bhk-21 cells Search Results


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    Size 1 vial Price 337 0 Molecule Name bhk 21wi 2 cells for generating vsv
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    Hamster Syrian kidney Susceptible to a range of viruses including adenovirus 25 vaccinia herpes simplex reovirus 3 vesicular stomatitis rubella pseudorabies
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    99
    ATCC bhk 21 cells
    Establishment of a stable cell line expressing the Kunjin virus (KUNV) packaging system of capsid (C), precursor membrane (prM), and envelope (E), C-prM-E. ( A ) Schematic illustration of the gene construct expressing C-prM-E having G418 antibiotic resistance. Gene expression is driven by the CAG promoter. The sequence coding C-prM-E was fused with an internal ribosome entry site (IRES) sequence and the neomycin/kanamycin resistance (NeoR/KanR) gene, followed by the polyadenylation signal (pA). To generate baby hamster kidney cells <t>(BHK)-21</t> stably expressing KUNV C-prM-E, the gene construct was transfected into BHK-21 cells, followed by selection for transfected cells with G418. Cells were then separated into single cells and grown as individual clones. The schema was generated by using the Biorender web tool. ( B ) Immunoblotting of the cell lysates of each clone (1–11) to examine the expression level of E protein. The expression levels were normalized using the endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein as control. ( C ) Immunofluorescence staining of cells from clone 9 with the E antibody. The nucleus was counterstained by DAPI (blue). The bar scales represent 20 µm.
    Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/ATCC
    Average 99 stars, based on 2964 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2021-01
    99/100 stars
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    99
    Thermo Fisher bhk 21 cells
    The subcellular localization of the FMDV 2B protein in <t>BHK-21</t> cells. BHK-21 cells were transfected with pEGFPN1 (A) and pEGFPN1-2B (B) and evaluated using a LSCM. The blue fluorescence represents the nucleus, the green fluorescence represents the GFP protein or GFP-2B protein, and the red fluorescence represents the ER.
    Bhk 21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Thermo Fisher
    Average 99 stars, based on 3496 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2021-01
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    92
    Bio-Rad bhk 21 cells
    Translation of the genomic vRNA correlates with viral capping efficiency. (A) Schematic diagram of the SINV nanoluciferase reporter used in this study. UTR, untranslated region. (B) <t>BHK-21</t> cells were infected with either the parental wild-type strain or an individual SINV capping mutant nanoluciferase reporter strain. The level of nanoluciferase activity was quantified at the indicated times post-infection. (C) The nanoluciferase activity, as reported in panel B, normalized to wild-type expression at each individual time point to enable readers to identify differences in translation. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using analysis of variance (ANOVA) followed by post hoc statistical analyses by Student's t test.
    Bhk 21 Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Bio-Rad
    Average 92 stars, based on 517 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2021-01
    92/100 stars
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    93
    ATCC baby hamster kidney bhk 21 cells
    Translation of the genomic vRNA correlates with viral capping efficiency. (A) Schematic diagram of the SINV nanoluciferase reporter used in this study. UTR, untranslated region. (B) <t>BHK-21</t> cells were infected with either the parental wild-type strain or an individual SINV capping mutant nanoluciferase reporter strain. The level of nanoluciferase activity was quantified at the indicated times post-infection. (C) The nanoluciferase activity, as reported in panel B, normalized to wild-type expression at each individual time point to enable readers to identify differences in translation. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using analysis of variance (ANOVA) followed by post hoc statistical analyses by Student's t test.
    Baby Hamster Kidney Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk 21 cells/product/ATCC
    Average 93 stars, based on 342 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk 21 cells - by Bioz Stars, 2021-01
    93/100 stars
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    90
    Mirus Bio bhk 21 cells
    A recombinant alphavirus with EEEV replication machinery efficiently replicates in the absence of expression of either G3BPs or FXRs. Parental NIH 3T3 cells and their G3bp dKO and Fxr tKO derivatives were infected with VEEV/nsP3-Cherry (A), CHIKV/nsP3-Cherry (B), and EEE/nsP3-Cherry/SINV (C) at an MOI of 0.1 PFU/cell. Media were harvested at 7 h p.i., and virus titers were determined by plaque assay on <t>BHK-21</t> cells. The experiment was repeated three times with reproducible results. SPs, structural proteins; n.d., the titer was below the limit of detection, which was 50 PFU/ml.
    Bhk 21 Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 90/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Mirus Bio
    Average 90 stars, based on 104 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2021-01
    90/100 stars
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    92
    Corning Life Sciences bhk 21 cells
    Time-of-addition assay. ( a ) Schematic representation of the experimental design. <t>BHK-21</t> cells were treated with 10 μM PGG at the indicated times. ( b ) The viral P protein expression level was analyzed by Western blotting at 48 h p.i. ( c ) The relative expression levels of viral P, normalizing to that of α-tubulin. ( d ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted. Ctr is the abbreviation of control. (* p
    Bhk 21 Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Corning Life Sciences
    Average 92 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2021-01
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    91
    ATCC baby hamster kidney bhk21 cells
    Analysis of fusion-promotion and cleavage-promoting activities of the HN mutant proteins. (A) HN mutant plasmid and F plasmid were cotransfected into <t>BHK21</t> cells for 36 h, and then the fusion regions were photographed on an inverted microscope with 400-fold amplification, Bar = 20 μm. (B) The areas of 40 fusion regions were separately measured by using the software ImageJ as the fusion index of these HN mutant proteins. (C) After 36 h of cotransfection, the amount of each HN or F (F 0 and F 1 ) protein was determined by Western blot analysis using the HA or Flag label-specific mouse primary antibody and corresponding secondary antibody, and then determined the fusion-promotion activity of HN mutant proteins. (D) Those results of Western blot were quantified by densitometry using the ImageJ software. ∗ P
    Baby Hamster Kidney Bhk21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 91/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk21 cells/product/ATCC
    Average 91 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk21 cells - by Bioz Stars, 2021-01
    91/100 stars
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    The cells are positive for porcine circovirus PCV antigens The cells are positive for keratin by immunoperoxidase staining
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    Image Search Results


    Establishment of a stable cell line expressing the Kunjin virus (KUNV) packaging system of capsid (C), precursor membrane (prM), and envelope (E), C-prM-E. ( A ) Schematic illustration of the gene construct expressing C-prM-E having G418 antibiotic resistance. Gene expression is driven by the CAG promoter. The sequence coding C-prM-E was fused with an internal ribosome entry site (IRES) sequence and the neomycin/kanamycin resistance (NeoR/KanR) gene, followed by the polyadenylation signal (pA). To generate baby hamster kidney cells (BHK)-21 stably expressing KUNV C-prM-E, the gene construct was transfected into BHK-21 cells, followed by selection for transfected cells with G418. Cells were then separated into single cells and grown as individual clones. The schema was generated by using the Biorender web tool. ( B ) Immunoblotting of the cell lysates of each clone (1–11) to examine the expression level of E protein. The expression levels were normalized using the endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein as control. ( C ) Immunofluorescence staining of cells from clone 9 with the E antibody. The nucleus was counterstained by DAPI (blue). The bar scales represent 20 µm.

    Journal: Microorganisms

    Article Title: Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

    doi: 10.3390/microorganisms8121890

    Figure Lengend Snippet: Establishment of a stable cell line expressing the Kunjin virus (KUNV) packaging system of capsid (C), precursor membrane (prM), and envelope (E), C-prM-E. ( A ) Schematic illustration of the gene construct expressing C-prM-E having G418 antibiotic resistance. Gene expression is driven by the CAG promoter. The sequence coding C-prM-E was fused with an internal ribosome entry site (IRES) sequence and the neomycin/kanamycin resistance (NeoR/KanR) gene, followed by the polyadenylation signal (pA). To generate baby hamster kidney cells (BHK)-21 stably expressing KUNV C-prM-E, the gene construct was transfected into BHK-21 cells, followed by selection for transfected cells with G418. Cells were then separated into single cells and grown as individual clones. The schema was generated by using the Biorender web tool. ( B ) Immunoblotting of the cell lysates of each clone (1–11) to examine the expression level of E protein. The expression levels were normalized using the endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein as control. ( C ) Immunofluorescence staining of cells from clone 9 with the E antibody. The nucleus was counterstained by DAPI (blue). The bar scales represent 20 µm.

    Article Snippet: Cell Culture The Baby hamster kidney cell line (BHK-21) (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, New York, NY, USA) containing 1 g/L glucose (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin–streptomycin (Gibco), and 1% nonessential amino acids (Gibco) at 37 °C in 5% CO2.

    Techniques: Stable Transfection, Expressing, Construct, Sequencing, Transfection, Selection, Clone Assay, Generated, Immunofluorescence, Staining

    KUNV RVPs infect cells in a single round. ( A ) Schematic illustration of the process of RVP infection of BHK-21 cells. Hereby, the absence of a packaging system in the infected cells limits additional RVP production. ( B ) Gene copy numbers of the KUNV replicon in cell lysates after first- and second-round infection and uninfected control lysates as measured by qPCR. The experiments were conducted independently three times with two technical repeats. The p values are indicated using *** p

    Journal: Microorganisms

    Article Title: Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

    doi: 10.3390/microorganisms8121890

    Figure Lengend Snippet: KUNV RVPs infect cells in a single round. ( A ) Schematic illustration of the process of RVP infection of BHK-21 cells. Hereby, the absence of a packaging system in the infected cells limits additional RVP production. ( B ) Gene copy numbers of the KUNV replicon in cell lysates after first- and second-round infection and uninfected control lysates as measured by qPCR. The experiments were conducted independently three times with two technical repeats. The p values are indicated using *** p

    Article Snippet: Cell Culture The Baby hamster kidney cell line (BHK-21) (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, New York, NY, USA) containing 1 g/L glucose (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin–streptomycin (Gibco), and 1% nonessential amino acids (Gibco) at 37 °C in 5% CO2.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Immunofluorescence labeling of BHK-21 C-prM-E cells after transfection with the KUNV replicons expressing EBOV GP or VP40 proteins ( A , B ), respectively, followed by two cycles of RVP infections of BHK-21 cells. The cells were visualized with the antibodies anti-EBOV GP (green), EBOV VP40 (green) ( A , B ), respectively, and the antibodies anti-dsRNA (red) and KUNV NS1 (red). The nucleus was counterstained with DAPI (blue). Bar scales represent 20 µm.

    Journal: Microorganisms

    Article Title: Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

    doi: 10.3390/microorganisms8121890

    Figure Lengend Snippet: Immunofluorescence labeling of BHK-21 C-prM-E cells after transfection with the KUNV replicons expressing EBOV GP or VP40 proteins ( A , B ), respectively, followed by two cycles of RVP infections of BHK-21 cells. The cells were visualized with the antibodies anti-EBOV GP (green), EBOV VP40 (green) ( A , B ), respectively, and the antibodies anti-dsRNA (red) and KUNV NS1 (red). The nucleus was counterstained with DAPI (blue). Bar scales represent 20 µm.

    Article Snippet: Cell Culture The Baby hamster kidney cell line (BHK-21) (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, New York, NY, USA) containing 1 g/L glucose (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin–streptomycin (Gibco), and 1% nonessential amino acids (Gibco) at 37 °C in 5% CO2.

    Techniques: Immunofluorescence, Labeling, Transfection, Expressing

    Transmission electron microscopy images of the RVP production system. ( A – D ) Images representing the C-prM-E stable BHK-21 cell line transfected with the KUNV replicon construct. ( A , C ) represent different subcellular areas, whereas ( B , D ) show higher magnification images of the yellow boxes indicated in the ( A ) and the ( C ), respectively. ( E ) The image represents BHK-21 cells used as control. ( F ) The image of cell culture supernatants from the C-prM-E stable cells transfected with the KUNV replicon. ER: the endoplasmic reticulum; Nu: nucleus; M: mitochondria; CM/PC: convoluted membranes, paracrystalline structure; Vp: vesicle packets; Ve: virus-induced vesicles in the ER; VLP/RVP: virus-like particle, reporter virus-like particles; Ri: ribosome. Scale bars represent 500 nm in ( A , C , E ) and 100 nm in ( B , D , F ).

    Journal: Microorganisms

    Article Title: Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

    doi: 10.3390/microorganisms8121890

    Figure Lengend Snippet: Transmission electron microscopy images of the RVP production system. ( A – D ) Images representing the C-prM-E stable BHK-21 cell line transfected with the KUNV replicon construct. ( A , C ) represent different subcellular areas, whereas ( B , D ) show higher magnification images of the yellow boxes indicated in the ( A ) and the ( C ), respectively. ( E ) The image represents BHK-21 cells used as control. ( F ) The image of cell culture supernatants from the C-prM-E stable cells transfected with the KUNV replicon. ER: the endoplasmic reticulum; Nu: nucleus; M: mitochondria; CM/PC: convoluted membranes, paracrystalline structure; Vp: vesicle packets; Ve: virus-induced vesicles in the ER; VLP/RVP: virus-like particle, reporter virus-like particles; Ri: ribosome. Scale bars represent 500 nm in ( A , C , E ) and 100 nm in ( B , D , F ).

    Article Snippet: Cell Culture The Baby hamster kidney cell line (BHK-21) (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, New York, NY, USA) containing 1 g/L glucose (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin–streptomycin (Gibco), and 1% nonessential amino acids (Gibco) at 37 °C in 5% CO2.

    Techniques: Transmission Assay, Electron Microscopy, Transfection, Construct, Cell Culture

    The subcellular localization of the FMDV 2B protein in BHK-21 cells. BHK-21 cells were transfected with pEGFPN1 (A) and pEGFPN1-2B (B) and evaluated using a LSCM. The blue fluorescence represents the nucleus, the green fluorescence represents the GFP protein or GFP-2B protein, and the red fluorescence represents the ER.

    Journal: PLoS ONE

    Article Title: Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

    doi: 10.1371/journal.pone.0125828

    Figure Lengend Snippet: The subcellular localization of the FMDV 2B protein in BHK-21 cells. BHK-21 cells were transfected with pEGFPN1 (A) and pEGFPN1-2B (B) and evaluated using a LSCM. The blue fluorescence represents the nucleus, the green fluorescence represents the GFP protein or GFP-2B protein, and the red fluorescence represents the ER.

    Article Snippet: Subcellular localization of the 2B protein in BHK-21 cells The plasmid pEGFPN1 and the recombinant plasmid pEGFPN1-2B were transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, California, USA).

    Techniques: Transfection, Fluorescence

    The pore-forming activity of the 2B protein. (A) Purified Sumo-2B protein was incubated with a glutaraldehyde cross-linker at the indicated concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0, and 3.0 mM). The monomers and oligomers of the 2B protein were detected by immunoblot analysis with an anti-His monoclonal antibody. (B) BHK-21 cells were transfected with pXJ-FLAG-2B or pXJ-2B-HA. The cell lysates were subjected to immunoprecipitation using anti-HA antibodies.

    Journal: PLoS ONE

    Article Title: Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

    doi: 10.1371/journal.pone.0125828

    Figure Lengend Snippet: The pore-forming activity of the 2B protein. (A) Purified Sumo-2B protein was incubated with a glutaraldehyde cross-linker at the indicated concentrations (0, 0.1, 0.5, 1.0, 1.5, 2.0, and 3.0 mM). The monomers and oligomers of the 2B protein were detected by immunoblot analysis with an anti-His monoclonal antibody. (B) BHK-21 cells were transfected with pXJ-FLAG-2B or pXJ-2B-HA. The cell lysates were subjected to immunoprecipitation using anti-HA antibodies.

    Article Snippet: Subcellular localization of the 2B protein in BHK-21 cells The plasmid pEGFPN1 and the recombinant plasmid pEGFPN1-2B were transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, California, USA).

    Techniques: Activity Assay, Purification, Incubation, Transfection, Immunoprecipitation

    The effect of amantadine on the release of FMDV virions. BHK-21 cell cultures were infected with FMDV (MOI/0.1) and then treated with amantadine. The virus in the supernatant was collected at 4 hours post-infection. The virus titer was determined by TCID50. Asterisks indicate significant differences between the indicated samples (*P

    Journal: PLoS ONE

    Article Title: Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

    doi: 10.1371/journal.pone.0125828

    Figure Lengend Snippet: The effect of amantadine on the release of FMDV virions. BHK-21 cell cultures were infected with FMDV (MOI/0.1) and then treated with amantadine. The virus in the supernatant was collected at 4 hours post-infection. The virus titer was determined by TCID50. Asterisks indicate significant differences between the indicated samples (*P

    Article Snippet: Subcellular localization of the 2B protein in BHK-21 cells The plasmid pEGFPN1 and the recombinant plasmid pEGFPN1-2B were transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, California, USA).

    Techniques: Infection

    The effects of the 2B protein on the Ca 2+ content and membrane integrity in host cells. Untreated BHK-21 cells (A) and cells transfected with pXJ-2B-HA (B) were stained with Fluo-3 AM and propidium iodide (PI) at 12 hours post-transfection. The increase in intracellular Ca 2+ (Fluo-3 AM) is shown in Q4. This increase was associated with a change in the PI in BHK-21 cells. A histogram was constructed to reveal the changes in the intracellular Ca 2+ concentration (C). Asterisks indicate significant differences between the indicated samples (*P

    Journal: PLoS ONE

    Article Title: Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

    doi: 10.1371/journal.pone.0125828

    Figure Lengend Snippet: The effects of the 2B protein on the Ca 2+ content and membrane integrity in host cells. Untreated BHK-21 cells (A) and cells transfected with pXJ-2B-HA (B) were stained with Fluo-3 AM and propidium iodide (PI) at 12 hours post-transfection. The increase in intracellular Ca 2+ (Fluo-3 AM) is shown in Q4. This increase was associated with a change in the PI in BHK-21 cells. A histogram was constructed to reveal the changes in the intracellular Ca 2+ concentration (C). Asterisks indicate significant differences between the indicated samples (*P

    Article Snippet: Subcellular localization of the 2B protein in BHK-21 cells The plasmid pEGFPN1 and the recombinant plasmid pEGFPN1-2B were transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, California, USA).

    Techniques: Transfection, Staining, Construct, Concentration Assay

    Autophagy induced by the 2B protein in BHK-21 cells and H1299 cells. Four groups of BHK-21 cells were cultured with different treatments, and the LC3-I and LC3-II levels were determined by Western blot analysis with an anti-LC3 antibody (A). Untreated cells (B, F), cells transfected with pCS2 (C, G) or pCS2-2B (D, H), or treated with rapamycin (E, I) were cultured and observed under a LSCM to evaluate LC3 aggregation using IFA in BHK-21 cells (B-E) or based on the green and red fluorescence in H1299 cells (F-I). The blue fluorescence represents the nucleus, the green fluorescence represents the LC3 protein, and the red fluorescence represents the RFP-2B protein.

    Journal: PLoS ONE

    Article Title: Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

    doi: 10.1371/journal.pone.0125828

    Figure Lengend Snippet: Autophagy induced by the 2B protein in BHK-21 cells and H1299 cells. Four groups of BHK-21 cells were cultured with different treatments, and the LC3-I and LC3-II levels were determined by Western blot analysis with an anti-LC3 antibody (A). Untreated cells (B, F), cells transfected with pCS2 (C, G) or pCS2-2B (D, H), or treated with rapamycin (E, I) were cultured and observed under a LSCM to evaluate LC3 aggregation using IFA in BHK-21 cells (B-E) or based on the green and red fluorescence in H1299 cells (F-I). The blue fluorescence represents the nucleus, the green fluorescence represents the LC3 protein, and the red fluorescence represents the RFP-2B protein.

    Article Snippet: Subcellular localization of the 2B protein in BHK-21 cells The plasmid pEGFPN1 and the recombinant plasmid pEGFPN1-2B were transfected into BHK-21 cells using Lipofectamine 2000 (Invitrogen, California, USA).

    Techniques: Cell Culture, Western Blot, Transfection, Immunofluorescence, Fluorescence

    Replication activities of various DENV2 reporter replicons containing NS2A mutations. (A) Schematic representation of the DNA-launched DENV2 reporter replicon pCMV-DV2Rep, which was used for a transient replicon assay. The transcriptional expression of the replicon RNA was under the control of the CMVmin promoter, and the processing of the 3′ terminus of the transcript was ensured by the inclusion of HDV ribozyme sequences. The 5′ UTR (left black line), the N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site (black box), a neomycin resistance gene (Neo), an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) element (gray box), the C-terminal 24 amino acids of E (E24), all of the NS protein regions (NS1 to NS5), the 3′ UTR (right black line), the HDV ribozyme sequence, and the SV40 poly(A) signal sequence are indicated. (B) Replication kinetics of the WT DENV replicon. BHK21 cells were transfected with WT or NS5 mutant replicon plasmid. The luciferase activity of the transfected cells was monitored at 24, 48, 72, and 96 h posttransfection. The error bars represent the standard errors of the mean (SEM) of the results from three independent experiments. (C) Replication activities of transient expression of DNA-launched wild-type and NS2A mutant replicons in BHK21 cells. BHK21 cells were transfected with WT or NS2A mutant replicon plasmids. At 24 h and 72 h posttransfection, the luciferase activities of the transfected cells were measured. Replication efficiency was calculated by determination of the ratio of luciferase activity obtained at 72 h to the average value obtained from all replicon constructs at 24 h posttransfection and compared with that of the WT replicon. The numbers above the bars represent the percentages of luciferase activity relative to the activity of the WT replicon (which was considered to be 100%), and the error bars represent the SEM of the results from three independent experiments. (D) There is no apparent effect of mutations within NM5, NM7, NM9, NM17, NM18, or NM19 on the translation of viral RNAs. BHK21 cells were cotransfected with the pSV-beta-Galactosidase vector and DNA-launched WT or NS2A mutant replicon plasmids. At 24 h posttransfection, the luciferase activity (normalized to β-galactosidase activity) of the transfected cells was measured and normalized to the activity in cells transfected with the WT replicon plasmid, which was set at 100%. The error bars represent the SEM of the results from three independent experiments. (E) Replication activities of transient expression of RNA-launched wild-type and NS2A mutant replicons in BHK21 cells. At 4 h and 48 h posttransfection, the luciferase activities of the transfected cells were measured. Replication efficiency was calculated by determination of the ratio of the luciferase activity obtained at 48 h to the value obtained at 4 h posttransfection and compared with that of the WT replicon. The numbers above the bars represent the percentages of luciferase activity relative to the activity of the WT replicon (which was considered to be 100%), and the error bars represent the SEM of the results from three independent experiments.

    Journal: Journal of Virology

    Article Title: Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

    doi: 10.1128/JVI.01836-16

    Figure Lengend Snippet: Replication activities of various DENV2 reporter replicons containing NS2A mutations. (A) Schematic representation of the DNA-launched DENV2 reporter replicon pCMV-DV2Rep, which was used for a transient replicon assay. The transcriptional expression of the replicon RNA was under the control of the CMVmin promoter, and the processing of the 3′ terminus of the transcript was ensured by the inclusion of HDV ribozyme sequences. The 5′ UTR (left black line), the N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site (black box), a neomycin resistance gene (Neo), an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) element (gray box), the C-terminal 24 amino acids of E (E24), all of the NS protein regions (NS1 to NS5), the 3′ UTR (right black line), the HDV ribozyme sequence, and the SV40 poly(A) signal sequence are indicated. (B) Replication kinetics of the WT DENV replicon. BHK21 cells were transfected with WT or NS5 mutant replicon plasmid. The luciferase activity of the transfected cells was monitored at 24, 48, 72, and 96 h posttransfection. The error bars represent the standard errors of the mean (SEM) of the results from three independent experiments. (C) Replication activities of transient expression of DNA-launched wild-type and NS2A mutant replicons in BHK21 cells. BHK21 cells were transfected with WT or NS2A mutant replicon plasmids. At 24 h and 72 h posttransfection, the luciferase activities of the transfected cells were measured. Replication efficiency was calculated by determination of the ratio of luciferase activity obtained at 72 h to the average value obtained from all replicon constructs at 24 h posttransfection and compared with that of the WT replicon. The numbers above the bars represent the percentages of luciferase activity relative to the activity of the WT replicon (which was considered to be 100%), and the error bars represent the SEM of the results from three independent experiments. (D) There is no apparent effect of mutations within NM5, NM7, NM9, NM17, NM18, or NM19 on the translation of viral RNAs. BHK21 cells were cotransfected with the pSV-beta-Galactosidase vector and DNA-launched WT or NS2A mutant replicon plasmids. At 24 h posttransfection, the luciferase activity (normalized to β-galactosidase activity) of the transfected cells was measured and normalized to the activity in cells transfected with the WT replicon plasmid, which was set at 100%. The error bars represent the SEM of the results from three independent experiments. (E) Replication activities of transient expression of RNA-launched wild-type and NS2A mutant replicons in BHK21 cells. At 4 h and 48 h posttransfection, the luciferase activities of the transfected cells were measured. Replication efficiency was calculated by determination of the ratio of the luciferase activity obtained at 48 h to the value obtained at 4 h posttransfection and compared with that of the WT replicon. The numbers above the bars represent the percentages of luciferase activity relative to the activity of the WT replicon (which was considered to be 100%), and the error bars represent the SEM of the results from three independent experiments.

    Article Snippet: RNAs were transfected into BHK21 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

    Techniques: Expressing, Luciferase, Sequencing, Transfection, Mutagenesis, Plasmid Preparation, Activity Assay, Construct

    Translation of the genomic vRNA correlates with viral capping efficiency. (A) Schematic diagram of the SINV nanoluciferase reporter used in this study. UTR, untranslated region. (B) BHK-21 cells were infected with either the parental wild-type strain or an individual SINV capping mutant nanoluciferase reporter strain. The level of nanoluciferase activity was quantified at the indicated times post-infection. (C) The nanoluciferase activity, as reported in panel B, normalized to wild-type expression at each individual time point to enable readers to identify differences in translation. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using analysis of variance (ANOVA) followed by post hoc statistical analyses by Student's t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Translation of the genomic vRNA correlates with viral capping efficiency. (A) Schematic diagram of the SINV nanoluciferase reporter used in this study. UTR, untranslated region. (B) BHK-21 cells were infected with either the parental wild-type strain or an individual SINV capping mutant nanoluciferase reporter strain. The level of nanoluciferase activity was quantified at the indicated times post-infection. (C) The nanoluciferase activity, as reported in panel B, normalized to wild-type expression at each individual time point to enable readers to identify differences in translation. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using analysis of variance (ANOVA) followed by post hoc statistical analyses by Student's t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Infection, Mutagenesis, Activity Assay, Expressing

    Altering viral capping efficiency negatively impacts viral infection. (A) Multi-step growth kinetics of the individual capping mutants and parental wild-type SINV as observed in BHK-21 cells infected at an MOI of 0.5 PFU/cell. Statistical significance was determined by analysis of the area under the curve. (B) Plaque morphology of wild-type SINV and capping mutant viruses in BHK-21 cells overlaid with 0.5% solution of Avicel at 24 h post-infection. (C) Graph indicating the average plaque diameter of the mutant viruses relative to wild-type SINV. Size was determined by ImageJ software (NIH). (D) Cell viability of BHK-21 cells infected with the individual capping mutants at 24 hpi relative to mock-infected BHK-21 cells. All quantitative data shown represent means of results from at least three independent biological replicates, with error bars representing standard deviations of the means. Statistical significance, as indicated within each panel, was determined by Student's t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Altering viral capping efficiency negatively impacts viral infection. (A) Multi-step growth kinetics of the individual capping mutants and parental wild-type SINV as observed in BHK-21 cells infected at an MOI of 0.5 PFU/cell. Statistical significance was determined by analysis of the area under the curve. (B) Plaque morphology of wild-type SINV and capping mutant viruses in BHK-21 cells overlaid with 0.5% solution of Avicel at 24 h post-infection. (C) Graph indicating the average plaque diameter of the mutant viruses relative to wild-type SINV. Size was determined by ImageJ software (NIH). (D) Cell viability of BHK-21 cells infected with the individual capping mutants at 24 hpi relative to mock-infected BHK-21 cells. All quantitative data shown represent means of results from at least three independent biological replicates, with error bars representing standard deviations of the means. Statistical significance, as indicated within each panel, was determined by Student's t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Infection, Mutagenesis, Software

    Analysis of SINV particle production. (A) BHK-21 cells were infected with either wild-type SINV or an individual capping mutant at an MOI of 5 PFU/cell. At 24 hpi, the total number of viral particles produced was measured using qRT-PCR. Data shown represent means of results from at least 6 independent biological samples. (B) Quantitative determination of the composition of the encapsidated viral RNAs in mature extracellular viral particles. Samples of virus-containing supernatants were assessed to determine the absolute quantities of the genomic and subgenomic RNAs via standard curve qRT-PCR. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance data, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Analysis of SINV particle production. (A) BHK-21 cells were infected with either wild-type SINV or an individual capping mutant at an MOI of 5 PFU/cell. At 24 hpi, the total number of viral particles produced was measured using qRT-PCR. Data shown represent means of results from at least 6 independent biological samples. (B) Quantitative determination of the composition of the encapsidated viral RNAs in mature extracellular viral particles. Samples of virus-containing supernatants were assessed to determine the absolute quantities of the genomic and subgenomic RNAs via standard curve qRT-PCR. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance data, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Infection, Mutagenesis, Produced, Quantitative RT-PCR

    Altering vRNA capping efficiency impacts early RNA synthesis. (A) BHK-21 cells were infected with either wild-type parental SINV or an individual capping mutant virus at an MOI of 5 PFU/cell. At 2 h post-infection, the total cellular RNA was extracted and assessed for the absolute quantities of the genomic, subgenomic, and minus-strand vRNAs by qRT-PCR. (B and C) Identical to panel A, with the exception that the time points represent 4 and 8 h post-infection, respectively. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance data, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test. The P values determined by Student's t test are represented as follows: *, P

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Altering vRNA capping efficiency impacts early RNA synthesis. (A) BHK-21 cells were infected with either wild-type parental SINV or an individual capping mutant virus at an MOI of 5 PFU/cell. At 2 h post-infection, the total cellular RNA was extracted and assessed for the absolute quantities of the genomic, subgenomic, and minus-strand vRNAs by qRT-PCR. (B and C) Identical to panel A, with the exception that the time points represent 4 and 8 h post-infection, respectively. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance data, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test. The P values determined by Student's t test are represented as follows: *, P

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Infection, Mutagenesis, Quantitative RT-PCR

    Subgenomic gene expression is unaffected by altering SINV vRNA capping. (A) BHK-21 cells were either mock treated or infected with wild-type SINV or an individual capping mutant at an MOI of 10 PFU/cell. At 14 hpi, the cells were pulsed with L-AHA for a period of 2 h. The cells were then harvested, and equal cell volumes of cell lysate were analyzed by SDS-PAGE and fluorescent imaging. The data shown are representative of results from three independent biological replicates. (B) Densitometric quantification of the SINV capsid protein, with intensity relative to wild-type SINV shown. (C) Densitometric quantification of the host actin protein, with intensity relative to wild-type SINV shown. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance was determined by Student's t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Subgenomic gene expression is unaffected by altering SINV vRNA capping. (A) BHK-21 cells were either mock treated or infected with wild-type SINV or an individual capping mutant at an MOI of 10 PFU/cell. At 14 hpi, the cells were pulsed with L-AHA for a period of 2 h. The cells were then harvested, and equal cell volumes of cell lysate were analyzed by SDS-PAGE and fluorescent imaging. The data shown are representative of results from three independent biological replicates. (B) Densitometric quantification of the SINV capsid protein, with intensity relative to wild-type SINV shown. (C) Densitometric quantification of the host actin protein, with intensity relative to wild-type SINV shown. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significance was determined by Student's t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Expressing, Infection, Mutagenesis, SDS Page, Imaging

    Point mutations in the nsP1 protein of SINV alter 5′ vRNA capping efficiency. (A) Amino acid sequence alignment of selected alphavirus nsP1 proteins. The individual nsP1 protein sequences of Sindbis virus (ViPR accession no. U38305 ), Venezuelan equine encephalitis virus (VEEV; ViPR accession no. L01443 ), chikungunya virus (CHIKV; ViPR accession no. DQ443544 ), and Ross River virus (RRV; ViPR accession no. GQ433359 ) were aligned by the use of Clustal Omega. (B) An ITASSER-predicted structure of the SINV nsP1 protein. Amino acid residues of importance are highlighted as follows: red, Y286; green, D355; blue, N376; cyan, amphipathic helix; pink, residues involved in methyltransferase activities (including H39, which binds to the m7 GMP residue). (C) Graph depicting the relative quantities of XRN-1-resistant vRNA isolated from viral particles produced by BHK-21 cells infected with wild-type SINV or the individual capping mutants at 24 hpi. (D) Identical to panel C, with the exception that the viral genomic RNAs were enzymatically decapped concurrently with XRN-1 treatment. All quantitative data shown represent means of data from a minimum of three independent biological replicates utilizing 3 independent particle preparations, with the error bars indicating standard deviations of the means. The P values indicated on the figure were determined by Student’s t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: Point mutations in the nsP1 protein of SINV alter 5′ vRNA capping efficiency. (A) Amino acid sequence alignment of selected alphavirus nsP1 proteins. The individual nsP1 protein sequences of Sindbis virus (ViPR accession no. U38305 ), Venezuelan equine encephalitis virus (VEEV; ViPR accession no. L01443 ), chikungunya virus (CHIKV; ViPR accession no. DQ443544 ), and Ross River virus (RRV; ViPR accession no. GQ433359 ) were aligned by the use of Clustal Omega. (B) An ITASSER-predicted structure of the SINV nsP1 protein. Amino acid residues of importance are highlighted as follows: red, Y286; green, D355; blue, N376; cyan, amphipathic helix; pink, residues involved in methyltransferase activities (including H39, which binds to the m7 GMP residue). (C) Graph depicting the relative quantities of XRN-1-resistant vRNA isolated from viral particles produced by BHK-21 cells infected with wild-type SINV or the individual capping mutants at 24 hpi. (D) Identical to panel C, with the exception that the viral genomic RNAs were enzymatically decapped concurrently with XRN-1 treatment. All quantitative data shown represent means of data from a minimum of three independent biological replicates utilizing 3 independent particle preparations, with the error bars indicating standard deviations of the means. The P values indicated on the figure were determined by Student’s t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Sequencing, Isolation, Produced, Infection

    nsP2 protein levels are impacted by mutation of the nsP1 protein. (A) BHK-21 cells were infected with wild-type SINV or an individual capping mutant at an MOI of 5 PFU/cell and assessed by Western blotting to determine the abundance of nsP2 at 8 hpi. Actin is shown as a loading control. (B) Densitometric quantification of the nsP2 protein normalized to actin levels at 8 hpi. (C and D) Western blots and densitometry analyses identical to those described for panels A and B, respectively, with the exception that the timing of the assay coincided with 12 hpi. (E and F) Western blots and densitometry analyses identical to those described for panels A and B, respectively, with the exception that the timing of the assay coincided with 16 hpi. The Western blot images shown are representative of results of at least three independent biological replicates. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test.

    Journal: mBio

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

    doi: 10.1128/mBio.02342-18

    Figure Lengend Snippet: nsP2 protein levels are impacted by mutation of the nsP1 protein. (A) BHK-21 cells were infected with wild-type SINV or an individual capping mutant at an MOI of 5 PFU/cell and assessed by Western blotting to determine the abundance of nsP2 at 8 hpi. Actin is shown as a loading control. (B) Densitometric quantification of the nsP2 protein normalized to actin levels at 8 hpi. (C and D) Western blots and densitometry analyses identical to those described for panels A and B, respectively, with the exception that the timing of the assay coincided with 12 hpi. (E and F) Western blots and densitometry analyses identical to those described for panels A and B, respectively, with the exception that the timing of the assay coincided with 16 hpi. The Western blot images shown are representative of results of at least three independent biological replicates. All the quantitative data shown represent means of results from three independent biological replicates, with the error bars representing standard deviations of the means. Statistical significances, as indicated in the figure, were first determined using ANOVA followed by post hoc statistical analyses by Student's t test.

    Article Snippet: Briefly, 2.8 × 106 BHK-21 cells were electroporated with 10 μg of in vitro -transcribed RNA using a single pulse at 1.5 kV, 25 mA, and 200 Ω from a Gene Pulser Xcell system (Bio-Rad) as previously described ( ).

    Techniques: Mutagenesis, Infection, Western Blot

    A recombinant alphavirus with EEEV replication machinery efficiently replicates in the absence of expression of either G3BPs or FXRs. Parental NIH 3T3 cells and their G3bp dKO and Fxr tKO derivatives were infected with VEEV/nsP3-Cherry (A), CHIKV/nsP3-Cherry (B), and EEE/nsP3-Cherry/SINV (C) at an MOI of 0.1 PFU/cell. Media were harvested at 7 h p.i., and virus titers were determined by plaque assay on BHK-21 cells. The experiment was repeated three times with reproducible results. SPs, structural proteins; n.d., the titer was below the limit of detection, which was 50 PFU/ml.

    Journal: Journal of Virology

    Article Title: Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly

    doi: 10.1128/JVI.00371-17

    Figure Lengend Snippet: A recombinant alphavirus with EEEV replication machinery efficiently replicates in the absence of expression of either G3BPs or FXRs. Parental NIH 3T3 cells and their G3bp dKO and Fxr tKO derivatives were infected with VEEV/nsP3-Cherry (A), CHIKV/nsP3-Cherry (B), and EEE/nsP3-Cherry/SINV (C) at an MOI of 0.1 PFU/cell. Media were harvested at 7 h p.i., and virus titers were determined by plaque assay on BHK-21 cells. The experiment was repeated three times with reproducible results. SPs, structural proteins; n.d., the titer was below the limit of detection, which was 50 PFU/ml.

    Article Snippet: Wild-type EEEV FL93 and its HVD mutants were rescued in BHK-21 cells using the TransIT-mRNA reagent according to the manufacturer's instructions (Mirus).

    Techniques: Recombinant, Expressing, Infection, Plaque Assay

    EEEV nsP3 HVD interactions with G3BPs and FXRs and other host proteins determine the rates of virus replication. (A) Schematic representation of the EEEV genome and modifications introduced into the nsP3 HVD. (B) The indicated cell lines were infected with the designed viruses at an MOI of 1 PFU/cell. Then, the cells were washed 5 times with complete medium and incubated for 7 h (NIH 3T3 and Vero cells) or 8 h (BHK-21 cells). The titers of harvested samples were determined by plaque assay on BHK-21 cells. n.d., the titer was below the limit of detection, which was 50 PFU/ml.

    Journal: Journal of Virology

    Article Title: Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly

    doi: 10.1128/JVI.00371-17

    Figure Lengend Snippet: EEEV nsP3 HVD interactions with G3BPs and FXRs and other host proteins determine the rates of virus replication. (A) Schematic representation of the EEEV genome and modifications introduced into the nsP3 HVD. (B) The indicated cell lines were infected with the designed viruses at an MOI of 1 PFU/cell. Then, the cells were washed 5 times with complete medium and incubated for 7 h (NIH 3T3 and Vero cells) or 8 h (BHK-21 cells). The titers of harvested samples were determined by plaque assay on BHK-21 cells. n.d., the titer was below the limit of detection, which was 50 PFU/ml.

    Article Snippet: Wild-type EEEV FL93 and its HVD mutants were rescued in BHK-21 cells using the TransIT-mRNA reagent according to the manufacturer's instructions (Mirus).

    Techniques: Infection, Incubation, Plaque Assay

    Time-of-addition assay. ( a ) Schematic representation of the experimental design. BHK-21 cells were treated with 10 μM PGG at the indicated times. ( b ) The viral P protein expression level was analyzed by Western blotting at 48 h p.i. ( c ) The relative expression levels of viral P, normalizing to that of α-tubulin. ( d ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted. Ctr is the abbreviation of control. (* p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: Time-of-addition assay. ( a ) Schematic representation of the experimental design. BHK-21 cells were treated with 10 μM PGG at the indicated times. ( b ) The viral P protein expression level was analyzed by Western blotting at 48 h p.i. ( c ) The relative expression levels of viral P, normalizing to that of α-tubulin. ( d ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted. Ctr is the abbreviation of control. (* p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Expressing, Western Blot

    PGG inhibits RABV adsorption and entry. For viral adsorption or entry assay, BHK-21 cells were treated with PGG for 1 h. ( a ) The vira1 P gene mRNA expression level was detected by qRT-PCR; ( b ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted; ( c ) The expression level of viral P protein was analyzed by Western blotting; ( d ) The relative expression levels of viral P, normalizing to that of α-tubulin. (** p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: PGG inhibits RABV adsorption and entry. For viral adsorption or entry assay, BHK-21 cells were treated with PGG for 1 h. ( a ) The vira1 P gene mRNA expression level was detected by qRT-PCR; ( b ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted; ( c ) The expression level of viral P protein was analyzed by Western blotting; ( d ) The relative expression levels of viral P, normalizing to that of α-tubulin. (** p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Adsorption, Expressing, Quantitative RT-PCR, Western Blot

    PGG inhibits viral yields and viral protein synthesis. Infected BHK-21 cells were treated with PGG or IPS at various time points. ( a ) Growth kinetics of infected BHK-21 cells during a 48-h period p.i. after treatment with PGG or IPS; ( b ) The viral P protein expression level was analyzed by Western blotting during a 48-h period p.i.; ( c ) The relative expression levels of viral P, normalizing to that of α-tubulin. (* p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: PGG inhibits viral yields and viral protein synthesis. Infected BHK-21 cells were treated with PGG or IPS at various time points. ( a ) Growth kinetics of infected BHK-21 cells during a 48-h period p.i. after treatment with PGG or IPS; ( b ) The viral P protein expression level was analyzed by Western blotting during a 48-h period p.i.; ( c ) The relative expression levels of viral P, normalizing to that of α-tubulin. (* p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Infection, Expressing, Western Blot

    PGG inhibits the replication of RABV via suppression of mTOR-dependent autophagy. BHK-21 cells were pretreated with rapamycin (1 μM) or MHY1485 (5 μM) for 1 h, then infected with CVS-11 and PGG, rapamycin or MHY1485 respectively or in combination for 24 h. ( a , c ) autophagy markers and P protein were analyzed by Western blotting. ( b , d ) The relative expression levels of SQSTM1, LC3 and viral P, normalizing to that of α-tubulin. ( e ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted. (* p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: PGG inhibits the replication of RABV via suppression of mTOR-dependent autophagy. BHK-21 cells were pretreated with rapamycin (1 μM) or MHY1485 (5 μM) for 1 h, then infected with CVS-11 and PGG, rapamycin or MHY1485 respectively or in combination for 24 h. ( a , c ) autophagy markers and P protein were analyzed by Western blotting. ( b , d ) The relative expression levels of SQSTM1, LC3 and viral P, normalizing to that of α-tubulin. ( e ) The virus titers of culture supernatants were determined by TCID 50 assay and plotted. (* p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Infection, Western Blot, Expressing

    PGG inhibits the replication of RABV. Infected BHK-21 cells were treated with PGG or IPS for 48 h, and ( a ) the viral particles were detected by direct fluorescent antibody (DFA) assay; apple-green fluorescence represents fluorescein isothiocyanate (FITC)-labeled RABV, scale bar = 100 μm ( b ) The viral P gene mRNA expression level was detected by qRT-PCR. ( c ) The viral proteins’ expression levels were analyzed by Western blotting. ( d ) The relative expression levels of viral P, M, N, and G, normalizing to that of α-tubulin. ( e ) The viral P protein expression level was detected by in-cell Western assay; red fluorescence represents RABV P protein, scale bar = 100 μm ( f ) The relative expression levels of viral P, normalizing to that of the control. (* p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: PGG inhibits the replication of RABV. Infected BHK-21 cells were treated with PGG or IPS for 48 h, and ( a ) the viral particles were detected by direct fluorescent antibody (DFA) assay; apple-green fluorescence represents fluorescein isothiocyanate (FITC)-labeled RABV, scale bar = 100 μm ( b ) The viral P gene mRNA expression level was detected by qRT-PCR. ( c ) The viral proteins’ expression levels were analyzed by Western blotting. ( d ) The relative expression levels of viral P, M, N, and G, normalizing to that of α-tubulin. ( e ) The viral P protein expression level was detected by in-cell Western assay; red fluorescence represents RABV P protein, scale bar = 100 μm ( f ) The relative expression levels of viral P, normalizing to that of the control. (* p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Infection, Direct Fluorescent Antibody Test, Fluorescence, Labeling, Expressing, Quantitative RT-PCR, Western Blot, In-Cell ELISA

    PGG inhibits the replication of RABV via mTOR-associated autophagy. ( a ) mTOR pathway-associated proteins and autophagy markers were analyzed by Western blotting, after CVS-infected BHK-21 cells treated with PGG for 24 h. ( b ) The relative expression levels of mTOR, p-mTOR, SQSTM1, LC3, and viral P protein, normalizing to that of α-tubulin. BHK-21 cells were pretreated with rapamycin (1 or 2 μM) or MHY1485 (5 or 10 μM) for 1 h, then infected with CVS-11 and cultured in medium containing rapamycin or MHY1485 for 24 h. ( c , e ) p-mTOR, autophagy markers, and P protein were analyzed by Western blotting. ( d , f ) The relative expression levels of p-mTOR, SQSTM1, LC3, and viral P, normalizing to that of α-tubulin. (* p

    Journal: Viruses

    Article Title: Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

    doi: 10.3390/v10040201

    Figure Lengend Snippet: PGG inhibits the replication of RABV via mTOR-associated autophagy. ( a ) mTOR pathway-associated proteins and autophagy markers were analyzed by Western blotting, after CVS-infected BHK-21 cells treated with PGG for 24 h. ( b ) The relative expression levels of mTOR, p-mTOR, SQSTM1, LC3, and viral P protein, normalizing to that of α-tubulin. BHK-21 cells were pretreated with rapamycin (1 or 2 μM) or MHY1485 (5 or 10 μM) for 1 h, then infected with CVS-11 and cultured in medium containing rapamycin or MHY1485 for 24 h. ( c , e ) p-mTOR, autophagy markers, and P protein were analyzed by Western blotting. ( d , f ) The relative expression levels of p-mTOR, SQSTM1, LC3, and viral P, normalizing to that of α-tubulin. (* p

    Article Snippet: BHK-21 cells were propagated in Minimum Essential Medium (MEM; Corning Inc., Corning, NY, USA) supplemented with 5% fetal bovine serum (FBS; Corning), 100 U/mL penicillin G, and 100 µg/mL streptomycin.

    Techniques: Western Blot, Infection, Expressing, Cell Culture

    Analysis of fusion-promotion and cleavage-promoting activities of the HN mutant proteins. (A) HN mutant plasmid and F plasmid were cotransfected into BHK21 cells for 36 h, and then the fusion regions were photographed on an inverted microscope with 400-fold amplification, Bar = 20 μm. (B) The areas of 40 fusion regions were separately measured by using the software ImageJ as the fusion index of these HN mutant proteins. (C) After 36 h of cotransfection, the amount of each HN or F (F 0 and F 1 ) protein was determined by Western blot analysis using the HA or Flag label-specific mouse primary antibody and corresponding secondary antibody, and then determined the fusion-promotion activity of HN mutant proteins. (D) Those results of Western blot were quantified by densitometry using the ImageJ software. ∗ P

    Journal: Poultry Science

    Article Title: Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

    doi: 10.1016/j.psj.2020.04.014

    Figure Lengend Snippet: Analysis of fusion-promotion and cleavage-promoting activities of the HN mutant proteins. (A) HN mutant plasmid and F plasmid were cotransfected into BHK21 cells for 36 h, and then the fusion regions were photographed on an inverted microscope with 400-fold amplification, Bar = 20 μm. (B) The areas of 40 fusion regions were separately measured by using the software ImageJ as the fusion index of these HN mutant proteins. (C) After 36 h of cotransfection, the amount of each HN or F (F 0 and F 1 ) protein was determined by Western blot analysis using the HA or Flag label-specific mouse primary antibody and corresponding secondary antibody, and then determined the fusion-promotion activity of HN mutant proteins. (D) Those results of Western blot were quantified by densitometry using the ImageJ software. ∗ P

    Article Snippet: Chicken embryo fibroblasts ( DF-1 cells) and baby hamster kidney ( BHK21 ) cells were obtained from the American Type Culture Collection, preserved in our laboratory, and grown in Dulbecco's Modified Eagle Medium ( DMEM ; Gibco, Grand Island, NY) with 10% fetal bovine serum ( FBS ; Gibco, Grand Island, NY).

    Techniques: Mutagenesis, Plasmid Preparation, Inverted Microscopy, Amplification, Software, Cotransfection, Western Blot, Activity Assay

    Analysis of HAd and NA activities of HN mutant proteins. HN mutant plasmid was transfected into BHK21 cells for 24 h, and then determined for HAd activity (A) using HAd assay, and NA activity (B) using neuraminidase assay kit, respectively. ∗ P

    Journal: Poultry Science

    Article Title: Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

    doi: 10.1016/j.psj.2020.04.014

    Figure Lengend Snippet: Analysis of HAd and NA activities of HN mutant proteins. HN mutant plasmid was transfected into BHK21 cells for 24 h, and then determined for HAd activity (A) using HAd assay, and NA activity (B) using neuraminidase assay kit, respectively. ∗ P

    Article Snippet: Chicken embryo fibroblasts ( DF-1 cells) and baby hamster kidney ( BHK21 ) cells were obtained from the American Type Culture Collection, preserved in our laboratory, and grown in Dulbecco's Modified Eagle Medium ( DMEM ; Gibco, Grand Island, NY) with 10% fetal bovine serum ( FBS ; Gibco, Grand Island, NY).

    Techniques: Mutagenesis, Plasmid Preparation, Transfection, Activity Assay

    Analysis of HAd and NA activities between NDV-Blackbird and NDV-Dove. BHK21 cells were inoculated with 2 viruses at an MOI of 2 for 8 hpi and then determined for hemadsorption (HAd) (A) using HAd assay and neuraminidase (NA) activity (B) using neuraminidase assay kit, respectively. ∗∗∗ P

    Journal: Poultry Science

    Article Title: Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

    doi: 10.1016/j.psj.2020.04.014

    Figure Lengend Snippet: Analysis of HAd and NA activities between NDV-Blackbird and NDV-Dove. BHK21 cells were inoculated with 2 viruses at an MOI of 2 for 8 hpi and then determined for hemadsorption (HAd) (A) using HAd assay and neuraminidase (NA) activity (B) using neuraminidase assay kit, respectively. ∗∗∗ P

    Article Snippet: Chicken embryo fibroblasts ( DF-1 cells) and baby hamster kidney ( BHK21 ) cells were obtained from the American Type Culture Collection, preserved in our laboratory, and grown in Dulbecco's Modified Eagle Medium ( DMEM ; Gibco, Grand Island, NY) with 10% fetal bovine serum ( FBS ; Gibco, Grand Island, NY).

    Techniques: Activity Assay

    Analysis of cell surface expression efficiency of the HN mutant proteins. (A) The expression of the HN mutant proteins was detected by immunofluorescence assay (IFA). The HN mutant plasmid was transfected into BHK21 cells for 24 h, then the cells were incubated with primary antibody of anti-HA and secondary antibody of anti-Fluorescein Isothiocyanate (FITC), and then cells were photographed on a fluorescent inverted microscope with 100-fold magnification, Bar = 100 μm. (B) The surface expression efficiency of the HN mutant protein was detected by flow cytometry (FCM) assay. The HN mutant plasmid was transfected into BHK21 cells for 24 h, and cells were incubated with primary antibody and secondary antibody as mentioned previously, and then cell surface fluorescence intensity was measured by using a flow cytometer.

    Journal: Poultry Science

    Article Title: Screening and mechanistic study of key sites of the hemagglutinin-neuraminidase protein related to the virulence of Newcastle disease virus

    doi: 10.1016/j.psj.2020.04.014

    Figure Lengend Snippet: Analysis of cell surface expression efficiency of the HN mutant proteins. (A) The expression of the HN mutant proteins was detected by immunofluorescence assay (IFA). The HN mutant plasmid was transfected into BHK21 cells for 24 h, then the cells were incubated with primary antibody of anti-HA and secondary antibody of anti-Fluorescein Isothiocyanate (FITC), and then cells were photographed on a fluorescent inverted microscope with 100-fold magnification, Bar = 100 μm. (B) The surface expression efficiency of the HN mutant protein was detected by flow cytometry (FCM) assay. The HN mutant plasmid was transfected into BHK21 cells for 24 h, and cells were incubated with primary antibody and secondary antibody as mentioned previously, and then cell surface fluorescence intensity was measured by using a flow cytometer.

    Article Snippet: Chicken embryo fibroblasts ( DF-1 cells) and baby hamster kidney ( BHK21 ) cells were obtained from the American Type Culture Collection, preserved in our laboratory, and grown in Dulbecco's Modified Eagle Medium ( DMEM ; Gibco, Grand Island, NY) with 10% fetal bovine serum ( FBS ; Gibco, Grand Island, NY).

    Techniques: Expressing, Mutagenesis, Immunofluorescence, Plasmid Preparation, Transfection, Incubation, Inverted Microscopy, Flow Cytometry, Fluorescence