Western blot analysis of anti-MERS-COV rS1 IgY antibodies. (Left) The S1 protein of MERS-COV was subjected to SDS-PAGE under reducing conditions; (Right) Western blot analysis of the anti-S1 IgY antibody response. SDS gels were electrically transferred onto nitrocellulose membranes and probed with IgY from immunized and nonimmunized hens (marker: molecular maker; lane A: S1-immunized IgY; lane B: adjuvant-immunized IgY). The strips were processed separately and pasted beside each other for documentation.

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Western blot analysis of anti-MERS-COV rS1 IgY antibodies. (Left) The S1 protein of MERS-COV was subjected to SDS-PAGE under reducing conditions; (Right) Western blot analysis of the anti-S1 IgY antibody response. SDS gels were electrically transferred onto nitrocellulose membranes and probed with IgY from immunized and nonimmunized hens (marker: molecular maker; lane A: S1-immunized IgY; lane B: adjuvant-immunized IgY). The strips were processed separately and pasted beside each other for documentation.

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Western Blot, SDS Page, Marker

    Evaluation of the neutralizing potential of anti-S1 IgY antibodies, using plaque reduction neutralization test. ( A ) MERS-CoV (MOI 0.01) was incubated with different concentrations of anti-S1 IgY antibodies and added to Vero E6 cells. After virus adsorption, agar medium was added to the Vero E6 cells, and the plaques that formed were stained with crystal violet, each IgY concentration was tested in triplicate. ( B ) Percent inhibition of anti-S1 IgY antibodies with different concentrations. The best fit equation is:

    Journal: Vaccines

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model

    doi: 10.3390/vaccines8040634

    Figure Lengend Snippet: Evaluation of the neutralizing potential of anti-S1 IgY antibodies, using plaque reduction neutralization test. ( A ) MERS-CoV (MOI 0.01) was incubated with different concentrations of anti-S1 IgY antibodies and added to Vero E6 cells. After virus adsorption, agar medium was added to the Vero E6 cells, and the plaques that formed were stained with crystal violet, each IgY concentration was tested in triplicate. ( B ) Percent inhibition of anti-S1 IgY antibodies with different concentrations. The best fit equation is:

    Article Snippet: MERS-CoV antigens were detected utilizing a polymer-based detection system (Nichirei-Histofine Simple Stain Mouse MAX PO(R); Nichirei) with a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological Inc., Beijing, China).

    Techniques: Plaque Reduction Neutralization Test, Incubation, Adsorption, Staining, Concentration Assay, Inhibition