bhk 21 cells  (ATCC)


Bioz Verified Symbol ATCC is a verified supplier
Bioz Manufacturer Symbol ATCC manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    2254 62 2
    Description:

    Catalog Number:
    crl-8544
    Price:
    None
    Applications:
    This line was derived from BHK-21(c-13) (see ATCC CCL-10).This line has been used as a host for transformation with expression vectors containing selectable and amplifiable marker DNAs (e.
    Buy from Supplier


    Structured Review

    ATCC bhk 21 cells
    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on <t>BHK-21</t> cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    https://www.bioz.com/result/bhk 21 cells/product/ATCC
    Average 99 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice"

    Article Title: Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice

    Journal: Journal of Virology

    doi:

    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.
    Figure Legend Snippet: S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    Techniques Used: Mouse Assay, Infection, Dissection, Titration, Plaque Assay

    2) Product Images from "A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease"

    Article Title: A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01281-13

    BP13944 suppresses the viral yields of DENV-2. Mock- and compound-pretreated BHK-21 cells were infected with DENV-2 at an MOI of 0.1 (filled circles) or 1.0 (open circles) in the absence or presence of BP13944 and analyzed to determine the viral yield in the culture medium at 72 h postinfection. Error bars indicate the standard errors of the means (SEM) from three independent experiments.
    Figure Legend Snippet: BP13944 suppresses the viral yields of DENV-2. Mock- and compound-pretreated BHK-21 cells were infected with DENV-2 at an MOI of 0.1 (filled circles) or 1.0 (open circles) in the absence or presence of BP13944 and analyzed to determine the viral yield in the culture medium at 72 h postinfection. Error bars indicate the standard errors of the means (SEM) from three independent experiments.

    Techniques Used: Infection

    BP13944 inhibits all four serotypes of DENV. BHK-21 cells were incubated with 8 μM BP13944 and infected with the four serotypes of DENV at an MOI of 0.1. The viral yield in culture medium was determined via plaque formation assays at 72 h postinfection. The mean values and SEM from three independent experiments are plotted. **, P
    Figure Legend Snippet: BP13944 inhibits all four serotypes of DENV. BHK-21 cells were incubated with 8 μM BP13944 and infected with the four serotypes of DENV at an MOI of 0.1. The viral yield in culture medium was determined via plaque formation assays at 72 h postinfection. The mean values and SEM from three independent experiments are plotted. **, P

    Techniques Used: Incubation, Infection

    BP13944 selectively inhibits the viral yield and NS2B/NS3 protease activity of DNEV. BHK-21 cells infected with JEV or DENV-2 at an MOI of 0.1 were maintained in the absence or presence of 12 μM BP13944. The viral yields in the culture medium at 72 h postinfection were determined via plaque formation assays. The mean values and SEM from three independent experiments are plotted. ***, P
    Figure Legend Snippet: BP13944 selectively inhibits the viral yield and NS2B/NS3 protease activity of DNEV. BHK-21 cells infected with JEV or DENV-2 at an MOI of 0.1 were maintained in the absence or presence of 12 μM BP13944. The viral yields in the culture medium at 72 h postinfection were determined via plaque formation assays. The mean values and SEM from three independent experiments are plotted. ***, P

    Techniques Used: Activity Assay, Infection

    The E66G substitution within the DENV NS3 protease domain in the DENV replicon, infectious cDNA clone, or NS2B/NS3 protease results in BP13944 resistance. (A) BP13944 susceptibility of the parental and E66G substitution DENV replicon. The curves were fitted based on the transient replication activity assay as percentages of the control value (DMSO-treated controls). BHK-21 cells were electroporated with equal amounts (100 ng/2 × 10 5 cells) of parental or E66G substitution replicon RNA and treated with BP13944 at the indicated concentrations. (B) Resistance analyses of the recombinant parental and E66G substitution DENV. BHK-21 cells were infected with parental and E66G substitution DENVs at an MOI of 0.1 and treated with BP13944 at the indicated concentrations. The EC 50 s were determined by calculating the viral yield at 72 h postinfection via plaque formation assays. (C) BP13944 susceptibility of the parental and E66G substitution NS2B/NS3 proteases. The curves were fitted based on the observed enzyme activity as a percentage of the control value (DMSO treated). The IC 50 s of the parental and E66G substitution NS2B/NS3 proteases, corresponding to a 50% reduction in the protease activity assay, were calculated as 22.63 ± 0.74 μM and 71.10 ± 0.79 μM, respectively. (D) Multiplication kinetics of parental and E66G substitution DENVs in BHK-21 cells. Growth curves were conducted at an MOI of 0.1, and the limit of detection was ≦10 PFU/ml. The viral yields in culture medium at each time point were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted.
    Figure Legend Snippet: The E66G substitution within the DENV NS3 protease domain in the DENV replicon, infectious cDNA clone, or NS2B/NS3 protease results in BP13944 resistance. (A) BP13944 susceptibility of the parental and E66G substitution DENV replicon. The curves were fitted based on the transient replication activity assay as percentages of the control value (DMSO-treated controls). BHK-21 cells were electroporated with equal amounts (100 ng/2 × 10 5 cells) of parental or E66G substitution replicon RNA and treated with BP13944 at the indicated concentrations. (B) Resistance analyses of the recombinant parental and E66G substitution DENV. BHK-21 cells were infected with parental and E66G substitution DENVs at an MOI of 0.1 and treated with BP13944 at the indicated concentrations. The EC 50 s were determined by calculating the viral yield at 72 h postinfection via plaque formation assays. (C) BP13944 susceptibility of the parental and E66G substitution NS2B/NS3 proteases. The curves were fitted based on the observed enzyme activity as a percentage of the control value (DMSO treated). The IC 50 s of the parental and E66G substitution NS2B/NS3 proteases, corresponding to a 50% reduction in the protease activity assay, were calculated as 22.63 ± 0.74 μM and 71.10 ± 0.79 μM, respectively. (D) Multiplication kinetics of parental and E66G substitution DENVs in BHK-21 cells. Growth curves were conducted at an MOI of 0.1, and the limit of detection was ≦10 PFU/ml. The viral yields in culture medium at each time point were determined by plaque formation assays. The mean values and SEM from three independent experiments are plotted.

    Techniques Used: Activity Assay, Recombinant, Infection

    Effect of BP13944 on the replication of DENV-2 viral RNA. BHK-21 cells infected with DENV-2 at an MOI of 0.1 and 1.0 were maintained in the absence (open bars) or presence (filled bars) of 12 μM BP13944 for 72 h. The accumulation of positive-strand DENV-2 RNA in the cells was determined via TaqMan fluorogenic quantitative RT-PCR. RNA copy numbers were determined using standards, which were measured with a spectrophotometer. The mean values and SEM from three independent experiments are plotted. **, P
    Figure Legend Snippet: Effect of BP13944 on the replication of DENV-2 viral RNA. BHK-21 cells infected with DENV-2 at an MOI of 0.1 and 1.0 were maintained in the absence (open bars) or presence (filled bars) of 12 μM BP13944 for 72 h. The accumulation of positive-strand DENV-2 RNA in the cells was determined via TaqMan fluorogenic quantitative RT-PCR. RNA copy numbers were determined using standards, which were measured with a spectrophotometer. The mean values and SEM from three independent experiments are plotted. **, P

    Techniques Used: Infection, Quantitative RT-PCR, Spectrophotometry

    3) Product Images from "Murine Gammaherpesvirus 68 Lacking gp150 Shows Defective Virion Release but Establishes Normal Latency In Vivo"

    Article Title: Murine Gammaherpesvirus 68 Lacking gp150 Shows Defective Virion Release but Establishes Normal Latency In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.10.5103-5112.2004

    Interactions between MHV-68 and GAGs. (A) BHK-21 (fibroblast), NS0 (myeloma), and RAW264.7 (monocyte-macrophage) cells were infected at 0.1, 1, or 10 PFU per cell as indicated and analyzed for GFP expression 16 h later. (B) BHK-21, NS0, and RAW264.7 cells
    Figure Legend Snippet: Interactions between MHV-68 and GAGs. (A) BHK-21 (fibroblast), NS0 (myeloma), and RAW264.7 (monocyte-macrophage) cells were infected at 0.1, 1, or 10 PFU per cell as indicated and analyzed for GFP expression 16 h later. (B) BHK-21, NS0, and RAW264.7 cells

    Techniques Used: Infection, Expressing

    Electron microscopy of virus-infected cells. BHK-21 cells were infected (16 h; multiplicity of infection, 5) with WT, M7 − FRT, or M7REV viruses as indicated. The pictures shown are representative of at least 10 sections per sample. The heterogeneity
    Figure Legend Snippet: Electron microscopy of virus-infected cells. BHK-21 cells were infected (16 h; multiplicity of infection, 5) with WT, M7 − FRT, or M7REV viruses as indicated. The pictures shown are representative of at least 10 sections per sample. The heterogeneity

    Techniques Used: Electron Microscopy, Infection

    In vitro growth of gp150-deficient viruses. Subconfluent MEF (A to D) or BHK-21 cell (E to F) monolayers were infected for 2 h at a multiplicity of infection of 5 or 0.01, acid washed, and incubated in complete medium. At the indicated times, titers of
    Figure Legend Snippet: In vitro growth of gp150-deficient viruses. Subconfluent MEF (A to D) or BHK-21 cell (E to F) monolayers were infected for 2 h at a multiplicity of infection of 5 or 0.01, acid washed, and incubated in complete medium. At the indicated times, titers of

    Techniques Used: In Vitro, Infection, Incubation

    Fluid phase virus spread. (A) A total of 4 × 10 5 infected (multiplicity of infection, 0.01) and 4 × 10 5 uninfected BHK-21 cells were seeded on opposite sides of a 6-cm-diameter dish, separated by a physical barrier but connected by medium
    Figure Legend Snippet: Fluid phase virus spread. (A) A total of 4 × 10 5 infected (multiplicity of infection, 0.01) and 4 × 10 5 uninfected BHK-21 cells were seeded on opposite sides of a 6-cm-diameter dish, separated by a physical barrier but connected by medium

    Techniques Used: Infection

    Virus binding and penetration and effect of virion multiplicity on infection. (A) BHK-21 cells were infected for 2 h with dilutions of GFP + viruses and analyzed for GFP expression by flow cytometry 16 h later. (B and C) Subconfluent BHK-21 cell
    Figure Legend Snippet: Virus binding and penetration and effect of virion multiplicity on infection. (A) BHK-21 cells were infected for 2 h with dilutions of GFP + viruses and analyzed for GFP expression by flow cytometry 16 h later. (B and C) Subconfluent BHK-21 cell

    Techniques Used: Binding Assay, Infection, Expressing, Flow Cytometry, Cytometry

    4) Product Images from "Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects"

    Article Title: Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects

    Journal: Journal of Virology

    doi: 10.1128/JVI.06615-11

    Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.
    Figure Legend Snippet: Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.

    Techniques Used: In Vitro, Mutagenesis, Plaque Assay, Produced

    pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.
    Figure Legend Snippet: pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.

    Techniques Used: In Vitro, Mutagenesis, Lysis, SDS Page, Construct, Western Blot, Migration

    5) Product Images from "Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects"

    Article Title: Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects

    Journal: Journal of Virology

    doi: 10.1128/JVI.06615-11

    Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.
    Figure Legend Snippet: Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.

    Techniques Used: In Vitro, Mutagenesis, Plaque Assay, Produced

    pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.
    Figure Legend Snippet: pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.

    Techniques Used: In Vitro, Mutagenesis, Lysis, SDS Page, Construct, Western Blot, Migration

    6) Product Images from "Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects"

    Article Title: Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects

    Journal: Journal of Virology

    doi: 10.1128/JVI.06615-11

    Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.
    Figure Legend Snippet: Production of infectious virus particles by the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, the medium was harvested and titers were determined by standard plaque assay procedure. The experiments to obtain growth curves were performed twice for each E2 Cys mutant. The results from one representative experiment are shown. (A) Growth curves of the SINV E2 Cys mutants. ■, wild-type SINV; △, SINV E2 C19S; ♢, SINV E2 C201S; □, SINV E2 C220S. SINV E2 C16S produced no detectable infectious particles. (B) Growth curves of the RRV E2 Cys mutants. ■, wild-type RRV; ○, RRV E2 C19S; △, RRV E2 C22S; ♢, RRV E2 C201S; □, RRV E2 C220S.

    Techniques Used: In Vitro, Mutagenesis, Plaque Assay, Produced

    pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.
    Figure Legend Snippet: pE2 processing characterization of the SINV and RRV E2 Cys mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or mutant viral RNA. At the indicated time points, cells were lysed with BHK cell lysis buffer. Cell lysates were solubilized in reducing SDS sample buffer. The solubilized cell lysates were analyzed by SDS-PAGE and probed with polyclonal antibodies recognizing pE2/E2 (top blot for each construct) and capsid (bottom blot for each construct) by Western blot. Migration of pE2, E2, and capsid bands is indicated to the right of each blot. Migration of molecular mass standards is indicated to the left of each blot. (A) Wild-type SINV and RRV; (B) pE2 furin cleavage mutants of SINV and RRV; (C and D) domain A mutants; (E and F) domain B mutants.

    Techniques Used: In Vitro, Mutagenesis, Lysis, SDS Page, Construct, Western Blot, Migration

    7) Product Images from "Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2"

    Article Title: Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    Journal: Journal of Virology

    doi: 10.1128/JVI.03213-14

    Effects of the identified mutations on the synthesis of positive-strand RNAs and the nsP2 protein in cells transfected with CHIKV replicon RNAs. (A and B) Northern blot analysis was performed using total RNAs extracted from BHK-21 (A) or Huh7 (B) cell
    Figure Legend Snippet: Effects of the identified mutations on the synthesis of positive-strand RNAs and the nsP2 protein in cells transfected with CHIKV replicon RNAs. (A and B) Northern blot analysis was performed using total RNAs extracted from BHK-21 (A) or Huh7 (B) cell

    Techniques Used: Transfection, Northern Blot

    Analysis of the subcellular localization of nsP2 in BHK-21 cells infected with wt or mutant versions of CHIKV. (A) The localization of nsP2 in infected cells was analyzed by immunofluorescence microscopy. BHK-21 cells were infected with wt CHIKV or CHIKV-5A-PG
    Figure Legend Snippet: Analysis of the subcellular localization of nsP2 in BHK-21 cells infected with wt or mutant versions of CHIKV. (A) The localization of nsP2 in infected cells was analyzed by immunofluorescence microscopy. BHK-21 cells were infected with wt CHIKV or CHIKV-5A-PG

    Techniques Used: Infection, Mutagenesis, Immunofluorescence, Microscopy

    Development of stable BHK-21 CHIKV replicon cell lines.
    Figure Legend Snippet: Development of stable BHK-21 CHIKV replicon cell lines.

    Techniques Used:

    Schematic presentation of the CHIKV replicons and the survival of cell cultures transfected with the original and mutant replicons. Upper panels, design of ChikvRep and ChikvRepRluc, used to transfect BHK-21 (A) and Huh7 cells (B), respectively. The positions
    Figure Legend Snippet: Schematic presentation of the CHIKV replicons and the survival of cell cultures transfected with the original and mutant replicons. Upper panels, design of ChikvRep and ChikvRepRluc, used to transfect BHK-21 (A) and Huh7 cells (B), respectively. The positions

    Techniques Used: Transfection, Mutagenesis

    8) Product Images from "Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2"

    Article Title: Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    Journal: Journal of Virology

    doi: 10.1128/JVI.03213-14

    Effects of the identified mutations on the synthesis of positive-strand RNAs and the nsP2 protein in cells transfected with CHIKV replicon RNAs. (A and B) Northern blot analysis was performed using total RNAs extracted from BHK-21 (A) or Huh7 (B) cell
    Figure Legend Snippet: Effects of the identified mutations on the synthesis of positive-strand RNAs and the nsP2 protein in cells transfected with CHIKV replicon RNAs. (A and B) Northern blot analysis was performed using total RNAs extracted from BHK-21 (A) or Huh7 (B) cell

    Techniques Used: Transfection, Northern Blot

    Analysis of the subcellular localization of nsP2 in BHK-21 cells infected with wt or mutant versions of CHIKV. (A) The localization of nsP2 in infected cells was analyzed by immunofluorescence microscopy. BHK-21 cells were infected with wt CHIKV or CHIKV-5A-PG
    Figure Legend Snippet: Analysis of the subcellular localization of nsP2 in BHK-21 cells infected with wt or mutant versions of CHIKV. (A) The localization of nsP2 in infected cells was analyzed by immunofluorescence microscopy. BHK-21 cells were infected with wt CHIKV or CHIKV-5A-PG

    Techniques Used: Infection, Mutagenesis, Immunofluorescence, Microscopy

    Development of stable BHK-21 CHIKV replicon cell lines.
    Figure Legend Snippet: Development of stable BHK-21 CHIKV replicon cell lines.

    Techniques Used:

    Schematic presentation of the CHIKV replicons and the survival of cell cultures transfected with the original and mutant replicons. Upper panels, design of ChikvRep and ChikvRepRluc, used to transfect BHK-21 (A) and Huh7 cells (B), respectively. The positions
    Figure Legend Snippet: Schematic presentation of the CHIKV replicons and the survival of cell cultures transfected with the original and mutant replicons. Upper panels, design of ChikvRep and ChikvRepRluc, used to transfect BHK-21 (A) and Huh7 cells (B), respectively. The positions

    Techniques Used: Transfection, Mutagenesis

    9) Product Images from "Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication"

    Article Title: Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.8.5129-5141.2005

    Viral gene expression and DNA replication of the ORF45-null mutant. (A and B) BHK-21 cells were infected with the wt or ORF45-null (45STOP) virus at an MOI of 500 genome copies per cell. Cellular RNAs were isolated at the indicated times postinfection
    Figure Legend Snippet: Viral gene expression and DNA replication of the ORF45-null mutant. (A and B) BHK-21 cells were infected with the wt or ORF45-null (45STOP) virus at an MOI of 500 genome copies per cell. Cellular RNAs were isolated at the indicated times postinfection

    Techniques Used: Expressing, Mutagenesis, Infection, Isolation

    Deficiency in and complementation of viral replication for the ORF45-null mutant. (A) BHK-21 cells were transfected with wt MHV-68(BAC) plus plasmid pEGFP-C1 (panels 1, 4, 1′, and 4′), 45STOP plus pEGFP-C1 (panels 2, 5, 2′, and
    Figure Legend Snippet: Deficiency in and complementation of viral replication for the ORF45-null mutant. (A) BHK-21 cells were transfected with wt MHV-68(BAC) plus plasmid pEGFP-C1 (panels 1, 4, 1′, and 4′), 45STOP plus pEGFP-C1 (panels 2, 5, 2′, and

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation

    Analysis of functional domain(s) of ORF45 in viral replication. (A) BHK-21 cells were mock transfected or cotransfected with the wt, ORF45-null (45STOP), or null-revertant (45STOP.R) BAC DNA plus an expression plasmid containing GFP or GFP fused to full-length
    Figure Legend Snippet: Analysis of functional domain(s) of ORF45 in viral replication. (A) BHK-21 cells were mock transfected or cotransfected with the wt, ORF45-null (45STOP), or null-revertant (45STOP.R) BAC DNA plus an expression plasmid containing GFP or GFP fused to full-length

    Techniques Used: Functional Assay, Transfection, BAC Assay, Expressing, Plasmid Preparation

    10) Product Images from "Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia"

    Article Title: Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.15-0318

    Replication kinetics of two different strains of chikungunya virus (CHIKV) infectious clones in the midguts of Aedes aegypti. Virus titers were measured by plaque assay in BHK-21 cells and refer to plaque forming units per milliliter of homogenate. Error
    Figure Legend Snippet: Replication kinetics of two different strains of chikungunya virus (CHIKV) infectious clones in the midguts of Aedes aegypti. Virus titers were measured by plaque assay in BHK-21 cells and refer to plaque forming units per milliliter of homogenate. Error

    Techniques Used: Clone Assay, Plaque Assay

    11) Product Images from "A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization"

    Article Title: A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization

    Journal: Journal of Virology

    doi: 10.1128/JVI.03116-13

    Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P
    Figure Legend Snippet: Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P

    Techniques Used: Inhibition, Adsorption, Incubation, Infection, Immunofluorescence

    12) Product Images from "A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization"

    Article Title: A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization

    Journal: Journal of Virology

    doi: 10.1128/JVI.03116-13

    Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P
    Figure Legend Snippet: Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P

    Techniques Used: Inhibition, Adsorption, Incubation, Infection, Immunofluorescence

    13) Product Images from "Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells"

    Article Title: Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells

    Journal: Journal of Virology

    doi:

    Analysis of SIN RNAs in Pur r cell lines. (A) Pur r cell lines were labeled for 18 h with [ 3 H]uridine (30 μCi/ml) in the presence of dactinomycin (1 μg/ml). To provide a control for replicon genomic and subgenomic RNAs, BHK-21 cells were infected at a high multiplicity (10 infectious units/cell) with packaged TSG/PAC replicons (lane 5). (B) Cells were superinfected or infected with Toto1101 (10 PFU/cell). Infected cells were also labeled as described above for 18 h beginning 1 h postinfection. The Pur r cell lines analyzed were S22 (lanes 1 and 6), S1 (lanes 2 and 7), S24 (lanes 3 and 8), S25 (lanes 4 and 9), and BHK-21 cells (lane 10). RNAs were extracted and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes 1 to 4 contain RNAs from 5 × 10 5 cells, and the gel was fluorographed and exposed for 60 h. Lanes 5 to 10 contain RNAs isolated from 10 5 cells, and the exposure time for these samples was only 6 h. The positions of replicon and SIN genomic and subgenomic RNAs are indicated on the right of panel B.
    Figure Legend Snippet: Analysis of SIN RNAs in Pur r cell lines. (A) Pur r cell lines were labeled for 18 h with [ 3 H]uridine (30 μCi/ml) in the presence of dactinomycin (1 μg/ml). To provide a control for replicon genomic and subgenomic RNAs, BHK-21 cells were infected at a high multiplicity (10 infectious units/cell) with packaged TSG/PAC replicons (lane 5). (B) Cells were superinfected or infected with Toto1101 (10 PFU/cell). Infected cells were also labeled as described above for 18 h beginning 1 h postinfection. The Pur r cell lines analyzed were S22 (lanes 1 and 6), S1 (lanes 2 and 7), S24 (lanes 3 and 8), S25 (lanes 4 and 9), and BHK-21 cells (lane 10). RNAs were extracted and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes 1 to 4 contain RNAs from 5 × 10 5 cells, and the gel was fluorographed and exposed for 60 h. Lanes 5 to 10 contain RNAs isolated from 10 5 cells, and the exposure time for these samples was only 6 h. The positions of replicon and SIN genomic and subgenomic RNAs are indicated on the right of panel B.

    Techniques Used: Labeling, Infection, Agarose Gel Electrophoresis, Isolation

    Transfection of naive BHK cells with RNA from the Pur r S1 cell line. BHK-21 cells were electroporated with poly(A) + RNA isolated from 2 × 10 6 cells of the S1 Pur r cell line (dish 1), total RNA isolated from 5 × 10 5 cells of the S1 line (dish 2), or poly(A) + RNA isolated from 1 × 10 6 BHK-21 cells (dish 3). Puromycin (10 μg/ml) was added to the medium at 16 h post-transfection. Pur r foci were stained with crystal violet after 5 days (dish 1) or 7 days (dishes 2 and 3).
    Figure Legend Snippet: Transfection of naive BHK cells with RNA from the Pur r S1 cell line. BHK-21 cells were electroporated with poly(A) + RNA isolated from 2 × 10 6 cells of the S1 Pur r cell line (dish 1), total RNA isolated from 5 × 10 5 cells of the S1 line (dish 2), or poly(A) + RNA isolated from 1 × 10 6 BHK-21 cells (dish 3). Puromycin (10 μg/ml) was added to the medium at 16 h post-transfection. Pur r foci were stained with crystal violet after 5 days (dish 1) or 7 days (dishes 2 and 3).

    Techniques Used: Transfection, Isolation, Staining

    14) Product Images from "Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells"

    Article Title: Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells

    Journal: Journal of Virology

    doi:

    Analysis of SIN RNAs in Pur r cell lines. (A) Pur r cell lines were labeled for 18 h with [ 3 H]uridine (30 μCi/ml) in the presence of dactinomycin (1 μg/ml). To provide a control for replicon genomic and subgenomic RNAs, BHK-21 cells were infected at a high multiplicity (10 infectious units/cell) with packaged TSG/PAC replicons (lane 5). (B) Cells were superinfected or infected with Toto1101 (10 PFU/cell). Infected cells were also labeled as described above for 18 h beginning 1 h postinfection. The Pur r cell lines analyzed were S22 (lanes 1 and 6), S1 (lanes 2 and 7), S24 (lanes 3 and 8), S25 (lanes 4 and 9), and BHK-21 cells (lane 10). RNAs were extracted and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes 1 to 4 contain RNAs from 5 × 10 5 cells, and the gel was fluorographed and exposed for 60 h. Lanes 5 to 10 contain RNAs isolated from 10 5 cells, and the exposure time for these samples was only 6 h. The positions of replicon and SIN genomic and subgenomic RNAs are indicated on the right of panel B.
    Figure Legend Snippet: Analysis of SIN RNAs in Pur r cell lines. (A) Pur r cell lines were labeled for 18 h with [ 3 H]uridine (30 μCi/ml) in the presence of dactinomycin (1 μg/ml). To provide a control for replicon genomic and subgenomic RNAs, BHK-21 cells were infected at a high multiplicity (10 infectious units/cell) with packaged TSG/PAC replicons (lane 5). (B) Cells were superinfected or infected with Toto1101 (10 PFU/cell). Infected cells were also labeled as described above for 18 h beginning 1 h postinfection. The Pur r cell lines analyzed were S22 (lanes 1 and 6), S1 (lanes 2 and 7), S24 (lanes 3 and 8), S25 (lanes 4 and 9), and BHK-21 cells (lane 10). RNAs were extracted and analyzed by agarose gel electrophoresis as described in Materials and Methods. Lanes 1 to 4 contain RNAs from 5 × 10 5 cells, and the gel was fluorographed and exposed for 60 h. Lanes 5 to 10 contain RNAs isolated from 10 5 cells, and the exposure time for these samples was only 6 h. The positions of replicon and SIN genomic and subgenomic RNAs are indicated on the right of panel B.

    Techniques Used: Labeling, Infection, Agarose Gel Electrophoresis, Isolation

    Transfection of naive BHK cells with RNA from the Pur r S1 cell line. BHK-21 cells were electroporated with poly(A) + RNA isolated from 2 × 10 6 cells of the S1 Pur r cell line (dish 1), total RNA isolated from 5 × 10 5 cells of the S1 line (dish 2), or poly(A) + RNA isolated from 1 × 10 6 BHK-21 cells (dish 3). Puromycin (10 μg/ml) was added to the medium at 16 h post-transfection. Pur r foci were stained with crystal violet after 5 days (dish 1) or 7 days (dishes 2 and 3).
    Figure Legend Snippet: Transfection of naive BHK cells with RNA from the Pur r S1 cell line. BHK-21 cells were electroporated with poly(A) + RNA isolated from 2 × 10 6 cells of the S1 Pur r cell line (dish 1), total RNA isolated from 5 × 10 5 cells of the S1 line (dish 2), or poly(A) + RNA isolated from 1 × 10 6 BHK-21 cells (dish 3). Puromycin (10 μg/ml) was added to the medium at 16 h post-transfection. Pur r foci were stained with crystal violet after 5 days (dish 1) or 7 days (dishes 2 and 3).

    Techniques Used: Transfection, Isolation, Staining

    15) Product Images from "Acidity/Alkalinity of Japanese Encephalitis Virus E Protein Residue 138 Alters Neurovirulence in Mice"

    Article Title: Acidity/Alkalinity of Japanese Encephalitis Virus E Protein Residue 138 Alters Neurovirulence in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.00108-18

    Multistep growth curves of the E47 mutant virus. BHK-21 cells were inoculated with JEVs at an MOI of 0.01. The supernatant of infected cells was harvested at the indicated time points and used to determine the viral titers in BHK-21 cells. The data are presented as means ± SD from three independent experiments. * indicates a significant difference between vJEHEN and the E47 mutants vHN47A, vHN47D, and vHN47K (***, P
    Figure Legend Snippet: Multistep growth curves of the E47 mutant virus. BHK-21 cells were inoculated with JEVs at an MOI of 0.01. The supernatant of infected cells was harvested at the indicated time points and used to determine the viral titers in BHK-21 cells. The data are presented as means ± SD from three independent experiments. * indicates a significant difference between vJEHEN and the E47 mutants vHN47A, vHN47D, and vHN47K (***, P

    Techniques Used: Mutagenesis, Infection

    16) Product Images from "A Protein Critical for a Theiler's Virus-Induced Immune System-Mediated Demyelinating Disease Has a Cell Type-Specific Antiapoptotic Effect and a Key Role in Virus Persistence"

    Article Title: A Protein Critical for a Theiler's Virus-Induced Immune System-Mediated Demyelinating Disease Has a Cell Type-Specific Antiapoptotic Effect and a Key Role in Virus Persistence

    Journal: Journal of Virology

    doi:

    DAL*-1 virus infection of P388D1 cells causes a premature shutoff of viral and cellular protein synthesis. Shown is an autoradiogram of P388D1 and BHK-21 cells that were either mock infected or infected with wild-type DA or DAL*-1 virus and then radiolabeled at 3 to 5 h p.i. (A), 6 to 8 h p.i. (B), or 10 to 18 h p.i. (C and D). Cells were harvested at the end of the period of radiolabeling. M, the left-most lane in panel A, is a marker lane for the viral proteins and contains radiolabeled BHK-21 cells infected with DA virus.
    Figure Legend Snippet: DAL*-1 virus infection of P388D1 cells causes a premature shutoff of viral and cellular protein synthesis. Shown is an autoradiogram of P388D1 and BHK-21 cells that were either mock infected or infected with wild-type DA or DAL*-1 virus and then radiolabeled at 3 to 5 h p.i. (A), 6 to 8 h p.i. (B), or 10 to 18 h p.i. (C and D). Cells were harvested at the end of the period of radiolabeling. M, the left-most lane in panel A, is a marker lane for the viral proteins and contains radiolabeled BHK-21 cells infected with DA virus.

    Techniques Used: Infection, Radioactivity, Marker

    17) Product Images from "A Chikungunya Virus trans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells"

    Article Title: A Chikungunya Virus trans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00152-18

    Western blot analysis of proteins expressed by CHIKV WA upon virus rescue (A) or during and after adaptation to growth at 37°C (B). BHK-21 cells were transfected with in vitro -synthesized transcripts of wt pICRES1 and pICRES1 WA using electroporation, and C6/36 cells were transfected using Lipofectamine 2000. At 24 h p.t. (wt virus in BHK-21 cells) or at 72 h p.t. (all other samples), transfected cells were collected and lysed by boiling in SDS gel-loading buffer. BHK-21 cells harvested after collection of P1 and P5 stocks of CHIKV WA adapted to growth at 37°C were also collected and lysed as described above. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected using antibodies against CHIKV nsP1 and capsid protein. In lysates of BHK-21 cells, antibody against β-actin was used to detect the loading control (for C6/36 cells, the corresponding antibody was not available).
    Figure Legend Snippet: Western blot analysis of proteins expressed by CHIKV WA upon virus rescue (A) or during and after adaptation to growth at 37°C (B). BHK-21 cells were transfected with in vitro -synthesized transcripts of wt pICRES1 and pICRES1 WA using electroporation, and C6/36 cells were transfected using Lipofectamine 2000. At 24 h p.t. (wt virus in BHK-21 cells) or at 72 h p.t. (all other samples), transfected cells were collected and lysed by boiling in SDS gel-loading buffer. BHK-21 cells harvested after collection of P1 and P5 stocks of CHIKV WA adapted to growth at 37°C were also collected and lysed as described above. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected using antibodies against CHIKV nsP1 and capsid protein. In lysates of BHK-21 cells, antibody against β-actin was used to detect the loading control (for C6/36 cells, the corresponding antibody was not available).

    Techniques Used: Western Blot, Transfection, In Vitro, Synthesized, Electroporation, SDS-Gel, SDS Page

    18) Product Images from "Control of the Rescue and Replication of Semliki Forest Virus Recombinants by the Insertion of miRNA Target Sequences"

    Article Title: Control of the Rescue and Replication of Semliki Forest Virus Recombinants by the Insertion of miRNA Target Sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075802

    Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets. (A) General design of the vectors; the same symbols as in Fig. 3A are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and He La (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p
    Figure Legend Snippet: Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets. (A) General design of the vectors; the same symbols as in Fig. 3A are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and He La (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p

    Techniques Used: Infection, Recombinant, Standard Deviation

    Northern blot analysis of SFV RNAs in transfected cells. BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.
    Figure Legend Snippet: Northern blot analysis of SFV RNAs in transfected cells. BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.

    Techniques Used: Northern Blot, Transfection, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Activated Clotting Time Assay

    Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors. BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to Let-7 , miR-17 and miR-19 (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.
    Figure Legend Snippet: Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors. BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to Let-7 , miR-17 and miR-19 (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.

    Techniques Used: Expressing, Recombinant, Activity Assay, Luciferase

    Insertion of miR-1cl and miR-cl2 cassettes suppresses Gluc expression from a non-viral vector. (A) Schematic representation of non-viral expression vector (above) and corresponding mRNA (below). The plasmid backbone of the vector is not shown. CMV, immediate-early promoter of human cytomegalovirus; Rz, negative strand ribozyme of hepatitis delta virus (cleavage site is indicated with bent arrow); p(A), early polyadenylation signal of simian virus 40; (A)n, poly(A) tail. The 5′ and 3′ UTRs of the transcribed mRNA correspond to those of SFV4. Effects of miR-cl1 target sequences on Gluc expression in transfected BHK-21 (B) or HeLa cells (C) and effects of miR-cl2 target sequences on Gluc expression in transfected BHK-21 (D) or HeLa cells (E) were analysed as follows. Cells were transfected by electroporation, and Gluc activity in the growth medium was measured at times indicated on the horizontal axes. The results were normalised to Gluc activity in the growth media of cells transfected with the Gluc expression vector lacking an miRNA target site; the corresponding activity was set at 100% (shown on vertical axes). In all panels, open, black and grey columns represent normalised data for sponge design, perfect design and native design cassettes, respectively. Each column represents an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p
    Figure Legend Snippet: Insertion of miR-1cl and miR-cl2 cassettes suppresses Gluc expression from a non-viral vector. (A) Schematic representation of non-viral expression vector (above) and corresponding mRNA (below). The plasmid backbone of the vector is not shown. CMV, immediate-early promoter of human cytomegalovirus; Rz, negative strand ribozyme of hepatitis delta virus (cleavage site is indicated with bent arrow); p(A), early polyadenylation signal of simian virus 40; (A)n, poly(A) tail. The 5′ and 3′ UTRs of the transcribed mRNA correspond to those of SFV4. Effects of miR-cl1 target sequences on Gluc expression in transfected BHK-21 (B) or HeLa cells (C) and effects of miR-cl2 target sequences on Gluc expression in transfected BHK-21 (D) or HeLa cells (E) were analysed as follows. Cells were transfected by electroporation, and Gluc activity in the growth medium was measured at times indicated on the horizontal axes. The results were normalised to Gluc activity in the growth media of cells transfected with the Gluc expression vector lacking an miRNA target site; the corresponding activity was set at 100% (shown on vertical axes). In all panels, open, black and grey columns represent normalised data for sponge design, perfect design and native design cassettes, respectively. Each column represents an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Electroporation, Activity Assay, Standard Deviation

    19) Product Images from "Control of the Rescue and Replication of Semliki Forest Virus Recombinants by the Insertion of miRNA Target Sequences"

    Article Title: Control of the Rescue and Replication of Semliki Forest Virus Recombinants by the Insertion of miRNA Target Sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075802

    Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets. (A) General design of the vectors; the same symbols as in Fig. 3A are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and He La (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p
    Figure Legend Snippet: Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets. (A) General design of the vectors; the same symbols as in Fig. 3A are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and He La (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p

    Techniques Used: Infection, Recombinant, Standard Deviation

    Northern blot analysis of SFV RNAs in transfected cells. BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.
    Figure Legend Snippet: Northern blot analysis of SFV RNAs in transfected cells. BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.

    Techniques Used: Northern Blot, Transfection, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Activated Clotting Time Assay

    Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors. BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to Let-7 , miR-17 and miR-19 (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.
    Figure Legend Snippet: Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors. BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to Let-7 , miR-17 and miR-19 (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.

    Techniques Used: Expressing, Recombinant, Activity Assay, Luciferase

    Insertion of miR-1cl and miR-cl2 cassettes suppresses Gluc expression from a non-viral vector. (A) Schematic representation of non-viral expression vector (above) and corresponding mRNA (below). The plasmid backbone of the vector is not shown. CMV, immediate-early promoter of human cytomegalovirus; Rz, negative strand ribozyme of hepatitis delta virus (cleavage site is indicated with bent arrow); p(A), early polyadenylation signal of simian virus 40; (A)n, poly(A) tail. The 5′ and 3′ UTRs of the transcribed mRNA correspond to those of SFV4. Effects of miR-cl1 target sequences on Gluc expression in transfected BHK-21 (B) or HeLa cells (C) and effects of miR-cl2 target sequences on Gluc expression in transfected BHK-21 (D) or HeLa cells (E) were analysed as follows. Cells were transfected by electroporation, and Gluc activity in the growth medium was measured at times indicated on the horizontal axes. The results were normalised to Gluc activity in the growth media of cells transfected with the Gluc expression vector lacking an miRNA target site; the corresponding activity was set at 100% (shown on vertical axes). In all panels, open, black and grey columns represent normalised data for sponge design, perfect design and native design cassettes, respectively. Each column represents an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p
    Figure Legend Snippet: Insertion of miR-1cl and miR-cl2 cassettes suppresses Gluc expression from a non-viral vector. (A) Schematic representation of non-viral expression vector (above) and corresponding mRNA (below). The plasmid backbone of the vector is not shown. CMV, immediate-early promoter of human cytomegalovirus; Rz, negative strand ribozyme of hepatitis delta virus (cleavage site is indicated with bent arrow); p(A), early polyadenylation signal of simian virus 40; (A)n, poly(A) tail. The 5′ and 3′ UTRs of the transcribed mRNA correspond to those of SFV4. Effects of miR-cl1 target sequences on Gluc expression in transfected BHK-21 (B) or HeLa cells (C) and effects of miR-cl2 target sequences on Gluc expression in transfected BHK-21 (D) or HeLa cells (E) were analysed as follows. Cells were transfected by electroporation, and Gluc activity in the growth medium was measured at times indicated on the horizontal axes. The results were normalised to Gluc activity in the growth media of cells transfected with the Gluc expression vector lacking an miRNA target site; the corresponding activity was set at 100% (shown on vertical axes). In all panels, open, black and grey columns represent normalised data for sponge design, perfect design and native design cassettes, respectively. Each column represents an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Electroporation, Activity Assay, Standard Deviation

    20) Product Images from "Synergistic role of micronemal proteins in Toxoplasma gondii virulence"

    Article Title: Synergistic role of micronemal proteins in Toxoplasma gondii virulence

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041672

    Identification of critical residues in the CBL domain of MIC3. (A) Representation of conserved residues in the CB type 1 domain (PROSITE PDOC00025, CB type 1 domain signature and profile) and the CBL domain of T. gondii MIC3 (CAB56644). The topological arrangement of the four disulfide bonds is indicated by brackets. Conserved residues of the binding site are in red letters and well-conserved positions are in blue letters. C, cysteine; S, serine; P, proline; X, any amino acid; Φ, aromatic amino acid. All amino acids are contiguous, but the sequences are represented with a carriage return before each cysteine. (B) IF of mutated MIC3 proteins in transfected cells. BHK-21 cells were transfected with plasmids allowing expression of MIC3 or mutated MIC3 tagged with V5 epitope and coexpression of the green fluorescent protein. Cytoplasmic fluorescence of green fluorescent protein allows the monitoring of transfected cells. MIC3 was visualized with mAb anti-V5 (red) after permeabilization (Perm, intracellular MIC3) or without permeabilization (No perm, surface-bound MIC 3). Secretion was monitored by Western blotting. (d, e, and f). Equivalent fractions of cells (pellet) and corresponding supernatants (SN) collected 18 h after transfection were probed with mAb anti-V5. Three phenotypes were obtained: the recombinant protein is retained in the secretory pathway (exemplified by Y141 in a and d); the protein is secreted and binds to the surface of transfected cells (exemplified by R-MIC3 in b and e); and the protein is secreted but does not bind to the surface of transfected cells (exemplified by F128A in c and f). Adhesive proteins show a stronger signal in the pellet, resulting from their surface association, in contrast to nonadhesive R-MIC3 where a stronger signal is obtained in the supernatant.
    Figure Legend Snippet: Identification of critical residues in the CBL domain of MIC3. (A) Representation of conserved residues in the CB type 1 domain (PROSITE PDOC00025, CB type 1 domain signature and profile) and the CBL domain of T. gondii MIC3 (CAB56644). The topological arrangement of the four disulfide bonds is indicated by brackets. Conserved residues of the binding site are in red letters and well-conserved positions are in blue letters. C, cysteine; S, serine; P, proline; X, any amino acid; Φ, aromatic amino acid. All amino acids are contiguous, but the sequences are represented with a carriage return before each cysteine. (B) IF of mutated MIC3 proteins in transfected cells. BHK-21 cells were transfected with plasmids allowing expression of MIC3 or mutated MIC3 tagged with V5 epitope and coexpression of the green fluorescent protein. Cytoplasmic fluorescence of green fluorescent protein allows the monitoring of transfected cells. MIC3 was visualized with mAb anti-V5 (red) after permeabilization (Perm, intracellular MIC3) or without permeabilization (No perm, surface-bound MIC 3). Secretion was monitored by Western blotting. (d, e, and f). Equivalent fractions of cells (pellet) and corresponding supernatants (SN) collected 18 h after transfection were probed with mAb anti-V5. Three phenotypes were obtained: the recombinant protein is retained in the secretory pathway (exemplified by Y141 in a and d); the protein is secreted and binds to the surface of transfected cells (exemplified by R-MIC3 in b and e); and the protein is secreted but does not bind to the surface of transfected cells (exemplified by F128A in c and f). Adhesive proteins show a stronger signal in the pellet, resulting from their surface association, in contrast to nonadhesive R-MIC3 where a stronger signal is obtained in the supernatant.

    Techniques Used: Binding Assay, Transfection, Expressing, Fluorescence, Western Blot, Recombinant

    21) Product Images from "Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome"

    Article Title: Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2011/926258

    pMHV-68(BAC)v and MHV-68(loxP) displayed similar growth curves to wild-type MHV-68 in vitro . BHK-21 cells were infected with each virus at an MOI of 0.05. The viral titers in cells and the supernatants harvested at various times after infection were determined by plaque assays.
    Figure Legend Snippet: pMHV-68(BAC)v and MHV-68(loxP) displayed similar growth curves to wild-type MHV-68 in vitro . BHK-21 cells were infected with each virus at an MOI of 0.05. The viral titers in cells and the supernatants harvested at various times after infection were determined by plaque assays.

    Techniques Used: In Vitro, Infection

    The genomic structures of the MHV-68 recombinant viruses and the MHV-68 BAC plasmid. (a) Schematic diagram of the construction of the MHV-68 BAC virus. Shown is the left end of the viral genome encompassing terminal repeats and ORFs M1–M3. The BAC vector sequence and a puromycin expression cassette was recombined into the locus where the EGFP expression cassette was inserted in MHV-68(EGFP). MHV-68(BAC) was selected for the loss of the EGFP expression. The pMHV-68(BAC) plasmid was obtained by electroporating the circular genome of MHV-68(BAC) into DH10B cells. Following transfection of pMHV-68(BAC) into BHK-21 cells, the reconstituted virus, pMHV-68(BAC)v, was generated. The structures of pMHV-68(BAC) and pMHV-68(BAC)v are identical to MHV-68(BAC), except that pMHV-68(BAC) is circular with termini fused. To remove the BAC insert flanking by loxP sites, pMHV-68(BAC)v was co-transfected with a Cre recombinase expression plasmid and MHV-68(loxP) was isolated. The sites of Hind III are indicated with H and the ones of EcoR I were indicated with E. (b) Restriction enzyme analysis of the genome of MHV-68 recombinant viruses and the MHV-68 BAC plasmid. Each DNA sample was digested with either EcoR I or Hind III. Lane 1 is virion DNA of wild-type MHV-68; lane 2 is virion DNA of MHV-68(BAC); lane 3 is the plasmid DNA of pMHV-68(BAC); lane 4 is virion DNA of pMHV-68(BAC)v; lane 5 is virion DNA of MHV-68(loxP) and lane 6 is virion DNA of MHV-68(EGFP). The wild-type fragments which were eliminated due to the insertion of the BAC sequence were marked with asterisks. The fragments marked with a, b, d, and e, were generated because of the BAC insert. The fragments marked with c and f, resulted from the removal of BAC, 200 bp larger than the fragments indicated with asterisks. The fragments marked with F were from the fused termini of the circular pMHV-68(BAC). On the bottom of each agarose gel picture is the corresponding Southern blot using the probe spanning nt 51–6298. The laddering patterns in EcoR I-digested samples were due to various number of repeats on the linear virion DNA but not present in pMHV-68(BAC) (lane 3).
    Figure Legend Snippet: The genomic structures of the MHV-68 recombinant viruses and the MHV-68 BAC plasmid. (a) Schematic diagram of the construction of the MHV-68 BAC virus. Shown is the left end of the viral genome encompassing terminal repeats and ORFs M1–M3. The BAC vector sequence and a puromycin expression cassette was recombined into the locus where the EGFP expression cassette was inserted in MHV-68(EGFP). MHV-68(BAC) was selected for the loss of the EGFP expression. The pMHV-68(BAC) plasmid was obtained by electroporating the circular genome of MHV-68(BAC) into DH10B cells. Following transfection of pMHV-68(BAC) into BHK-21 cells, the reconstituted virus, pMHV-68(BAC)v, was generated. The structures of pMHV-68(BAC) and pMHV-68(BAC)v are identical to MHV-68(BAC), except that pMHV-68(BAC) is circular with termini fused. To remove the BAC insert flanking by loxP sites, pMHV-68(BAC)v was co-transfected with a Cre recombinase expression plasmid and MHV-68(loxP) was isolated. The sites of Hind III are indicated with H and the ones of EcoR I were indicated with E. (b) Restriction enzyme analysis of the genome of MHV-68 recombinant viruses and the MHV-68 BAC plasmid. Each DNA sample was digested with either EcoR I or Hind III. Lane 1 is virion DNA of wild-type MHV-68; lane 2 is virion DNA of MHV-68(BAC); lane 3 is the plasmid DNA of pMHV-68(BAC); lane 4 is virion DNA of pMHV-68(BAC)v; lane 5 is virion DNA of MHV-68(loxP) and lane 6 is virion DNA of MHV-68(EGFP). The wild-type fragments which were eliminated due to the insertion of the BAC sequence were marked with asterisks. The fragments marked with a, b, d, and e, were generated because of the BAC insert. The fragments marked with c and f, resulted from the removal of BAC, 200 bp larger than the fragments indicated with asterisks. The fragments marked with F were from the fused termini of the circular pMHV-68(BAC). On the bottom of each agarose gel picture is the corresponding Southern blot using the probe spanning nt 51–6298. The laddering patterns in EcoR I-digested samples were due to various number of repeats on the linear virion DNA but not present in pMHV-68(BAC) (lane 3).

    Techniques Used: Recombinant, BAC Assay, Plasmid Preparation, Sequencing, Expressing, Transfection, Generated, Isolation, Agarose Gel Electrophoresis, Southern Blot

    22) Product Images from "Ability of the Encephalitic Arbovirus Semliki Forest Virus To Cross the Blood-Brain Barrier Is Determined by the Charge of the E2 Glycoprotein"

    Article Title: Ability of the Encephalitic Arbovirus Semliki Forest Virus To Cross the Blood-Brain Barrier Is Determined by the Charge of the E2 Glycoprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.03645-14

    L10 is a heterogeneous stock containing viruses with three different plaque phenotypes. (A) Plaque assay of L10 on BHK-21 cells using an agar overlay. Small (S), medium (M), and large (L) plaques are labeled. (B) Three SNPs were identified (Piledriver) in E2 of the L10 high-throughput sequencing data. The nucleotide position in the reference genome and the occurrence of each of the four bases as a percentage are shown. Coverage was > 10,000. The plaque-purified small and large plaque viruses from the biological stock of L10 (5,000 PFU) were i.p. inoculated into groups ( n = 3 to 4) of 4- to 5-week-old mice, and at 1 and 2 days postinfection blood (C and D) and brain (E and F) virus titers were determined by plaque assay. PBSA was included as a control, and no virus was detected. Each symbol represents an individual mouse. The line depicts the mean titers for each group. The dotted line depicts the limit of detection of the assay. Significant difference is shown by an asterisk ( P
    Figure Legend Snippet: L10 is a heterogeneous stock containing viruses with three different plaque phenotypes. (A) Plaque assay of L10 on BHK-21 cells using an agar overlay. Small (S), medium (M), and large (L) plaques are labeled. (B) Three SNPs were identified (Piledriver) in E2 of the L10 high-throughput sequencing data. The nucleotide position in the reference genome and the occurrence of each of the four bases as a percentage are shown. Coverage was > 10,000. The plaque-purified small and large plaque viruses from the biological stock of L10 (5,000 PFU) were i.p. inoculated into groups ( n = 3 to 4) of 4- to 5-week-old mice, and at 1 and 2 days postinfection blood (C and D) and brain (E and F) virus titers were determined by plaque assay. PBSA was included as a control, and no virus was detected. Each symbol represents an individual mouse. The line depicts the mean titers for each group. The dotted line depicts the limit of detection of the assay. Significant difference is shown by an asterisk ( P

    Techniques Used: Plaque Assay, Labeling, Next-Generation Sequencing, Purification, Mouse Assay

    23) Product Images from "Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication"

    Article Title: Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.00469-16

    Replication-defective 3D insertions can be recovered using wild-type helper replicon. (A) Cartoon of the FMDV replicon, including a schematic of the 3D sequence with conserved functional regions highlighted. The seven conserved motifs are colored as follows: A, red; B, green; C, yellow; D, light blue; E, orange; and F, dark blue. The region shown to interact with dsRNA is shaded in black. Complementation experiments involved cotransfecting mCherry replicons containing replication-defective 3D insertions, indicated with arrows, with wild-type ptGFP helper replicons or yeast tRNA. BHK-21 cells were seeded into 24-well plates and allowed to adhere for 16 h. Cells were cotransfected with mCherry replicons bearing replication-defective 3D insertions, a motif C active-site 3D mutation (3D-GNN), or a wild-type (wt) mCherry replicon control, together with wild-type ptGFP helper replicon or yeast tRNA as a negative control. Expression of mCherry (B) and ptGFP (C) was monitored hourly over a 24-h period. Data are represented as mean positive cells per well ± SD at 8 h posttransfection. Significance between plus ptGFP and plus yeast tRNA control ( n = 3): *, P
    Figure Legend Snippet: Replication-defective 3D insertions can be recovered using wild-type helper replicon. (A) Cartoon of the FMDV replicon, including a schematic of the 3D sequence with conserved functional regions highlighted. The seven conserved motifs are colored as follows: A, red; B, green; C, yellow; D, light blue; E, orange; and F, dark blue. The region shown to interact with dsRNA is shaded in black. Complementation experiments involved cotransfecting mCherry replicons containing replication-defective 3D insertions, indicated with arrows, with wild-type ptGFP helper replicons or yeast tRNA. BHK-21 cells were seeded into 24-well plates and allowed to adhere for 16 h. Cells were cotransfected with mCherry replicons bearing replication-defective 3D insertions, a motif C active-site 3D mutation (3D-GNN), or a wild-type (wt) mCherry replicon control, together with wild-type ptGFP helper replicon or yeast tRNA as a negative control. Expression of mCherry (B) and ptGFP (C) was monitored hourly over a 24-h period. Data are represented as mean positive cells per well ± SD at 8 h posttransfection. Significance between plus ptGFP and plus yeast tRNA control ( n = 3): *, P

    Techniques Used: Sequencing, Functional Assay, Mutagenesis, Negative Control, Expressing

    Replication-defective mutations in 3D motifs A and C can be recovered using wild-type helper replicon. (A) Cartoon of the FMDV replicon bearing replication-defective polymerase point mutations and a simplified version of the mCherry helper replicon (see Fig. 1 for full schematic). The schematic of the 3D polymerase gene is shown with the conserved motifs A to F highlighted. Arrows indicate the locations of mutated residues. Complementation assays involved cotransfection of ptGFP replicons bearing 3D point mutations with wild-type mCherry helper replicon or yeast tRNA. BHK-21 cells seeded into 24-well plates were cotransfected with ptGFP replicons harboring replication-defective 3D point mutations or a wild-type control, together with wild-type mCherry helper replicon or yeast tRNA as a negative control. Expression of ptGFP (B and D) and mCherry (C and E) were monitored hourly over a 24-h period. Data show mean positive cells per well ± SD (B and C) or mean total fluorescent intensity per well ± SD (D and E) at 8 h posttransfection. Significance between plus mCherry and plus yeast tRNA control ( n = 2): *, P
    Figure Legend Snippet: Replication-defective mutations in 3D motifs A and C can be recovered using wild-type helper replicon. (A) Cartoon of the FMDV replicon bearing replication-defective polymerase point mutations and a simplified version of the mCherry helper replicon (see Fig. 1 for full schematic). The schematic of the 3D polymerase gene is shown with the conserved motifs A to F highlighted. Arrows indicate the locations of mutated residues. Complementation assays involved cotransfection of ptGFP replicons bearing 3D point mutations with wild-type mCherry helper replicon or yeast tRNA. BHK-21 cells seeded into 24-well plates were cotransfected with ptGFP replicons harboring replication-defective 3D point mutations or a wild-type control, together with wild-type mCherry helper replicon or yeast tRNA as a negative control. Expression of ptGFP (B and D) and mCherry (C and E) were monitored hourly over a 24-h period. Data show mean positive cells per well ± SD (B and C) or mean total fluorescent intensity per well ± SD (D and E) at 8 h posttransfection. Significance between plus mCherry and plus yeast tRNA control ( n = 2): *, P

    Techniques Used: Cotransfection, Negative Control, Expressing

    Expression of 3D alone is not sufficient for recovery of 3D mutations. (A) Western blot analysis of helper construct DNA transfected into BSR-T7 cells. Cells were seeded into 12-well plates, allowed to adhere for 16 h, and transfected with DNA constructs as indicated; protein was harvested at 12 h posttransfection. Lysates were analyzed by Western blotting for 3D and GAPDH expression. (B) For reciprocal complementation, BHK-21 cells seeded into 24-well plates were cotransfected with ptGFP replicons bearing the 3D-GNN replication-defective mutation or wild-type control, together with RNA transcripts expressing 3D alone or the entire 2A-3D polyprotein. Cotransfections were also performed with wild-type mCherry or mCherry-ΔS replicon transcripts and the equivalent 3D-GNN mutant constructs. Expression of ptGFP was monitored hourly over a 24-h period. Data represent mean positive cells per well ± SD at 8 h posttransfection. Significance compared to yeast tRNA control ( n = 3): *, P
    Figure Legend Snippet: Expression of 3D alone is not sufficient for recovery of 3D mutations. (A) Western blot analysis of helper construct DNA transfected into BSR-T7 cells. Cells were seeded into 12-well plates, allowed to adhere for 16 h, and transfected with DNA constructs as indicated; protein was harvested at 12 h posttransfection. Lysates were analyzed by Western blotting for 3D and GAPDH expression. (B) For reciprocal complementation, BHK-21 cells seeded into 24-well plates were cotransfected with ptGFP replicons bearing the 3D-GNN replication-defective mutation or wild-type control, together with RNA transcripts expressing 3D alone or the entire 2A-3D polyprotein. Cotransfections were also performed with wild-type mCherry or mCherry-ΔS replicon transcripts and the equivalent 3D-GNN mutant constructs. Expression of ptGFP was monitored hourly over a 24-h period. Data represent mean positive cells per well ± SD at 8 h posttransfection. Significance compared to yeast tRNA control ( n = 3): *, P

    Techniques Used: Expressing, Western Blot, Construct, Transfection, Mutagenesis

    Replication-defective mutations to the 5′ UTR improve recovery of 3D mutations. (A) Schematic of the mutant ptGFP FMDV replicon genome and a simplified version of the mCherry genomes used in the reciprocal complementation assay. BHK-21 cells seeded into 24-well plates were cotransfected with replication-defective 3D mutant ptGFP replicons or wild-type control, together with mCherry replicons containing either an entire S-fragment deletion (ΔS) (B and D) or a replication-defective CRE mutation (CRE A1G ) (C and E). Wild-type mCherry helper replicon and yeast tRNA cotransfections were included as controls. Expression of ptGFP (B and C) and mCherry (D and E) was monitored hourly over a 24-h period. Data represent mean positive cells per well ± SD at 8 h posttransfection. Significance of differences between treatment and yeast tRNA negative control ( n = 2): *, P
    Figure Legend Snippet: Replication-defective mutations to the 5′ UTR improve recovery of 3D mutations. (A) Schematic of the mutant ptGFP FMDV replicon genome and a simplified version of the mCherry genomes used in the reciprocal complementation assay. BHK-21 cells seeded into 24-well plates were cotransfected with replication-defective 3D mutant ptGFP replicons or wild-type control, together with mCherry replicons containing either an entire S-fragment deletion (ΔS) (B and D) or a replication-defective CRE mutation (CRE A1G ) (C and E). Wild-type mCherry helper replicon and yeast tRNA cotransfections were included as controls. Expression of ptGFP (B and C) and mCherry (D and E) was monitored hourly over a 24-h period. Data represent mean positive cells per well ± SD at 8 h posttransfection. Significance of differences between treatment and yeast tRNA negative control ( n = 2): *, P

    Techniques Used: Mutagenesis, Expressing, Negative Control

    Replication-defective 3D mutations do not affect RNA structure or 3D polyprotein processing. (A) Replicon RNAs containing codon-modified 3D were transfected into BHK-21 cells along with control replicons, and ptGFP expression was monitored hourly over 24 h. Data show mean numbers ± SD of ptGFP-positive cells per well at 8 h posttransfection ( n = 2). (B) Effect of nocodazole on complementation. BHK-21 cells treated with 5 μM nocodazole or left untreated (see Materials and Methods for details) were cotransfected with wild-type or 3D-GNN ptGFP replicon RNA together with helper mCherry replicon or yeast tRNA negative control. ptGFP expression was monitored for 24 h. Data show normalized numbers of ptGFP-positive cells per well ± standard errors of the means (SEM) at 8 h posttransfection. Significance between results for untreated samples and samples treated with 5 μM nocodazole ( n = 3) was determined: *, P
    Figure Legend Snippet: Replication-defective 3D mutations do not affect RNA structure or 3D polyprotein processing. (A) Replicon RNAs containing codon-modified 3D were transfected into BHK-21 cells along with control replicons, and ptGFP expression was monitored hourly over 24 h. Data show mean numbers ± SD of ptGFP-positive cells per well at 8 h posttransfection ( n = 2). (B) Effect of nocodazole on complementation. BHK-21 cells treated with 5 μM nocodazole or left untreated (see Materials and Methods for details) were cotransfected with wild-type or 3D-GNN ptGFP replicon RNA together with helper mCherry replicon or yeast tRNA negative control. ptGFP expression was monitored for 24 h. Data show normalized numbers of ptGFP-positive cells per well ± standard errors of the means (SEM) at 8 h posttransfection. Significance between results for untreated samples and samples treated with 5 μM nocodazole ( n = 3) was determined: *, P

    Techniques Used: Modification, Transfection, Expressing, Negative Control

    Cartoon of the FMDV replicons used in complementation assays. Schematics of the FMDV subgenomic replicons used in this study. Complementation experiments involved simultaneous cotransfection of BHK-21 cells with equal amounts of replication-defective “mutant” RNA with helper replicon or yeast tRNA as a negative control. Replication of mutant and helper constructs was subsequently monitored by differential expression of fluorescent reporter transgenes encoding ptGFP and mCherry. Expression of ptGFP (A and B) and mCherry (C and D) was monitored hourly over a 24-h period. (A and C) Data are typically represented as mean positive cells per well ± standard deviations (SD) at 8 h posttransfection. (B and D) Time course of fluorescence expression over 20 h from a subset of samples ( n = 3). The point mutation to the 3D active-site motif C (3D-GNN) has previously been shown to be recovered in trans by cotransfection with wild-type replicon.
    Figure Legend Snippet: Cartoon of the FMDV replicons used in complementation assays. Schematics of the FMDV subgenomic replicons used in this study. Complementation experiments involved simultaneous cotransfection of BHK-21 cells with equal amounts of replication-defective “mutant” RNA with helper replicon or yeast tRNA as a negative control. Replication of mutant and helper constructs was subsequently monitored by differential expression of fluorescent reporter transgenes encoding ptGFP and mCherry. Expression of ptGFP (A and B) and mCherry (C and D) was monitored hourly over a 24-h period. (A and C) Data are typically represented as mean positive cells per well ± standard deviations (SD) at 8 h posttransfection. (B and D) Time course of fluorescence expression over 20 h from a subset of samples ( n = 3). The point mutation to the 3D active-site motif C (3D-GNN) has previously been shown to be recovered in trans by cotransfection with wild-type replicon.

    Techniques Used: Cotransfection, Negative Control, Mutagenesis, Construct, Expressing, Fluorescence

    Wild-type helper 3D colocalizes with replication-defective mutant 3D. (A) Replicon RNAs containing C-terminal epitope labels were transfected into BHK-21 cells, and ptGFP expression was monitored hourly over a 24-h period. Data show mean numbers ± SD of ptGFP-positive cells per well at 8 h posttransfection ( n = 2). (B) BHK-21 cells were cotransfected with FLAG-tagged mutant 3D replicons and HA-tagged 3D helper replicons, fixed at 4 h posttransfection, and labeled for FLAG-Alexa Fluor 568 (pseudocolored green) and HA-Alexa Fluor 647 (pseudocolored red) as described in Materials and Methods. Cell nuclei were counterstained with DAPI (blue). Images were captured on a Zeiss LSM-880 confocal microscope with Airyscan. Zoomed images represent the boxed areas on the merged images (bar, 20 μM). (C) Quantification of FLAG versus HA colocalization by confocal immunofluorescence. Fluorescent intensity of 3D-FLAG (green) versus 3D-HA (red) was plotted as two-dimensional (2D) scatterplots, and the number of costained pixels was determined by a 4-area gating set using controls. Data points show the percentages of coexpressing pixels from 8 randomly selected cells after cotransfection. Horizontal lines represents mean values ± SEM.
    Figure Legend Snippet: Wild-type helper 3D colocalizes with replication-defective mutant 3D. (A) Replicon RNAs containing C-terminal epitope labels were transfected into BHK-21 cells, and ptGFP expression was monitored hourly over a 24-h period. Data show mean numbers ± SD of ptGFP-positive cells per well at 8 h posttransfection ( n = 2). (B) BHK-21 cells were cotransfected with FLAG-tagged mutant 3D replicons and HA-tagged 3D helper replicons, fixed at 4 h posttransfection, and labeled for FLAG-Alexa Fluor 568 (pseudocolored green) and HA-Alexa Fluor 647 (pseudocolored red) as described in Materials and Methods. Cell nuclei were counterstained with DAPI (blue). Images were captured on a Zeiss LSM-880 confocal microscope with Airyscan. Zoomed images represent the boxed areas on the merged images (bar, 20 μM). (C) Quantification of FLAG versus HA colocalization by confocal immunofluorescence. Fluorescent intensity of 3D-FLAG (green) versus 3D-HA (red) was plotted as two-dimensional (2D) scatterplots, and the number of costained pixels was determined by a 4-area gating set using controls. Data points show the percentages of coexpressing pixels from 8 randomly selected cells after cotransfection. Horizontal lines represents mean values ± SEM.

    Techniques Used: Mutagenesis, Transfection, Expressing, Labeling, Microscopy, Immunofluorescence, Cotransfection

    24) Product Images from "Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice"

    Article Title: Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173352

    p24 Gag production by: ( A ) DNA vaccines in HEK cells. HEK cells were transfected with 4μg of DNA-Gag M or DNA-Gag N and samples taken at the indicated time points. ( B ) Recombinant MVA in BHK-21 and HeLa cell lines. Permissive (BHK-21) and non-permissive (HeLa) cell lines were infected at an MOI of 0.1 with MVA-Gag M and samples taken at the indicated time points. Gag p24 was detected using an ELISA assay.
    Figure Legend Snippet: p24 Gag production by: ( A ) DNA vaccines in HEK cells. HEK cells were transfected with 4μg of DNA-Gag M or DNA-Gag N and samples taken at the indicated time points. ( B ) Recombinant MVA in BHK-21 and HeLa cell lines. Permissive (BHK-21) and non-permissive (HeLa) cell lines were infected at an MOI of 0.1 with MVA-Gag M and samples taken at the indicated time points. Gag p24 was detected using an ELISA assay.

    Techniques Used: Transfection, Recombinant, Infection, Enzyme-linked Immunosorbent Assay

    Electron micrographs of VLP formation in HEK293 cells transfected with (A) DNA-Gag M , (B) DNA-Gag N and (C) BHK-21 cells infected with MVA-Gag M 30 hours post transfection or infection. HEK293 cells were transfected with 4μg of the DNA vaccines and BHK-21 cells were infected with MVA-Gag M at an MOI of 1. The VLPs are indicated by arrows. The scale bars represent 200nm.
    Figure Legend Snippet: Electron micrographs of VLP formation in HEK293 cells transfected with (A) DNA-Gag M , (B) DNA-Gag N and (C) BHK-21 cells infected with MVA-Gag M 30 hours post transfection or infection. HEK293 cells were transfected with 4μg of the DNA vaccines and BHK-21 cells were infected with MVA-Gag M at an MOI of 1. The VLPs are indicated by arrows. The scale bars represent 200nm.

    Techniques Used: Transfection, Infection

    25) Product Images from "Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies"

    Article Title: Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0856-8

    Inhibitory effect of anti-E mAbs on DV1 infection of C6/36, Vero, and BHK-21 cells. C6/36, Vero, and BHK21 cells were infected with DV1 in the absence or presence of DV1-E1 (dark column) or DV1-E2 (gray column). Mouse IgG (w/o mAb; white column) was used as the control for evaluation the inhibitory effects of two anti-E mAbs. After incubation at 37 °C for 48 h, the cells were harvested and the viral titers were determined by FFA. The data are the means and standard deviations from three independent experiments
    Figure Legend Snippet: Inhibitory effect of anti-E mAbs on DV1 infection of C6/36, Vero, and BHK-21 cells. C6/36, Vero, and BHK21 cells were infected with DV1 in the absence or presence of DV1-E1 (dark column) or DV1-E2 (gray column). Mouse IgG (w/o mAb; white column) was used as the control for evaluation the inhibitory effects of two anti-E mAbs. After incubation at 37 °C for 48 h, the cells were harvested and the viral titers were determined by FFA. The data are the means and standard deviations from three independent experiments

    Techniques Used: Infection, Incubation

    26) Product Images from "Porcine epidemic diarrhea virus S1 protein is the critical inducer of apoptosis"

    Article Title: Porcine epidemic diarrhea virus S1 protein is the critical inducer of apoptosis

    Journal: Virology Journal

    doi: 10.1186/s12985-018-1078-4

    TGEV, IBV, CoCoV, SARS and MERS CoV Spike Protein S1 Could also Induce Apoptosis in Vero-E6 cells. TGEV, IBV, CCoV, SARS and MERS S1 genes were cloned or synthesized in fusion with EGFP gene and were transfected into VERO-E6, 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. ( a ) TGEV, IBV, CCoV, SARS and MERS S1 genes were correctly constructed in fusion with EGFP. The empty vector pEGFP-N1 and the recombinant plasmids were all transfected into confluent BHK-21 monolayer cells separately. 24 h later, bright green fluorescence was observed from the all the recombinant plasmids transfected cells. ( b ) The results showed that TGEV, IBV, CCoV, SARS and MERS S1 Protein Could Induce Apoptosis in Vero-E6 cells. ( c )Percentages of annexin-V-PE positive cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p
    Figure Legend Snippet: TGEV, IBV, CoCoV, SARS and MERS CoV Spike Protein S1 Could also Induce Apoptosis in Vero-E6 cells. TGEV, IBV, CCoV, SARS and MERS S1 genes were cloned or synthesized in fusion with EGFP gene and were transfected into VERO-E6, 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. ( a ) TGEV, IBV, CCoV, SARS and MERS S1 genes were correctly constructed in fusion with EGFP. The empty vector pEGFP-N1 and the recombinant plasmids were all transfected into confluent BHK-21 monolayer cells separately. 24 h later, bright green fluorescence was observed from the all the recombinant plasmids transfected cells. ( b ) The results showed that TGEV, IBV, CCoV, SARS and MERS S1 Protein Could Induce Apoptosis in Vero-E6 cells. ( c )Percentages of annexin-V-PE positive cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p

    Techniques Used: Clone Assay, Synthesized, Transfection, Fluorescence, FACS, Construct, Plasmid Preparation, Recombinant

    PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in VERO-E6 even in BHK-21 cells. pEGFP-N1 and the recombinant plasmids expressing CV777 or BJ2011 S1 protein were transfected into confluent Vero-E6 or BHK-21 monolayer cells respectively. 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. ( a ) PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in Vero-E6 even in BHK-21 cells. ( b ) Percentages of annexin-V-PE positive Vero-E6 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. ( c ) Percentages of annexin-V-PE positive BHK-21 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p
    Figure Legend Snippet: PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in VERO-E6 even in BHK-21 cells. pEGFP-N1 and the recombinant plasmids expressing CV777 or BJ2011 S1 protein were transfected into confluent Vero-E6 or BHK-21 monolayer cells respectively. 48 h later, the cells were harvested and analyzed by Fluorescence-activated cells sorter (FACS), GFP-positive cells were gated for apoptosis analysis. ( a ) PEDV CV777 and new-emerging strain BJ2011 S1 protein Could also induce apoptosis in Vero-E6 even in BHK-21 cells. ( b ) Percentages of annexin-V-PE positive Vero-E6 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. ( c ) Percentages of annexin-V-PE positive BHK-21 Cells from gated cells are shown with graphs. Results are representative of three independent experiments. (*, p

    Techniques Used: Recombinant, Expressing, Transfection, Fluorescence, FACS

    27) Product Images from "Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic"

    Article Title: Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.17.9278-9286.2003

    Analysis of plaque morphology and virus replication in BHK-21 cells. (A) The originally designed SIN-83S virus, stock prepared from plaque-purified virus, which formed large plaques, designated SIN-83 lp, and SIN-83 virus containing the S 795 →T mutation in nsP2 found in SIN-83 lp were titrated on monolayers of BHK-21 cells. Plaques were allowed to develop for 36 h prior to fixation and staining. (B) BHK-21 cells were infected with SIN-83S, SIN-83 lp, and SIN-83 viruses at an MOI of 5 PFU/cell. At the indicated times, media were replaced, and virus titers were determined as described in the Materials and Methods. The experiment was performed twice with very similar results.
    Figure Legend Snippet: Analysis of plaque morphology and virus replication in BHK-21 cells. (A) The originally designed SIN-83S virus, stock prepared from plaque-purified virus, which formed large plaques, designated SIN-83 lp, and SIN-83 virus containing the S 795 →T mutation in nsP2 found in SIN-83 lp were titrated on monolayers of BHK-21 cells. Plaques were allowed to develop for 36 h prior to fixation and staining. (B) BHK-21 cells were infected with SIN-83S, SIN-83 lp, and SIN-83 viruses at an MOI of 5 PFU/cell. At the indicated times, media were replaced, and virus titers were determined as described in the Materials and Methods. The experiment was performed twice with very similar results.

    Techniques Used: Purification, Mutagenesis, Staining, Infection

    Analysis of protein synthesis and replication of virus-specific RNAs in infected cells. BHK-21 cells were infected with VEE TC-83, SIN-83, and SIN Toto 1101 viruses at an MOI of 10 PFU/cell. (A) At 12 h postinfection, the cells were labeled with [ 35 S]methionine and analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel as described in Materials and Methods. The additional 35 S-labeled protein bands in the lysate of SIN-83-infected cells are marked by stars. (B) At 2 h postinfection, medium in the wells was replaced by 1 ml of Alpha MEM supplemented with 10% FBS, dactinomycin (1 μg/ml), and [ 3 H]uridine (20 μCi/ml). After 3 h of incubation at 37°C, RNAs were isolated from the cells and analyzed by agarose gel electrophoresis as described in Materials and Methods.
    Figure Legend Snippet: Analysis of protein synthesis and replication of virus-specific RNAs in infected cells. BHK-21 cells were infected with VEE TC-83, SIN-83, and SIN Toto 1101 viruses at an MOI of 10 PFU/cell. (A) At 12 h postinfection, the cells were labeled with [ 35 S]methionine and analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel as described in Materials and Methods. The additional 35 S-labeled protein bands in the lysate of SIN-83-infected cells are marked by stars. (B) At 2 h postinfection, medium in the wells was replaced by 1 ml of Alpha MEM supplemented with 10% FBS, dactinomycin (1 μg/ml), and [ 3 H]uridine (20 μCi/ml). After 3 h of incubation at 37°C, RNAs were isolated from the cells and analyzed by agarose gel electrophoresis as described in Materials and Methods.

    Techniques Used: Infection, Labeling, Incubation, Isolation, Agarose Gel Electrophoresis

    28) Product Images from "RNA Interference-Mediated In Vivo Silencing of Fas Ligand as a Strategy for the Enhancement of DNA Vaccine Potency"

    Article Title: RNA Interference-Mediated In Vivo Silencing of Fas Ligand as a Strategy for the Enhancement of DNA Vaccine Potency

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2007.059

    Detection of siRNA-mediated gene knockdown in vitro , using a bioluminescence imaging system. BHK-21 cells were transfected with 0.01 μg of pcDNA3-Luc and DNA encoding luciferase-specific shRNA (pRS-Luc) in amounts of 0.00, 0.01, 0.03, 0.09, 0.27, and 0.81 μg at 24- and 48-hr time points. Cells transfected with pcDNA3-Luc and pRS vector alone (0.00 μg of pRS-Luc) were used as the control. ( A ) Luminescent images of transfected cells at 24 hr ( left ) and 48 hr ( right ) posttransfection. ( B ) Line graph depicting the luminescence intensities over time of cells transfected with 0.01 μg of pcDNA3-Luc and various amounts of pRS-Luc. Results are expressed as the mean luminescence intensity and SD. Note : Luciferase expression in shRNA-transfected cells was suppressed in a dose-dependent manner and was still substantially reduced after 120 hr.
    Figure Legend Snippet: Detection of siRNA-mediated gene knockdown in vitro , using a bioluminescence imaging system. BHK-21 cells were transfected with 0.01 μg of pcDNA3-Luc and DNA encoding luciferase-specific shRNA (pRS-Luc) in amounts of 0.00, 0.01, 0.03, 0.09, 0.27, and 0.81 μg at 24- and 48-hr time points. Cells transfected with pcDNA3-Luc and pRS vector alone (0.00 μg of pRS-Luc) were used as the control. ( A ) Luminescent images of transfected cells at 24 hr ( left ) and 48 hr ( right ) posttransfection. ( B ) Line graph depicting the luminescence intensities over time of cells transfected with 0.01 μg of pcDNA3-Luc and various amounts of pRS-Luc. Results are expressed as the mean luminescence intensity and SD. Note : Luciferase expression in shRNA-transfected cells was suppressed in a dose-dependent manner and was still substantially reduced after 120 hr.

    Techniques Used: In Vitro, Imaging, Transfection, Luciferase, shRNA, Plasmid Preparation, Expressing

    Flow cytometric analysis demonstrating expression of FasL in BHK-21 cells in vitro. BHK-21 cells were transfected with FasL DNA and DNA encoding either nonspecific shRNA or FasL-specific shRNA. Cells were then stained with FasL-specific antibody or an isotype control (data not shown) and subjected to flow cytometric analysis to characterize FasL expression. Cells that did not receive FasL transfection were measured as a negative control. ( A ) Flow cytometric data representative of three separate analyses. The number shown in each histogram provides an indication of the percentage of gated cells staining positive for FasL. ( B ) Bar graph depicting relative FasL expression. Data are shown as means and SD. p Values were calculated by Student t test (* p
    Figure Legend Snippet: Flow cytometric analysis demonstrating expression of FasL in BHK-21 cells in vitro. BHK-21 cells were transfected with FasL DNA and DNA encoding either nonspecific shRNA or FasL-specific shRNA. Cells were then stained with FasL-specific antibody or an isotype control (data not shown) and subjected to flow cytometric analysis to characterize FasL expression. Cells that did not receive FasL transfection were measured as a negative control. ( A ) Flow cytometric data representative of three separate analyses. The number shown in each histogram provides an indication of the percentage of gated cells staining positive for FasL. ( B ) Bar graph depicting relative FasL expression. Data are shown as means and SD. p Values were calculated by Student t test (* p

    Techniques Used: Flow Cytometry, Expressing, In Vitro, Transfection, shRNA, Staining, Negative Control

    29) Product Images from "RNA Interference-Mediated In Vivo Silencing of Fas Ligand as a Strategy for the Enhancement of DNA Vaccine Potency"

    Article Title: RNA Interference-Mediated In Vivo Silencing of Fas Ligand as a Strategy for the Enhancement of DNA Vaccine Potency

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2007.059

    Detection of siRNA-mediated gene knockdown in vitro , using a bioluminescence imaging system. BHK-21 cells were transfected with 0.01 μg of pcDNA3-Luc and DNA encoding luciferase-specific shRNA (pRS-Luc) in amounts of 0.00, 0.01, 0.03, 0.09, 0.27, and 0.81 μg at 24- and 48-hr time points. Cells transfected with pcDNA3-Luc and pRS vector alone (0.00 μg of pRS-Luc) were used as the control. ( A ) Luminescent images of transfected cells at 24 hr ( left ) and 48 hr ( right ) posttransfection. ( B ) Line graph depicting the luminescence intensities over time of cells transfected with 0.01 μg of pcDNA3-Luc and various amounts of pRS-Luc. Results are expressed as the mean luminescence intensity and SD. Note : Luciferase expression in shRNA-transfected cells was suppressed in a dose-dependent manner and was still substantially reduced after 120 hr.
    Figure Legend Snippet: Detection of siRNA-mediated gene knockdown in vitro , using a bioluminescence imaging system. BHK-21 cells were transfected with 0.01 μg of pcDNA3-Luc and DNA encoding luciferase-specific shRNA (pRS-Luc) in amounts of 0.00, 0.01, 0.03, 0.09, 0.27, and 0.81 μg at 24- and 48-hr time points. Cells transfected with pcDNA3-Luc and pRS vector alone (0.00 μg of pRS-Luc) were used as the control. ( A ) Luminescent images of transfected cells at 24 hr ( left ) and 48 hr ( right ) posttransfection. ( B ) Line graph depicting the luminescence intensities over time of cells transfected with 0.01 μg of pcDNA3-Luc and various amounts of pRS-Luc. Results are expressed as the mean luminescence intensity and SD. Note : Luciferase expression in shRNA-transfected cells was suppressed in a dose-dependent manner and was still substantially reduced after 120 hr.

    Techniques Used: In Vitro, Imaging, Transfection, Luciferase, shRNA, Plasmid Preparation, Expressing

    Flow cytometric analysis demonstrating expression of FasL in BHK-21 cells in vitro. BHK-21 cells were transfected with FasL DNA and DNA encoding either nonspecific shRNA or FasL-specific shRNA. Cells were then stained with FasL-specific antibody or an isotype control (data not shown) and subjected to flow cytometric analysis to characterize FasL expression. Cells that did not receive FasL transfection were measured as a negative control. ( A ) Flow cytometric data representative of three separate analyses. The number shown in each histogram provides an indication of the percentage of gated cells staining positive for FasL. ( B ) Bar graph depicting relative FasL expression. Data are shown as means and SD. p Values were calculated by Student t test (* p
    Figure Legend Snippet: Flow cytometric analysis demonstrating expression of FasL in BHK-21 cells in vitro. BHK-21 cells were transfected with FasL DNA and DNA encoding either nonspecific shRNA or FasL-specific shRNA. Cells were then stained with FasL-specific antibody or an isotype control (data not shown) and subjected to flow cytometric analysis to characterize FasL expression. Cells that did not receive FasL transfection were measured as a negative control. ( A ) Flow cytometric data representative of three separate analyses. The number shown in each histogram provides an indication of the percentage of gated cells staining positive for FasL. ( B ) Bar graph depicting relative FasL expression. Data are shown as means and SD. p Values were calculated by Student t test (* p

    Techniques Used: Flow Cytometry, Expressing, In Vitro, Transfection, shRNA, Staining, Negative Control

    30) Product Images from "Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿"

    Article Title: Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿

    Journal:

    doi: 10.1128/JVI.01017-07

    Selection of adaptive mutations in BHK-21 cells.
    Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

    Techniques Used: Selection

    Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,
    Figure Legend Snippet: Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,

    Techniques Used: Selection, Cell Culture

    Selection of adaptive mutations in BHK-21 cells.
    Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

    Techniques Used: Selection

    31) Product Images from "Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿"

    Article Title: Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿

    Journal:

    doi: 10.1128/JVI.01017-07

    Selection of adaptive mutations in BHK-21 cells.
    Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

    Techniques Used: Selection

    Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,
    Figure Legend Snippet: Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,

    Techniques Used: Selection, Cell Culture

    Selection of adaptive mutations in BHK-21 cells.
    Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

    Techniques Used: Selection

    32) Product Images from "Role for the Phosphatidylinositol 3-Kinase-Akt-TOR Pathway during Sindbis Virus Replication in Arthropods"

    Article Title: Role for the Phosphatidylinositol 3-Kinase-Akt-TOR Pathway during Sindbis Virus Replication in Arthropods

    Journal: Journal of Virology

    doi: 10.1128/JVI.06625-11

    Differential 4E-BP1 phosphorylation post-SINV infection in mosquito and vertebrate cells. (A) Western blot with anti-p4E-BP1 antibody and anti-4E-BP1 antibody on whole-cell extracts of C6/36 cells. Cells were either mock infected or infected with SINV for 6 h before being harvested for analysis. (B) Western blot with anti-p4E-BP1 antibody on whole-cell extracts of C6/36 cells. Cells were infected or mock infected in the presence and absence of the TOR inhibitor. (C and D) Western blot with anti-p4E-BP1 and anti-4E-BP1 antibody on whole-cell extracts of BHK-21 (C) and 293 (D) cells. Cells were mock infected, infected with SINV for 6 h, and infected with SINV for 6 h and treated with the TOR inhibitor before being harvested for analysis. Relative quantities of p4E-BP1 standardized to the total 4E-BP1 signal are shown. β-Actin was used as the loading control.
    Figure Legend Snippet: Differential 4E-BP1 phosphorylation post-SINV infection in mosquito and vertebrate cells. (A) Western blot with anti-p4E-BP1 antibody and anti-4E-BP1 antibody on whole-cell extracts of C6/36 cells. Cells were either mock infected or infected with SINV for 6 h before being harvested for analysis. (B) Western blot with anti-p4E-BP1 antibody on whole-cell extracts of C6/36 cells. Cells were infected or mock infected in the presence and absence of the TOR inhibitor. (C and D) Western blot with anti-p4E-BP1 and anti-4E-BP1 antibody on whole-cell extracts of BHK-21 (C) and 293 (D) cells. Cells were mock infected, infected with SINV for 6 h, and infected with SINV for 6 h and treated with the TOR inhibitor before being harvested for analysis. Relative quantities of p4E-BP1 standardized to the total 4E-BP1 signal are shown. β-Actin was used as the loading control.

    Techniques Used: Infection, Western Blot

    33) Product Images from "Adaptation of Saffold Virus 2 for High-Titer Growth in Mammalian Cells ▿"

    Article Title: Adaptation of Saffold Virus 2 for High-Titer Growth in Mammalian Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00265-11

    SAFV-2 plaque morphology, cell association, and sialic acid binding. (A and B) Plaques produced in BHK-21 cells by SAFV-2 strain GDVII after 3 days and BeAn after 4 days of incubation, respectively. (C) SAFV-2 plaques in HeLa cells after 4 days of incubation.
    Figure Legend Snippet: SAFV-2 plaque morphology, cell association, and sialic acid binding. (A and B) Plaques produced in BHK-21 cells by SAFV-2 strain GDVII after 3 days and BeAn after 4 days of incubation, respectively. (C) SAFV-2 plaques in HeLa cells after 4 days of incubation.

    Techniques Used: Binding Assay, Produced, Incubation

    34) Product Images from "Activation of the Alphavirus Spike Protein Is Suppressed by Bound E3 ▿"

    Article Title: Activation of the Alphavirus Spike Protein Is Suppressed by Bound E3 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00130-11

    E3 binding to SFV wt . A sparse, an almost confluent, and a dense BHK-21 cell culture were infected with SFV wt and incubated for 4 h before being labeled with [ 35 S]Cys for 20 h. After this, the media with the released virus were collected. The pH values
    Figure Legend Snippet: E3 binding to SFV wt . A sparse, an almost confluent, and a dense BHK-21 cell culture were infected with SFV wt and incubated for 4 h before being labeled with [ 35 S]Cys for 20 h. After this, the media with the released virus were collected. The pH values

    Techniques Used: Binding Assay, Cell Culture, Infection, Incubation, Labeling

    PK-cleaved E3 is released from SFV SQL -infected cells in a pH-dependent way. BHK-21 cell cultures were infected with SFV SQL and SFV wt . The SFV SQL -infected cells were treated with 5 to 50 μg/ml PK in pH 6.1 buffer for 10 min at 20°C. After
    Figure Legend Snippet: PK-cleaved E3 is released from SFV SQL -infected cells in a pH-dependent way. BHK-21 cell cultures were infected with SFV SQL and SFV wt . The SFV SQL -infected cells were treated with 5 to 50 μg/ml PK in pH 6.1 buffer for 10 min at 20°C. After

    Techniques Used: Infection

    E3 association with the spike suppresses spike-mediated cell-cell fusion. (A to F) One set of SFV wt - and SFV SQL -infected BHK-21 cells was washed with pH 7.4, 5.5, or 4.5 buffer and then further incubated in pH 7.4 medium for 2 h. After this, the cultures
    Figure Legend Snippet: E3 association with the spike suppresses spike-mediated cell-cell fusion. (A to F) One set of SFV wt - and SFV SQL -infected BHK-21 cells was washed with pH 7.4, 5.5, or 4.5 buffer and then further incubated in pH 7.4 medium for 2 h. After this, the cultures

    Techniques Used: Infection, Incubation

    35) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.214189

    Mutagenesis of cavities A and B using an infectious cDNA clone of DENV-2. A and C , analysis of viral E protein synthesis and plaque morphology for cavity A and B mutant viruses, respectively. BHK-21 cells were transfected with WT and mutant genome-length
    Figure Legend Snippet: Mutagenesis of cavities A and B using an infectious cDNA clone of DENV-2. A and C , analysis of viral E protein synthesis and plaque morphology for cavity A and B mutant viruses, respectively. BHK-21 cells were transfected with WT and mutant genome-length

    Techniques Used: Mutagenesis, Transfection

    Site-directed mutagenesis of Trp-859 in cavity B. BHK-21 cells were transfected with WT and mutant genome-length RNAs (10 μg) and analyzed for viral E protein synthesis by IFA at 72 h post-transfection. The engineered mutant nucleotides are underlined
    Figure Legend Snippet: Site-directed mutagenesis of Trp-859 in cavity B. BHK-21 cells were transfected with WT and mutant genome-length RNAs (10 μg) and analyzed for viral E protein synthesis by IFA at 72 h post-transfection. The engineered mutant nucleotides are underlined

    Techniques Used: Mutagenesis, Transfection, Immunofluorescence

    36) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.214189

    Mutagenesis of cavities A and B using an infectious cDNA clone of DENV-2. A and C , analysis of viral E protein synthesis and plaque morphology for cavity A and B mutant viruses, respectively. BHK-21 cells were transfected with WT and mutant genome-length
    Figure Legend Snippet: Mutagenesis of cavities A and B using an infectious cDNA clone of DENV-2. A and C , analysis of viral E protein synthesis and plaque morphology for cavity A and B mutant viruses, respectively. BHK-21 cells were transfected with WT and mutant genome-length

    Techniques Used: Mutagenesis, Transfection

    Site-directed mutagenesis of Trp-859 in cavity B. BHK-21 cells were transfected with WT and mutant genome-length RNAs (10 μg) and analyzed for viral E protein synthesis by IFA at 72 h post-transfection. The engineered mutant nucleotides are underlined
    Figure Legend Snippet: Site-directed mutagenesis of Trp-859 in cavity B. BHK-21 cells were transfected with WT and mutant genome-length RNAs (10 μg) and analyzed for viral E protein synthesis by IFA at 72 h post-transfection. The engineered mutant nucleotides are underlined

    Techniques Used: Mutagenesis, Transfection, Immunofluorescence

    37) Product Images from "Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection ▿"

    Article Title: Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02058-10

    Viral and host protein syntheses are affected by the amino-terminal mutations. BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 3, 7, and 11 h (A) or 7, 17, and 23 h (B). After 1 h of labeling, cells were
    Figure Legend Snippet: Viral and host protein syntheses are affected by the amino-terminal mutations. BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 3, 7, and 11 h (A) or 7, 17, and 23 h (B). After 1 h of labeling, cells were

    Techniques Used: Infection, Labeling

    nsP4 amino-terminal domain mutations affect viral growth. (A) Plaque morphology of select mutants. BHK-21 cells were infected with virus stocks rescued from transfections and stained with crystal violet at 40 hpi. (B) Growth kinetics in vertebrate cells.
    Figure Legend Snippet: nsP4 amino-terminal domain mutations affect viral growth. (A) Plaque morphology of select mutants. BHK-21 cells were infected with virus stocks rescued from transfections and stained with crystal violet at 40 hpi. (B) Growth kinetics in vertebrate cells.

    Techniques Used: Infection, Transfection, Staining

    Second-site changes restore viral protein expression and host translation shutoff. (A) BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 7 hpi. After 1 h of labeling, cells were harvested and postnuclear
    Figure Legend Snippet: Second-site changes restore viral protein expression and host translation shutoff. (A) BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 7 hpi. After 1 h of labeling, cells were harvested and postnuclear

    Techniques Used: Expressing, Infection, Labeling

    Select amino-terminal mutations each affect the synthesis of different viral RNAs. BHK-21 cells were infected at an MOI of 10 PFU/cell, treated with ActD, and labeled with [ 3 H]uridine from 4 to 6 hpi (A) or 8 to 10 hpi (B), after which RNA was harvested.
    Figure Legend Snippet: Select amino-terminal mutations each affect the synthesis of different viral RNAs. BHK-21 cells were infected at an MOI of 10 PFU/cell, treated with ActD, and labeled with [ 3 H]uridine from 4 to 6 hpi (A) or 8 to 10 hpi (B), after which RNA was harvested.

    Techniques Used: Infection, Labeling

    38) Product Images from "Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection ▿"

    Article Title: Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02058-10

    Viral and host protein syntheses are affected by the amino-terminal mutations. BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 3, 7, and 11 h (A) or 7, 17, and 23 h (B). After 1 h of labeling, cells were
    Figure Legend Snippet: Viral and host protein syntheses are affected by the amino-terminal mutations. BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 3, 7, and 11 h (A) or 7, 17, and 23 h (B). After 1 h of labeling, cells were

    Techniques Used: Infection, Labeling

    nsP4 amino-terminal domain mutations affect viral growth. (A) Plaque morphology of select mutants. BHK-21 cells were infected with virus stocks rescued from transfections and stained with crystal violet at 40 hpi. (B) Growth kinetics in vertebrate cells.
    Figure Legend Snippet: nsP4 amino-terminal domain mutations affect viral growth. (A) Plaque morphology of select mutants. BHK-21 cells were infected with virus stocks rescued from transfections and stained with crystal violet at 40 hpi. (B) Growth kinetics in vertebrate cells.

    Techniques Used: Infection, Transfection, Staining

    Second-site changes restore viral protein expression and host translation shutoff. (A) BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 7 hpi. After 1 h of labeling, cells were harvested and postnuclear
    Figure Legend Snippet: Second-site changes restore viral protein expression and host translation shutoff. (A) BHK-21 cells infected at an MOI of 10 PFU/cell were labeled with [ 35 S]cysteine and methionine at 7 hpi. After 1 h of labeling, cells were harvested and postnuclear

    Techniques Used: Expressing, Infection, Labeling

    Select amino-terminal mutations each affect the synthesis of different viral RNAs. BHK-21 cells were infected at an MOI of 10 PFU/cell, treated with ActD, and labeled with [ 3 H]uridine from 4 to 6 hpi (A) or 8 to 10 hpi (B), after which RNA was harvested.
    Figure Legend Snippet: Select amino-terminal mutations each affect the synthesis of different viral RNAs. BHK-21 cells were infected at an MOI of 10 PFU/cell, treated with ActD, and labeled with [ 3 H]uridine from 4 to 6 hpi (A) or 8 to 10 hpi (B), after which RNA was harvested.

    Techniques Used: Infection, Labeling

    39) Product Images from "The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner"

    Article Title: The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner

    Journal: Journal of Virology

    doi: 10.1128/JVI.01805-12

    Infectivities of SINV and RRV chimeras. (A and B) Production of infectious virus by SINV and RRV chimeras, respectively. BHK-21 cells were electroporated with in vitro -transcribed wild-type or chimeric viral RNA. At the indicated time points, the medium
    Figure Legend Snippet: Infectivities of SINV and RRV chimeras. (A and B) Production of infectious virus by SINV and RRV chimeras, respectively. BHK-21 cells were electroporated with in vitro -transcribed wild-type or chimeric viral RNA. At the indicated time points, the medium

    Techniques Used: In Vitro

    Characterization of RRV E3 in SINV pocket mutants. (A) Infectivities of RRV E3 in SINV pocket mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or chimeric viral RNA. At 25 h postelectroporation, the medium was harvested and
    Figure Legend Snippet: Characterization of RRV E3 in SINV pocket mutants. (A) Infectivities of RRV E3 in SINV pocket mutants. BHK-21 cells were electroporated with in vitro -transcribed wild-type or chimeric viral RNA. At 25 h postelectroporation, the medium was harvested and

    Techniques Used: In Vitro

    Intracellular and cell surface spike glycoprotein expression in cells infected with RRV chimeras. (A) Pulse-chase analysis probing for intracellular spike glycoprotein expression. BHK-21 cells were electroporated with in vitro -transcribed wild-type or
    Figure Legend Snippet: Intracellular and cell surface spike glycoprotein expression in cells infected with RRV chimeras. (A) Pulse-chase analysis probing for intracellular spike glycoprotein expression. BHK-21 cells were electroporated with in vitro -transcribed wild-type or

    Techniques Used: Expressing, Infection, Pulse Chase, In Vitro

    40) Product Images from "Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence"

    Article Title: Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

    Journal: Journal of Virology

    doi:

    Viral RNA synthesis during early infection measured by scintillation counting. BHK-21 monolayers (solid bars) and 72-h-cultured rat cerebellar granule neurons (open bars) were infected with parental strains and recombinants at 20 PFU per cell. [ 3 H]uridine was added at 3 h postinfection, and cells were lysed 3 h later in 10% SDS. Actinomycin D was used to inhibit cellular RNA synthesis. Control cells (C) were uninfected. The star indicates a value below 500 cpm.
    Figure Legend Snippet: Viral RNA synthesis during early infection measured by scintillation counting. BHK-21 monolayers (solid bars) and 72-h-cultured rat cerebellar granule neurons (open bars) were infected with parental strains and recombinants at 20 PFU per cell. [ 3 H]uridine was added at 3 h postinfection, and cells were lysed 3 h later in 10% SDS. Actinomycin D was used to inhibit cellular RNA synthesis. Control cells (C) were uninfected. The star indicates a value below 500 cpm.

    Techniques Used: Infection, Cell Culture

    Related Articles

    Centrifugation:

    Article Title: Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice
    Article Snippet: .. The tissue homogenate was then clarified by centrifugation and assayed for infectious virus by plaque assay on BHK-21 cells (ATCC CRL 8544) as previously described ( ). ..

    Plaque Assay:

    Article Title: Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice
    Article Snippet: .. The tissue homogenate was then clarified by centrifugation and assayed for infectious virus by plaque assay on BHK-21 cells (ATCC CRL 8544) as previously described ( ). ..

    Isolation:

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300
    Article Snippet: .. BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine. .. Cloning of the cDNA fragment carrying the wild-type tax gene or the tax gene carrying the F1 mutation (Ser113 and Leu128 converted into Ala and Pro, respectively) in the pSFV3 vector ( ) was described previously ( ).

    Infection:

    Article Title: CpG-Containing Oligonucleotides Are Efficient Adjuvants for Induction of Protective Antiviral Immune Responses with T-Cell Peptide Vaccines
    Article Snippet: .. VSV Indiana seeds (Mudd-Summers isolate), originally obtained from D. Kolakofsky, University of Geneva, were grown on BHK-21 cells (ATCC CRL 8544) infected at low multiplicity and plaqued on Vero cells. .. Vaccinia virus expressing VSV-G (VV-INDG ) was a generous gift of B. Moss (Laboratory of Viral Diseases, National Institutes of Health, Bethesda, Md.) ( ).

    Cell Culture:

    Article Title: Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons
    Article Snippet: .. Baby hamster kidney (BHK) cells, BHK21 (CCL10(13), CRL8544, ATCC) were cultured in Glasgow’s modified Eagle’s medium (Life Technologies) supplemented with 5% FCS, 2 m m l -glutamine, 10% tryptose-phosphate broth, and penicillin/streptomycin. .. Semliki Forest virus infection and metabolic studies.

    Modification:

    Article Title: Differential Transcriptional Activation by Human T-Cell Leukemia Virus Type 1 Tax Mutants Is Mediated by Distinct Interactions with CREB Binding Protein and p300
    Article Snippet: .. BHK21 cells (hamster kidney, ATCC CRL 8544) were grown in Glasgow minimum essential medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% tryptose phosphate, 20 mM HEPES buffer, and 5% fetal bovine serum (FBS); HOS cells (human osteogenic sarcoma, ATCC CRL 1543) were grown in Dulbecco modified Eagle medium (Gibco BRL) supplemented with 10% FBS; and MT2 cells (human T-cell leukemia cells isolated from cord blood lymphocytes cocultured with cells from a patient with T-cell leukemia, obtained from the National Institutes of Health AIDS Research and Reagent Program) were grown in RPMI medium (Gibco BRL) supplemented with 10% FBS, 100 U of penicillin G sodium salt per ml, 100 μg of streptomycin sulfate per ml, and 2 mM glutamine. .. Cloning of the cDNA fragment carrying the wild-type tax gene or the tax gene carrying the F1 mutation (Ser113 and Leu128 converted into Ala and Pro, respectively) in the pSFV3 vector ( ) was described previously ( ).

    Article Title: Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons
    Article Snippet: .. Baby hamster kidney (BHK) cells, BHK21 (CCL10(13), CRL8544, ATCC) were cultured in Glasgow’s modified Eagle’s medium (Life Technologies) supplemented with 5% FCS, 2 m m l -glutamine, 10% tryptose-phosphate broth, and penicillin/streptomycin. .. Semliki Forest virus infection and metabolic studies.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • bhk21  (ATCC)
    99
    ATCC bhk21
    Tumor regression activities of SV-WT, SV-ΔDLP, and AURAV. (A) PKR levels in <t>BHK21</t> cells after type I IFN treatment analyzed by Western blotting using anti-PKR (D-20) and eIF2α antibodies (top). The bottom panel shows an IFN-mediated antiviral response assay against AURAV in BHK21 cells. The estimated IC 50 for IFN-α was 200 U/ml, whereas IFN-β showed no effect on BHK21 cells. (B) Subcutaneous tumors of BHK21 cells were induced in SCID mice, and when indicated (red arrowheads), mice were inoculated intraperitoneally with two doses of 10 7 PFU of the indicated virus ( n = 5 per group). Tumor size was measured every 3 days and plotted; bars show the 95% confidence interval (CI). At the end of the experiment, mice were sacrificed and tumors were extracted and split into two parts. (C) One part was used to prepare cell extracts for viral yield estimation (top) and to detect the presence of viral antigen by Western blot (bottom). A representative photograph of tumor size is shown (middle). (D) The other part of the tumor biopsies was fixed and subjected to TUNEL (green), coupled to IF with anti-SV capsid protein in order to detect infected cells (red). Note that regions expressing viral antigens also showed an intense TUNEL staining. Note that antibodies raised against SV capsid cross-react with AURAV capsid protein in Western blotting but not in IF.
    Bhk21, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk21/product/ATCC
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    bhk21 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    85
    ATCC treatments bhk 21 cells
    Cytotoxic effect of Fibrillar BSAs . Cell viability was determined by the MTT assay. (A) <t>BHK-21</t> cells were treated for 8 h with various concentrations of G-BSA, BSA-S75, BSA-S200, BSA-Zwit, or BSA-HW55S as indicated in serum-free medium. (B) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 in serum-free medium for 8 h. (C) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 or F-BSA (BSA-S200) in serum-free medium for 24 h. (D) BHK-21 cells were treated with or without increasing concentrations of G-BSA (BSA) for 1 h, then incubated with 0.5 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Data are means ± S.D. (n = 3) of percentage of cell survival as determined by the MTT assays.
    Treatments Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/treatments bhk 21 cells/product/ATCC
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    treatments bhk 21 cells - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    Tumor regression activities of SV-WT, SV-ΔDLP, and AURAV. (A) PKR levels in BHK21 cells after type I IFN treatment analyzed by Western blotting using anti-PKR (D-20) and eIF2α antibodies (top). The bottom panel shows an IFN-mediated antiviral response assay against AURAV in BHK21 cells. The estimated IC 50 for IFN-α was 200 U/ml, whereas IFN-β showed no effect on BHK21 cells. (B) Subcutaneous tumors of BHK21 cells were induced in SCID mice, and when indicated (red arrowheads), mice were inoculated intraperitoneally with two doses of 10 7 PFU of the indicated virus ( n = 5 per group). Tumor size was measured every 3 days and plotted; bars show the 95% confidence interval (CI). At the end of the experiment, mice were sacrificed and tumors were extracted and split into two parts. (C) One part was used to prepare cell extracts for viral yield estimation (top) and to detect the presence of viral antigen by Western blot (bottom). A representative photograph of tumor size is shown (middle). (D) The other part of the tumor biopsies was fixed and subjected to TUNEL (green), coupled to IF with anti-SV capsid protein in order to detect infected cells (red). Note that regions expressing viral antigens also showed an intense TUNEL staining. Note that antibodies raised against SV capsid cross-react with AURAV capsid protein in Western blotting but not in IF.

    Journal: Journal of Virology

    Article Title: Naturally Occurring and Engineered Alphaviruses Sensitive to Double-Stranded-RNA-Activated Protein Kinase Show Restricted Translation in Mammalian Cells, Increased Sensitivity to Interferon, and Marked Oncotropism

    doi: 10.1128/JVI.01630-19

    Figure Lengend Snippet: Tumor regression activities of SV-WT, SV-ΔDLP, and AURAV. (A) PKR levels in BHK21 cells after type I IFN treatment analyzed by Western blotting using anti-PKR (D-20) and eIF2α antibodies (top). The bottom panel shows an IFN-mediated antiviral response assay against AURAV in BHK21 cells. The estimated IC 50 for IFN-α was 200 U/ml, whereas IFN-β showed no effect on BHK21 cells. (B) Subcutaneous tumors of BHK21 cells were induced in SCID mice, and when indicated (red arrowheads), mice were inoculated intraperitoneally with two doses of 10 7 PFU of the indicated virus ( n = 5 per group). Tumor size was measured every 3 days and plotted; bars show the 95% confidence interval (CI). At the end of the experiment, mice were sacrificed and tumors were extracted and split into two parts. (C) One part was used to prepare cell extracts for viral yield estimation (top) and to detect the presence of viral antigen by Western blot (bottom). A representative photograph of tumor size is shown (middle). (D) The other part of the tumor biopsies was fixed and subjected to TUNEL (green), coupled to IF with anti-SV capsid protein in order to detect infected cells (red). Note that regions expressing viral antigens also showed an intense TUNEL staining. Note that antibodies raised against SV capsid cross-react with AURAV capsid protein in Western blotting but not in IF.

    Article Snippet: The rest of the cells were purchased from ATCC and include the following: BHK21, HeLa, U87, U373, Hep3B, Huh7.5, PLC/PRF/5, HepG2, Jurkat, MCF7, Panc1, HCT116, and HT29.

    Techniques: Western Blot, Mouse Assay, TUNEL Assay, Infection, Expressing, Staining

    Replication of AURAV in tumor cell lines. (A) Comparative analysis of virus replication in BHK21, Huh7.5, HeLa, and U87 cell lines. Viral yields were titrated 2 to 3 days after infection. Data represent the mean ± SD from three independent experiments. (B) Cell killing associated with replication of AURAV and SVs in different cell lines. In all cases, cells were infected at an MOI of 0.1 PFU/cell and analyzed 3 to 4 days later. Data are the mean of two independent experiments. Cell lines that promoted AURAV replication and cell killing are in bold. Those cell lines derived from hepatic tumors are marked with asterisks. (C) Analysis of viral capsid (C) accumulation (top) and eIF2 phosphorylation (middle) in HCC cell lines infected with AURAV. Cells were infected at a high MOI (25 PFU/cell) and analyzed at 6 and 8 h postinoculation. For comparison, an extract of MEFs infected with AURAV was included. To detect the AURAV capsid protein, a 3-fold more extract of infected MEFs was loaded in the gel (top right). (D) Cell killing associated with virus replication in 3D cultures of Huh7 cells. Huh7 cells were embedded in Matrigel for 6 days and then were infected with 10 5 PFU of the indicated virus, and the cultures were fixed and stained with DAPI 5 days later. Virus-induced spheroid breakage and nuclear condensation suggestive of cell death are marked with arrows. Dotted lines demarcate intact spheroids.

    Journal: Journal of Virology

    Article Title: Naturally Occurring and Engineered Alphaviruses Sensitive to Double-Stranded-RNA-Activated Protein Kinase Show Restricted Translation in Mammalian Cells, Increased Sensitivity to Interferon, and Marked Oncotropism

    doi: 10.1128/JVI.01630-19

    Figure Lengend Snippet: Replication of AURAV in tumor cell lines. (A) Comparative analysis of virus replication in BHK21, Huh7.5, HeLa, and U87 cell lines. Viral yields were titrated 2 to 3 days after infection. Data represent the mean ± SD from three independent experiments. (B) Cell killing associated with replication of AURAV and SVs in different cell lines. In all cases, cells were infected at an MOI of 0.1 PFU/cell and analyzed 3 to 4 days later. Data are the mean of two independent experiments. Cell lines that promoted AURAV replication and cell killing are in bold. Those cell lines derived from hepatic tumors are marked with asterisks. (C) Analysis of viral capsid (C) accumulation (top) and eIF2 phosphorylation (middle) in HCC cell lines infected with AURAV. Cells were infected at a high MOI (25 PFU/cell) and analyzed at 6 and 8 h postinoculation. For comparison, an extract of MEFs infected with AURAV was included. To detect the AURAV capsid protein, a 3-fold more extract of infected MEFs was loaded in the gel (top right). (D) Cell killing associated with virus replication in 3D cultures of Huh7 cells. Huh7 cells were embedded in Matrigel for 6 days and then were infected with 10 5 PFU of the indicated virus, and the cultures were fixed and stained with DAPI 5 days later. Virus-induced spheroid breakage and nuclear condensation suggestive of cell death are marked with arrows. Dotted lines demarcate intact spheroids.

    Article Snippet: The rest of the cells were purchased from ATCC and include the following: BHK21, HeLa, U87, U373, Hep3B, Huh7.5, PLC/PRF/5, HepG2, Jurkat, MCF7, Panc1, HCT116, and HT29.

    Techniques: Infection, Derivative Assay, Staining

    Dye transfer directed by the wt or mutated F and HPIV3 HN proteins. R18-labeled RBCs were bound at 4°C to BHK-21 cells coexpressing the wt or mutanted F and HN proteins. Cells were incubated for 60 min at 37°C to allow membrane fusion, images were acquired by using fluorescent microscopy. (A) Representative images of dye transfer. White arrows indicate the lipid mixing events. (B) Quantification of the events of dye transfer. The extent of dye tranfer is expressed as the average number of R18 lipid dye tranfer events of six microscopic fields. The means and standard errors are from six microscopic fields (** P

    Journal: PLoS ONE

    Article Title: Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

    doi: 10.1371/journal.pone.0136474

    Figure Lengend Snippet: Dye transfer directed by the wt or mutated F and HPIV3 HN proteins. R18-labeled RBCs were bound at 4°C to BHK-21 cells coexpressing the wt or mutanted F and HN proteins. Cells were incubated for 60 min at 37°C to allow membrane fusion, images were acquired by using fluorescent microscopy. (A) Representative images of dye transfer. White arrows indicate the lipid mixing events. (B) Quantification of the events of dye transfer. The extent of dye tranfer is expressed as the average number of R18 lipid dye tranfer events of six microscopic fields. The means and standard errors are from six microscopic fields (** P

    Article Snippet: Cells and viruses BHK-21 cells, obtained from the American Type Culture Collection, were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1% glutamine, 10% fetal calf serum (FCS) (Gibco), and 1% penicillin-streptomycin (Invitrogen).

    Techniques: Labeling, Incubation, Microscopy

    Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins. After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (* P

    Journal: PLoS ONE

    Article Title: Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

    doi: 10.1371/journal.pone.0136474

    Figure Lengend Snippet: Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins. After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (* P

    Article Snippet: Cells and viruses BHK-21 cells, obtained from the American Type Culture Collection, were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 1% glutamine, 10% fetal calf serum (FCS) (Gibco), and 1% penicillin-streptomycin (Invitrogen).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Staining, Giemsa Stain, Produced, Standard Deviation

    Ovarian cancer cells respond to type I interferon and produce IFN upon viral infection. Ovarian cancer cell lines were seeded at 5 × 10 4 and pretreated with 0, 20, 100 or 500 Units of universal type I IFN for 24 hours. After 24 hours, cells were infected with VSV-GP at an MOI of 0.1 ( a ) or 10 ( b ). 24 hours post infection supernatants were collected and titrated on BHK21 cells using a TCID 50 assay. A cell line was defined as responsive (marked with #), when a difference of more than 2-log in viral titer with and without IFN pretreatment was observed. Bars represent the mean ± SEM of at least two independent experiments using duplicates. ( c ) For an indirect type I IFN secretion analysis, cell lines were infected with an MOI of 3 of the single-cycle infective, attenuated VSV*M Q ΔG-GP virus or left uninfected. Supernatants, potentially containing IFN or other antiviral biomolecules, were collected 24 hours after infection. For analysis of biologically active IFN, Vero cells or L929 cells (for analysis of human or murine ovarian cancer cell lines respectively) were incubated either with the supernatant of noninfected cells (SN) or with potentially IFN containing supernatant from virus infected cells (VSV-SN). After 24 hours preincubation, cells were infected with an MOI of 1 of VSV-GP and the supernatant was collected 24 hours post infection. Supernatants were titrated on IFN-nonresponsive G62 cells using a TCID 50 assay to determine virus replication. Cells were defined as IFN producing cells when the difference in viral titers was more than 2-logs between SN and VSV-SN treated cells. Vero cells were used as negative control for cells not producing IFN. Bars represent the mean ± SEM of at least two independent experiments using triplicate samples. IFN, interferon; MOI, multiplicity of infection; SEM, standard error of mean; TCID 50 , 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

    Journal: Molecular Therapy Oncolytics

    Article Title: Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy

    doi: 10.1038/mto.2016.21

    Figure Lengend Snippet: Ovarian cancer cells respond to type I interferon and produce IFN upon viral infection. Ovarian cancer cell lines were seeded at 5 × 10 4 and pretreated with 0, 20, 100 or 500 Units of universal type I IFN for 24 hours. After 24 hours, cells were infected with VSV-GP at an MOI of 0.1 ( a ) or 10 ( b ). 24 hours post infection supernatants were collected and titrated on BHK21 cells using a TCID 50 assay. A cell line was defined as responsive (marked with #), when a difference of more than 2-log in viral titer with and without IFN pretreatment was observed. Bars represent the mean ± SEM of at least two independent experiments using duplicates. ( c ) For an indirect type I IFN secretion analysis, cell lines were infected with an MOI of 3 of the single-cycle infective, attenuated VSV*M Q ΔG-GP virus or left uninfected. Supernatants, potentially containing IFN or other antiviral biomolecules, were collected 24 hours after infection. For analysis of biologically active IFN, Vero cells or L929 cells (for analysis of human or murine ovarian cancer cell lines respectively) were incubated either with the supernatant of noninfected cells (SN) or with potentially IFN containing supernatant from virus infected cells (VSV-SN). After 24 hours preincubation, cells were infected with an MOI of 1 of VSV-GP and the supernatant was collected 24 hours post infection. Supernatants were titrated on IFN-nonresponsive G62 cells using a TCID 50 assay to determine virus replication. Cells were defined as IFN producing cells when the difference in viral titers was more than 2-logs between SN and VSV-SN treated cells. Vero cells were used as negative control for cells not producing IFN. Bars represent the mean ± SEM of at least two independent experiments using triplicate samples. IFN, interferon; MOI, multiplicity of infection; SEM, standard error of mean; TCID 50 , 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

    Article Snippet: Cell lines BHK21 cells (American Type Culture Collection, Manassas, VA) were maintained in Glasgow minimum essential medium (GMEM) (Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Cölbe, Germany), 5% tryptose phosphate broth (Gibco, Carlsbad, CA), 100 units/ml penicillin (Gibco), and 0.1 mg/ml streptomycin (Gibco).

    Techniques: Infection, Incubation, Negative Control

    Ovarian cancer cell lines are efficiently infected and killed by VSV-GP. ( a ) Tropism of VSV*ΔG-GP and VSV*ΔG-G was determined for different human ovarian cancer cell lines as well as a murine cancer cell line (ID8) and a benign cell line (HOSE). Cells were infected with serial dilutions of VSV*ΔG-GP or VSV*ΔG-G. As a control in each experiment, BHK21 cells were infected in the same way. Sixteen hours post infection the percentage of GFP positive cells was determined by flow cytometry and the titer for both viruses on each cell line was calculated. Titers are given relative to the reference cell line BHK21. Bars represent the mean ± SEM (standard error of mean) of one representative of at least two independent experiments using duplicate or triplicate samples. For the control cell line, BHK21, the mean and SEM of one representative experiment is shown. ( b ) For replication kinetics, cells were infected with an MOI of 0.1 of either VSV or VSV-GP. One hour after infection, inoculum was removed, cells were washed with PBS and fresh medium was added. Twenty four hours post infection, the supernatant was collected and titrated on BHK21 cells using TCID 50 assay. Data represent mean ± SEM of at least n = 2 independent experiments using duplicates. The limit of detection for the TCID 50 assay (3.16 TCID 50 /ml) is shown as dashed line in the graph. For the killing assay, ovarian cancer cell lines were seeded as monolayers and cells were infected with an MOI of 0.1 of VSV or VSV-GP ( c ). Viability was determined at indicated time points using WST-1 assay. As a reference, PBS treated cells were used. The human benign cell line HOSE was infected with an MOI of 1 and viability was determined after 72 hours. Bars represent mean ± SEM of one representative experiment of at least two independent experiments performed in decaplicates or dodecaplicates. MOI, multiplicity of infection; PBS, phosphate buffered saline; TCID 50 , 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

    Journal: Molecular Therapy Oncolytics

    Article Title: Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy

    doi: 10.1038/mto.2016.21

    Figure Lengend Snippet: Ovarian cancer cell lines are efficiently infected and killed by VSV-GP. ( a ) Tropism of VSV*ΔG-GP and VSV*ΔG-G was determined for different human ovarian cancer cell lines as well as a murine cancer cell line (ID8) and a benign cell line (HOSE). Cells were infected with serial dilutions of VSV*ΔG-GP or VSV*ΔG-G. As a control in each experiment, BHK21 cells were infected in the same way. Sixteen hours post infection the percentage of GFP positive cells was determined by flow cytometry and the titer for both viruses on each cell line was calculated. Titers are given relative to the reference cell line BHK21. Bars represent the mean ± SEM (standard error of mean) of one representative of at least two independent experiments using duplicate or triplicate samples. For the control cell line, BHK21, the mean and SEM of one representative experiment is shown. ( b ) For replication kinetics, cells were infected with an MOI of 0.1 of either VSV or VSV-GP. One hour after infection, inoculum was removed, cells were washed with PBS and fresh medium was added. Twenty four hours post infection, the supernatant was collected and titrated on BHK21 cells using TCID 50 assay. Data represent mean ± SEM of at least n = 2 independent experiments using duplicates. The limit of detection for the TCID 50 assay (3.16 TCID 50 /ml) is shown as dashed line in the graph. For the killing assay, ovarian cancer cell lines were seeded as monolayers and cells were infected with an MOI of 0.1 of VSV or VSV-GP ( c ). Viability was determined at indicated time points using WST-1 assay. As a reference, PBS treated cells were used. The human benign cell line HOSE was infected with an MOI of 1 and viability was determined after 72 hours. Bars represent mean ± SEM of one representative experiment of at least two independent experiments performed in decaplicates or dodecaplicates. MOI, multiplicity of infection; PBS, phosphate buffered saline; TCID 50 , 50% tissue culture infective dose; VSV, vesicular stomatitis virus.

    Article Snippet: Cell lines BHK21 cells (American Type Culture Collection, Manassas, VA) were maintained in Glasgow minimum essential medium (GMEM) (Gibco, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Cölbe, Germany), 5% tryptose phosphate broth (Gibco, Carlsbad, CA), 100 units/ml penicillin (Gibco), and 0.1 mg/ml streptomycin (Gibco).

    Techniques: Infection, Flow Cytometry, Cytometry, WST-1 Assay

    Cytotoxic effect of Fibrillar BSAs . Cell viability was determined by the MTT assay. (A) BHK-21 cells were treated for 8 h with various concentrations of G-BSA, BSA-S75, BSA-S200, BSA-Zwit, or BSA-HW55S as indicated in serum-free medium. (B) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 in serum-free medium for 8 h. (C) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 or F-BSA (BSA-S200) in serum-free medium for 24 h. (D) BHK-21 cells were treated with or without increasing concentrations of G-BSA (BSA) for 1 h, then incubated with 0.5 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Data are means ± S.D. (n = 3) of percentage of cell survival as determined by the MTT assays.

    Journal: BMC Biotechnology

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    doi: 10.1186/1472-6750-9-2

    Figure Lengend Snippet: Cytotoxic effect of Fibrillar BSAs . Cell viability was determined by the MTT assay. (A) BHK-21 cells were treated for 8 h with various concentrations of G-BSA, BSA-S75, BSA-S200, BSA-Zwit, or BSA-HW55S as indicated in serum-free medium. (B) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 in serum-free medium for 8 h. (C) BHK-21 cells were treated with increasing concentrations of Aβ 25–35 or F-BSA (BSA-S200) in serum-free medium for 24 h. (D) BHK-21 cells were treated with or without increasing concentrations of G-BSA (BSA) for 1 h, then incubated with 0.5 μM F-BSA (BSA-S200) in serum-free medium for 8 h. Data are means ± S.D. (n = 3) of percentage of cell survival as determined by the MTT assays.

    Article Snippet: Cell lines and treatments BHK-21 cells (baby hamster kidney; ATCC CRL-1632) and T47D cells (human breast duct carcinoma; ATCC HTB-133) were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: MTT Assay, Incubation

    Evaluation of the apoptotic effect of fibrillar BSA . (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ 25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under

    Journal: BMC Biotechnology

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    doi: 10.1186/1472-6750-9-2

    Figure Lengend Snippet: Evaluation of the apoptotic effect of fibrillar BSA . (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ 25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under "Methods". Data are the mean ± SD of three experiments.

    Article Snippet: Cell lines and treatments BHK-21 cells (baby hamster kidney; ATCC CRL-1632) and T47D cells (human breast duct carcinoma; ATCC HTB-133) were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Incubation, Fluorescence, Microscopy, Staining, Cell Culture, Activity Assay

    Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.

    Journal: BMC Biotechnology

    Article Title: Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    doi: 10.1186/1472-6750-9-2

    Figure Lengend Snippet: Fibrillar BSA induced cytotoxicity via the integrin/FAK/Akt pathway . (A) BHK-21 cells were treated with 3 μM F-BSA (BSA-S200) in serum-free medium for the indicated time, and cell lysates were analyzed by western blotting with anti-phospho-FAK(Tyr576/577), anti-phospho-FAK(Tyr397), and anti-phospho-Akt (p-Akt) antibodies. (B) BHK-21 cells were pre-treated for 30 min with or without 1 μM goat IgG or 1 μM goat anti-integrin α5β1 antibody as indicated, then treated with 3 μM F-BSA (BSA-S200) in serum-free medium for 15 min. Cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) and anti-phospho-GSK-3β (p-GSK-3β) antibodies. (C) BHK-21 cells were treated with increasing concentrations of G-BSA (BSA) in serum-free medium as indicated, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody. (D) BHK-21 cells were treated with or without 1 μM anti-integrin α5β1 antibody in serum-free medium for 30 min, and cell lysates were analyzed by western blotting with anti-phospho-Akt (p-Akt) antibody.

    Article Snippet: Cell lines and treatments BHK-21 cells (baby hamster kidney; ATCC CRL-1632) and T47D cells (human breast duct carcinoma; ATCC HTB-133) were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Western Blot