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  • chikv  (ATCC)
    94
    ATCC chikv
    Viral infection in SPCS1 −/− cells a–c . Alphaviruses replicate and are processed efficiently in 293T cells in the absence of expression of SPCS1. a . SINV infection in control and SPCS1 −/− clonal cells. Cells were infected (MOI of 0.01) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. b . Control and SPCS1 −/− gene-edited 293T cells were infected with SINV. At the indicated time, lysates were prepared, electrophoresed and Western blotted with anti-SINV E2 ascites fluid (ATCC VR-1248AF). c . Control or SPCS1 −/− 293T cells were infected with <t>CHIKV</t> (MOI of 5). 8 h later, cells were labeled for 30 min with 35 S cysteine-methionine. Excess cold cysteine-methionine was added for indicated chase times (0, 1 or 4 h). An uninfected control established the specificity of the immunoprecipitation. After 35 S labeling, lysates were prepared and immunoprecipitated with anti-E2 MAb (CHK-48). Immunoprecipitates were left untreated (blank) or treated with Endo H (E) or PNGase F (P) for 1 h at 37°C prior to SDS-PAGE and fluorography. d . Sequencing of SPCS1 alleles in gene-edited 293T and Huh7 cell clones after puromycin selection and limiting dilution cloning. The sgRNA targeting site and the “PAM” sequences are highlighted at the top of WT gene, and the sequence of edited alleles are indicated. e . Western blotting of bulk-selected or clonal (clone #7) Huh7.5 cells (control and SPCS1 sgRNA selected) for expression of SPCS1 (~12 kDa). f . WNV, HCV, ZIKV, or <t>JEV</t> (Bennett strain) infection in control and SPCS1-deficient Huh7.5 cells. Cells were infected at an MOI of 0.01 (WNV, ZIKV, JEV) or 1 (HCV) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. g . Control or SPCS1 −/− Huh7.5 cells were infected at an MOI of 150 for 45 h with a pathogenic JEV isolate (Bennett strain). Lysates were blotted with an anti-JEV E MAb. Higher molecular weight bands (E hi and E med ) that reacted specifically with the anti-E MAb are indicated. One representative experiment of two is shown and loading controls (β-actin) are included. For gel source data, see Supplementary Figure 1 .
    Chikv, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH chikungunya virus chikv biomol
    Viral infection in SPCS1 −/− cells a–c . Alphaviruses replicate and are processed efficiently in 293T cells in the absence of expression of SPCS1. a . SINV infection in control and SPCS1 −/− clonal cells. Cells were infected (MOI of 0.01) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. b . Control and SPCS1 −/− gene-edited 293T cells were infected with SINV. At the indicated time, lysates were prepared, electrophoresed and Western blotted with anti-SINV E2 ascites fluid (ATCC VR-1248AF). c . Control or SPCS1 −/− 293T cells were infected with <t>CHIKV</t> (MOI of 5). 8 h later, cells were labeled for 30 min with 35 S cysteine-methionine. Excess cold cysteine-methionine was added for indicated chase times (0, 1 or 4 h). An uninfected control established the specificity of the immunoprecipitation. After 35 S labeling, lysates were prepared and immunoprecipitated with anti-E2 MAb (CHK-48). Immunoprecipitates were left untreated (blank) or treated with Endo H (E) or PNGase F (P) for 1 h at 37°C prior to SDS-PAGE and fluorography. d . Sequencing of SPCS1 alleles in gene-edited 293T and Huh7 cell clones after puromycin selection and limiting dilution cloning. The sgRNA targeting site and the “PAM” sequences are highlighted at the top of WT gene, and the sequence of edited alleles are indicated. e . Western blotting of bulk-selected or clonal (clone #7) Huh7.5 cells (control and SPCS1 sgRNA selected) for expression of SPCS1 (~12 kDa). f . WNV, HCV, ZIKV, or <t>JEV</t> (Bennett strain) infection in control and SPCS1-deficient Huh7.5 cells. Cells were infected at an MOI of 0.01 (WNV, ZIKV, JEV) or 1 (HCV) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. g . Control or SPCS1 −/− Huh7.5 cells were infected at an MOI of 150 for 45 h with a pathogenic JEV isolate (Bennett strain). Lysates were blotted with an anti-JEV E MAb. Higher molecular weight bands (E hi and E med ) that reacted specifically with the anti-E MAb are indicated. One representative experiment of two is shown and loading controls (β-actin) are included. For gel source data, see Supplementary Figure 1 .
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    93
    BEI Resources chikungunya virus chikv chikv
    Viral infection in SPCS1 −/− cells a–c . Alphaviruses replicate and are processed efficiently in 293T cells in the absence of expression of SPCS1. a . SINV infection in control and SPCS1 −/− clonal cells. Cells were infected (MOI of 0.01) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. b . Control and SPCS1 −/− gene-edited 293T cells were infected with SINV. At the indicated time, lysates were prepared, electrophoresed and Western blotted with anti-SINV E2 ascites fluid (ATCC VR-1248AF). c . Control or SPCS1 −/− 293T cells were infected with <t>CHIKV</t> (MOI of 5). 8 h later, cells were labeled for 30 min with 35 S cysteine-methionine. Excess cold cysteine-methionine was added for indicated chase times (0, 1 or 4 h). An uninfected control established the specificity of the immunoprecipitation. After 35 S labeling, lysates were prepared and immunoprecipitated with anti-E2 MAb (CHK-48). Immunoprecipitates were left untreated (blank) or treated with Endo H (E) or PNGase F (P) for 1 h at 37°C prior to SDS-PAGE and fluorography. d . Sequencing of SPCS1 alleles in gene-edited 293T and Huh7 cell clones after puromycin selection and limiting dilution cloning. The sgRNA targeting site and the “PAM” sequences are highlighted at the top of WT gene, and the sequence of edited alleles are indicated. e . Western blotting of bulk-selected or clonal (clone #7) Huh7.5 cells (control and SPCS1 sgRNA selected) for expression of SPCS1 (~12 kDa). f . WNV, HCV, ZIKV, or <t>JEV</t> (Bennett strain) infection in control and SPCS1-deficient Huh7.5 cells. Cells were infected at an MOI of 0.01 (WNV, ZIKV, JEV) or 1 (HCV) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. g . Control or SPCS1 −/− Huh7.5 cells were infected at an MOI of 150 for 45 h with a pathogenic JEV isolate (Bennett strain). Lysates were blotted with an anti-JEV E MAb. Higher molecular weight bands (E hi and E med ) that reacted specifically with the anti-E MAb are indicated. One representative experiment of two is shown and loading controls (β-actin) are included. For gel source data, see Supplementary Figure 1 .
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    91
    EUROIMMUN chikungunya virus chikv
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
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    85
    ATCC chikv strain s27
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikv Strain S27, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    EUROIMMUN chikungunya virus chikv immunofluorescence assay
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikungunya Virus Chikv Immunofluorescence Assay, supplied by EUROIMMUN, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Themis Bioscience chikungunya virus
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
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    91
    CTK Biotech chikungunya virus
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikungunya Virus, supplied by CTK Biotech, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Prospec chikungunya virus chikv type e1 envelope protein
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikungunya Virus Chikv Type E1 Envelope Protein, supplied by Prospec, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GenWay chikv
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikv, supplied by GenWay, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    chikv  (Abcam)
    90
    Abcam chikv
    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second <t>chikungunya</t> fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.
    Chikv, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti chikv igm
    Comparison of <t>EILV/CHIKV</t> and Abcam human anti-CHIKV <t>IgM-capture</t> ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.
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    TIB MOLBIOL lightmix kit chikungunya virus light cycler
    Comparison of <t>EILV/CHIKV</t> and Abcam human anti-CHIKV <t>IgM-capture</t> ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.
    Lightmix Kit Chikungunya Virus Light Cycler, supplied by TIB MOLBIOL, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATCC chikv antigen
    Comparison of <t>EILV/CHIKV</t> and Abcam human anti-CHIKV <t>IgM-capture</t> ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.
    Chikv Antigen, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche lightmix chikungunya virus kit
    Comparison of <t>EILV/CHIKV</t> and Abcam human anti-CHIKV <t>IgM-capture</t> ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.
    Lightmix Chikungunya Virus Kit, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IBT Bioservices anti chikungunya virus coat protein antibody
    Comparison of <t>EILV/CHIKV</t> and Abcam human anti-CHIKV <t>IgM-capture</t> ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.
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    90
    Lifespan Biosciences anti chikungunya virus mouse antibody
    Progression of lesions in IRF 3/7 -/- -/- mice infected with <t>chikungunya</t> virus [mice were not evaluated on Day 3 post-infection].
    Anti Chikungunya Virus Mouse Antibody, supplied by Lifespan Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oxford Nanopore high titer chikungunya virus
    Progression of lesions in IRF 3/7 -/- -/- mice infected with <t>chikungunya</t> virus [mice were not evaluated on Day 3 post-infection].
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    Image Search Results


    Viral infection in SPCS1 −/− cells a–c . Alphaviruses replicate and are processed efficiently in 293T cells in the absence of expression of SPCS1. a . SINV infection in control and SPCS1 −/− clonal cells. Cells were infected (MOI of 0.01) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. b . Control and SPCS1 −/− gene-edited 293T cells were infected with SINV. At the indicated time, lysates were prepared, electrophoresed and Western blotted with anti-SINV E2 ascites fluid (ATCC VR-1248AF). c . Control or SPCS1 −/− 293T cells were infected with CHIKV (MOI of 5). 8 h later, cells were labeled for 30 min with 35 S cysteine-methionine. Excess cold cysteine-methionine was added for indicated chase times (0, 1 or 4 h). An uninfected control established the specificity of the immunoprecipitation. After 35 S labeling, lysates were prepared and immunoprecipitated with anti-E2 MAb (CHK-48). Immunoprecipitates were left untreated (blank) or treated with Endo H (E) or PNGase F (P) for 1 h at 37°C prior to SDS-PAGE and fluorography. d . Sequencing of SPCS1 alleles in gene-edited 293T and Huh7 cell clones after puromycin selection and limiting dilution cloning. The sgRNA targeting site and the “PAM” sequences are highlighted at the top of WT gene, and the sequence of edited alleles are indicated. e . Western blotting of bulk-selected or clonal (clone #7) Huh7.5 cells (control and SPCS1 sgRNA selected) for expression of SPCS1 (~12 kDa). f . WNV, HCV, ZIKV, or JEV (Bennett strain) infection in control and SPCS1-deficient Huh7.5 cells. Cells were infected at an MOI of 0.01 (WNV, ZIKV, JEV) or 1 (HCV) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. g . Control or SPCS1 −/− Huh7.5 cells were infected at an MOI of 150 for 45 h with a pathogenic JEV isolate (Bennett strain). Lysates were blotted with an anti-JEV E MAb. Higher molecular weight bands (E hi and E med ) that reacted specifically with the anti-E MAb are indicated. One representative experiment of two is shown and loading controls (β-actin) are included. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: A CRISPR screen defines a signal peptide processing pathway required by flaviviruses

    doi: 10.1038/nature18625

    Figure Lengend Snippet: Viral infection in SPCS1 −/− cells a–c . Alphaviruses replicate and are processed efficiently in 293T cells in the absence of expression of SPCS1. a . SINV infection in control and SPCS1 −/− clonal cells. Cells were infected (MOI of 0.01) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. b . Control and SPCS1 −/− gene-edited 293T cells were infected with SINV. At the indicated time, lysates were prepared, electrophoresed and Western blotted with anti-SINV E2 ascites fluid (ATCC VR-1248AF). c . Control or SPCS1 −/− 293T cells were infected with CHIKV (MOI of 5). 8 h later, cells were labeled for 30 min with 35 S cysteine-methionine. Excess cold cysteine-methionine was added for indicated chase times (0, 1 or 4 h). An uninfected control established the specificity of the immunoprecipitation. After 35 S labeling, lysates were prepared and immunoprecipitated with anti-E2 MAb (CHK-48). Immunoprecipitates were left untreated (blank) or treated with Endo H (E) or PNGase F (P) for 1 h at 37°C prior to SDS-PAGE and fluorography. d . Sequencing of SPCS1 alleles in gene-edited 293T and Huh7 cell clones after puromycin selection and limiting dilution cloning. The sgRNA targeting site and the “PAM” sequences are highlighted at the top of WT gene, and the sequence of edited alleles are indicated. e . Western blotting of bulk-selected or clonal (clone #7) Huh7.5 cells (control and SPCS1 sgRNA selected) for expression of SPCS1 (~12 kDa). f . WNV, HCV, ZIKV, or JEV (Bennett strain) infection in control and SPCS1-deficient Huh7.5 cells. Cells were infected at an MOI of 0.01 (WNV, ZIKV, JEV) or 1 (HCV) and supernatants were harvested and analyzed by FFA. The results are the average of two independent experiments performed in triplicate. g . Control or SPCS1 −/− Huh7.5 cells were infected at an MOI of 150 for 45 h with a pathogenic JEV isolate (Bennett strain). Lysates were blotted with an anti-JEV E MAb. Higher molecular weight bands (E hi and E med ) that reacted specifically with the anti-E MAb are indicated. One representative experiment of two is shown and loading controls (β-actin) are included. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Cells then were rinsed one additional time with Perm buffer, transferred to a U-bottom plate, and incubated for 1 h at 4°C with 1 µg/ml of the following virus-specific antibodies: WNV (human E16 ); DENV2 (mouse E18 ); JEV (mouse E18 ); YFV (mouse E60 ); CHIKV (CHK-11 ); SINV (ascites fluid, ATCC VR-1248AF), LACV (807-31 and 807-33, gift of A. Pekosz, Baltimore, MD).

    Techniques: Infection, Expressing, Western Blot, Labeling, Immunoprecipitation, SDS Page, Sequencing, Clone Assay, Selection, Molecular Weight

    SPCS1 is required for flavivirus protein processing and infection a . Western blotting of SPCS1 −/− 293T cells. b . Cells were transfected with YFV-luciferase replicon RNA (WT GDD or loss-of-function GVD). Firefly luciferase activity was measured and normalized to intracellular protein levels. The data reflects the average of two to three independent experiments performed in duplicate. c–h . Cells were infected with WNV ( c, h ), DENV-2 ( d ), JEV ( e ), YFV ( f ) or ZIKV ( g ), and viral yield measured. In h , cells were trans-complemented with an SPCS1 or control plasmid. Results are the average of two to three independent experiments performed in triplicate. i–k . Cells were infected with CHIKV (alphavirus), RVFV (bunyavirus), or VSV (rhabdovirus) and viral yield was measured. Results are the average of two to three independent experiments performed in triplicate. l . The polyprotein processing strategy of flaviviruses 13 . Red and blue arrows indicate sites of cleavage by host and viral (NS2B-NS3) proteases, respectively. m–o . Control or SPCS1 −/− 293T ( m , o ) or Huh7.5 ( n ) cells were infected with WNV ( m, o ) or JEV ( n ). Lysates were blotted with ( m ) anti-WNV E, ( n ) anti-JEV E, or ( o ) anti-WNV NS1 MAbs. Higher molecular weight bands (E hi , E med , and NS1 hi ) that react with anti-flavivirus MAbs are indicated. One experiment of three is shown. p . 293T cells were infected with CHIKV or RVFV. Lysates were blotted with anti-CHIKV E2 or anti-RVFV Gn mAbs. One experiment of two is shown. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: A CRISPR screen defines a signal peptide processing pathway required by flaviviruses

    doi: 10.1038/nature18625

    Figure Lengend Snippet: SPCS1 is required for flavivirus protein processing and infection a . Western blotting of SPCS1 −/− 293T cells. b . Cells were transfected with YFV-luciferase replicon RNA (WT GDD or loss-of-function GVD). Firefly luciferase activity was measured and normalized to intracellular protein levels. The data reflects the average of two to three independent experiments performed in duplicate. c–h . Cells were infected with WNV ( c, h ), DENV-2 ( d ), JEV ( e ), YFV ( f ) or ZIKV ( g ), and viral yield measured. In h , cells were trans-complemented with an SPCS1 or control plasmid. Results are the average of two to three independent experiments performed in triplicate. i–k . Cells were infected with CHIKV (alphavirus), RVFV (bunyavirus), or VSV (rhabdovirus) and viral yield was measured. Results are the average of two to three independent experiments performed in triplicate. l . The polyprotein processing strategy of flaviviruses 13 . Red and blue arrows indicate sites of cleavage by host and viral (NS2B-NS3) proteases, respectively. m–o . Control or SPCS1 −/− 293T ( m , o ) or Huh7.5 ( n ) cells were infected with WNV ( m, o ) or JEV ( n ). Lysates were blotted with ( m ) anti-WNV E, ( n ) anti-JEV E, or ( o ) anti-WNV NS1 MAbs. Higher molecular weight bands (E hi , E med , and NS1 hi ) that react with anti-flavivirus MAbs are indicated. One experiment of three is shown. p . 293T cells were infected with CHIKV or RVFV. Lysates were blotted with anti-CHIKV E2 or anti-RVFV Gn mAbs. One experiment of two is shown. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Cells then were rinsed one additional time with Perm buffer, transferred to a U-bottom plate, and incubated for 1 h at 4°C with 1 µg/ml of the following virus-specific antibodies: WNV (human E16 ); DENV2 (mouse E18 ); JEV (mouse E18 ); YFV (mouse E60 ); CHIKV (CHK-11 ); SINV (ascites fluid, ATCC VR-1248AF), LACV (807-31 and 807-33, gift of A. Pekosz, Baltimore, MD).

    Techniques: Infection, Western Blot, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Molecular Weight

    Gene editing of SEC11A and SEC11C do not susbtantively impact infection of several enveloped viruses ( Top ) 293T cells were administered the indicated sgRNA and isolated in bulk after puromycin drug selection. Western blotting confirmed gene editing of SEC11A ( left , 20 kDa) or SEC11C ( middle , 22 kDa). No difference in levels or migration pattern of WNV E was observed in SEC11A or SEC11C gene edited cells ( right ) after WNV infection at an MOI of 200 for 24 h. Spaces between the Western blots indicate cropping to remove lanes that were not relevant to this experiment. ( Bottom ) Control or gene-edited 293T cells were infected with viruses and supernatants were harvested after infection for titration. Left . WNV (MOI of 0.01, 72 h) or YFV (MOI of 1, 72 h); Right , SINV (MOI of 0.01, 72 h), CHIKV (MOI of 0.01, 36 h), VSV (MOI of 0.01, 36 h), or RVFV (MOI of 1, 72 h). Results are representative of two independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: A CRISPR screen defines a signal peptide processing pathway required by flaviviruses

    doi: 10.1038/nature18625

    Figure Lengend Snippet: Gene editing of SEC11A and SEC11C do not susbtantively impact infection of several enveloped viruses ( Top ) 293T cells were administered the indicated sgRNA and isolated in bulk after puromycin drug selection. Western blotting confirmed gene editing of SEC11A ( left , 20 kDa) or SEC11C ( middle , 22 kDa). No difference in levels or migration pattern of WNV E was observed in SEC11A or SEC11C gene edited cells ( right ) after WNV infection at an MOI of 200 for 24 h. Spaces between the Western blots indicate cropping to remove lanes that were not relevant to this experiment. ( Bottom ) Control or gene-edited 293T cells were infected with viruses and supernatants were harvested after infection for titration. Left . WNV (MOI of 0.01, 72 h) or YFV (MOI of 1, 72 h); Right , SINV (MOI of 0.01, 72 h), CHIKV (MOI of 0.01, 36 h), VSV (MOI of 0.01, 36 h), or RVFV (MOI of 1, 72 h). Results are representative of two independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Cells then were rinsed one additional time with Perm buffer, transferred to a U-bottom plate, and incubated for 1 h at 4°C with 1 µg/ml of the following virus-specific antibodies: WNV (human E16 ); DENV2 (mouse E18 ); JEV (mouse E18 ); YFV (mouse E60 ); CHIKV (CHK-11 ); SINV (ascites fluid, ATCC VR-1248AF), LACV (807-31 and 807-33, gift of A. Pekosz, Baltimore, MD).

    Techniques: Infection, Isolation, Selection, Western Blot, Migration, Titration

    MAb IM-CKV063 strongly neutralizes CHIKV. Anti-CHIKV MAbs were tested for the ability to neutralize the infectivity of reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 (A) or the VSV envelope (B). Viruses were preincubated with MAbs, as described in the Materials and Methods section, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point is the mean of two replicates, and data are representative of those from at least two independent experiments. (C) IM-CKV063 was tested for neutralization of additional alphavirus envelope proteins pseudotyped onto reporter HIV isolates. Data points represent the mean of three replicates, and data are representative of those from two independent experiments. (D) Live CHIKV was preincubated with MAbs before addition to Vero cells. The S27 strain of CHIKV was used in all experiments. Strain 37997 was also tested against IM-CKV065. Infectivity was assessed after 72 h using a PRNT. Data points represent the mean and standard deviation of two to three replicates and are representative of those from at least two individual experiments. Neut, neutralization.

    Journal: Journal of Virology

    Article Title: Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

    doi: 10.1128/JVI.01943-14

    Figure Lengend Snippet: MAb IM-CKV063 strongly neutralizes CHIKV. Anti-CHIKV MAbs were tested for the ability to neutralize the infectivity of reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 (A) or the VSV envelope (B). Viruses were preincubated with MAbs, as described in the Materials and Methods section, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point is the mean of two replicates, and data are representative of those from at least two independent experiments. (C) IM-CKV063 was tested for neutralization of additional alphavirus envelope proteins pseudotyped onto reporter HIV isolates. Data points represent the mean of three replicates, and data are representative of those from two independent experiments. (D) Live CHIKV was preincubated with MAbs before addition to Vero cells. The S27 strain of CHIKV was used in all experiments. Strain 37997 was also tested against IM-CKV065. Infectivity was assessed after 72 h using a PRNT. Data points represent the mean and standard deviation of two to three replicates and are representative of those from at least two individual experiments. Neut, neutralization.

    Article Snippet: Replication-competent CHIKV S27 (ATCC vr-64), a strain originally isolated in 1953 from a patient in East Africa, was grown in Vero cells.

    Techniques: Infection, Expressing, Luciferase, Neutralization, Plaque Reduction Neutralization Test, Standard Deviation

    Temperature-dependent effects of CHIKV MAb neutralization. The effect of temperature on MAb neutralization is shown for a representative nonneutralizing MAb (IM-CKV061) (A), a moderately neutralizing MAb (IM-CKV065) (B), and a strongly neutralizing MAb (IM-CKV063) (C). Reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 were preincubated with MAbs at the indicated temperatures, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point represents the mean and range of two replicates within an experiment. Data shown are representative of those from two independent experiments. For IM-CKV065 and IM-CKV063, there were no statistically significant differences in log EC 50 at different temperatures ( P > 0.05). IM-CKV061 did not reach 50% neutralization irrespective of temperature.

    Journal: Journal of Virology

    Article Title: Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

    doi: 10.1128/JVI.01943-14

    Figure Lengend Snippet: Temperature-dependent effects of CHIKV MAb neutralization. The effect of temperature on MAb neutralization is shown for a representative nonneutralizing MAb (IM-CKV061) (A), a moderately neutralizing MAb (IM-CKV065) (B), and a strongly neutralizing MAb (IM-CKV063) (C). Reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 were preincubated with MAbs at the indicated temperatures, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point represents the mean and range of two replicates within an experiment. Data shown are representative of those from two independent experiments. For IM-CKV065 and IM-CKV063, there were no statistically significant differences in log EC 50 at different temperatures ( P > 0.05). IM-CKV061 did not reach 50% neutralization irrespective of temperature.

    Article Snippet: Replication-competent CHIKV S27 (ATCC vr-64), a strain originally isolated in 1953 from a patient in East Africa, was grown in Vero cells.

    Techniques: Neutralization, Infection, Expressing, Luciferase

    Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second chikungunya fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Evaluation of Chikungunya Diagnostic Assays: Differences in Sensitivity of Serology Assays in Two Independent Outbreaks

    doi: 10.1371/journal.pntd.0000753

    Figure Lengend Snippet: Longitudinal variation of the sensitivity of IgM assays in the first seven days after fever onset. 1a: overall sensitivity based on samples collected from first and second chikungunya fever outbreaks in January and May to September 2008, respectively; 1b: sensitivity based on samples collected from the first outbreak due to CHIKV-A226 strain; 1c: sensitivity based on samples collected from the second outbreak due to CHIKV-226V strain. The numbers in brackets along the x-axis represent the number of samples tested for each day after onset of fever.

    Article Snippet: Using sera collected in outbreaks caused by two variants of Chikungunya virus (A226 and 226V), we tested 2 commercial IgM tests (CTK lateral flow rapid test and EUROIMMUN IFA) alongside our in-house IgM assays (using both variants of the virus).

    Techniques:

    Comparison of EILV/CHIKV and Abcam human anti-CHIKV IgM-capture ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

    doi: 10.1371/journal.pntd.0004119

    Figure Lengend Snippet: Comparison of EILV/CHIKV and Abcam human anti-CHIKV IgM-capture ELISA methods. (A) raw OD values or (B) signal-to-noise ratios (calculated by dividing positive sample OD values by mean negative control OD value) produced by samples of varying degrees of seropositivity were compared. Low-positive samples had PRNT 50 ≤80 (n = 4), medium-positive samples had PRNT 50 160–320 (n = 4), and high-positive samples had PRNT 50 > 640 (n = 24). Mean and standard deviations are reported. A 2-way ANOVA with Tukey’s test was used to compare absorbance values and signal-to-noise ratios within each ELISA method. 2-way ANOVA with Bonferroni’s test was used to compare absorbance values and signal-to-noise ratios between each ELISA method.

    Article Snippet: Commercially available anti-CHIKV IgM (ab177848, Lot: GR195090-3, Abcam, Cambridge, MA) and anti-CHIKV IgG Human ELISA Kits (ab177835, Lot: GR148047-1, Abcam) were used according to manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Produced, Plaque Reduction Neutralization Test

    Human anti-CHIKV antibody-capture ELISAs utilizing EILV/CHIKV as antigen to detect either (A) IgM or (B) IgG antibodies (both measured over a range of serum dilutions, red). Human serum samples that were antibody-positive for either DENV or VEEV but negative for CHIKV by HI were included as negative controls. Mean and standard deviations of 2 replicates are reported.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

    doi: 10.1371/journal.pntd.0004119

    Figure Lengend Snippet: Human anti-CHIKV antibody-capture ELISAs utilizing EILV/CHIKV as antigen to detect either (A) IgM or (B) IgG antibodies (both measured over a range of serum dilutions, red). Human serum samples that were antibody-positive for either DENV or VEEV but negative for CHIKV by HI were included as negative controls. Mean and standard deviations of 2 replicates are reported.

    Article Snippet: Commercially available anti-CHIKV IgM (ab177848, Lot: GR195090-3, Abcam, Cambridge, MA) and anti-CHIKV IgG Human ELISA Kits (ab177835, Lot: GR148047-1, Abcam) were used according to manufacturer’s instructions.

    Techniques:

    Progression of lesions in IRF 3/7 -/- -/- mice infected with chikungunya virus [mice were not evaluated on Day 3 post-infection].

    Journal: PLoS ONE

    Article Title: Novel Lesions of Bones and Joints Associated with Chikungunya Virus Infection in Two Mouse Models of Disease: New Insights into Disease Pathogenesis

    doi: 10.1371/journal.pone.0155243

    Figure Lengend Snippet: Progression of lesions in IRF 3/7 -/- -/- mice infected with chikungunya virus [mice were not evaluated on Day 3 post-infection].

    Article Snippet: Numerous attempts were made to develop an immunohistochemistry protocol using a commercially available anti-chikungunya virus mouse antibody (LifeSpan BioSciences, Inc., Seattle, WA) and Mouse on Mouse peroxidase and fluorescein kits (Vector Laboratories, Burlingame, CA) (data not shown).

    Techniques: Mouse Assay, Infection

    Progression of lesions in C57BL/6J mice infected with chikungunya virus.

    Journal: PLoS ONE

    Article Title: Novel Lesions of Bones and Joints Associated with Chikungunya Virus Infection in Two Mouse Models of Disease: New Insights into Disease Pathogenesis

    doi: 10.1371/journal.pone.0155243

    Figure Lengend Snippet: Progression of lesions in C57BL/6J mice infected with chikungunya virus.

    Article Snippet: Numerous attempts were made to develop an immunohistochemistry protocol using a commercially available anti-chikungunya virus mouse antibody (LifeSpan BioSciences, Inc., Seattle, WA) and Mouse on Mouse peroxidase and fluorescein kits (Vector Laboratories, Burlingame, CA) (data not shown).

    Techniques: Mouse Assay, Infection