bovine viral diarrhea virus  (ATCC)


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    Name:
    Bovine viral diarrhea virus 1
    Description:
    Applications REFERENCE STRAIN Western Hemisphere Committee on Non primate Animal Virus Characterization WHO
    Catalog Number:
    VR-534
    Price:
    None
    Applications:
    REFERENCE STRAIN: Western Hemisphere Committee on Non-primate Animal Virus Characterization (WHO).
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    Structured Review

    ATCC bovine viral diarrhea virus
    Applications REFERENCE STRAIN Western Hemisphere Committee on Non primate Animal Virus Characterization WHO
    https://www.bioz.com/result/bovine viral diarrhea virus/product/ATCC
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bovine viral diarrhea virus - by Bioz Stars, 2021-07
    95/100 stars

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    Related Articles

    Expressing:

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: After incubation with α-HA-MAb 12CA5 and peroxidase-conjugated anti-mouse IgG, signals were revealed by chemiluminescence. .. A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C. .. The numbers of infected cells were determined by immunohistochemical detection with an MAb to viral E2 protein (D5) or to BHV-1 (121/3/3), respectively, or a polyclonal α-SinV serum at 12 to 16 h postinfection (p.i.).

    Incubation:

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: After incubation with α-HA-MAb 12CA5 and peroxidase-conjugated anti-mouse IgG, signals were revealed by chemiluminescence. .. A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C. .. The numbers of infected cells were determined by immunohistochemical detection with an MAb to viral E2 protein (D5) or to BHV-1 (121/3/3), respectively, or a polyclonal α-SinV serum at 12 to 16 h postinfection (p.i.).

    Activity Assay:

    Article Title: A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea Virus In Vitro
    Article Snippet: Plasmid pMAL-p5X (New England BioLabs, Ipswich, MA) was used to clone the BVDV E2 gene for expression of a maltose binding protein (MBP)-E2 fusion protein as an ELISA coating agent to titrate anti-BVDV E2-specific antibodies. .. The BVDV NADL strain (ATCC VR-534) was used to amplify the BVDV E2 gene and also as a challenge strain in viral neutralizing activity assays. .. The K99 major structural subunit gene fanC was amplified in a PCR with primers FanCNheI-F2 (5′-ATGATG GCTAGC ACACTCCTAGCTATTATCTTAGGT-3′; NheI restriction site underlined) and FanCEagI-R (5′-TCATCGATA CGGCCG CAATGTAA-3′; EagI site underlined) and pK99 plasmid as the DNA template.

    other:

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: A total of 5 × 105 MDBK cells were inoculated with 2 × 102 PFU of BVDV strain NADL for 1 h at 4°C and washed with prewarmed citrate-phosphate buffer at the indicated pH in the presence or absence of 10 mM DTT for 2 min at 37°C.

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: A total of 5 × 105 MDBK cells were inoculated with 2 × 105 PFU of BVDV strain NADL for 90 min at 37°C in the presence or absence of 0.45 M sucrose, followed by the addition of DMEM containing 0.2 μM bafilomycin A1.

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: BVDV strain NADL was adsorbed to MDBK cells for 1 h at 4°C, followed by the addition of different concentrations of chlorpromazine.

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: A total of 5 × 105 MDBK cells were inoculated with 2 × 102 PFU of SinV or BVDV strain NADL for 1 h at 4°C.

    Adsorption:

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry
    Article Snippet: The number of infected cells at pH 7.0 without DTT and bafilomycin A1 was set to 100%, and the fusion activity was calculated as a percentage of the control value. .. Different methods were applied to determine the route of entry used by BVDV strain NADL after adsorption to its cellular receptor CD46bov. ..

    Mouse Assay:

    Article Title: A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea Virus In Vitro
    Article Snippet: Antibodies in serum samples of the immunized mice prevented BVDV from causing cytopathic effects in MDBK cells ( ). .. The BVDV NADL strain mixed with the serum sample pooled from the immunized mice (up to a 1:640 dilution), when used to infect MDBK cells, did not cause cytopathic effects to the MDBK cells. .. There was no noticeable difference between the cells without treatment ( ) and those incubated with a mixture of BVDV and the serum sample of the immunized mice until the serum sample of the immunized mice was diluted to over 1:640 ( and ).

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  • 95
    ATCC bvdv nadl strain
    Antibody neutralization against <t>BVDV</t> viral infection. (A) Normal MDBK cells grown in cell culture medium. (B) MDBK cells incubated with BVDV <t>NADL</t> virus pretreated with the serum sample (1:320 dilution) pooled from mice immunized with the FanC-STa-E2 fusion.
    Bvdv Nadl Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bvdv  (ATCC)
    93
    ATCC bvdv
    Comparison of virus derived from <t>pACNR/NADL</t> and wt <t>BVDV</t> NADL. (A) Plaques were visualized by crystal violet staining at 3 days postinfection (NADL) or post-RNA transfection (ACNR/NADL) as described in Materials and Methods. The mock-transfected monolayer was treated the same as the transfected monolayer except that no RNA was present in the transfection mixture. (B) MDBK cells were infected with either wt NADL or ACNR/NADL at an MOI (as determined on MDBK monolayers) of either 0.1 or 1 PFU/cell, washed, and harvested at the indicated times postinfection. Titers were determined by plaque assay on MDBK monolayers as described in Materials and Methods. (C) MDBK cells were mock infected or infected with wt NADL or ACNR/NADL at an MOI of 1 PFU/cell. At 20 h postinfection, proteins were labeled for 4 h with Expre 35 S 35 S label (NEN) and lysed, and BVDV-specific proteins were immunoprecipitated with a polyclonal anti-BVDV serum (α49). Proteins were separated by SDS-PAGE (8% gel) and visualized by fluorography. Molecular mass markers are indicated at the left; BVDV-specific proteins, identified by size and, in some cases, by immunoreactivity with region-specific antisera (data not shown), are indicated at the right.
    Bvdv, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATCC bovine rotavirus ncdv g6p6
    Inhibition of the RhoA/ROCK/MLC signaling pathway affects <t>rotavirus</t> infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or <t>NCDV</t> (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p
    Bovine Rotavirus Ncdv G6p6, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Antibody neutralization against BVDV viral infection. (A) Normal MDBK cells grown in cell culture medium. (B) MDBK cells incubated with BVDV NADL virus pretreated with the serum sample (1:320 dilution) pooled from mice immunized with the FanC-STa-E2 fusion.

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea Virus In Vitro

    doi: 10.1128/CVI.00249-13

    Figure Lengend Snippet: Antibody neutralization against BVDV viral infection. (A) Normal MDBK cells grown in cell culture medium. (B) MDBK cells incubated with BVDV NADL virus pretreated with the serum sample (1:320 dilution) pooled from mice immunized with the FanC-STa-E2 fusion.

    Article Snippet: The BVDV NADL strain (ATCC VR-534) was used to amplify the BVDV E2 gene and also as a challenge strain in viral neutralizing activity assays.

    Techniques: Neutralization, Infection, Cell Culture, Incubation, Mouse Assay

    Fusion from without of BVDV in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain NADL and briefly shifted to 37°C at the indicated pH in the presence or absence of 10 mM DTT; virus uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Higher concentrations of DTT could not be used due to high cell toxicity. The numbers of infectious centers were determined 12 to 16 h p.i. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Fusion from without of BVDV in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain NADL and briefly shifted to 37°C at the indicated pH in the presence or absence of 10 mM DTT; virus uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Higher concentrations of DTT could not be used due to high cell toxicity. The numbers of infectious centers were determined 12 to 16 h p.i. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques:

    pH stability of BVDV. A total of 2 × 10 6 PFU of BVDV strain NADL were incubated in citrate-phosphate buffers of a defined pH (pH 3.0 to 7.0) in the presence of 10 mM DTT for 15 min at 25°C and titrated on MDBK cells. The same experiment was performed in the absence of DTT, but only the infectivity after treatment at pH 3.0 and 7.0 is indicated. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: pH stability of BVDV. A total of 2 × 10 6 PFU of BVDV strain NADL were incubated in citrate-phosphate buffers of a defined pH (pH 3.0 to 7.0) in the presence of 10 mM DTT for 15 min at 25°C and titrated on MDBK cells. The same experiment was performed in the absence of DTT, but only the infectivity after treatment at pH 3.0 and 7.0 is indicated. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Incubation, Infection

    Effect of chlorpromazine and β-MCD on BVDV and BHV-1 infection. BVDV NADL (dark gray bars) or BHV-1 (light gray bars) was adsorbed to MDBK cells, and the effects of chlorpromazine and β-MCD on infection were investigated. Susceptibility to BVDV infection was decreased up to 1,000-fold, whereas BHV-1 infection was inhibited five-fold by β-MCD but not by chlorpromazine. The columns represent mean values of duplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Effect of chlorpromazine and β-MCD on BVDV and BHV-1 infection. BVDV NADL (dark gray bars) or BHV-1 (light gray bars) was adsorbed to MDBK cells, and the effects of chlorpromazine and β-MCD on infection were investigated. Susceptibility to BVDV infection was decreased up to 1,000-fold, whereas BHV-1 infection was inhibited five-fold by β-MCD but not by chlorpromazine. The columns represent mean values of duplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Infection

    Expression of Dyn K44A reduces susceptibility to BVDV infection. (a) Immunoblot of MDBK Tet on dynamin-overexpressing cell lines. Crude cell lysates from equal numbers of cells grown in the presence or absence of 10 μg of doxycycline/ml were separated. After induction, a 99-kDa band of each HA-tagged protein is visible. (b) Inhibition of BVDV NADL/SinV infection by overexpression of dominant-negative Dyn K44A . Each indicated cell line was tested for its susceptibility to BVDV or SinV infection by inoculation with 2 × 10 5 PFU of BVDV strain NADL or SinV, respectively. MDBK cells overexpressing mutant dynamin after induction with doxycycline exhibited a 10-fold- reduced susceptibility compared to MDBK cells. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Expression of Dyn K44A reduces susceptibility to BVDV infection. (a) Immunoblot of MDBK Tet on dynamin-overexpressing cell lines. Crude cell lysates from equal numbers of cells grown in the presence or absence of 10 μg of doxycycline/ml were separated. After induction, a 99-kDa band of each HA-tagged protein is visible. (b) Inhibition of BVDV NADL/SinV infection by overexpression of dominant-negative Dyn K44A . Each indicated cell line was tested for its susceptibility to BVDV or SinV infection by inoculation with 2 × 10 5 PFU of BVDV strain NADL or SinV, respectively. MDBK cells overexpressing mutant dynamin after induction with doxycycline exhibited a 10-fold- reduced susceptibility compared to MDBK cells. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Expressing, Infection, Inhibition, Over Expression, Dominant Negative Mutation, Mutagenesis

    Effect of different inhibitors of endosomal acidification on BVDV and BHV-1 infection. Directly after adsorption of BVDV NADL (dark gray bars) or BHV-1 (light gray bars) to MDBK cells, different inhibitors of endosomal acidification (bafilomycin A1, chloroquine, or ammonium chloride) were applied to determine the pH dependence of viral entry. Each inhibitor of endosomal acidification blocks BVDV infection in a concentration-dependent manner, whereas BHV-1 infection is not affected. The columns represent mean values of duplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Effect of different inhibitors of endosomal acidification on BVDV and BHV-1 infection. Directly after adsorption of BVDV NADL (dark gray bars) or BHV-1 (light gray bars) to MDBK cells, different inhibitors of endosomal acidification (bafilomycin A1, chloroquine, or ammonium chloride) were applied to determine the pH dependence of viral entry. Each inhibitor of endosomal acidification blocks BVDV infection in a concentration-dependent manner, whereas BHV-1 infection is not affected. The columns represent mean values of duplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Infection, Adsorption, Concentration Assay

    Fusion from without of BVDV and SinV. MDBK cells were inoculated with BVDV strain NADL or SinV, respectively, for 1 h at 4°C. Medium was replaced by prewarmed buffers of the indicated pH, followed by incubation for 2 min at 37°C. Viral uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Since fusion from without is cell type specific, SinV was used as control. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Fusion from without of BVDV and SinV. MDBK cells were inoculated with BVDV strain NADL or SinV, respectively, for 1 h at 4°C. Medium was replaced by prewarmed buffers of the indicated pH, followed by incubation for 2 min at 37°C. Viral uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Since fusion from without is cell type specific, SinV was used as control. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Incubation

    Comparison of CxxC motifs from different viruses. A decapeptide of BVDV strain NADL and CSFV Alfort E2 containing the CxxC motif was aligned to the corresponding sequence in rubella virus E1 (GI:33415288) and Mo-MuLV gPr80 glycosylated envelope polyprotein (GI:18448745). The numbers denote the position of the decapeptide in the respective protein; the conserved CxxC motif is indicated above the sequence. Conserved cysteine residues are boxed.

    Journal: Journal of Virology

    Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

    doi: 10.1128/JVI.79.7.4191-4200.2005

    Figure Lengend Snippet: Comparison of CxxC motifs from different viruses. A decapeptide of BVDV strain NADL and CSFV Alfort E2 containing the CxxC motif was aligned to the corresponding sequence in rubella virus E1 (GI:33415288) and Mo-MuLV gPr80 glycosylated envelope polyprotein (GI:18448745). The numbers denote the position of the decapeptide in the respective protein; the conserved CxxC motif is indicated above the sequence. Conserved cysteine residues are boxed.

    Article Snippet: A total of 5 × 105 MDBK cells or MDBK cells expressing either form of dynamin were grown in the absence or presence of 10 μg of doxycycline/ml for 24 h. A total of 2 × 105 PFU of BVDV strain NADL or SinV was adsorbed for 1 h at 4°C and washed with DMEM, followed by incubation at 37°C.

    Techniques: Sequencing

    Comparison of virus derived from pACNR/NADL and wt BVDV NADL. (A) Plaques were visualized by crystal violet staining at 3 days postinfection (NADL) or post-RNA transfection (ACNR/NADL) as described in Materials and Methods. The mock-transfected monolayer was treated the same as the transfected monolayer except that no RNA was present in the transfection mixture. (B) MDBK cells were infected with either wt NADL or ACNR/NADL at an MOI (as determined on MDBK monolayers) of either 0.1 or 1 PFU/cell, washed, and harvested at the indicated times postinfection. Titers were determined by plaque assay on MDBK monolayers as described in Materials and Methods. (C) MDBK cells were mock infected or infected with wt NADL or ACNR/NADL at an MOI of 1 PFU/cell. At 20 h postinfection, proteins were labeled for 4 h with Expre 35 S 35 S label (NEN) and lysed, and BVDV-specific proteins were immunoprecipitated with a polyclonal anti-BVDV serum (α49). Proteins were separated by SDS-PAGE (8% gel) and visualized by fluorography. Molecular mass markers are indicated at the left; BVDV-specific proteins, identified by size and, in some cases, by immunoreactivity with region-specific antisera (data not shown), are indicated at the right.

    Journal: Journal of Virology

    Article Title: Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

    doi:

    Figure Lengend Snippet: Comparison of virus derived from pACNR/NADL and wt BVDV NADL. (A) Plaques were visualized by crystal violet staining at 3 days postinfection (NADL) or post-RNA transfection (ACNR/NADL) as described in Materials and Methods. The mock-transfected monolayer was treated the same as the transfected monolayer except that no RNA was present in the transfection mixture. (B) MDBK cells were infected with either wt NADL or ACNR/NADL at an MOI (as determined on MDBK monolayers) of either 0.1 or 1 PFU/cell, washed, and harvested at the indicated times postinfection. Titers were determined by plaque assay on MDBK monolayers as described in Materials and Methods. (C) MDBK cells were mock infected or infected with wt NADL or ACNR/NADL at an MOI of 1 PFU/cell. At 20 h postinfection, proteins were labeled for 4 h with Expre 35 S 35 S label (NEN) and lysed, and BVDV-specific proteins were immunoprecipitated with a polyclonal anti-BVDV serum (α49). Proteins were separated by SDS-PAGE (8% gel) and visualized by fluorography. Molecular mass markers are indicated at the left; BVDV-specific proteins, identified by size and, in some cases, by immunoreactivity with region-specific antisera (data not shown), are indicated at the right.

    Article Snippet: The NADL strain of BVDV was obtained from the American Type Culture Collection, plaque purified, and amplified by growth in MDBK cells.

    Techniques: Derivative Assay, Staining, Transfection, Infection, Plaque Assay, Labeling, Immunoprecipitation, SDS Page

    Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. ( a ) Total viral RNA was determined by real-time RT-PCR. ( b ) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). ( c and d ) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection, Expressing, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Produced, Western Blot

    The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. ( a and b ) Confluent MDCK monolayers were mock-infected or infected with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. ( c and d ) Confluent MDCK monolayers were pretreated with or without ( c ) RhoA inhibitor (CT04) or ( d ) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Activation Assay, Infection, Pull Down Assay, Western Blot, Expressing

    Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10) for 5, 60, or 120 min. ( a and b ) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. ( c and d ) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection, Positive Control, Marker, Microscopy

    Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. ( a and b ) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain ( a ) DS-1 or ( b ) NCDV (10 μg/ml) for the indicated time points. Cell lysates were subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. ( c and d ) Confluent MDCK monolayers were either mock-treated or pretreated with ( c ) RhoA inhibitor CT04 or ( d ) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Activation Assay, Incubation, Recombinant, Pull Down Assay, Western Blot, Expressing, Purification

    Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores tight junction resistance in rotavirus-infected polarized epithelial cells. ( a and b ) MDCK monolayers grown on transwell filters were pretreated with or without RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a ) DS-1 or ( b ) NCDV (MOI = 10), followed by determination of TER at the indicated time points. Net TER was calculated by subtracting the background (membrane filter without cells) and multiplying the resistance (Ω) by the area (0.33 cm 2 ) of the filter. All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean from three independent experiments.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Inhibition, Infection

    Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Rotavirus infection-induced phosphorylation of MLC in polarized epithelial cells is an early event. ( a and b ) Human RVA DS-1 and bovine RVA NCDV strains (MOI = 10) were inoculated into confluent MDCK monolayers, and cells were harvested at the indicated time points. Cell lysates were subjected to western blot analysis to determine expression levels of phosphorylated MLC (pMLC) and MLC2 using the relevant antibodies. GAPDH was used as a loading control. The intensity of pMLC relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Infection, Western Blot, Expressing

    Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.

    Journal: Scientific Reports

    Article Title: Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells

    doi: 10.1038/s41598-018-32352-y

    Figure Lengend Snippet: Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. ( a – d ) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain ( a and b ) DS-1 or ( c and d ) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. ( b and d ) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.

    Article Snippet: The human rotavirus DS-1 (G2P1B[4]) and bovine rotavirus NCDV (G6P6[1]) strains purchased from ATCC were preactivated with 10 μg/ml crystalized trypsin (Cat. No. 27250-018; Gibco, Fort Worth, TX, USA) and propagated in MA104 cells as described previously .

    Techniques: Confocal Microscopy, Fluorescence