bvdv strain nadl  (ATCC)


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    Name:
    Bovine viral diarrhea virus 1
    Description:

    Catalog Number:
    VR-1422
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    Structured Review

    ATCC bvdv strain nadl
    Fusion from without of <t>BVDV</t> in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain <t>NADL</t> 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.

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    Images

    1) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    2) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    3) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    4) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    5) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    6) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    7) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    8) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    9) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

    10) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.7.4191-4200.2005

    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.
    Figure Legend 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.

    Techniques Used:

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Sequencing

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    Article Snippet: .. Bovine viruses Bovine herpesvirus-4 type 4 (BoHV-4-A-EGFPΔTK) [ ] and bovine viral diarrhea virus (BVDV, strain NADL, ATCC) were propagated by infecting confluent monolayers of bovine embryo kidney [(BS CL-94) BEK] or Madin Darby Bovine Kidney cells [(ATCC: CCL-22) MDBK] at a multiplicity of infection (MOI) of 0.5 50% tissue culture infectious doses (TCID50 ) per cell and maintained in MEM (ThermoFisher Scientific) with 2% FBS (ThermoFisher Scientific) for 2 h. The medium was then removed and replaced by fresh MEM containing 10% FBS. ..

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    Article Snippet: .. Viruses and Viral Amplification BoHV-4-A-CMV-NiV-FΔTK, BoHV-4-A-CMV-NiV-GΔTK, BoHV-4EGFP∆TK, and BoHV-4-A were propagated by infecting confluent monolayers of BEK cells at a multiplicity of infection (MOI) of 0.5 tissue culture infectious doses 50 (TCID50 ) per cell and maintained in medium with only 2% FBS for 2 h. Bovine herpesvirus-1 (BoHV-1, strain Oregon, ATCC VR-2066) and bovine viral diarrhea virus (BVDV, strain NADL, ATCC VR-1422) were propagated by infecting confluent monolayers of MDBK cells at an MOI of 0.5 and maintained in complete EMEM with 2% FBS for 2 h. Medium was replaced with fresh cEMEM. ..

    other:

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  • 94
    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: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bvdv nadl strain/product/ATCC
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    93
    ATCC bvdv strain nadl
    Fusion from without of <t>BVDV</t> in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain <t>NADL</t> 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.
    Bvdv Strain Nadl, 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
    https://www.bioz.com/result/bvdv strain nadl/product/ATCC
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bvdv strain nadl - by Bioz Stars, 2021-09
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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

    The NS5B C terminus conditions the production of infectious viral progeny. (A) Infectivity of in vitro-transcribed RNA from WT BVDV and NS5B mutant genomes determined by infectious center assay. BHK-21 cells were electroporated with RNA transcripts from pNADLp15 or from p5Bgdd, p5B-741, or p5B-L726P serially diluted and plated with additional fresh MDBK cells with agar overlay. (B) Immunofluorescence assay of cells electroporated with in vitro-transcribed RNA. RNA transcripts (2 μg) from 5Bgdd (1), mutant 5B-741 (3), 5B-L726P (5), N-ΔINS ncBVDV (7), and WT BVDV NADLp15 (9) were electroporated into RD420 cells and RNA replication was monitored by IIF using anti-E2 antibodies. Release of infectious virus was assessed by harvesting the culture medium (panels 1, 3, 5, 7, and 9) at 24 hpe, inoculating into fresh RD420 cells, and monitoring by IIF using anti-E2 antibodies (panels 2, 4, 6, 8, and 10, respectively). (C) Northern blot analysis of total RNA from cells electroporated with RNA from NS5B mutants. (a) Total RNA from BHK-21 cells electroporated with 5Bgdd (1), 5B-741 (2), 5B-L726P (3), N-ΔINS (4), and N-p15 (5) was analyzed, and 28S rRNA is indicated as a control. (b) The transferred RNA was hybridized to a 32 ). (D) Western blot analysis to demonstrate expression of structural and NS proteins in electroporated cells with mutant NS5B RNA. Panels: 1, 5Bgdd; 2, 5B741; 3, L726P; 4, N-ΔINS; 5, NADLp15. The total protein was resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-E2 and anti-NS3 MAbs, and signal was developed with a chemiluminescence detection system. Molecular mass markers are indicated (in kilodaltons). (E) Plaque phenotype of NS5B mutants. Plaques formed by revertant i-5B-741rev (a), the virus recovered from the engineered mutant i-5B-L726P (b), or WT BVDV strain NADL (c) on bovine cells infected for 60 h. The average diameters of the plaques are indicated.

    Journal: Journal of Virology

    Article Title: Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly †

    doi: 10.1128/JVI.78.18.9612-9623.2004

    Figure Lengend Snippet: The NS5B C terminus conditions the production of infectious viral progeny. (A) Infectivity of in vitro-transcribed RNA from WT BVDV and NS5B mutant genomes determined by infectious center assay. BHK-21 cells were electroporated with RNA transcripts from pNADLp15 or from p5Bgdd, p5B-741, or p5B-L726P serially diluted and plated with additional fresh MDBK cells with agar overlay. (B) Immunofluorescence assay of cells electroporated with in vitro-transcribed RNA. RNA transcripts (2 μg) from 5Bgdd (1), mutant 5B-741 (3), 5B-L726P (5), N-ΔINS ncBVDV (7), and WT BVDV NADLp15 (9) were electroporated into RD420 cells and RNA replication was monitored by IIF using anti-E2 antibodies. Release of infectious virus was assessed by harvesting the culture medium (panels 1, 3, 5, 7, and 9) at 24 hpe, inoculating into fresh RD420 cells, and monitoring by IIF using anti-E2 antibodies (panels 2, 4, 6, 8, and 10, respectively). (C) Northern blot analysis of total RNA from cells electroporated with RNA from NS5B mutants. (a) Total RNA from BHK-21 cells electroporated with 5Bgdd (1), 5B-741 (2), 5B-L726P (3), N-ΔINS (4), and N-p15 (5) was analyzed, and 28S rRNA is indicated as a control. (b) The transferred RNA was hybridized to a 32 ). (D) Western blot analysis to demonstrate expression of structural and NS proteins in electroporated cells with mutant NS5B RNA. Panels: 1, 5Bgdd; 2, 5B741; 3, L726P; 4, N-ΔINS; 5, NADLp15. The total protein was resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-E2 and anti-NS3 MAbs, and signal was developed with a chemiluminescence detection system. Molecular mass markers are indicated (in kilodaltons). (E) Plaque phenotype of NS5B mutants. Plaques formed by revertant i-5B-741rev (a), the virus recovered from the engineered mutant i-5B-L726P (b), or WT BVDV strain NADL (c) on bovine cells infected for 60 h. The average diameters of the plaques are indicated.

    Article Snippet: The cytopathogenic (cp) BVDV strain NADL was obtained from the American Type Culture Collection ( ).

    Techniques: Infection, In Vitro, Mutagenesis, Immunofluorescence, Northern Blot, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis