mers cov nucleoprotein np antibody rabbit pab  (Sino Biological)


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    Name:
    MERS CoV Nucleoprotein NP Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Nucleoprotein NP Catalog 40068 V08B AFS88943 1 Met1 Asp413 Total IgG was purified by Protein A affinity chromatography
    Catalog Number:
    40068-RP01
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    MERS CoV
    Applications:
    ELISA
    Immunogen:
    Recombinant MERS-CoV (NCoV / Novel coronavirus) Nucleoprotein / NP protein (Catalog#40068-V08B)
    Product Aliases:
    Anti-coronavirus NP Antibody, Anti-coronavirus Nucleocapsid Antibody, Anti-coronavirus Nucleoprotein Antibody, Anti-cov np Antibody, Anti-ncov NP Antibody, Anti-novel coronavirus Nucleoprotein Antibody, Anti-NP Antibody, Anti-Nucleocapsid Antibody, Anti-Nucleoprotein Antibody
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological mers cov nucleoprotein np antibody rabbit pab
    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
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Nucleoprotein NP Catalog 40068 V08B AFS88943 1 Met1 Asp413 Total IgG was purified by Protein A affinity chromatography
    https://www.bioz.com/result/mers cov nucleoprotein np antibody rabbit pab/product/Sino Biological
    Average 93 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
    93/100 stars

    Images

    1) Product Images from "Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64264-1

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: Infection, Transfection, Negative Control, Western Blot

    2) Product Images from "Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model"

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

    Journal: Vaccines

    doi: 10.3390/vaccines8040634

    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).
    Figure Legend 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).

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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:
    Figure Legend 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:

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

    3) Product Images from "Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64264-1

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: Infection, Transfection, Negative Control, Western Blot

    4) Product Images from "Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells"

    Article Title: Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells

    Journal: The Journal of General Virology

    doi: 10.1099/jgv.0.000351

    Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of
    Figure Legend Snippet: Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of

    Techniques Used: Expressing, Infection, Western Blot

    5) Product Images from "Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells"

    Article Title: Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells

    Journal: The Journal of General Virology

    doi: 10.1099/jgv.0.000351

    Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of
    Figure Legend Snippet: Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of

    Techniques Used: Expressing, Infection, Western Blot

    6) Product Images from "Acute Respiratory Infection in Human Dipeptidyl Peptidase 4-Transgenic Mice Infected with Middle East Respiratory Syndrome Coronavirus"

    Article Title: Acute Respiratory Infection in Human Dipeptidyl Peptidase 4-Transgenic Mice Infected with Middle East Respiratory Syndrome Coronavirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.01818-18

    Susceptibility of adult human dipeptidyl peptidase 4 (hDPP4)-transgenic mice to MERS-CoV infection. Tg2, hDPP4 +/− transgenic mouse line 2; non-Tg, hDPP4 −/− mouse. (A) The body weight of 25-week-old mice was monitored daily after intranasal inoculation with MERS-CoV ( n = 6 Tg2 mice and n = 7 non-Tg mice). *, P
    Figure Legend Snippet: Susceptibility of adult human dipeptidyl peptidase 4 (hDPP4)-transgenic mice to MERS-CoV infection. Tg2, hDPP4 +/− transgenic mouse line 2; non-Tg, hDPP4 −/− mouse. (A) The body weight of 25-week-old mice was monitored daily after intranasal inoculation with MERS-CoV ( n = 6 Tg2 mice and n = 7 non-Tg mice). *, P

    Techniques Used: Transgenic Assay, Mouse Assay, Infection

    Histopathological changes in the lungs of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. Representative images of lungs from hDPP4 +/− transgenic mouse line 2 on days 1, 3, 5, 7, 14, and 35 postinoculation. Mild but progressive interstitial infiltration was seen within 7 days postinoculation (dpi) (left column, A, D, G, J, M, and P). IHC staining of sequential sections revealed abundant MERS-CoV antigen-positive cells in affected areas (middle column, B, E, H, K, N, and Q). Severe inflammation, with many mononuclear cells in the alveolar spaces and regenerated type II pneumocytes in the alveolar wall, was observed within 7 days p.i. (right column, C, F, I, L, O, and R). Scale bars: 100 μm (left and middle columns), 50 μm (right column), and 20 μm (insets of middle column). HE, hematoxylin and eosin staining; IHC, immunohistochemistry using an anti-MERS-CoV nucleocapsid protein polyclonal antibody; Ag, antigen.
    Figure Legend Snippet: Histopathological changes in the lungs of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. Representative images of lungs from hDPP4 +/− transgenic mouse line 2 on days 1, 3, 5, 7, 14, and 35 postinoculation. Mild but progressive interstitial infiltration was seen within 7 days postinoculation (dpi) (left column, A, D, G, J, M, and P). IHC staining of sequential sections revealed abundant MERS-CoV antigen-positive cells in affected areas (middle column, B, E, H, K, N, and Q). Severe inflammation, with many mononuclear cells in the alveolar spaces and regenerated type II pneumocytes in the alveolar wall, was observed within 7 days p.i. (right column, C, F, I, L, O, and R). Scale bars: 100 μm (left and middle columns), 50 μm (right column), and 20 μm (insets of middle column). HE, hematoxylin and eosin staining; IHC, immunohistochemistry using an anti-MERS-CoV nucleocapsid protein polyclonal antibody; Ag, antigen.

    Techniques Used: Transgenic Assay, Mouse Assay, Immunohistochemistry, Staining

    Identification of cells infiltrating the lung of Tg2 mice infected with MERS-CoV. Representative images of lungs from 10-week-old (young) and 25-week-old (adult) hDPP4 +/− transgenic mice (line 2) on day 7 postinoculation (p.i.). Infiltrating cells were positive for Iba-1 (green) or CD3 (brown). Bar, 20 μm.: Hematoxylin and eosin staining (HE) was used for the images in the upper panels, and anti-Iba-1 polyclonal antibody and anti-CD3 monoclonal antibody were used for IHC in the lower panels.
    Figure Legend Snippet: Identification of cells infiltrating the lung of Tg2 mice infected with MERS-CoV. Representative images of lungs from 10-week-old (young) and 25-week-old (adult) hDPP4 +/− transgenic mice (line 2) on day 7 postinoculation (p.i.). Infiltrating cells were positive for Iba-1 (green) or CD3 (brown). Bar, 20 μm.: Hematoxylin and eosin staining (HE) was used for the images in the upper panels, and anti-Iba-1 polyclonal antibody and anti-CD3 monoclonal antibody were used for IHC in the lower panels.

    Techniques Used: Mouse Assay, Infection, Transgenic Assay, Staining, Immunohistochemistry

    Cytokine and chemokine levels and expression of type I interferon (IFN) genes in the lungs of Tg2 mice infected with MERS-CoV. Cytokine and chemokine levels in lung samples from 10-week-old (A) and 25-week-old (B) mice are shown. Tg2 mice were inoculated with MERS-CoV or cell culture medium containing 2% FBS. Lungs were collected at the indicated times post-viral inoculation ( n = 3 to 4 mice per time point). Data represent the means ± standard deviations. A dotted line denotes the detection limit of the assay. *, P
    Figure Legend Snippet: Cytokine and chemokine levels and expression of type I interferon (IFN) genes in the lungs of Tg2 mice infected with MERS-CoV. Cytokine and chemokine levels in lung samples from 10-week-old (A) and 25-week-old (B) mice are shown. Tg2 mice were inoculated with MERS-CoV or cell culture medium containing 2% FBS. Lungs were collected at the indicated times post-viral inoculation ( n = 3 to 4 mice per time point). Data represent the means ± standard deviations. A dotted line denotes the detection limit of the assay. *, P

    Techniques Used: Expressing, Mouse Assay, Infection, Cell Culture

    Histopathological changes in the brain of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. (A, D, and G) Sagittal sections of the head, including the nasal cavity, olfactory bulb, and brain, of a Tg2 mouse infected with MERS-CoV (images taken at 3, 7, and 35 days p.i. [dpi]). Right panels show the brain cortex from samples from panels A, D, and G, respectively, with hematoxylin and eosin staining (B, E, and F) and immunohistochemical analysis of MERS-CoV antigen (C, F, I). Neither lesions nor MERS-CoV antigen-positive cells were detected in the brain. Scale bars, 1 mm (A, D, and G) and 20 μm (B, C, E, F, H, and I).
    Figure Legend Snippet: Histopathological changes in the brain of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. (A, D, and G) Sagittal sections of the head, including the nasal cavity, olfactory bulb, and brain, of a Tg2 mouse infected with MERS-CoV (images taken at 3, 7, and 35 days p.i. [dpi]). Right panels show the brain cortex from samples from panels A, D, and G, respectively, with hematoxylin and eosin staining (B, E, and F) and immunohistochemical analysis of MERS-CoV antigen (C, F, I). Neither lesions nor MERS-CoV antigen-positive cells were detected in the brain. Scale bars, 1 mm (A, D, and G) and 20 μm (B, C, E, F, H, and I).

    Techniques Used: Transgenic Assay, Mouse Assay, Infection, Staining, Immunohistochemistry

    Double-immunofluorescence images taken at 1 day p.i. showing human dipeptidyl peptidase 4 (hDPP4) (green) and MERS-CoV antigen (red) in the lungs of Tg2 mice infected with MERS-CoV. Viral antigen-positive cells in the lungs were hDPP4-positive bronchiolar epithelial cells (upper panels) and alveolar epithelial cells (lower panels). Original magnification, ×600.
    Figure Legend Snippet: Double-immunofluorescence images taken at 1 day p.i. showing human dipeptidyl peptidase 4 (hDPP4) (green) and MERS-CoV antigen (red) in the lungs of Tg2 mice infected with MERS-CoV. Viral antigen-positive cells in the lungs were hDPP4-positive bronchiolar epithelial cells (upper panels) and alveolar epithelial cells (lower panels). Original magnification, ×600.

    Techniques Used: Immunofluorescence, Mouse Assay, Infection

    Histopathological changes in the lungs of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. Representative histopathological images of the lungs from 25-week-old Tg2 mice at 1, 3, 5, 7, 14, and 35 days post-MERS-CoV infection. Images in the left (A, D, G, J, M, and P) and right (C, F, I, L, O, and R) columns show time-dependent recruitment of inflammatory cells to the lung. Marked inflammatory cell infiltration was noted at 7 days postinoculation (dpi) in panels J and L. Images in the middle column (B, E, H, K, N, and Q) show immunohistochemical staining for MERS-CoV antigen (Ag). Scale bars: 100 μm (left and middle columns), 50 μm (right column), and 20 μm (insets of middle column). HE, hematoxylin and eosin staining; IHC, immunohistochemistry using an anti-MERS-CoV nucleocapsid protein polyclonal antibody.
    Figure Legend Snippet: Histopathological changes in the lungs of human dipeptidyl peptidase 4 (hDPP4)-transgenic mice inoculated with MERS-CoV. Representative histopathological images of the lungs from 25-week-old Tg2 mice at 1, 3, 5, 7, 14, and 35 days post-MERS-CoV infection. Images in the left (A, D, G, J, M, and P) and right (C, F, I, L, O, and R) columns show time-dependent recruitment of inflammatory cells to the lung. Marked inflammatory cell infiltration was noted at 7 days postinoculation (dpi) in panels J and L. Images in the middle column (B, E, H, K, N, and Q) show immunohistochemical staining for MERS-CoV antigen (Ag). Scale bars: 100 μm (left and middle columns), 50 μm (right column), and 20 μm (insets of middle column). HE, hematoxylin and eosin staining; IHC, immunohistochemistry using an anti-MERS-CoV nucleocapsid protein polyclonal antibody.

    Techniques Used: Transgenic Assay, Mouse Assay, Infection, Immunohistochemistry, Staining

    Related Articles

    Immunohistochemistry:

    Article Title: Acute Respiratory Infection in Human Dipeptidyl Peptidase 4-Transgenic Mice Infected with Middle East Respiratory Syndrome Coronavirus
    Article Snippet: To obtain animal tissues, mice were anesthetized and perfused with 2 ml of 10% phosphate-buffered formalin, and the lungs, liver, spleen, kidney, heart, gastrointestinal tract, salivary glands, and brain tissue were harvested and fixed. .. For IHC, antigen retrieval of formalin-fixed mouse tissue sections was performed by autoclaving at 121°C for 10 min in retrieval solution at pH 6.0 (Nichirei, Tokyo, Japan). hDPP4 and MERS-CoV antigens were detected using a standard immunoperoxidase method and a goat anti-hDPP4 antibody (R & D Systems) and a rabbit anti-MERS-CoV nucleocapsid antibody (40068-RP01; Sino Biological, Inc., Beijing, China). .. For double staining of CD3 (T cells) and Iba-1 (macrophages) antigen, we used a rabbit anti-human CD3 antibody (790-4341; Ventana Medical System, Inc., Tucson, AZ) and a rabbit anti-human Iba-1 antibody (019-19741; Wako Pure Chemical Industries, Ltd., Osaka, Japan).

    other:

    Article Title: Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells
    Article Snippet: Commercial rabbit antibodies against MERS-CoV N protein (1 : 1000 dilution; Sino Biological), human DPP4 (1 : 1000 dilution; AbCam) and actin (1 : 500 dilution; Santa Cruz Biotechnology) were used according to the manufacturer's instructions.

    Incubation:

    Article Title: Enterokinase Enhances Influenza A Virus Infection by Activating Trypsinogen in Human Cell Lines
    Article Snippet: After 1 h of incubation, an equal volume of 1.2% Avicel (colloidal cellulose, Sigma-Aldrich #435244,) containing E-MEM, 10% fetal bovine serum, and 90 μg/ml N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biomedical Co.) was added to the wells. .. The plate was mixed by tapping, and incubated at 37°C for 24 h. The plated cells were fixed with 3.7% formaldehyde and immuno-stained with an anti-IAV nucleoprotein antibody (Sino Biological Inc.) and TrueBlue peroxidase substrate (KPL) to count the foci. .. cDNA cloning and reverse transcription (RT)–PCR Total mRNAs were extracted from the human cells with the standard acid guanidinium thiocyanate–phenol–chloroform method, and the cDNAs were synthesized with an oligo(dT)18 primer and ProtoScript II Reverse Transcriptase (New England Biolabs Inc., Ipswich, MA USA).

    Staining:

    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.

    Article Title: Anti-S1 MERS-COV IgY Specific Antibodies Decreases Lung Inflammation and Viral Antigen Positive Cells in the Human Transgenic Mouse Model
    Article Snippet: Formalin-fixed paraffin-embedded tissue sections were autoclaved at 121 °C for 10 min in retrieval solution at pH 6.0 (Nichirei Biosciences Inc., Tokyo, Japan) for antigen retrieval in preparation for immunohistochemistry (IHC). .. 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). .. Peroxidase activity was detected with 3,3′-diaminobenzidine (Sigma-Aldrich), and hematoxylin was used for counterstaining.

    Article Title: Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein
    Article Snippet: .. At 48 h after MERS-CoV infection, cells were fixed and stained with anti-MERS-CoV N protein antibody (Sino Biological Inc.) to count the viral plaques. .. 2.10 Enzyme-linked immunosorbent assay (ELISA)RBD-, S1-, S2-, and SΔTM-specific antibody in the hybridoma culture supernatant was measured by ELISA as described previously ( ; ).

    Infection:

    Article Title: Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein
    Article Snippet: .. At 48 h after MERS-CoV infection, cells were fixed and stained with anti-MERS-CoV N protein antibody (Sino Biological Inc.) to count the viral plaques. .. 2.10 Enzyme-linked immunosorbent assay (ELISA)RBD-, S1-, S2-, and SΔTM-specific antibody in the hybridoma culture supernatant was measured by ELISA as described previously ( ; ).

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    Sino Biological mers cov nucleoprotein np antibody rabbit pab
    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
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    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

    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

    Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of

    Journal: The Journal of General Virology

    Article Title: Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells

    doi: 10.1099/jgv.0.000351

    Figure Lengend Snippet: Quantification of MERS-CoV and SARS-CoV N protein expression during infection. Cell lysates from MDDC, macrophage, Calu-3 and Vero E6 infection experiments collected at the indicated time points were analysed by Western blotting for the expression of

    Article Snippet: Commercial rabbit antibodies against MERS-CoV N protein (1 : 1000 dilution; Sino Biological), human DPP4 (1 : 1000 dilution; AbCam) and actin (1 : 500 dilution; Santa Cruz Biotechnology) were used according to the manufacturer's instructions.

    Techniques: Expressing, Infection, Western Blot