bhk 21 cells  (Roche)


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    Structured Review

    Roche bhk 21 cells
    Fig. 3. Overexpressed FRB–WASp418–502 induces F-actin clusters in <t>BHK-21</t> cells. ( A ) Lysates of RBL-2H3 cell line expressing FRB–WASp418–502, or BHK-21 cells overexpressing FRB–WASp418–502 or Scar-WA were run on 12% SDS–PAGE (9 µg total protein per lane) and blotted on to PVDF. Blots were revealed using mouse anti-myc tag antibody (clone 9E10) and the ECL procedure. The signal in RBL-2H3 cells is ∼5-fold less than in BHK-21 cells (compare lanes 1 and 3). Taking into consideration that ∼10% of BHK-21 cells expressed the FRB–WASp418–502 protein, we estimate that the level of expression of FRB–WASp418–502 is ∼50-fold lower in the RBL-2H3 stable cell line compared with that in BHK-21 cells. ( B – D ) BHK-21 cells expressing the Scar-WA domain. ( E – G ) BHK-21 cells expressing the FRB–WASp418–502 construct. (B and E) Localization of myc-tagged constructs as revealed using anti-myc tag antibody. (C and F) Distribution of F-actin. Arrows point at clusters of WASp or Scar C-terminal domains and F-actin. Arrowheads show non-transfected cells. (D and G) Superimposition of anti-myc and F-actin images. Bar: 10 µm.
    Bhk 21 Cells, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A WASp-VASP complex regulates actin polymerization at the plasma membrane"

    Article Title: A WASp-VASP complex regulates actin polymerization at the plasma membrane

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.20.5603

    Fig. 3. Overexpressed FRB–WASp418–502 induces F-actin clusters in BHK-21 cells. ( A ) Lysates of RBL-2H3 cell line expressing FRB–WASp418–502, or BHK-21 cells overexpressing FRB–WASp418–502 or Scar-WA were run on 12% SDS–PAGE (9 µg total protein per lane) and blotted on to PVDF. Blots were revealed using mouse anti-myc tag antibody (clone 9E10) and the ECL procedure. The signal in RBL-2H3 cells is ∼5-fold less than in BHK-21 cells (compare lanes 1 and 3). Taking into consideration that ∼10% of BHK-21 cells expressed the FRB–WASp418–502 protein, we estimate that the level of expression of FRB–WASp418–502 is ∼50-fold lower in the RBL-2H3 stable cell line compared with that in BHK-21 cells. ( B – D ) BHK-21 cells expressing the Scar-WA domain. ( E – G ) BHK-21 cells expressing the FRB–WASp418–502 construct. (B and E) Localization of myc-tagged constructs as revealed using anti-myc tag antibody. (C and F) Distribution of F-actin. Arrows point at clusters of WASp or Scar C-terminal domains and F-actin. Arrowheads show non-transfected cells. (D and G) Superimposition of anti-myc and F-actin images. Bar: 10 µm.
    Figure Legend Snippet: Fig. 3. Overexpressed FRB–WASp418–502 induces F-actin clusters in BHK-21 cells. ( A ) Lysates of RBL-2H3 cell line expressing FRB–WASp418–502, or BHK-21 cells overexpressing FRB–WASp418–502 or Scar-WA were run on 12% SDS–PAGE (9 µg total protein per lane) and blotted on to PVDF. Blots were revealed using mouse anti-myc tag antibody (clone 9E10) and the ECL procedure. The signal in RBL-2H3 cells is ∼5-fold less than in BHK-21 cells (compare lanes 1 and 3). Taking into consideration that ∼10% of BHK-21 cells expressed the FRB–WASp418–502 protein, we estimate that the level of expression of FRB–WASp418–502 is ∼50-fold lower in the RBL-2H3 stable cell line compared with that in BHK-21 cells. ( B – D ) BHK-21 cells expressing the Scar-WA domain. ( E – G ) BHK-21 cells expressing the FRB–WASp418–502 construct. (B and E) Localization of myc-tagged constructs as revealed using anti-myc tag antibody. (C and F) Distribution of F-actin. Arrows point at clusters of WASp or Scar C-terminal domains and F-actin. Arrowheads show non-transfected cells. (D and G) Superimposition of anti-myc and F-actin images. Bar: 10 µm.

    Techniques Used: Expressing, SDS Page, Stable Transfection, Construct, Transfection

    2) Product Images from "Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿"

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿

    Journal:

    doi: 10.1128/JVI.01011-08

    Analysis of G3BP1 and G3BP2 relocalization in cells infected with SINV/nsP3Cherry variant. The stable cell line, expressing G3BP1/GFP or BHK-21 cells transiently expressing G3BP2/GFP (see Materials and Methods for details), were infected with SINV/nsP3Cherry
    Figure Legend Snippet: Analysis of G3BP1 and G3BP2 relocalization in cells infected with SINV/nsP3Cherry variant. The stable cell line, expressing G3BP1/GFP or BHK-21 cells transiently expressing G3BP2/GFP (see Materials and Methods for details), were infected with SINV/nsP3Cherry

    Techniques Used: Infection, Variant Assay, Stable Transfection, Expressing

    Analysis of nsP3/GFP distribution in different fractions of cell lysates. BHK-21 cells were infected with packaged SINrep(nsP3GFP) at an MOI of 20 inf.u./cell. At 6 h postinfection, cells were harvested and homogenized. A nucleus-containing (NUC) fraction
    Figure Legend Snippet: Analysis of nsP3/GFP distribution in different fractions of cell lysates. BHK-21 cells were infected with packaged SINrep(nsP3GFP) at an MOI of 20 inf.u./cell. At 6 h postinfection, cells were harvested and homogenized. A nucleus-containing (NUC) fraction

    Techniques Used: Infection

    Localization of dsRNA- and nsP3/GFP-containing protein complexes in BHK-21 cells infected with SINV/nsP3GFP. (A) BHK-21 cells infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell. At 4 h postinfection, the cells were fixed with 3% paraformaldehyde,
    Figure Legend Snippet: Localization of dsRNA- and nsP3/GFP-containing protein complexes in BHK-21 cells infected with SINV/nsP3GFP. (A) BHK-21 cells infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell. At 4 h postinfection, the cells were fixed with 3% paraformaldehyde,

    Techniques Used: Infection

    Analysis of the colocalization of nsP3/GFP and different, membrane-containing cellular organelles. (a and b) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell and stained with FM4-64FX reagent as described in Materials and
    Figure Legend Snippet: Analysis of the colocalization of nsP3/GFP and different, membrane-containing cellular organelles. (a and b) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell and stained with FM4-64FX reagent as described in Materials and

    Techniques Used: Infection, Staining

    Identification of the viral and cellular components in the nsP3-containing protein complexes. (A) Schematic representation of the SINV replicons used in the study. (B) BHK-21 cell were infected with the indicated, packaged replicons at an MOI of 20 infectious
    Figure Legend Snippet: Identification of the viral and cellular components in the nsP3-containing protein complexes. (A) Schematic representation of the SINV replicons used in the study. (B) BHK-21 cell were infected with the indicated, packaged replicons at an MOI of 20 infectious

    Techniques Used: Infection

    3) Product Images from "Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells"

    Article Title: Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00709-13

    Growth properties of gB FCS − viruses. (A) Low-MOI growth curve. BHK-21 cells were infected with eGFP + WT virus and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant viruses at an MOI of 0.05 eGFP units/cell. At the indicated times p.i., the cells were lysed by 1 cycle of freeze-thawing and titrated on BHK-21 cells by counting eGFP + cells by flow cytometry. Each point shows the mean ± standard error of the mean from 3 wells. The dashed line shows the sensitivity threshold of virus titration. The titers of the gB FCS − viruses (ΔFCSv1, ΔFCSv2, and ΔFCSv3) were significantly lower than those of the gB FCS + viruses (WT, ΔFCSv1 R, ΔFCSv2 R, and ΔFCSv3 R) on days 3 ( P
    Figure Legend Snippet: Growth properties of gB FCS − viruses. (A) Low-MOI growth curve. BHK-21 cells were infected with eGFP + WT virus and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant viruses at an MOI of 0.05 eGFP units/cell. At the indicated times p.i., the cells were lysed by 1 cycle of freeze-thawing and titrated on BHK-21 cells by counting eGFP + cells by flow cytometry. Each point shows the mean ± standard error of the mean from 3 wells. The dashed line shows the sensitivity threshold of virus titration. The titers of the gB FCS − viruses (ΔFCSv1, ΔFCSv2, and ΔFCSv3) were significantly lower than those of the gB FCS + viruses (WT, ΔFCSv1 R, ΔFCSv2 R, and ΔFCSv3 R) on days 3 ( P

    Techniques Used: Infection, Mutagenesis, Flow Cytometry, Cytometry, Titration

    Susceptibility of gB FCS − viruses to gB-directed neutralization. eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses (MOI of 0.5 eGFP units/cell) were incubated (2 h, RT) with the gB-specific neutralizing MAbs SC-9E8 and SC-9A5 blocking viral membrane fusion or the gH/gL-specific MAb 8F10 blocking cell binding. The viruses were then added to BHK-21 cells and incubated for 18 h at 37°C in the presence of 100 μg/ml PAA. The eGFP + cells were enumerated by flow cytometry and are shown relative to untreated virus. Each point shows the mean ± standard error of the mean from 3 wells. The gB FCS − viruses (ΔFCSv1 and ΔFCSv3) were neutralized significantly better than the gB FCS + viruses (WT and ΔFCSv1 R) with MAb SC-9E8 at antibody doses of 16.7 ( P
    Figure Legend Snippet: Susceptibility of gB FCS − viruses to gB-directed neutralization. eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses (MOI of 0.5 eGFP units/cell) were incubated (2 h, RT) with the gB-specific neutralizing MAbs SC-9E8 and SC-9A5 blocking viral membrane fusion or the gH/gL-specific MAb 8F10 blocking cell binding. The viruses were then added to BHK-21 cells and incubated for 18 h at 37°C in the presence of 100 μg/ml PAA. The eGFP + cells were enumerated by flow cytometry and are shown relative to untreated virus. Each point shows the mean ± standard error of the mean from 3 wells. The gB FCS − viruses (ΔFCSv1 and ΔFCSv3) were neutralized significantly better than the gB FCS + viruses (WT and ΔFCSv1 R) with MAb SC-9E8 at antibody doses of 16.7 ( P

    Techniques Used: Neutralization, Mutagenesis, Incubation, Blocking Assay, Binding Assay, Flow Cytometry, Cytometry

    Antigenic conformation of uncleaved gB. (A) BHK-21 cells were infected with eGFP + WT virus and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant viruses at an MOI of 2 PFU/cell. Eighteen hours later, cells were stained with MAbs BN-1A7 and SC-9E8 recognizing prefusion gB, MAb MG-1A12 recognizing postfusion gB, MAb T2C12 recognizing gH/gL, and MAb BN-3A4 recognizing gp150. Bound antibody was detected with an Alexa Fluor 633-conjugated secondary antibody and flow cytometry (open histograms). The gray histograms show staining with secondary antibody only. Detection of gH/gL and gp150 served as the control for equivalent infection of cells. Equivalent data were obtained in a repeat experiment. (B) NMuMG cells were incubated with eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 15 eGFP units/cell (2 h, 4°C). After 3 washes in ice-cold PBS to remove unbound virions, the cells were fixed either directly or after further incubations at 37°C to allow virion endocytosis. The cells were then incubated with MAb BN-1A7 recognizing prefusion gB and MAb MG-1A12 recognizing postfusion gB. Bound antibody was detected with an alkaline phosphatase-conjugated secondary antibody, incubation with p -nitrophenyl phosphate substrate, and measurement of the absorbance at 405 nm ( A 405 ). The bars show means ± standard errors of the means from 6 wells. Equivalent data were obtained in 3 further experiments.
    Figure Legend Snippet: Antigenic conformation of uncleaved gB. (A) BHK-21 cells were infected with eGFP + WT virus and ΔFCSv1, ΔFCSv2, and ΔFCSv3 mutant and revertant viruses at an MOI of 2 PFU/cell. Eighteen hours later, cells were stained with MAbs BN-1A7 and SC-9E8 recognizing prefusion gB, MAb MG-1A12 recognizing postfusion gB, MAb T2C12 recognizing gH/gL, and MAb BN-3A4 recognizing gp150. Bound antibody was detected with an Alexa Fluor 633-conjugated secondary antibody and flow cytometry (open histograms). The gray histograms show staining with secondary antibody only. Detection of gH/gL and gp150 served as the control for equivalent infection of cells. Equivalent data were obtained in a repeat experiment. (B) NMuMG cells were incubated with eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 15 eGFP units/cell (2 h, 4°C). After 3 washes in ice-cold PBS to remove unbound virions, the cells were fixed either directly or after further incubations at 37°C to allow virion endocytosis. The cells were then incubated with MAb BN-1A7 recognizing prefusion gB and MAb MG-1A12 recognizing postfusion gB. Bound antibody was detected with an alkaline phosphatase-conjugated secondary antibody, incubation with p -nitrophenyl phosphate substrate, and measurement of the absorbance at 405 nm ( A 405 ). The bars show means ± standard errors of the means from 6 wells. Equivalent data were obtained in 3 further experiments.

    Techniques Used: Infection, Mutagenesis, Staining, Flow Cytometry, Cytometry, Incubation

    Kinetics of virus entry in fibroblasts, epithelial cells, and macrophages. (A) BHK-21 fibroblasts were exposed at 37°C to eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 1 eGFP units/cell in the presence of 100 μg/ml PAA for the times indicated on the x axis. The cells were then washed with PBS to remove unbound virions or with phosphate-citrate buffer (pH 3) (acid wash) to inactivate all extracellular virions, followed by incubation at 37°C in the presence of 100 μg/ml PAA until 24 h p.i. The proportion of eGFP + cells was then determined by flow cytometry. Each point shows the mean ± standard error of the mean from 3 wells. The incubation times required for half-maximal infection levels ( t 50% ) were as follows (mean ± standard error of the mean): PBS wash, 2 ± 0.05 h for gB FCS + (WT and ΔFCSv1 R) and 2.3 ± 0.1 h for gB FCS − (ΔFCSv1 and ΔFCSv3) viruses; acid wash, 4.1 ± 0.08 h for gB FCS + and 4.7 ± 0.15 h for gB FCS − viruses. For acid wash, the difference in t 50% values was statistically significant ( P
    Figure Legend Snippet: Kinetics of virus entry in fibroblasts, epithelial cells, and macrophages. (A) BHK-21 fibroblasts were exposed at 37°C to eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 1 eGFP units/cell in the presence of 100 μg/ml PAA for the times indicated on the x axis. The cells were then washed with PBS to remove unbound virions or with phosphate-citrate buffer (pH 3) (acid wash) to inactivate all extracellular virions, followed by incubation at 37°C in the presence of 100 μg/ml PAA until 24 h p.i. The proportion of eGFP + cells was then determined by flow cytometry. Each point shows the mean ± standard error of the mean from 3 wells. The incubation times required for half-maximal infection levels ( t 50% ) were as follows (mean ± standard error of the mean): PBS wash, 2 ± 0.05 h for gB FCS + (WT and ΔFCSv1 R) and 2.3 ± 0.1 h for gB FCS − (ΔFCSv1 and ΔFCSv3) viruses; acid wash, 4.1 ± 0.08 h for gB FCS + and 4.7 ± 0.15 h for gB FCS − viruses. For acid wash, the difference in t 50% values was statistically significant ( P

    Techniques Used: Mutagenesis, Incubation, Flow Cytometry, Cytometry, Infection

    Comparative analysis of virus entry in fibroblasts, epithelial cells, myeloma cells, macrophages, and DCs. (A and B) BHK-21 fibroblasts (A) or NMuMG epithelial cells (B) were infected with eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 0.5 eGFP units/cell. After incubation in the presence of 100 μg/ml PAA for 18 h at 37°C, the proportion of eGFP + cells was determined by flow cytometry. The bars show the means ± standard errors of the means from 3 wells. Equivalent data were obtained in 3 further experiments. (C) NS0 myeloma cells were infected as for panel A but at an MOI of 100 eGFP units/cell. The bars show the means ± standard errors of the means from 3 wells. The values for the gB FCS − viruses (ΔFCSv1 and ΔFCSv3) were significantly lower than those for the gB FCS + viruses (WT and ΔFCSv1 R) ( P
    Figure Legend Snippet: Comparative analysis of virus entry in fibroblasts, epithelial cells, myeloma cells, macrophages, and DCs. (A and B) BHK-21 fibroblasts (A) or NMuMG epithelial cells (B) were infected with eGFP + WT, ΔFCSv1 and ΔFCSv3 mutant, and ΔFCSv1 revertant viruses at an MOI of 0.5 eGFP units/cell. After incubation in the presence of 100 μg/ml PAA for 18 h at 37°C, the proportion of eGFP + cells was determined by flow cytometry. The bars show the means ± standard errors of the means from 3 wells. Equivalent data were obtained in 3 further experiments. (C) NS0 myeloma cells were infected as for panel A but at an MOI of 100 eGFP units/cell. The bars show the means ± standard errors of the means from 3 wells. The values for the gB FCS − viruses (ΔFCSv1 and ΔFCSv3) were significantly lower than those for the gB FCS + viruses (WT and ΔFCSv1 R) ( P

    Techniques Used: Infection, Mutagenesis, Incubation, Flow Cytometry, Cytometry

    4) Product Images from "A Novel Neutralizing Antibody Targeting a Unique Cross-Reactive Epitope on the hi Loop of Domain II of the Envelope Protein Protects Mice against Duck Tembusu Virus"

    Article Title: A Novel Neutralizing Antibody Targeting a Unique Cross-Reactive Epitope on the hi Loop of Domain II of the Envelope Protein Protects Mice against Duck Tembusu Virus

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1901352

    Broad cross-reactivity of mAb 1G2 to flaviviruses. BHK-21 cells infected with DTMUV and JEV or transfected with pCDNA-Ew and pCDNA-Ez were probed with mAb 1G2 and imaged by confocal microscopy. The nuclei were counterstained with Hoechst stain.
    Figure Legend Snippet: Broad cross-reactivity of mAb 1G2 to flaviviruses. BHK-21 cells infected with DTMUV and JEV or transfected with pCDNA-Ew and pCDNA-Ez were probed with mAb 1G2 and imaged by confocal microscopy. The nuclei were counterstained with Hoechst stain.

    Techniques Used: Infection, Transfection, Confocal Microscopy, Staining

    E protein and mAb 1G2 epitope identification. ( A ) The migration of E protein on polyacrylamide gel stained by Coomassie Brilliant Blue. Purified fractions from DTMUV-infected (lane 1) or mock-infected BHK-21 cell lysates (lane 2). Protein m.w. marker (lane M1). E protein is indicated by the arrow. ( B ) Western blotting analysis of DTMUV-infected BHK-21 cells detected by 1G2. DTMUV-infected cell lysates treated with 2-ME (lane 1) or without 2-ME (lane 2); DTMUV-infected cell lysates boiled with 2-ME (lane 3) or without 2-ME (lane 4). Protein m.w. marker (lane M2). ( C ) E protein from E. coli –expressed pET30a-E detected by mouse anti-DTMUV polyserum. M3, m.w. marker. Lysate sediments from E. coli –expressed pET30a-E treated with (lane 1) or without 2-ME (lane 2); lysate supernatants of E. coli –expressed pET30a-E treated with 2-ME (lane 3). ( D ) E protein from the E. coli –expressed pET30a-E detected by 1G2. Lysate sediments from E. coli –expressed pET30a-E without 2-ME treatment (lane 1); lysate supernatants from E. coli –expressed pET30a-E without 2-ME treatment (lane 2). Lysate sediments from E. coli –expressed pET30a-E with 2-ME treatment (lane 3); lysate supernatants from E. coli –expressed pET30a-E with 2-ME treatment (lane 4).
    Figure Legend Snippet: E protein and mAb 1G2 epitope identification. ( A ) The migration of E protein on polyacrylamide gel stained by Coomassie Brilliant Blue. Purified fractions from DTMUV-infected (lane 1) or mock-infected BHK-21 cell lysates (lane 2). Protein m.w. marker (lane M1). E protein is indicated by the arrow. ( B ) Western blotting analysis of DTMUV-infected BHK-21 cells detected by 1G2. DTMUV-infected cell lysates treated with 2-ME (lane 1) or without 2-ME (lane 2); DTMUV-infected cell lysates boiled with 2-ME (lane 3) or without 2-ME (lane 4). Protein m.w. marker (lane M2). ( C ) E protein from E. coli –expressed pET30a-E detected by mouse anti-DTMUV polyserum. M3, m.w. marker. Lysate sediments from E. coli –expressed pET30a-E treated with (lane 1) or without 2-ME (lane 2); lysate supernatants of E. coli –expressed pET30a-E treated with 2-ME (lane 3). ( D ) E protein from the E. coli –expressed pET30a-E detected by 1G2. Lysate sediments from E. coli –expressed pET30a-E without 2-ME treatment (lane 1); lysate supernatants from E. coli –expressed pET30a-E without 2-ME treatment (lane 2). Lysate sediments from E. coli –expressed pET30a-E with 2-ME treatment (lane 3); lysate supernatants from E. coli –expressed pET30a-E with 2-ME treatment (lane 4).

    Techniques Used: Migration, Staining, Purification, Infection, Marker, Western Blot

    5) Product Images from "Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene"

    Article Title: Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00814-16

    Replication of HCoV-OC43 in response to drug treatment in BHK-21 cells. (A and B) Effect of chloroquine or ribavirin on the replication of HCoV-OC43-WT or rOC43-ns2DelRluc. BHK-21 cells seeded in 48-well plates were infected with HCoV-OC43-WT or rOC43-ns2DelRluc at an MOI of 0.01 for 2 h and subsequently treated with chloroquine or ribavirin at the indicated concentrations. At 72 h postinfection, supernatants were removed and subsequently analyzed for viral load by real-time quantitative RT-PCR. Error bars indicate means and standard deviations from three independent experiments. (C and D) Chloroquine or ribavirin inhibition of Rluc activity of rOC43-ns2DelRluc and cytotoxic effects. The inhibition assay was performed as described in Materials and Methods. Rluc activity of chloroquine- or ribavirin-treated cells was normalized to that of dimethyl sulfoxide (DMSO)-treated control cells and measured relative to that of DMSO-treated cells. Viable cell numbers were used to determine the percent cytotoxic effect in drug-treated cells relative to that in DMSO-treated cells. Error bars indicate means and standard deviations from three independent experiments. (E and F) Antiviral effect of chloroquine or ribavirin on HCoV-OC43-WT N protein synthesis.
    Figure Legend Snippet: Replication of HCoV-OC43 in response to drug treatment in BHK-21 cells. (A and B) Effect of chloroquine or ribavirin on the replication of HCoV-OC43-WT or rOC43-ns2DelRluc. BHK-21 cells seeded in 48-well plates were infected with HCoV-OC43-WT or rOC43-ns2DelRluc at an MOI of 0.01 for 2 h and subsequently treated with chloroquine or ribavirin at the indicated concentrations. At 72 h postinfection, supernatants were removed and subsequently analyzed for viral load by real-time quantitative RT-PCR. Error bars indicate means and standard deviations from three independent experiments. (C and D) Chloroquine or ribavirin inhibition of Rluc activity of rOC43-ns2DelRluc and cytotoxic effects. The inhibition assay was performed as described in Materials and Methods. Rluc activity of chloroquine- or ribavirin-treated cells was normalized to that of dimethyl sulfoxide (DMSO)-treated control cells and measured relative to that of DMSO-treated cells. Viable cell numbers were used to determine the percent cytotoxic effect in drug-treated cells relative to that in DMSO-treated cells. Error bars indicate means and standard deviations from three independent experiments. (E and F) Antiviral effect of chloroquine or ribavirin on HCoV-OC43-WT N protein synthesis.

    Techniques Used: Infection, Quantitative RT-PCR, Inhibition, Activity Assay

    Analysis of genetic stability of the reporter viruses. (A) Illustration of the virus passage procedure in BHK-21 cells. rHCoVs rescued from transfected cells were defined as P0. Culture supernatants from the transfected cells (P0) were added to naive cells to obtain passage 1 virus (P1). After 13 rounds of serial passages, the reporter viruses were passaged to P13. (B) Viral titers of reporter viruses during passages. Reporter viruses were passaged 13 times in BHK-21 cells, and the supernatants were collected from the virus-infected cells of each passage and titrated using the IFA-based viral titration assay. Data represent three independent experiments and are shown as means ± standard deviations. (C) Rluc activity of reporter viruses of each passage. BHK-21 cells were infected with reporter viruses (MOI = 0.01) of each passage in 48-well plates and assayed for the Rluc activity in RLU at 72 h postinfection. Data represent mean values from three independent experiments, with error bars representing the standard deviations of the means. (D) Analysis of genetic stability of the reporter viruses after several passages in BHK-21 cells. Viral RNA was extracted from culture supernatants of each passage, and RT-PCR was performed with a primer set flanking the Rluc gene. The resulting RT-PCR products were resolved by 1% agarose gel electrophoresis.
    Figure Legend Snippet: Analysis of genetic stability of the reporter viruses. (A) Illustration of the virus passage procedure in BHK-21 cells. rHCoVs rescued from transfected cells were defined as P0. Culture supernatants from the transfected cells (P0) were added to naive cells to obtain passage 1 virus (P1). After 13 rounds of serial passages, the reporter viruses were passaged to P13. (B) Viral titers of reporter viruses during passages. Reporter viruses were passaged 13 times in BHK-21 cells, and the supernatants were collected from the virus-infected cells of each passage and titrated using the IFA-based viral titration assay. Data represent three independent experiments and are shown as means ± standard deviations. (C) Rluc activity of reporter viruses of each passage. BHK-21 cells were infected with reporter viruses (MOI = 0.01) of each passage in 48-well plates and assayed for the Rluc activity in RLU at 72 h postinfection. Data represent mean values from three independent experiments, with error bars representing the standard deviations of the means. (D) Analysis of genetic stability of the reporter viruses after several passages in BHK-21 cells. Viral RNA was extracted from culture supernatants of each passage, and RT-PCR was performed with a primer set flanking the Rluc gene. The resulting RT-PCR products were resolved by 1% agarose gel electrophoresis.

    Techniques Used: Transfection, Infection, Immunofluorescence, Titration, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Characterization of reporter viruses using engineered accessary genes. (A) The N protein of recombinant HCoVs-OC43 (rHCoVs) examined by indirect immunofluorescence assay (IFA). At 72 h postinfection, virus-infected BHK-21 cells were incubated with anti-OC43-N mouse polyclonal antibodies and then stained with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG. Cells were analyzed under a fluorescence microscope. (B) Growth kinetics of rHCoVs. BHK-21 cells were infected with rHCoVs and HCoV-OC43-WT at a multiplicity of infection (MOI) of 0.01. Viral titers from culture supernatants at the indicated time points were determined by indirect IFA. Data represent three independent experiments and are shown as means ± standard deviations. (C) Complementation of rOC43-ns2DelRluc infection in BHK-21 cells expressing ns2. Cells were transfected with a plasmid expressing ns2-enhanced GFP (EGFP) or a control vector using the X-tremeGENE HP DNA transfection reagent, and the expression levels of ns2-EGFP were analyzed by Western blotting using anti-GFP antibody (left). After 24 h posttransfection, BHK-21 cells were infected with HCoV-OC43-WT or rOC43-ns2DelRluc at an MOI of 0.01. Cell supernatants were collected at 72 h postinfection, and the viral titers were determined by IFA (right). (D) Time course analysis of the reporter gene expression. The Rluc activity, represented as relative light units (RLU), was measured in BHK-21 cells infected with rHCoVs at the indicated time points (MOI = 0.01). Data represent three independent experiments and are shown as means ± standard deviations. (E and F) Western blot analysis of reporter gene expression. Proteins in cell lysates of BHK-21 cells infected with rHCoVs and HCoV-OC43-WT were analyzed by Western blotting using anti-OC43-N, anti-Rluc, and anti-β-actin antibodies. Cell lysates from uninfected cells (mock) served as a negative control. (G) Effect of the inserted reporter gene on subgenomic RNA (sgRNA) synthesis. At 72 h postinfection (MOI = 0.01), total cellular mRNAs were extracted and subjected to RT-PCR to determine the mRNA levels of ns2 (HCoV-OC43-WT), Rluc (rOC43-ns2DelRluc and rOC43-ns12.9StopRluc), ns2-Rluc (rOC43-ns2FusionRluc), and ns12.9-Rluc (rOC43-ns12.9FusionRluc). HCoV-OC43-WT was used as a control. Data were normalized to the levels of internal mouse GAPDH mRNA. Error bars indicate means and standard deviations from three independent experiments.
    Figure Legend Snippet: Characterization of reporter viruses using engineered accessary genes. (A) The N protein of recombinant HCoVs-OC43 (rHCoVs) examined by indirect immunofluorescence assay (IFA). At 72 h postinfection, virus-infected BHK-21 cells were incubated with anti-OC43-N mouse polyclonal antibodies and then stained with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG. Cells were analyzed under a fluorescence microscope. (B) Growth kinetics of rHCoVs. BHK-21 cells were infected with rHCoVs and HCoV-OC43-WT at a multiplicity of infection (MOI) of 0.01. Viral titers from culture supernatants at the indicated time points were determined by indirect IFA. Data represent three independent experiments and are shown as means ± standard deviations. (C) Complementation of rOC43-ns2DelRluc infection in BHK-21 cells expressing ns2. Cells were transfected with a plasmid expressing ns2-enhanced GFP (EGFP) or a control vector using the X-tremeGENE HP DNA transfection reagent, and the expression levels of ns2-EGFP were analyzed by Western blotting using anti-GFP antibody (left). After 24 h posttransfection, BHK-21 cells were infected with HCoV-OC43-WT or rOC43-ns2DelRluc at an MOI of 0.01. Cell supernatants were collected at 72 h postinfection, and the viral titers were determined by IFA (right). (D) Time course analysis of the reporter gene expression. The Rluc activity, represented as relative light units (RLU), was measured in BHK-21 cells infected with rHCoVs at the indicated time points (MOI = 0.01). Data represent three independent experiments and are shown as means ± standard deviations. (E and F) Western blot analysis of reporter gene expression. Proteins in cell lysates of BHK-21 cells infected with rHCoVs and HCoV-OC43-WT were analyzed by Western blotting using anti-OC43-N, anti-Rluc, and anti-β-actin antibodies. Cell lysates from uninfected cells (mock) served as a negative control. (G) Effect of the inserted reporter gene on subgenomic RNA (sgRNA) synthesis. At 72 h postinfection (MOI = 0.01), total cellular mRNAs were extracted and subjected to RT-PCR to determine the mRNA levels of ns2 (HCoV-OC43-WT), Rluc (rOC43-ns2DelRluc and rOC43-ns12.9StopRluc), ns2-Rluc (rOC43-ns2FusionRluc), and ns12.9-Rluc (rOC43-ns12.9FusionRluc). HCoV-OC43-WT was used as a control. Data were normalized to the levels of internal mouse GAPDH mRNA. Error bars indicate means and standard deviations from three independent experiments.

    Techniques Used: Recombinant, Immunofluorescence, Infection, Incubation, Staining, Labeling, Fluorescence, Microscopy, Expressing, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Negative Control, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome MaintenanceHow a Latent Virus Eludes Immune Defenses"

    Article Title: Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome MaintenanceHow a Latent Virus Eludes Immune Defenses

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0030120

    Modification of the MHV-68 Genome to Overcome cis- Acting Immune Evasion by ORF73 (A) An IRES element was inserted just downstream of ORF73, between its stop codon and that of M11. This allowed either three tandem CD8 + T cell epitopes (EPI) or GFP to be translated from the ORF73 mRNA. (B) DNA from BAC-cloned viral genomes (BAC) or virus-infected cells (VIR) was digested with NcoI, electrophoresed, transferred to nylon membranes, and blotted with a probe corresponding to the BamHI-G genomic fragment shown in (A). The predicted bands for WT virus were 1,021 bp, 3,121 bp, and 4,630 bp. The IRES-GFP insert introduced an NcoI site such that the WT 3,121-bp band was cut into 2,975-bp and 1,466-bp fragments. The NcoI site was lost from the IRES-EPI insert, such that the WT 3,121-bp band became a 3,861-bp band. (C) BHK-21 cells were infected (0.01 PFU/cell) with WT, GFP, or EPI viruses as indicated. Plaque titres of cell cultures are shown with time after infection. (D) H2 b MEF-1 cells or L929-K b cells were left uninfected (UI) or infected for 2 h with MHV-68 expressing either OVA under a strong lytic promoter (OVA) or the SIINFEKL epitope of OVA as part of the ORF73-IRES-EPI construct (EPI). B3Z cells were then added, and 18 h later their beta-galactosidase response was assayed using chlorophenol-red-beta- D -galactoside substrate. Mean ± SD values of triplicate cultures are shown. The data are from one or two equivalent experiments. (E) A20-syndecan-1 cells were infected (20 PFU/cell) with GFP − WT virus, WT virus with an HCMV IE1 promoter-driven GFP expression cassette (HCMV IE1-GFP), or with the ORF73-IRES-GFP virus. The numbers indicate the percentage of total cells in the gated region (GFP + ). Expression from the HCMV IE1 promoter is probably limited to lytic infection, whereas ORF73 is expressed in latency.
    Figure Legend Snippet: Modification of the MHV-68 Genome to Overcome cis- Acting Immune Evasion by ORF73 (A) An IRES element was inserted just downstream of ORF73, between its stop codon and that of M11. This allowed either three tandem CD8 + T cell epitopes (EPI) or GFP to be translated from the ORF73 mRNA. (B) DNA from BAC-cloned viral genomes (BAC) or virus-infected cells (VIR) was digested with NcoI, electrophoresed, transferred to nylon membranes, and blotted with a probe corresponding to the BamHI-G genomic fragment shown in (A). The predicted bands for WT virus were 1,021 bp, 3,121 bp, and 4,630 bp. The IRES-GFP insert introduced an NcoI site such that the WT 3,121-bp band was cut into 2,975-bp and 1,466-bp fragments. The NcoI site was lost from the IRES-EPI insert, such that the WT 3,121-bp band became a 3,861-bp band. (C) BHK-21 cells were infected (0.01 PFU/cell) with WT, GFP, or EPI viruses as indicated. Plaque titres of cell cultures are shown with time after infection. (D) H2 b MEF-1 cells or L929-K b cells were left uninfected (UI) or infected for 2 h with MHV-68 expressing either OVA under a strong lytic promoter (OVA) or the SIINFEKL epitope of OVA as part of the ORF73-IRES-EPI construct (EPI). B3Z cells were then added, and 18 h later their beta-galactosidase response was assayed using chlorophenol-red-beta- D -galactoside substrate. Mean ± SD values of triplicate cultures are shown. The data are from one or two equivalent experiments. (E) A20-syndecan-1 cells were infected (20 PFU/cell) with GFP − WT virus, WT virus with an HCMV IE1 promoter-driven GFP expression cassette (HCMV IE1-GFP), or with the ORF73-IRES-GFP virus. The numbers indicate the percentage of total cells in the gated region (GFP + ). Expression from the HCMV IE1 promoter is probably limited to lytic infection, whereas ORF73 is expressed in latency.

    Techniques Used: Modification, BAC Assay, Clone Assay, Infection, Expressing, Construct

    7) Product Images from "Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry"

    Article Title: Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-14

    Reduction of CD163 expression by ADAM17 reduces PRRSV entry. (A) Detection of porcine CD163 expression in BHK-21 cells. BHK-21 cells were transfected with pCAGGS/CD163 or pCAGGS vector for 24 h. Cell lysates were subjected to reducing SDS-PAGE before
    Figure Legend Snippet: Reduction of CD163 expression by ADAM17 reduces PRRSV entry. (A) Detection of porcine CD163 expression in BHK-21 cells. BHK-21 cells were transfected with pCAGGS/CD163 or pCAGGS vector for 24 h. Cell lysates were subjected to reducing SDS-PAGE before

    Techniques Used: Expressing, Transfection, Plasmid Preparation, SDS Page

    8) Product Images from "Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿"

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿

    Journal:

    doi: 10.1128/JVI.01011-08

    Analysis of G3BP1 and G3BP2 relocalization in cells infected with SINV/nsP3Cherry variant. The stable cell line, expressing G3BP1/GFP or BHK-21 cells transiently expressing G3BP2/GFP (see Materials and Methods for details), were infected with SINV/nsP3Cherry
    Figure Legend Snippet: Analysis of G3BP1 and G3BP2 relocalization in cells infected with SINV/nsP3Cherry variant. The stable cell line, expressing G3BP1/GFP or BHK-21 cells transiently expressing G3BP2/GFP (see Materials and Methods for details), were infected with SINV/nsP3Cherry

    Techniques Used: Infection, Variant Assay, Stable Transfection, Expressing

    Analysis of nsP3/GFP distribution in different fractions of cell lysates. BHK-21 cells were infected with packaged SINrep(nsP3GFP) at an MOI of 20 inf.u./cell. At 6 h postinfection, cells were harvested and homogenized. A nucleus-containing (NUC) fraction
    Figure Legend Snippet: Analysis of nsP3/GFP distribution in different fractions of cell lysates. BHK-21 cells were infected with packaged SINrep(nsP3GFP) at an MOI of 20 inf.u./cell. At 6 h postinfection, cells were harvested and homogenized. A nucleus-containing (NUC) fraction

    Techniques Used: Infection

    Localization of dsRNA- and nsP3/GFP-containing protein complexes in BHK-21 cells infected with SINV/nsP3GFP. (A) BHK-21 cells infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell. At 4 h postinfection, the cells were fixed with 3% paraformaldehyde,
    Figure Legend Snippet: Localization of dsRNA- and nsP3/GFP-containing protein complexes in BHK-21 cells infected with SINV/nsP3GFP. (A) BHK-21 cells infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell. At 4 h postinfection, the cells were fixed with 3% paraformaldehyde,

    Techniques Used: Infection

    Analysis of the colocalization of nsP3/GFP and different, membrane-containing cellular organelles. (a and b) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell and stained with FM4-64FX reagent as described in Materials and
    Figure Legend Snippet: Analysis of the colocalization of nsP3/GFP and different, membrane-containing cellular organelles. (a and b) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ∼20 PFU/cell and stained with FM4-64FX reagent as described in Materials and

    Techniques Used: Infection, Staining

    Identification of the viral and cellular components in the nsP3-containing protein complexes. (A) Schematic representation of the SINV replicons used in the study. (B) BHK-21 cell were infected with the indicated, packaged replicons at an MOI of 20 infectious
    Figure Legend Snippet: Identification of the viral and cellular components in the nsP3-containing protein complexes. (A) Schematic representation of the SINV replicons used in the study. (B) BHK-21 cell were infected with the indicated, packaged replicons at an MOI of 20 infectious

    Techniques Used: Infection

    9) Product Images from "Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H ▿"

    Article Title: Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H ▿

    Journal:

    doi: 10.1128/JVI.01616-06

    Decreased cell binding by gL-deficient MHV-68 mutants. A. BHK-21 cells were exposed to gL − or gL + viruses (2 PFU/cell) at 37°C for the times indicated and then fixed, permeabilized, and stained with the gN-specific MAb 3F7 plus
    Figure Legend Snippet: Decreased cell binding by gL-deficient MHV-68 mutants. A. BHK-21 cells were exposed to gL − or gL + viruses (2 PFU/cell) at 37°C for the times indicated and then fixed, permeabilized, and stained with the gN-specific MAb 3F7 plus

    Techniques Used: Binding Assay, Staining

    Growth of gL-deficient MHV-68 with allowance made for reduced cell binding. BHK-21 fibroblasts or NMuMG fibroblasts were infected at low multiplicity (0.01 PFU/cell) with gL − or gL + MHV-68 and then cultured without removing the virus inoculum.
    Figure Legend Snippet: Growth of gL-deficient MHV-68 with allowance made for reduced cell binding. BHK-21 fibroblasts or NMuMG fibroblasts were infected at low multiplicity (0.01 PFU/cell) with gL − or gL + MHV-68 and then cultured without removing the virus inoculum.

    Techniques Used: Binding Assay, Infection, Cell Culture

    Flow-cytometric identification of gL-dependent and gL-independent MAbs recognizing the MHV-68 gH. BHK-21 cells were infected (18 h, 2 PFU/cell) with wild-type MHV-68 (BHK + vir). CHO-gH cells stably express a glycosylphosphatidylinositol-linked
    Figure Legend Snippet: Flow-cytometric identification of gL-dependent and gL-independent MAbs recognizing the MHV-68 gH. BHK-21 cells were infected (18 h, 2 PFU/cell) with wild-type MHV-68 (BHK + vir). CHO-gH cells stably express a glycosylphosphatidylinositol-linked

    Techniques Used: Flow Cytometry, Infection, Stable Transfection

    Glycoprotein expression by gL-deficient MHV-68. A. BHK-21 cells were left uninfected (UI) or infected (1 PFU, 24 to 48 h) with gL − or gL + viruses as indicated. The cells were then trypsinized and stained for viral glycoproteins. nil, secondary
    Figure Legend Snippet: Glycoprotein expression by gL-deficient MHV-68. A. BHK-21 cells were left uninfected (UI) or infected (1 PFU, 24 to 48 h) with gL − or gL + viruses as indicated. The cells were then trypsinized and stained for viral glycoproteins. nil, secondary

    Techniques Used: Expressing, Infection, Staining

    Infectivity assays with gL-deficient MHV-68 mutants. A. BHK-21 cells were infected (0.01 PFU/cell, 1 h, 37°C) with wild-type (WT) or gL - DEL MHV-68. Infectious virus in replicate cultures was determined thereafter by plaque assay. B. BHK-21 cells
    Figure Legend Snippet: Infectivity assays with gL-deficient MHV-68 mutants. A. BHK-21 cells were infected (0.01 PFU/cell, 1 h, 37°C) with wild-type (WT) or gL - DEL MHV-68. Infectious virus in replicate cultures was determined thereafter by plaque assay. B. BHK-21 cells

    Techniques Used: Infection, Plaque Assay

    Tracking cell binding with eGFP-tagged MHV-68. A. BHK-21 cells were exposed to either wild-type (WT) or gL-deficient versions of MHV-68 with eGFP-tagged gM (2 PFU/cell) and then cultured at 37°C. The cells were washed with PBS or at low pH after
    Figure Legend Snippet: Tracking cell binding with eGFP-tagged MHV-68. A. BHK-21 cells were exposed to either wild-type (WT) or gL-deficient versions of MHV-68 with eGFP-tagged gM (2 PFU/cell) and then cultured at 37°C. The cells were washed with PBS or at low pH after

    Techniques Used: Binding Assay, Cell Culture

    10) Product Images from "Non-AUG-Initiated Internal Translation of the L* Protein of Theiler's Virus and Importance of This Protein for Viral Persistence"

    Article Title: Non-AUG-Initiated Internal Translation of the L* Protein of Theiler's Virus and Importance of This Protein for Viral Persistence

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.10665-10673.2002

    Detection by Western blotting of L* protein expression in infected BHK-21 cells. (A) Comparison of the amounts of L* protein produced by viruses with AUG or ACG: wild type virus DA1 and mutant viruses OV23, OV42, and OV46. (B) Comparison of the viruses in panel A and OV41, OV84, OV89, and OV90 mutant viruses. Note that samples from DA1- and OV42-infected cells were diluted 100-fold to allow a better molecular mass comparison. (C) Comparison of recombinant virus R2 and the corresponding stop codon mutant virus, OV57. The arrowhead points to a nonspecific band detected in both infected and mock-infected cell extracts. T-, mock-infected cell extract.
    Figure Legend Snippet: Detection by Western blotting of L* protein expression in infected BHK-21 cells. (A) Comparison of the amounts of L* protein produced by viruses with AUG or ACG: wild type virus DA1 and mutant viruses OV23, OV42, and OV46. (B) Comparison of the viruses in panel A and OV41, OV84, OV89, and OV90 mutant viruses. Note that samples from DA1- and OV42-infected cells were diluted 100-fold to allow a better molecular mass comparison. (C) Comparison of recombinant virus R2 and the corresponding stop codon mutant virus, OV57. The arrowhead points to a nonspecific band detected in both infected and mock-infected cell extracts. T-, mock-infected cell extract.

    Techniques Used: Western Blot, Expressing, Infection, Produced, Mutagenesis, Recombinant

    Mixed infections of fibroblast and macrophage cell lines. A mixture of two viruses (1:1) was prepared and used to infect BHK-21 and RAW264.7 (RAW) cells. RNA was extracted from infected cells after one to five passages of the virus mixture. As a control, RNA was extracted from the virus mixture before infection and from the parental virus stocks. The L* region was amplified by RT-PCR and digested with a restriction enzyme, allowing discrimination between the two viruses present in the mixture. (Left panels) Control analysis of the parental viruses and of the mixture used. (Right panels) Analysis of the virus mixture contained in the infected cells after one (P1) to five (P5) passages. Arrowheads indicate the fragments that are diagnostic of a given virus. (A) Competition between OV48, an AUG-to-ACG mutant, and OV42, a stop codon 93 mutant of DA1. Tru I was used to digest the PCR fragments. The sizes of the fragments are indicated in base pairs on the left. (B) Competition between GDVII and its stop codon derivative, OV47. Hsp 92II was used to digest the PCR fragments.
    Figure Legend Snippet: Mixed infections of fibroblast and macrophage cell lines. A mixture of two viruses (1:1) was prepared and used to infect BHK-21 and RAW264.7 (RAW) cells. RNA was extracted from infected cells after one to five passages of the virus mixture. As a control, RNA was extracted from the virus mixture before infection and from the parental virus stocks. The L* region was amplified by RT-PCR and digested with a restriction enzyme, allowing discrimination between the two viruses present in the mixture. (Left panels) Control analysis of the parental viruses and of the mixture used. (Right panels) Analysis of the virus mixture contained in the infected cells after one (P1) to five (P5) passages. Arrowheads indicate the fragments that are diagnostic of a given virus. (A) Competition between OV48, an AUG-to-ACG mutant, and OV42, a stop codon 93 mutant of DA1. Tru I was used to digest the PCR fragments. The sizes of the fragments are indicated in base pairs on the left. (B) Competition between GDVII and its stop codon derivative, OV47. Hsp 92II was used to digest the PCR fragments.

    Techniques Used: Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay, Mutagenesis, Polymerase Chain Reaction

    ). The 5′ end of the L* ORF, followed by a Bam HI restriction site, is fused in frame with the second codon of the firefly luciferase gene. The polyprotein and L* AUG initiation codons are shown in bold. The Kozak context sequence of the first L* AUG is underlined. The sequence of the firefly luciferase gene is italicized. The different codon combinations replacing AUG 1 and AUG 5 of L* are shown under the sequence. NC, noncoding region. Nucleotides of the L*-luciferase gene fusion are in uppercase. Nucleotides of the main ORF upstream of the L* ORF are in lowercase. (B) Luciferase activity expressed by the different constructs in BHK-21 cells. The bicistronic constructs shown in panel A were transfected into BHK-21 cells. At 24 h posttransfection, the level of expression of both luciferase genes was measured. The firefly/ Renilla luciferase expression ratio (construct:control) was calculated and normalized to the value obtained for the AUG 1 -AUG 5 combination. The histograms show means and standard deviations for at least three transfection experiments.
    Figure Legend Snippet: ). The 5′ end of the L* ORF, followed by a Bam HI restriction site, is fused in frame with the second codon of the firefly luciferase gene. The polyprotein and L* AUG initiation codons are shown in bold. The Kozak context sequence of the first L* AUG is underlined. The sequence of the firefly luciferase gene is italicized. The different codon combinations replacing AUG 1 and AUG 5 of L* are shown under the sequence. NC, noncoding region. Nucleotides of the L*-luciferase gene fusion are in uppercase. Nucleotides of the main ORF upstream of the L* ORF are in lowercase. (B) Luciferase activity expressed by the different constructs in BHK-21 cells. The bicistronic constructs shown in panel A were transfected into BHK-21 cells. At 24 h posttransfection, the level of expression of both luciferase genes was measured. The firefly/ Renilla luciferase expression ratio (construct:control) was calculated and normalized to the value obtained for the AUG 1 -AUG 5 combination. The histograms show means and standard deviations for at least three transfection experiments.

    Techniques Used: Luciferase, Sequencing, Activity Assay, Construct, Transfection, Expressing

    11) Product Images from "Herpes Simplex Virus 1 Targets the Murine Olfactory Neuroepithelium for Host Entry"

    Article Title: Herpes Simplex Virus 1 Targets the Murine Olfactory Neuroepithelium for Host Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01748-13

    HSV-1 infection of the infant olfactory neuroepithelium. (a) One- to two-week-old mice were infected i.n. with HSV-eGFP (10 6 PFU in 2 μl). Three days later, nose sections were stained for eGFP (green) and α-tubulin (red). Nuclei were stained with DAPI (blue). Light-gray-filled arrows show α-tubulin staining on the apical neuronal cilia; dark-gray-filled arrows show eGFP-positive neurons; white arrows show subepithelial eGFP-positive cells. (b) Mice were infected as described for panel a. Three days later, nose sections were stained for viral antigens (green) and α-tubulin (red). Nuclei were stained with DAPI (blue). The right-hand image shows the boxed region of the corresponding left-hand image at higher magnification. The gray-filled arrows show examples of eGFP-positive neuroepithelial cells. (c) One- to two-week-old BALB/c mice were infected i.n. with HSV-eGFP (10 6 PFU in 2 μl). One day later, noses were stained for eGFP (green), HSV-1 virion antigens (red), and α-tubulin (white). Nuclei were stained with DAPI (blue). Panels i, ii, and iii show different regions of nasal epithelia, either olfactory (OE) or respiratory (RE). Squamous epithelium showed no staining. The boxed regions are shown at higher magnification with individual channels below. (d) HSV-GFP and eGFP-expressing MuHV-4 were incubated with various concentrations of heparin (1 h, 37°C) and then added to BHK-21 cells (0.5 PFU/cell, 37°C). Eighteen hours later, cells were scored as eGFP positive or negative by flow cytometry. Each point shows means ± SD from 3 experiments, with 20,000 cells analyzed for each point in each experiment. The inhibition of HSV-1 infection by heparin was statistically significant at all doses of > 10 μg/ml ( P
    Figure Legend Snippet: HSV-1 infection of the infant olfactory neuroepithelium. (a) One- to two-week-old mice were infected i.n. with HSV-eGFP (10 6 PFU in 2 μl). Three days later, nose sections were stained for eGFP (green) and α-tubulin (red). Nuclei were stained with DAPI (blue). Light-gray-filled arrows show α-tubulin staining on the apical neuronal cilia; dark-gray-filled arrows show eGFP-positive neurons; white arrows show subepithelial eGFP-positive cells. (b) Mice were infected as described for panel a. Three days later, nose sections were stained for viral antigens (green) and α-tubulin (red). Nuclei were stained with DAPI (blue). The right-hand image shows the boxed region of the corresponding left-hand image at higher magnification. The gray-filled arrows show examples of eGFP-positive neuroepithelial cells. (c) One- to two-week-old BALB/c mice were infected i.n. with HSV-eGFP (10 6 PFU in 2 μl). One day later, noses were stained for eGFP (green), HSV-1 virion antigens (red), and α-tubulin (white). Nuclei were stained with DAPI (blue). Panels i, ii, and iii show different regions of nasal epithelia, either olfactory (OE) or respiratory (RE). Squamous epithelium showed no staining. The boxed regions are shown at higher magnification with individual channels below. (d) HSV-GFP and eGFP-expressing MuHV-4 were incubated with various concentrations of heparin (1 h, 37°C) and then added to BHK-21 cells (0.5 PFU/cell, 37°C). Eighteen hours later, cells were scored as eGFP positive or negative by flow cytometry. Each point shows means ± SD from 3 experiments, with 20,000 cells analyzed for each point in each experiment. The inhibition of HSV-1 infection by heparin was statistically significant at all doses of > 10 μg/ml ( P

    Techniques Used: Infection, Mouse Assay, Staining, Expressing, Incubation, Flow Cytometry, Cytometry, Inhibition

    12) Product Images from "The Murine Gammaherpesvirus 68 ORF27 Gene Product Contributes to Intercellular Viral Spread"

    Article Title: The Murine Gammaherpesvirus 68 ORF27 Gene Product Contributes to Intercellular Viral Spread

    Journal:

    doi: 10.1128/JVI.79.8.5059-5068.2005

    ORF27 mutant viruses showed impaired low-multiplicity growth in BHK-21 cells. (A) BHK-21 cells were infected at a low multiplicity (0.01 PFU/cell) with ORF27 + (WT and REV) and ORF27 − (FRT and STOP) viruses as indicated. The amounts of
    Figure Legend Snippet: ORF27 mutant viruses showed impaired low-multiplicity growth in BHK-21 cells. (A) BHK-21 cells were infected at a low multiplicity (0.01 PFU/cell) with ORF27 + (WT and REV) and ORF27 − (FRT and STOP) viruses as indicated. The amounts of

    Techniques Used: Mutagenesis, Infection

    The mature ORF27 gene product is a 48-kDa glycoprotein. (A) BHK-21 cells were infected with MHV-68 (2 PFU/cell), and 18 h later, were labeled with [ 35 S]cysteine-methionine for 1 h. ORF27 was then immunoprecipitated with the T6E11 or T8H3 MAb. Immunoprecipitations
    Figure Legend Snippet: The mature ORF27 gene product is a 48-kDa glycoprotein. (A) BHK-21 cells were infected with MHV-68 (2 PFU/cell), and 18 h later, were labeled with [ 35 S]cysteine-methionine for 1 h. ORF27 was then immunoprecipitated with the T6E11 or T8H3 MAb. Immunoprecipitations

    Techniques Used: Infection, Labeling, Immunoprecipitation

    Absence of gp48 expression after disruption of ORF27. (A) BHK-21 cells were infected (18 h, 2 PFU/cell) as indicated and assayed for cell surface gp48 expression by flow cytometric staining with the MAbs T8A11 and T6E11. The gp150-specific MAb T4G2 was
    Figure Legend Snippet: Absence of gp48 expression after disruption of ORF27. (A) BHK-21 cells were infected (18 h, 2 PFU/cell) as indicated and assayed for cell surface gp48 expression by flow cytometric staining with the MAbs T8A11 and T6E11. The gp150-specific MAb T4G2 was

    Techniques Used: Expressing, Infection, Flow Cytometry, Staining

    13) Product Images from "Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral LatencyA Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA ProteinsMolecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANA Research"

    Article Title: Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral LatencyA Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA ProteinsMolecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANA Research

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003673

    mLANA DNA binding is essential for virus persistence and the dorsal positive patch exerts a role in the expansion of GC B cells. ( A ) Amino acid substitutions in recombinant viruses (see also Figure S5 ). ( B ) Infection of BHK-21 cells at 0.01 p.f.u. per cell. Virus titres were determined by plaque assay. ( C ) Lungs from infected mice were removed and infectious viruses were titrated by plaque assay. ( D and E ) Quantification of latent infection in spleen by explant co-culture plaque assay (closed circles). Titres of infectious virus were determined in freeze/thawed splenocyte suspensions (open circles). Each circle represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. Mutant viruses are shown in panel D and revertant viruses in panel E. ( F and G ) Reciprocal frequencies of viral DNA-positive cells in total splenocytes ( F ) or GC B cells (CD19 + CD95 hi GL7 hi ) ( G ) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. ( H ) Identification of latently infected cells in spleens by in situ hybridization. Representative splenic sections from each group of viruses are shown. All images are magnified ×200. Dark staining indicates cells positive for virally encoded miRNAs (see also Table 4 ).
    Figure Legend Snippet: mLANA DNA binding is essential for virus persistence and the dorsal positive patch exerts a role in the expansion of GC B cells. ( A ) Amino acid substitutions in recombinant viruses (see also Figure S5 ). ( B ) Infection of BHK-21 cells at 0.01 p.f.u. per cell. Virus titres were determined by plaque assay. ( C ) Lungs from infected mice were removed and infectious viruses were titrated by plaque assay. ( D and E ) Quantification of latent infection in spleen by explant co-culture plaque assay (closed circles). Titres of infectious virus were determined in freeze/thawed splenocyte suspensions (open circles). Each circle represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. Mutant viruses are shown in panel D and revertant viruses in panel E. ( F and G ) Reciprocal frequencies of viral DNA-positive cells in total splenocytes ( F ) or GC B cells (CD19 + CD95 hi GL7 hi ) ( G ) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. ( H ) Identification of latently infected cells in spleens by in situ hybridization. Representative splenic sections from each group of viruses are shown. All images are magnified ×200. Dark staining indicates cells positive for virally encoded miRNAs (see also Table 4 ).

    Techniques Used: Binding Assay, Recombinant, Infection, Plaque Assay, Mouse Assay, Co-Culture Assay, Mutagenesis, Real-time Polymerase Chain Reaction, In Situ Hybridization, Staining

    14) Product Images from "Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus Inhibits the Antiviral Function of Interferon-Stimulated Gene 15"

    Article Title: Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus Inhibits the Antiviral Function of Interferon-Stimulated Gene 15

    Journal: Journal of Virology

    doi: 10.1128/JVI.06466-11

    Effect of ISG15 conjugation on PRRSV replication. BHK-21 cells were transfected with plasmids that express conjugation enzymes E1/E2/E3 and Flag-tagged ISG15 or ISG15 mutant (ISG15AA). Empty vector plasmid p3xFlag was used as a negative control. At 6
    Figure Legend Snippet: Effect of ISG15 conjugation on PRRSV replication. BHK-21 cells were transfected with plasmids that express conjugation enzymes E1/E2/E3 and Flag-tagged ISG15 or ISG15 mutant (ISG15AA). Empty vector plasmid p3xFlag was used as a negative control. At 6

    Techniques Used: Conjugation Assay, Transfection, Mutagenesis, Plasmid Preparation, Negative Control

    15) Product Images from "Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome MaintenanceHow a Latent Virus Eludes Immune Defenses"

    Article Title: Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome MaintenanceHow a Latent Virus Eludes Immune Defenses

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0030120

    Modification of the MHV-68 Genome to Overcome cis- Acting Immune Evasion by ORF73 (A) An IRES element was inserted just downstream of ORF73, between its stop codon and that of M11. This allowed either three tandem CD8 + T cell epitopes (EPI) or GFP to be translated from the ORF73 mRNA. (B) DNA from BAC-cloned viral genomes (BAC) or virus-infected cells (VIR) was digested with NcoI, electrophoresed, transferred to nylon membranes, and blotted with a probe corresponding to the BamHI-G genomic fragment shown in (A). The predicted bands for WT virus were 1,021 bp, 3,121 bp, and 4,630 bp. The IRES-GFP insert introduced an NcoI site such that the WT 3,121-bp band was cut into 2,975-bp and 1,466-bp fragments. The NcoI site was lost from the IRES-EPI insert, such that the WT 3,121-bp band became a 3,861-bp band. (C) BHK-21 cells were infected (0.01 PFU/cell) with WT, GFP, or EPI viruses as indicated. Plaque titres of cell cultures are shown with time after infection. (D) H2 b MEF-1 cells or L929-K b cells were left uninfected (UI) or infected for 2 h with MHV-68 expressing either OVA under a strong lytic promoter (OVA) or the SIINFEKL epitope of OVA as part of the ORF73-IRES-EPI construct (EPI). B3Z cells were then added, and 18 h later their beta-galactosidase response was assayed using chlorophenol-red-beta- D -galactoside substrate. Mean ± SD values of triplicate cultures are shown. The data are from one or two equivalent experiments. (E) A20-syndecan-1 cells were infected (20 PFU/cell) with GFP − WT virus, WT virus with an HCMV IE1 promoter-driven GFP expression cassette (HCMV IE1-GFP), or with the ORF73-IRES-GFP virus. The numbers indicate the percentage of total cells in the gated region (GFP + ). Expression from the HCMV IE1 promoter is probably limited to lytic infection, whereas ORF73 is expressed in latency.
    Figure Legend Snippet: Modification of the MHV-68 Genome to Overcome cis- Acting Immune Evasion by ORF73 (A) An IRES element was inserted just downstream of ORF73, between its stop codon and that of M11. This allowed either three tandem CD8 + T cell epitopes (EPI) or GFP to be translated from the ORF73 mRNA. (B) DNA from BAC-cloned viral genomes (BAC) or virus-infected cells (VIR) was digested with NcoI, electrophoresed, transferred to nylon membranes, and blotted with a probe corresponding to the BamHI-G genomic fragment shown in (A). The predicted bands for WT virus were 1,021 bp, 3,121 bp, and 4,630 bp. The IRES-GFP insert introduced an NcoI site such that the WT 3,121-bp band was cut into 2,975-bp and 1,466-bp fragments. The NcoI site was lost from the IRES-EPI insert, such that the WT 3,121-bp band became a 3,861-bp band. (C) BHK-21 cells were infected (0.01 PFU/cell) with WT, GFP, or EPI viruses as indicated. Plaque titres of cell cultures are shown with time after infection. (D) H2 b MEF-1 cells or L929-K b cells were left uninfected (UI) or infected for 2 h with MHV-68 expressing either OVA under a strong lytic promoter (OVA) or the SIINFEKL epitope of OVA as part of the ORF73-IRES-EPI construct (EPI). B3Z cells were then added, and 18 h later their beta-galactosidase response was assayed using chlorophenol-red-beta- D -galactoside substrate. Mean ± SD values of triplicate cultures are shown. The data are from one or two equivalent experiments. (E) A20-syndecan-1 cells were infected (20 PFU/cell) with GFP − WT virus, WT virus with an HCMV IE1 promoter-driven GFP expression cassette (HCMV IE1-GFP), or with the ORF73-IRES-GFP virus. The numbers indicate the percentage of total cells in the gated region (GFP + ). Expression from the HCMV IE1 promoter is probably limited to lytic infection, whereas ORF73 is expressed in latency.

    Techniques Used: Modification, BAC Assay, Clone Assay, Infection, Expressing, Construct

    Related Articles

    Transfection:

    Article Title: Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene
    Article Snippet: .. In brief, BHK-21 cells grown to 80% confluence were transfected with 4 μg of pBAC-OC43FL , pBAC-OC43-ns2DelRluc, pBAC-OC43-ns2FusionRluc, pBAC-OC43-ns12.9StopRluc, or pBAC-OC43-ns12.9FusionRluc using the X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer's instructions. .. After incubation for 6 h at 37°C in a humidified 5% CO2 incubator, the transfected cells were washed three times with DMEM and maintained in DMEM supplemented with 2% FBS for 72 h at 37°C and an additional 96 h at 33°C.

    Article Title: A WASp-VASP complex regulates actin polymerization at the plasma membrane
    Article Snippet: .. BHK-21 cells were transfected with Fugene 6 (Roche) according to the manufacturer’s instructions. .. Cells were fixed 40 h post-transfection and processed for immunofluorescence labelling with 9E10 anti-myc tag monoclonal antibody to recognize epitope-tagged FRB–WASp418–502 and Scar-WA ( ) constructs and with Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR) to label actin structures.

    Article Title: Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells
    Article Snippet: .. Mutant viruses were reconstituted by transfection of mutant BACs into BHK-21 cells using FuGENE 6 transfection reagent (Roche). .. To ascertain that the mutant viruses carried the correct mutations, DNA was extracted from virion preparations using the Wizard genomic DNA purification kit (Promega) and analyzed with the diagnostic PCR described above and by Southern blotting.

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Rab5a/Cherry- and Rab7/Cherry-expressing cell lines were generated by the transfection of pRab5a/Cherry or pRab7/Cherry, along with the pMAM/Neo (Clonetech) plasmid (10:1), into BHK-21 cells using a FuGene 6 reagent according to the manufacturer's instructions (Roche), followed by selection of the G418-resistant cells. .. To avoid a possible negative effect of overproduction of the proteins, only the cell colonies with a moderate level of expression were used in further experiments.

    Article Title: Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry
    Article Snippet: .. Marc-145 or BHK-21 cells were transfected with 2 μg of target plasmid pCAGGS/ADAM17, pCAGGS/CD163, and vector control pCAGGS using X-tremeGENE transfection reagent (Roche). .. At 24 h posttransfection, the cells were inoculated with PRRSV HuN4 at an MOI of 0.1, and the infection of PRRSV was detected by IFA as described above.

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Therefore, the corresponding plasmids were usually transfected into BHK-21 cells using FuGene 6 or Lipofectamine 2000 reagent according to the manufacturers' instructions (Roche and Invitrogen, respectively), and then, in 18 h, we employed puromycin selection. .. In standard experiments, ∼80% of the cells were Purr , and the untransfected cells died within 24 h after the start of puromycin selection.

    Article Title: A Novel Neutralizing Antibody Targeting a Unique Cross-Reactive Epitope on the hi Loop of Domain II of the Envelope Protein Protects Mice against Duck Tembusu Virus
    Article Snippet: .. For transfection of the plasmids pCDNA-Ew and pCDNA-Ez, monolayers of BHK-21 cells in 35-mm–diameter culture dishes were transfected with the plasmids by using the FuGENE HD Transfection Reagent (Roche Applied Science) according to the manufacturer’s instructions. .. Plaque reduction neutralization testA standard plaque reduction neutralization test (PRNT) was performed using 24-well plates as described previously ( ). mAb 1G2 was purified using protein G agarose (Invitrogen) according to the manufacturer’s instructions.

    Selection:

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Rab5a/Cherry- and Rab7/Cherry-expressing cell lines were generated by the transfection of pRab5a/Cherry or pRab7/Cherry, along with the pMAM/Neo (Clonetech) plasmid (10:1), into BHK-21 cells using a FuGene 6 reagent according to the manufacturer's instructions (Roche), followed by selection of the G418-resistant cells. .. To avoid a possible negative effect of overproduction of the proteins, only the cell colonies with a moderate level of expression were used in further experiments.

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Therefore, the corresponding plasmids were usually transfected into BHK-21 cells using FuGene 6 or Lipofectamine 2000 reagent according to the manufacturers' instructions (Roche and Invitrogen, respectively), and then, in 18 h, we employed puromycin selection. .. In standard experiments, ∼80% of the cells were Purr , and the untransfected cells died within 24 h after the start of puromycin selection.

    Mutagenesis:

    Article Title: Glycoprotein B Cleavage Is Important for Murid Herpesvirus 4 To Infect Myeloid Cells
    Article Snippet: .. Mutant viruses were reconstituted by transfection of mutant BACs into BHK-21 cells using FuGENE 6 transfection reagent (Roche). .. To ascertain that the mutant viruses carried the correct mutations, DNA was extracted from virion preparations using the Wizard genomic DNA purification kit (Promega) and analyzed with the diagnostic PCR described above and by Southern blotting.

    Generated:

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Rab5a/Cherry- and Rab7/Cherry-expressing cell lines were generated by the transfection of pRab5a/Cherry or pRab7/Cherry, along with the pMAM/Neo (Clonetech) plasmid (10:1), into BHK-21 cells using a FuGene 6 reagent according to the manufacturer's instructions (Roche), followed by selection of the G418-resistant cells. .. To avoid a possible negative effect of overproduction of the proteins, only the cell colonies with a moderate level of expression were used in further experiments.

    BAC Assay:

    Article Title: Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome MaintenanceHow a Latent Virus Eludes Immune Defenses
    Article Snippet: .. All BACs were reconstituted into infectious virus by transfecting 5 μg of BAC DNA into BHK-21 cells with Fugene-6 (Roche Diagnostics). .. The BAC cassette was removed by serial viral passage through NIH-3T3-CRE cells.

    Plasmid Preparation:

    Article Title: Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells ▿
    Article Snippet: .. Rab5a/Cherry- and Rab7/Cherry-expressing cell lines were generated by the transfection of pRab5a/Cherry or pRab7/Cherry, along with the pMAM/Neo (Clonetech) plasmid (10:1), into BHK-21 cells using a FuGene 6 reagent according to the manufacturer's instructions (Roche), followed by selection of the G418-resistant cells. .. To avoid a possible negative effect of overproduction of the proteins, only the cell colonies with a moderate level of expression were used in further experiments.

    Article Title: Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry
    Article Snippet: .. Marc-145 or BHK-21 cells were transfected with 2 μg of target plasmid pCAGGS/ADAM17, pCAGGS/CD163, and vector control pCAGGS using X-tremeGENE transfection reagent (Roche). .. At 24 h posttransfection, the cells were inoculated with PRRSV HuN4 at an MOI of 0.1, and the infection of PRRSV was detected by IFA as described above.

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    Roche confluent bhk 21 cells
    Infectious properties of WNV RRPs in Vero, HEK-293, <t>BHK-21</t> and SK-N-SH cells. ( a ) Expression of the GFP reporter gene in each cell line after the infection of equal amounts of RRPs. ( b ) Monolayers of each cell line in a six-well plate containing 10 5 to 10 6 cells/well were infected in parallel with 10-fold serial dilutions of WNV RRPs. Cells positive for GFP protein were counted at the appropriate dilution and the number of RRPs per 1 mL of supernatant were calculated.
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    Infectious properties of WNV RRPs in Vero, HEK-293, BHK-21 and SK-N-SH cells. ( a ) Expression of the GFP reporter gene in each cell line after the infection of equal amounts of RRPs. ( b ) Monolayers of each cell line in a six-well plate containing 10 5 to 10 6 cells/well were infected in parallel with 10-fold serial dilutions of WNV RRPs. Cells positive for GFP protein were counted at the appropriate dilution and the number of RRPs per 1 mL of supernatant were calculated.

    Journal: Scientific Reports

    Article Title: West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

    doi: 10.1038/s41598-017-03670-4

    Figure Lengend Snippet: Infectious properties of WNV RRPs in Vero, HEK-293, BHK-21 and SK-N-SH cells. ( a ) Expression of the GFP reporter gene in each cell line after the infection of equal amounts of RRPs. ( b ) Monolayers of each cell line in a six-well plate containing 10 5 to 10 6 cells/well were infected in parallel with 10-fold serial dilutions of WNV RRPs. Cells positive for GFP protein were counted at the appropriate dilution and the number of RRPs per 1 mL of supernatant were calculated.

    Article Snippet: A monolayer or about 90% confluent BHK-21 cells were transfected with the pCAG -WNV-CME plasmid using FuGENE HD transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Expressing, Infection

    Immunofluorescence and Western blotting analyses of C, prM and E protein expression in BWNV-CME cells. ( a ) Immunofluorescence analysis of BWNV-CME cells with monoclonal antibodies against the C, prM and E proteins. ( b ) BWNV-CME cell lysates were analysed by Western blotting with MAbs against C, prM and E. The cell lysates of BHK-21 cells were used as a negative control. The relative locations of the WNV E, C-PrM and PrM proteins are indicated on the right. Full-length blots are presented in Supplementary Figure 1 .

    Journal: Scientific Reports

    Article Title: West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

    doi: 10.1038/s41598-017-03670-4

    Figure Lengend Snippet: Immunofluorescence and Western blotting analyses of C, prM and E protein expression in BWNV-CME cells. ( a ) Immunofluorescence analysis of BWNV-CME cells with monoclonal antibodies against the C, prM and E proteins. ( b ) BWNV-CME cell lysates were analysed by Western blotting with MAbs against C, prM and E. The cell lysates of BHK-21 cells were used as a negative control. The relative locations of the WNV E, C-PrM and PrM proteins are indicated on the right. Full-length blots are presented in Supplementary Figure 1 .

    Article Snippet: A monolayer or about 90% confluent BHK-21 cells were transfected with the pCAG -WNV-CME plasmid using FuGENE HD transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Immunofluorescence, Western Blot, Expressing, Negative Control

    Production of RRPs from the WNV replicon plasmid by transfecting ( a ) BWNV-ME and ( b ) BWNV-CME cell lines. Green florescence was visualized when the replicon plasmid pWNVrepdCME-GFP was transfected into BWNV-ME cells (2), BHK-21 cells (11), BWNV-CME cells (10) or BWNV-ME cells together with the pCAG-WNV-C plasmid (1). The transfected cell supernatants were passaged onto fresh cell cultures as indicated by the arrows, and green florescence was observed only in the cells labelled 4 and 12. Three days post-inoculation, the supernatants of the cells infected first were used to inoculate the cells as indicated by the arrows for a second infection. Green florescence was analysed 72 h post-infection.

    Journal: Scientific Reports

    Article Title: West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

    doi: 10.1038/s41598-017-03670-4

    Figure Lengend Snippet: Production of RRPs from the WNV replicon plasmid by transfecting ( a ) BWNV-ME and ( b ) BWNV-CME cell lines. Green florescence was visualized when the replicon plasmid pWNVrepdCME-GFP was transfected into BWNV-ME cells (2), BHK-21 cells (11), BWNV-CME cells (10) or BWNV-ME cells together with the pCAG-WNV-C plasmid (1). The transfected cell supernatants were passaged onto fresh cell cultures as indicated by the arrows, and green florescence was observed only in the cells labelled 4 and 12. Three days post-inoculation, the supernatants of the cells infected first were used to inoculate the cells as indicated by the arrows for a second infection. Green florescence was analysed 72 h post-infection.

    Article Snippet: A monolayer or about 90% confluent BHK-21 cells were transfected with the pCAG -WNV-CME plasmid using FuGENE HD transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Plasmid Preparation, Transfection, Infection

    Schematic representation of WNV replicon constructs and packaging of WNV reporter replicon particles (RRPs). ( a ) The DNA based WNV replicon is under control of the CMV promoter. The replicon genome lacks the major coding sequence of the structural protein, C-prM-E, and the corresponding sequence was replaced with a GFP coding sequence following the FMDV 2A coding sequence. The replicon RNA with an authentic 5′ terminus is ensured by placing a hammerhead ribozyme sequence (HRr) before the first 5′ UTR nucleotide. The authentic 3′ terminus is ensured by the addition of a hepatitis delta virus ribozyme sequence (HDVr) after the last 3′ UTR nucleotide. To complement the structural proteins, a capsid protein expressing plasmid was constructed. The BWNV-ME cell line stably expressing the prM-E protein was generated via transfecting BHK-21 cells with the pCAG-WNV-ME plasmid. The BWNV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-WNV-CME plasmid. ( b ) WNV RRPs are packaged in two ways. First, the pCAG-WNV-C and replicon plasmids were transfected into BWNV-ME cells. The pCAG-WNV-C plasmid expressed protein C. The cell itself expressed prM and E protein. The replicon plasmid was transcribed by the CMV promoter into the replicon RNA expressing GFP and the non-structural replicase protein. The replicon RNA amplifies itself once again and three structural proteins package the replicon RNA into WNV RRPs which are secreted into the culture medium. The second way is to transfect BWNV-CME cells with only the replicon plasmid. The BWNV-CME cells express the C-prM-E polyprotein which is cleaved into the C, prM and E proteins by replicon RNA-encoded non-structural protease and endogenous cellular signal peptidase (SP). Then three structural proteins package the replicon RNA into RRPs which is secreted into the culture medium. When RRPs infect BHK-21 cells, the replicon RNA expresses GFP and non-structural proteins which amplify more RNA. However, no WNV structural proteins or additional RRPs produced in RRP-infected cells, thereby preventing further spread. When BWNV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs and the infection spreads in rounds similar to the wild type virus.

    Journal: Scientific Reports

    Article Title: West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

    doi: 10.1038/s41598-017-03670-4

    Figure Lengend Snippet: Schematic representation of WNV replicon constructs and packaging of WNV reporter replicon particles (RRPs). ( a ) The DNA based WNV replicon is under control of the CMV promoter. The replicon genome lacks the major coding sequence of the structural protein, C-prM-E, and the corresponding sequence was replaced with a GFP coding sequence following the FMDV 2A coding sequence. The replicon RNA with an authentic 5′ terminus is ensured by placing a hammerhead ribozyme sequence (HRr) before the first 5′ UTR nucleotide. The authentic 3′ terminus is ensured by the addition of a hepatitis delta virus ribozyme sequence (HDVr) after the last 3′ UTR nucleotide. To complement the structural proteins, a capsid protein expressing plasmid was constructed. The BWNV-ME cell line stably expressing the prM-E protein was generated via transfecting BHK-21 cells with the pCAG-WNV-ME plasmid. The BWNV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-WNV-CME plasmid. ( b ) WNV RRPs are packaged in two ways. First, the pCAG-WNV-C and replicon plasmids were transfected into BWNV-ME cells. The pCAG-WNV-C plasmid expressed protein C. The cell itself expressed prM and E protein. The replicon plasmid was transcribed by the CMV promoter into the replicon RNA expressing GFP and the non-structural replicase protein. The replicon RNA amplifies itself once again and three structural proteins package the replicon RNA into WNV RRPs which are secreted into the culture medium. The second way is to transfect BWNV-CME cells with only the replicon plasmid. The BWNV-CME cells express the C-prM-E polyprotein which is cleaved into the C, prM and E proteins by replicon RNA-encoded non-structural protease and endogenous cellular signal peptidase (SP). Then three structural proteins package the replicon RNA into RRPs which is secreted into the culture medium. When RRPs infect BHK-21 cells, the replicon RNA expresses GFP and non-structural proteins which amplify more RNA. However, no WNV structural proteins or additional RRPs produced in RRP-infected cells, thereby preventing further spread. When BWNV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs and the infection spreads in rounds similar to the wild type virus.

    Article Snippet: A monolayer or about 90% confluent BHK-21 cells were transfected with the pCAG -WNV-CME plasmid using FuGENE HD transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Construct, Sequencing, Expressing, Plasmid Preparation, Stable Transfection, Generated, Transfection, Produced, Infection

    Propagation characteristics of RRPs in BWNV-CME and BHK-21 cells. ( a ) The spread of green fluorescence in BWNV-CME cells infected with RRPs. In RRP-infected BHK-21 cells, the green fluorescence expressing cells did not increase from 24 h post-infection. The BWNV-CME and BHK-21 cells were seeded into 24-well plates and inoculated with RRPs for the indicated time periods. For detection of plaques, the infected cells were overlaid with Eagle’s medium containing 1.5% carboxymethyl cellulose and 1% foetal calf serum. The morphological changes of one plaque were observed with a fluorescent microscope. ( b ) WNV plaque formation in WNV-CME cells. Six days post-infection, the plates were stained with 0.1% crystal violet solution for 15 min at room temperature. The stain was discarded and the cells were rinsed with tap water.

    Journal: Scientific Reports

    Article Title: West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

    doi: 10.1038/s41598-017-03670-4

    Figure Lengend Snippet: Propagation characteristics of RRPs in BWNV-CME and BHK-21 cells. ( a ) The spread of green fluorescence in BWNV-CME cells infected with RRPs. In RRP-infected BHK-21 cells, the green fluorescence expressing cells did not increase from 24 h post-infection. The BWNV-CME and BHK-21 cells were seeded into 24-well plates and inoculated with RRPs for the indicated time periods. For detection of plaques, the infected cells were overlaid with Eagle’s medium containing 1.5% carboxymethyl cellulose and 1% foetal calf serum. The morphological changes of one plaque were observed with a fluorescent microscope. ( b ) WNV plaque formation in WNV-CME cells. Six days post-infection, the plates were stained with 0.1% crystal violet solution for 15 min at room temperature. The stain was discarded and the cells were rinsed with tap water.

    Article Snippet: A monolayer or about 90% confluent BHK-21 cells were transfected with the pCAG -WNV-CME plasmid using FuGENE HD transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Fluorescence, Infection, Expressing, Microscopy, Staining

    In vitro ). TR, terminal repeats. (b, c) Detection of viral proteins in infected BHK-21 (3 PFU/cell, 6 h) cellular lysates using Western blotting (WB). kLANA was detected with MAb LN53, and mLANA was detected with MAb 6A3. Blotting against the MuHV-4 M3 protein and actin was used to assess levels of infection and loading, respectively. In panel b, expression of vCyclin and YFP was confirmed with anti-vCyclin and anti-GFP immunoblotting, respectively. (d) Images (confocal slices) depicting localization of LANA proteins in BHK-21 cells infected as described for panel a. DNA was stained with DAPI. Bar, 10 μm. (e) Relative levels (ΔΔ C T ) of M11, M3, and ORF63 in infected BHK-21 cells (5 PFU/cell, 8 h) compared to uninfected cells, normalized to GAPDH. Bars indicate mean fold changes ± standard deviations (SD). (f) Growth curves in BHK-21 cells infected with 0.01 PFU/cell. Total virus titers were determined. Time zero indicates input of virus after washing inoculum. Virus titers did not differ significantly between infection groups (Kruskal-Wallis test).

    Journal: Journal of Virology

    Article Title: In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4

    doi: 10.1128/JVI.01251-18

    Figure Lengend Snippet: In vitro ). TR, terminal repeats. (b, c) Detection of viral proteins in infected BHK-21 (3 PFU/cell, 6 h) cellular lysates using Western blotting (WB). kLANA was detected with MAb LN53, and mLANA was detected with MAb 6A3. Blotting against the MuHV-4 M3 protein and actin was used to assess levels of infection and loading, respectively. In panel b, expression of vCyclin and YFP was confirmed with anti-vCyclin and anti-GFP immunoblotting, respectively. (d) Images (confocal slices) depicting localization of LANA proteins in BHK-21 cells infected as described for panel a. DNA was stained with DAPI. Bar, 10 μm. (e) Relative levels (ΔΔ C T ) of M11, M3, and ORF63 in infected BHK-21 cells (5 PFU/cell, 8 h) compared to uninfected cells, normalized to GAPDH. Bars indicate mean fold changes ± standard deviations (SD). (f) Growth curves in BHK-21 cells infected with 0.01 PFU/cell. Total virus titers were determined. Time zero indicates input of virus after washing inoculum. Virus titers did not differ significantly between infection groups (Kruskal-Wallis test).

    Article Snippet: Viruses were reconstituted by transfection of recombinant BAC DNA into BHK-21 cells using X-tremeGENE HP (Roche).

    Techniques: In Vitro, Infection, Western Blot, Expressing, Staining

    eGFP-tagged ORF58 decorates the membrane fronds. A. Schematic diagram of the MHV-68 ORF58 and ORF27 loci. Filled arrowheads show restriction sites created by eGFP tagging ORF58 or disrupting ORF27. Open arrowheads show genomic restriction sites. The thick lines show probe locations. B. Southern blots of wild-type (WT), eGFP-tagged ORF58 (G58) and ORF27-deficient eGFP-tagged ORF28 (G58/27 − ) viruses. The Eco RI digest plus Bam HI-C probe gave predicted wild-type and G58 virus fragments of 5611bp, 2703bp, 744bp and 1342bp (difficult to see because of limited overlap with the probe). The Eco RI-restricted oligonucleotide introduced into ORF27 at genomic co-ordinate 45480 converted the 5611bp band to 4075bp plus 1536bp. The Bgl II digest plus Sac I/ Sac I/ Eco RI probe gave predicted WT fragments of 5300bp, 5165bp, 2294bp and 3379bp. The eGFP insertion just upstream of ORF58 converted the 3379 band to 1193bp+2916bp. C. BHK-21 cells were left uninfected or infected (0.5 p.f.u./cell, 16 h) with ORF27 + or ORF27 − versions of the eGFP-ORF58 virus. The cells were then stained for gN (mAb 3F7), gp150 (mAb T1A1) or gp48 (mAb T8H3), each with phycoerythrin-conjugated goat anti-mouse IgG pAb. The data are from 1 of 3 equivalent experiments. D. NIH-3T3 cells were infected with eGFP-ORF58 MHV-68 (1 p.f.u./cell, 16 h), then fixed, permeabilized and stained for gp48 with mAb 6D10 plus Alexa568-conjugated goat anti-mouse IgG pAb. EGFP fluorescence was visualized directly. Nuclei were counterstained with DAPI. The zoomed images correspond to the boxed regions in the left-hand panels. The data are from 1 of 5 equivalent experiments. E. BHK-21 cells were infected (0.01 p.f.u./cell) with ORF27 + or ORF27 − versions of either untagged (wild-type) or eGFP-ORF58-tagged MHV-68. Virus titers were determined by plaque assay at the times indicated. F. NIH-3T3 cells were infected with eGFP-ORF58 MHV-68 (1 p.f.u./cell, 16h) then examined by time-lapse confocal microscopy. An infected (inf) cell is shown next to an uninfected (UI) or only recently infected neighbour to demonstrate eGFP + fronds reaching from one to the other. Movie S5 shows the difference between these cells in shape and motility. The findings were typical of > 100 cells examined.

    Journal: PLoS ONE

    Article Title: A Gamma-Herpesvirus Glycoprotein Complex Manipulates Actin to Promote Viral Spread

    doi: 10.1371/journal.pone.0001808

    Figure Lengend Snippet: eGFP-tagged ORF58 decorates the membrane fronds. A. Schematic diagram of the MHV-68 ORF58 and ORF27 loci. Filled arrowheads show restriction sites created by eGFP tagging ORF58 or disrupting ORF27. Open arrowheads show genomic restriction sites. The thick lines show probe locations. B. Southern blots of wild-type (WT), eGFP-tagged ORF58 (G58) and ORF27-deficient eGFP-tagged ORF28 (G58/27 − ) viruses. The Eco RI digest plus Bam HI-C probe gave predicted wild-type and G58 virus fragments of 5611bp, 2703bp, 744bp and 1342bp (difficult to see because of limited overlap with the probe). The Eco RI-restricted oligonucleotide introduced into ORF27 at genomic co-ordinate 45480 converted the 5611bp band to 4075bp plus 1536bp. The Bgl II digest plus Sac I/ Sac I/ Eco RI probe gave predicted WT fragments of 5300bp, 5165bp, 2294bp and 3379bp. The eGFP insertion just upstream of ORF58 converted the 3379 band to 1193bp+2916bp. C. BHK-21 cells were left uninfected or infected (0.5 p.f.u./cell, 16 h) with ORF27 + or ORF27 − versions of the eGFP-ORF58 virus. The cells were then stained for gN (mAb 3F7), gp150 (mAb T1A1) or gp48 (mAb T8H3), each with phycoerythrin-conjugated goat anti-mouse IgG pAb. The data are from 1 of 3 equivalent experiments. D. NIH-3T3 cells were infected with eGFP-ORF58 MHV-68 (1 p.f.u./cell, 16 h), then fixed, permeabilized and stained for gp48 with mAb 6D10 plus Alexa568-conjugated goat anti-mouse IgG pAb. EGFP fluorescence was visualized directly. Nuclei were counterstained with DAPI. The zoomed images correspond to the boxed regions in the left-hand panels. The data are from 1 of 5 equivalent experiments. E. BHK-21 cells were infected (0.01 p.f.u./cell) with ORF27 + or ORF27 − versions of either untagged (wild-type) or eGFP-ORF58-tagged MHV-68. Virus titers were determined by plaque assay at the times indicated. F. NIH-3T3 cells were infected with eGFP-ORF58 MHV-68 (1 p.f.u./cell, 16h) then examined by time-lapse confocal microscopy. An infected (inf) cell is shown next to an uninfected (UI) or only recently infected neighbour to demonstrate eGFP + fronds reaching from one to the other. Movie S5 shows the difference between these cells in shape and motility. The findings were typical of > 100 cells examined.

    Article Snippet: Viruses were titered by plaque assay on BHK-21 cells .

    Techniques: Hi-C, Infection, Staining, Fluorescence, Plaque Assay, Confocal Microscopy

    Relationship between membrane fronds and virions. A. BHK-21 cells infected with MHV-68 expressing eGFP-tagged gM (1 p.f.u./cell, 16 h) were examined by time-lapse confocal microscopy. The eGFP signal appears as black/gray. Each zoomed image corresponds to the boxed region of the corresponding overview. The punctate fluorescence of distal membrane fronds is seen to change with time. See also Movies S1 and S2 . B. BHK-21 cells were infected with gM-eGFP-tagged MHV-68 as in A. Time-lapse imaging then focussed on a single membrane process. Zoom 1 is the boxed region in the overview; zoom 2 is the boxed region of zoom 1, with its boxed region shown as a zoomed inset. Zoom 3 shows a region equivalent to the central part of zoom 2 at ×5 greater magnification. Again, its boxed region is shown as a further zoomed inset. The complete sets of zoom 2 and zoom 3 pictures make up Movies S3 and S4 . C. Stills from Movies S3 and S4 , which correspond to the zoom 2 and zoom 3 images in B, show the variation in eGFP + dot distribution at 10sec intervals. D. BHK-21 cells were infected with MHV-68 (1 p.f.u./cell, 16h) then fixed and processed for transmission electron microscopy. cyt = cytoplasm; ext = extracellular. Closed arrows show virions. Open arrows show the bases of membrane fronds.

    Journal: PLoS ONE

    Article Title: A Gamma-Herpesvirus Glycoprotein Complex Manipulates Actin to Promote Viral Spread

    doi: 10.1371/journal.pone.0001808

    Figure Lengend Snippet: Relationship between membrane fronds and virions. A. BHK-21 cells infected with MHV-68 expressing eGFP-tagged gM (1 p.f.u./cell, 16 h) were examined by time-lapse confocal microscopy. The eGFP signal appears as black/gray. Each zoomed image corresponds to the boxed region of the corresponding overview. The punctate fluorescence of distal membrane fronds is seen to change with time. See also Movies S1 and S2 . B. BHK-21 cells were infected with gM-eGFP-tagged MHV-68 as in A. Time-lapse imaging then focussed on a single membrane process. Zoom 1 is the boxed region in the overview; zoom 2 is the boxed region of zoom 1, with its boxed region shown as a zoomed inset. Zoom 3 shows a region equivalent to the central part of zoom 2 at ×5 greater magnification. Again, its boxed region is shown as a further zoomed inset. The complete sets of zoom 2 and zoom 3 pictures make up Movies S3 and S4 . C. Stills from Movies S3 and S4 , which correspond to the zoom 2 and zoom 3 images in B, show the variation in eGFP + dot distribution at 10sec intervals. D. BHK-21 cells were infected with MHV-68 (1 p.f.u./cell, 16h) then fixed and processed for transmission electron microscopy. cyt = cytoplasm; ext = extracellular. Closed arrows show virions. Open arrows show the bases of membrane fronds.

    Article Snippet: Viruses were titered by plaque assay on BHK-21 cells .

    Techniques: Infection, Expressing, Confocal Microscopy, Fluorescence, Imaging, Transmission Assay, Electron Microscopy

    Effect of the loss of SPCS1 function on propagation of JEV particles. (A) Sequencing of SPCS1 alleles in gene-edited HEK-293 cells after limiting-dilution cloning. The subgenomic RNA targeting site and protospacer adjacent motif (PAM) sequences are highlighted above the WT gene, and the sequences of edited alleles are indicated. Nucleotide triplet codons are indicated by shaded boxes. Gene editing resulting in insertions of the T nucleotide is indicated with a red arrow, and a nucleotide insertion resulting in a stop codon is indicated with a red box. (B) WT and SPCS1 KO HEK-293 cells were infected with JEV at an MOI of 0.5. At 48 hpi, cells were fixed and probed with JEV E protein-specific MAb by an immunofluorescence assay. Data from one experiment of three are shown. FITC, fluorescein isothiocyanate. (C) Cell infectivity examined with an HCS system. The data are the averages of results from three independent experiments performed in triplicate. (D and E) Cell cytopathic effects were observed at 72 hpi by microscopy (D) or crystal violet staining (E). Data from one experiment of three are shown. (F) Expression of the E and NS1 proteins in infected cells, analyzed by Western blotting with E protein- and NS1 protein-specific MAbs. Data from one experiment of two are shown. (G) Comparison of viral titers in the supernatants of WT and SPCS1 KO cells infected with JEV at an MOI of 0.01. At 12, 24, 48, and 72 hpi, the titer of infectious JEV was determined by plaque-forming assays on BHK-21 cells. The data are pooled from three experiments in duplicate. Statistical significance was determined by Student's t test (***, P

    Journal: Journal of Virology

    Article Title: Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

    doi: 10.1128/JVI.00197-18

    Figure Lengend Snippet: Effect of the loss of SPCS1 function on propagation of JEV particles. (A) Sequencing of SPCS1 alleles in gene-edited HEK-293 cells after limiting-dilution cloning. The subgenomic RNA targeting site and protospacer adjacent motif (PAM) sequences are highlighted above the WT gene, and the sequences of edited alleles are indicated. Nucleotide triplet codons are indicated by shaded boxes. Gene editing resulting in insertions of the T nucleotide is indicated with a red arrow, and a nucleotide insertion resulting in a stop codon is indicated with a red box. (B) WT and SPCS1 KO HEK-293 cells were infected with JEV at an MOI of 0.5. At 48 hpi, cells were fixed and probed with JEV E protein-specific MAb by an immunofluorescence assay. Data from one experiment of three are shown. FITC, fluorescein isothiocyanate. (C) Cell infectivity examined with an HCS system. The data are the averages of results from three independent experiments performed in triplicate. (D and E) Cell cytopathic effects were observed at 72 hpi by microscopy (D) or crystal violet staining (E). Data from one experiment of three are shown. (F) Expression of the E and NS1 proteins in infected cells, analyzed by Western blotting with E protein- and NS1 protein-specific MAbs. Data from one experiment of two are shown. (G) Comparison of viral titers in the supernatants of WT and SPCS1 KO cells infected with JEV at an MOI of 0.01. At 12, 24, 48, and 72 hpi, the titer of infectious JEV was determined by plaque-forming assays on BHK-21 cells. The data are pooled from three experiments in duplicate. Statistical significance was determined by Student's t test (***, P

    Article Snippet: Briefly, a monolayer of BHK-21 cells was transfected with the pCAG-J-CME plasmid by using X-tremeGENE HP DNA transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Sequencing, Clone Assay, Infection, Immunofluorescence, Microscopy, Staining, Expressing, Western Blot

    Generation of a cell line stably expressing the JEV C-prM-E protein and preparation of RRPs. (A) Schematic representation of the generation of a stable cell line expressing the JEV C-prM-E protein and the preparation of RRPs. The BJEV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-opti-JEV-CME plasmid, followed by cloning and selection with G418. To produce RRPs, BJEV-CME cells were transfected with a DNA-based WNV replicon under the control of the cytomegalovirus (CMV) promoter. The replicon genome lacks the major coding sequence of the structural protein C-prM-E, and the corresponding sequence was replaced with a GFP-coding sequence following the foot-and-mouth disease virus (FMDV) 2A coding sequence. The replicon plasmid was transcribed by the cytomegalovirus promoter into the replicon RNA expressing GFP and the nonstructural replicase protein. BJEV-CME cells express the C-prM-E polyprotein, which is cleaved into the C, prM, and E proteins by replicon RNA-encoded nonstructural protease and endogenous cellular signal peptidase (SP). The replicon RNA amplifies itself again, and the three structural proteins package the replicon RNA into RRPs, which are secreted into the culture medium. When RRPs infect other JEV-susceptible cells, such as BHK-21 cells, the replicon RNA expresses GFP and nonstructural proteins, which amplifies more RNA. However, no structural proteins or additional RRPs are produced in RRP-infected cells, thereby preventing further replication. When BJEV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs, and the infection spreads in rounds similar to those for the wild-type virus. UTR, untranslated region. (B) Cloned and selected stable cell lines were identified by an IFA with JEV E protein-specific MAb 5E7. (C) Production of RRPs from the WNV replicon plasmid by transfection of BJEV-CME cell lines. Green florescence was visualized when replicon plasmid pWNVrepdCME-GFP was transfected into BJEV-CME cells (first infection) and BHK-21 cells (second infection). Transfected-cell supernatants were passaged onto fresh cell cultures, as indicated by the arrows, and green florescence was observed only in BJEV-CME cells. At 3 days postinoculation, supernatants of cells infected first were used to inoculate cells, as indicated by the arrows, for a second infection, and green florescence was analyzed at 72 h postinfection. HRr, hammerhead ribozyme; HDVr, hepatitis delta virus ribozyme.

    Journal: Journal of Virology

    Article Title: Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

    doi: 10.1128/JVI.00197-18

    Figure Lengend Snippet: Generation of a cell line stably expressing the JEV C-prM-E protein and preparation of RRPs. (A) Schematic representation of the generation of a stable cell line expressing the JEV C-prM-E protein and the preparation of RRPs. The BJEV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-opti-JEV-CME plasmid, followed by cloning and selection with G418. To produce RRPs, BJEV-CME cells were transfected with a DNA-based WNV replicon under the control of the cytomegalovirus (CMV) promoter. The replicon genome lacks the major coding sequence of the structural protein C-prM-E, and the corresponding sequence was replaced with a GFP-coding sequence following the foot-and-mouth disease virus (FMDV) 2A coding sequence. The replicon plasmid was transcribed by the cytomegalovirus promoter into the replicon RNA expressing GFP and the nonstructural replicase protein. BJEV-CME cells express the C-prM-E polyprotein, which is cleaved into the C, prM, and E proteins by replicon RNA-encoded nonstructural protease and endogenous cellular signal peptidase (SP). The replicon RNA amplifies itself again, and the three structural proteins package the replicon RNA into RRPs, which are secreted into the culture medium. When RRPs infect other JEV-susceptible cells, such as BHK-21 cells, the replicon RNA expresses GFP and nonstructural proteins, which amplifies more RNA. However, no structural proteins or additional RRPs are produced in RRP-infected cells, thereby preventing further replication. When BJEV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs, and the infection spreads in rounds similar to those for the wild-type virus. UTR, untranslated region. (B) Cloned and selected stable cell lines were identified by an IFA with JEV E protein-specific MAb 5E7. (C) Production of RRPs from the WNV replicon plasmid by transfection of BJEV-CME cell lines. Green florescence was visualized when replicon plasmid pWNVrepdCME-GFP was transfected into BJEV-CME cells (first infection) and BHK-21 cells (second infection). Transfected-cell supernatants were passaged onto fresh cell cultures, as indicated by the arrows, and green florescence was observed only in BJEV-CME cells. At 3 days postinoculation, supernatants of cells infected first were used to inoculate cells, as indicated by the arrows, for a second infection, and green florescence was analyzed at 72 h postinfection. HRr, hammerhead ribozyme; HDVr, hepatitis delta virus ribozyme.

    Article Snippet: Briefly, a monolayer of BHK-21 cells was transfected with the pCAG-J-CME plasmid by using X-tremeGENE HP DNA transfection reagent (Roche Diagnostic GmbH, Mannheim, Germany).

    Techniques: Stable Transfection, Expressing, Generated, Plasmid Preparation, Clone Assay, Selection, Transfection, Sequencing, Produced, Infection, Immunofluorescence