chikv  (ATCC)


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    Name:
    Chikungunya virus
    Description:
    Applications Vector borne research
    Catalog Number:
    VR-64
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    Applications:
    Vector borne research
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    Structured Review

    ATCC chikv
    RISPR-Cas9-based gene editing screen Mouse 3T3 cells were transduced separately with two half libraries (A + B) comprising 130,209 sgRNAs, selected with puromycin, and then inoculated with <t>CHIKV-181/25-mKate2</t> (MOI of 1). One day later, mKate2-negative cells were sorted, and expanded in the presence of 2 μg/ml each of <t>CHK-152</t> and CHK-166 neutralizing mAbs. Several days later, cells were re-inoculated with CHIKV-181/25-mKate2 without neutralizing mAbs and re-sorted for mKate2-negative cells. This procedure was repeated one additional time. Afterwards, genomic DNA was harvested for sgRNA sequencing and compared to the parent library for abundance (see Supplemental Tables 1 and 2 ). b . Diagram of the mouse Mxra8 and human MXRA8 orthologs. The transcript indentification numbers and length of proteins are indicated to the right. Partial deletions in the isoforms 3 and 4 are shown as dashed lines. c . Phylogenetic tree of Mxra8 indicating genetic relationships. The Neighbor-Joining tree was constructed using MEGA 7. Scale bar shows the branch length. ( Right ) Identity (red) and similarity (yellow) matrix indicating the conservation of Mxra8 between species. The matrix was generated using MagGat 1.8.
    Applications Vector borne research
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    Average 94 stars, based on 1 article reviews
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    chikv - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Mxra8 is a receptor for multiple arthritogenic alphaviruses"

    Article Title: Mxra8 is a receptor for multiple arthritogenic alphaviruses

    Journal: Nature

    doi: 10.1038/s41586-018-0121-3

    RISPR-Cas9-based gene editing screen Mouse 3T3 cells were transduced separately with two half libraries (A + B) comprising 130,209 sgRNAs, selected with puromycin, and then inoculated with CHIKV-181/25-mKate2 (MOI of 1). One day later, mKate2-negative cells were sorted, and expanded in the presence of 2 μg/ml each of CHK-152 and CHK-166 neutralizing mAbs. Several days later, cells were re-inoculated with CHIKV-181/25-mKate2 without neutralizing mAbs and re-sorted for mKate2-negative cells. This procedure was repeated one additional time. Afterwards, genomic DNA was harvested for sgRNA sequencing and compared to the parent library for abundance (see Supplemental Tables 1 and 2 ). b . Diagram of the mouse Mxra8 and human MXRA8 orthologs. The transcript indentification numbers and length of proteins are indicated to the right. Partial deletions in the isoforms 3 and 4 are shown as dashed lines. c . Phylogenetic tree of Mxra8 indicating genetic relationships. The Neighbor-Joining tree was constructed using MEGA 7. Scale bar shows the branch length. ( Right ) Identity (red) and similarity (yellow) matrix indicating the conservation of Mxra8 between species. The matrix was generated using MagGat 1.8.
    Figure Legend Snippet: RISPR-Cas9-based gene editing screen Mouse 3T3 cells were transduced separately with two half libraries (A + B) comprising 130,209 sgRNAs, selected with puromycin, and then inoculated with CHIKV-181/25-mKate2 (MOI of 1). One day later, mKate2-negative cells were sorted, and expanded in the presence of 2 μg/ml each of CHK-152 and CHK-166 neutralizing mAbs. Several days later, cells were re-inoculated with CHIKV-181/25-mKate2 without neutralizing mAbs and re-sorted for mKate2-negative cells. This procedure was repeated one additional time. Afterwards, genomic DNA was harvested for sgRNA sequencing and compared to the parent library for abundance (see Supplemental Tables 1 and 2 ). b . Diagram of the mouse Mxra8 and human MXRA8 orthologs. The transcript indentification numbers and length of proteins are indicated to the right. Partial deletions in the isoforms 3 and 4 are shown as dashed lines. c . Phylogenetic tree of Mxra8 indicating genetic relationships. The Neighbor-Joining tree was constructed using MEGA 7. Scale bar shows the branch length. ( Right ) Identity (red) and similarity (yellow) matrix indicating the conservation of Mxra8 between species. The matrix was generated using MagGat 1.8.

    Techniques Used: Sequencing, Construct, Generated

    2) Product Images from "A CRISPR screen defines a signal peptide processing pathway required by flaviviruses"

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

    Journal: Nature

    doi: 10.1038/nature18625

    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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

    Techniques Used: 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 .
    Figure Legend 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 .

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

    3) Product Images from "Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses"

    Article Title: Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00299-16

    (A) Amplification plot for DENV/CHIKV multiplex assay (10-fold dilutions, triplicate), (B) The linear dynamic range of the multiplex RT-PCR is shown as gene copy number for DENV (5 × 10 1 to 5 × 10 6 ) and CHIKV (6 × 10 1 to 6 × 10 6 ).
    Figure Legend Snippet: (A) Amplification plot for DENV/CHIKV multiplex assay (10-fold dilutions, triplicate), (B) The linear dynamic range of the multiplex RT-PCR is shown as gene copy number for DENV (5 × 10 1 to 5 × 10 6 ) and CHIKV (6 × 10 1 to 6 × 10 6 ).

    Techniques Used: Amplification, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "Inactivation of Chikungunya virus by 1,5 iodonapthyl azide"

    Article Title: Inactivation of Chikungunya virus by 1,5 iodonapthyl azide

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-301

    Inactivation of CHIKV by INA: Inactivation of CHIKV by INA was evaluated as CPE, virus titer and viral RNA transcripts. A : BHK cells were infected with various test and control samples and virus induced CPE was observed. Cells were stained with 0.1% crystal violet dye in 4% neutral buffered formalin. No CPE was observed in cells infected with INA treated CHIKV in combination with UV irradiation and cells replicated normally as evident by the stain uptake by the cells similar to control cells without virus infection. Irradiation of CHIKV alone and treatment with DMSO (since INA stocks were made in DMSO) did not affect the infectivity as evident by the loss of cells in these wells. B : Virus titer was measured in the supernatant of the cells infected with non-inactivated and INA-inactivated CHIKV. No virus was detected in the supernatant of INA-inactivated CHIKV and treatment with INA alone did not affect the virus replication. C : Viral transcripts were detected in the supernatants of cells infected with the non-inactivated and INA-inactivated CHIKV. No viral transcripts were detected in the cells infected with CHIKV inactivated with 50, 100 and 200 μM dose of INA in combination with UV irradiation. D : Western Blot analysis of the CHIKV proteins showed low binding of anti-CHIKV polyclonal antibody to CHIKV upon inactivation with INA. 1 = CHIKV; 2 = CHIKV + UV Irradiation; 3 = CHIKV + INA (200 μM); 4 = CHIKV + INA (50 μM) + UV Irradiation; 5 = CHIKV + INA (100 μM) + UV Irradiation; 6 = CHIKV + INA(200 μM) + UV Irradiation; and 7 = INA (200 μM) alone. Irr = UV irradiation.
    Figure Legend Snippet: Inactivation of CHIKV by INA: Inactivation of CHIKV by INA was evaluated as CPE, virus titer and viral RNA transcripts. A : BHK cells were infected with various test and control samples and virus induced CPE was observed. Cells were stained with 0.1% crystal violet dye in 4% neutral buffered formalin. No CPE was observed in cells infected with INA treated CHIKV in combination with UV irradiation and cells replicated normally as evident by the stain uptake by the cells similar to control cells without virus infection. Irradiation of CHIKV alone and treatment with DMSO (since INA stocks were made in DMSO) did not affect the infectivity as evident by the loss of cells in these wells. B : Virus titer was measured in the supernatant of the cells infected with non-inactivated and INA-inactivated CHIKV. No virus was detected in the supernatant of INA-inactivated CHIKV and treatment with INA alone did not affect the virus replication. C : Viral transcripts were detected in the supernatants of cells infected with the non-inactivated and INA-inactivated CHIKV. No viral transcripts were detected in the cells infected with CHIKV inactivated with 50, 100 and 200 μM dose of INA in combination with UV irradiation. D : Western Blot analysis of the CHIKV proteins showed low binding of anti-CHIKV polyclonal antibody to CHIKV upon inactivation with INA. 1 = CHIKV; 2 = CHIKV + UV Irradiation; 3 = CHIKV + INA (200 μM); 4 = CHIKV + INA (50 μM) + UV Irradiation; 5 = CHIKV + INA (100 μM) + UV Irradiation; 6 = CHIKV + INA(200 μM) + UV Irradiation; and 7 = INA (200 μM) alone. Irr = UV irradiation.

    Techniques Used: Infection, Staining, Irradiation, Western Blot, Binding Assay

    Related Articles

    Incubation:

    Article Title: A CRISPR screen defines a signal peptide processing pathway required by flaviviruses
    Article Snippet: At the indicated times, cells were fixed with 1% paraformaldehyde (PFA) diluted in PBS for 20 min at room temperature and permeabilized with Perm buffer (HBSS (Invitrogen), 10 mM HEPES, 0.1% (w/v) saponin (Sigma), and 0.025% NaN3 (Sigma)) for 10 min at room temperature. .. 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). .. After washing, cells were incubated with an Alexa Fluor 647-conjugated goat anti-mouse or anti-human IgG (Invitrogen) for 1 h at 4°C.

    Article Title: Mxra8 is a receptor for multiple arthritogenic alphaviruses
    Article Snippet: .. Cells then were incubated for 30 min at room temperature with 1 μg/ml of the following virus-specific antibodies: CHIKV (mouse CHK-11 ), ONNV and MAYV (mouse CHK-48), RRV (human 1I9); SINV (ascites fluid, ATCC VR-1248AF), SFV (mouse CHK-124), VEEV (mouse 1A4A), WEEV (mouse 11A1), EEEV (mouse EEEV-10), WNV (human E16 ), EMCV (mouse serum). .. After washing, cells were incubated with 2 μg/ml of Alexa Fluor 647-conjugated goat anti-mouse or anti-human IgG (Invitrogen) for 30 min at room temperature.

    Isolation:

    Article Title: Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy
    Article Snippet: Infected target cells were lysed at 48 h postinfection, and lysates were assayed for luciferase activity (Promega, Madison, WI). .. Replication-competent CHIKV S27 (ATCC vr-64), a strain originally isolated in 1953 from a patient in East Africa, was grown in Vero cells. ..

    Produced:

    Article Title: Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and chikungunya viruses favour different entry routes
    Article Snippet: .. Viruses SINV and CHIKV viruses were produced on BHK cells [originally obtained from American Type Culture Collection (ATCC), #CC-L10], according to . .. The SINV-GFP backbone is from the hybrid TE12 strain, with non-structural and capsid regions from the laboratory-adapted Toto1101 strain and most of the envelope region from the NSV strain isolated after six intracerebral passages of AR339 in mice ( ).

    Transfection:

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus
    Article Snippet: To prepare virus for NGS, transfection supernatants were harvested at indicated times post-transfection and the virus titer was determined by standard plaque assay in Vero cells. .. Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27. .. For ICA, electroporated cells were seeded into 6-well plates, allowed to adhere for 4 h, covered with 1% low-melting agarose in complete medium (aMEM containing 10% FBS and 10 μg/ml gentamicin), and incubated for 72 h. To visualize plaques, monolayers were stained using neutral red and counted the following day.

    Expressing:

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus
    Article Snippet: To prepare virus for NGS, transfection supernatants were harvested at indicated times post-transfection and the virus titer was determined by standard plaque assay in Vero cells. .. Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27. .. For ICA, electroporated cells were seeded into 6-well plates, allowed to adhere for 4 h, covered with 1% low-melting agarose in complete medium (aMEM containing 10% FBS and 10 μg/ml gentamicin), and incubated for 72 h. To visualize plaques, monolayers were stained using neutral red and counted the following day.

    Immunofluorescence:

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus
    Article Snippet: To prepare virus for NGS, transfection supernatants were harvested at indicated times post-transfection and the virus titer was determined by standard plaque assay in Vero cells. .. Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27. .. For ICA, electroporated cells were seeded into 6-well plates, allowed to adhere for 4 h, covered with 1% low-melting agarose in complete medium (aMEM containing 10% FBS and 10 μg/ml gentamicin), and incubated for 72 h. To visualize plaques, monolayers were stained using neutral red and counted the following day.

    Western Blot:

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus
    Article Snippet: To prepare virus for NGS, transfection supernatants were harvested at indicated times post-transfection and the virus titer was determined by standard plaque assay in Vero cells. .. Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27. .. For ICA, electroporated cells were seeded into 6-well plates, allowed to adhere for 4 h, covered with 1% low-melting agarose in complete medium (aMEM containing 10% FBS and 10 μg/ml gentamicin), and incubated for 72 h. To visualize plaques, monolayers were stained using neutral red and counted the following day.

    Article Title: Inactivation of Chikungunya virus by 1,5 iodonapthyl azide
    Article Snippet: .. Viral proteins were detected by standard western blot procedure using polyclonal antibody (1:200) against CHIKV (CHIKV hyper-immune mouse ascitic sera, Catalog#VR64, ATCC, Manassas, VA) and alkaline phosphatase conjugated secondary antibody (1:25000 in 1XTBST, goat anti-mouse IgG, Catalog# AP124A, Chemicon International). .. Blot was developed using 10 ml substrate for alkaline phosphatase (Catalog# S3841, Promega Inc., Madison, WI) at room temperature for 15 min. A complete inactivation of CHIKV by INA was achieved in virus samples that were treated with INA in combination with UV irradiation.

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  • chikv  (ATCC)
    93
    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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC bhk 21 cells
    Effects of the identified mutations on the synthesis of positive-strand RNAs and the nsP2 protein in cells transfected with CHIKV replicon RNAs. (A and B) Northern blot analysis was performed using total RNAs extracted from <t>BHK-21</t> (A) or Huh7 (B) cell
    Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC chikv hyperimmune mouse ascitic fluid hmaf vr 1241af
    Transfection of immunization DNA (iDNA) plasmid p181/25-7 into CHO cells. A , The immunization DNA (iDNA) approach for launching chikungunya virus <t>(CHIKV)</t> live attenuated virus in eukaryotic cells is shown on the left. Indicated are the cytomegalovirus (CMV) promoter (open arrow), cell nucleus, and progeny virus. Expression of CHIKV antigens after transfection of iDNA plasmid is shown on the right, detected by immunofluorescence assay (IFA) 48 and 96 hours after transfection. Aliquots of transfected cells were seeded in 8-well chamber slides, fixed at indicated times in cold acetone, and processed by IFA, using mouse CHIKV-specific antibody, followed by fluorescein isothiocyanate–conjugated secondary antibody. B , Detection of CHIKV antigens in transfected CHO cells by Western blot (left) and in the growth medium by plaque assay 48 hours after transfection (middle). For comparison, plaque assay for the 181/25 virus vaccine (passage 1 in CHO cells) is shown. Right panel shows growth curve of p181/25-7 iDNA-derived virus (average of 3 experiments). Western blot was done using human convalescent-phase CHIKV-specific serum (lane 1) and CHIKV <t>HMAF</t> (lane 2). The PE2, E2, E1, and C antigens are indicated.
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    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

    Characterization of the CHIKV-7 vaccine virus and 181/25 virus by growth curve and next generation sequencing. (a) Growth curves of CHIKV-7 virus in iDNA-transfected Vero cells (solid line) and of 181/25 virus in infected Vero cells (dashed line). Vero

    Journal: Virology

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus

    doi: 10.1016/j.virol.2016.01.009

    Figure Lengend Snippet: Characterization of the CHIKV-7 vaccine virus and 181/25 virus by growth curve and next generation sequencing. (a) Growth curves of CHIKV-7 virus in iDNA-transfected Vero cells (solid line) and of 181/25 virus in infected Vero cells (dashed line). Vero

    Article Snippet: Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27.

    Techniques: Next-Generation Sequencing, Transfection, Infection

    Preparation and characterization of iDNA-launched CHIKV-7 vaccine virus. (a) Schematic depiction of the CHIKV-7 virus production in Vero cells transfected with iDNA plasmid. The full-length cDNA copy of CHIKV genome (solid line) was placed downstream

    Journal: Virology

    Article Title: Next Generation Sequencing of DNA-Launched Chikungunya Vaccine Virus

    doi: 10.1016/j.virol.2016.01.009

    Figure Lengend Snippet: Preparation and characterization of iDNA-launched CHIKV-7 vaccine virus. (a) Schematic depiction of the CHIKV-7 virus production in Vero cells transfected with iDNA plasmid. The full-length cDNA copy of CHIKV genome (solid line) was placed downstream

    Article Snippet: Aliquots of transfected cell suspension were taken for CHIKV expression tests by immunofluorescence assay (IFA), infectious center assay (ICA) and western blot using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-64 (ATCC VR-1241AF) prepared against Chikungunya strain S-27.

    Techniques: Transfection, Plasmid Preparation

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.03213-14

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

    Article Snippet: Hence, in BHK-21 cells (and/or in the context of the full-length CHIKV genome), these mutations reduced the infectivity of CHIKV containing the 5A, PG, and 5A-PG mutations.

    Techniques: Transfection, Northern Blot

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.03213-14

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

    Article Snippet: Hence, in BHK-21 cells (and/or in the context of the full-length CHIKV genome), these mutations reduced the infectivity of CHIKV containing the 5A, PG, and 5A-PG mutations.

    Techniques: Infection, Mutagenesis, Immunofluorescence, Microscopy

    Development of stable BHK-21 CHIKV replicon cell lines.

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.03213-14

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

    Article Snippet: Hence, in BHK-21 cells (and/or in the context of the full-length CHIKV genome), these mutations reduced the infectivity of CHIKV containing the 5A, PG, and 5A-PG mutations.

    Techniques:

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.03213-14

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

    Article Snippet: Hence, in BHK-21 cells (and/or in the context of the full-length CHIKV genome), these mutations reduced the infectivity of CHIKV containing the 5A, PG, and 5A-PG mutations.

    Techniques: Transfection, Mutagenesis

    Transfection of immunization DNA (iDNA) plasmid p181/25-7 into CHO cells. A , The immunization DNA (iDNA) approach for launching chikungunya virus (CHIKV) live attenuated virus in eukaryotic cells is shown on the left. Indicated are the cytomegalovirus (CMV) promoter (open arrow), cell nucleus, and progeny virus. Expression of CHIKV antigens after transfection of iDNA plasmid is shown on the right, detected by immunofluorescence assay (IFA) 48 and 96 hours after transfection. Aliquots of transfected cells were seeded in 8-well chamber slides, fixed at indicated times in cold acetone, and processed by IFA, using mouse CHIKV-specific antibody, followed by fluorescein isothiocyanate–conjugated secondary antibody. B , Detection of CHIKV antigens in transfected CHO cells by Western blot (left) and in the growth medium by plaque assay 48 hours after transfection (middle). For comparison, plaque assay for the 181/25 virus vaccine (passage 1 in CHO cells) is shown. Right panel shows growth curve of p181/25-7 iDNA-derived virus (average of 3 experiments). Western blot was done using human convalescent-phase CHIKV-specific serum (lane 1) and CHIKV HMAF (lane 2). The PE2, E2, E1, and C antigens are indicated.

    Journal: The Journal of Infectious Diseases

    Article Title: DNA Vaccine Initiates Replication of Live Attenuated Chikungunya Virus In Vitro and Elicits Protective Immune Response in Mice

    doi: 10.1093/infdis/jiu114

    Figure Lengend Snippet: Transfection of immunization DNA (iDNA) plasmid p181/25-7 into CHO cells. A , The immunization DNA (iDNA) approach for launching chikungunya virus (CHIKV) live attenuated virus in eukaryotic cells is shown on the left. Indicated are the cytomegalovirus (CMV) promoter (open arrow), cell nucleus, and progeny virus. Expression of CHIKV antigens after transfection of iDNA plasmid is shown on the right, detected by immunofluorescence assay (IFA) 48 and 96 hours after transfection. Aliquots of transfected cells were seeded in 8-well chamber slides, fixed at indicated times in cold acetone, and processed by IFA, using mouse CHIKV-specific antibody, followed by fluorescein isothiocyanate–conjugated secondary antibody. B , Detection of CHIKV antigens in transfected CHO cells by Western blot (left) and in the growth medium by plaque assay 48 hours after transfection (middle). For comparison, plaque assay for the 181/25 virus vaccine (passage 1 in CHO cells) is shown. Right panel shows growth curve of p181/25-7 iDNA-derived virus (average of 3 experiments). Western blot was done using human convalescent-phase CHIKV-specific serum (lane 1) and CHIKV HMAF (lane 2). The PE2, E2, E1, and C antigens are indicated.

    Article Snippet: Expression of CHIKV antigens in iDNA-transfected and virus-infected cells was detected by immunofluorescence assay (IFA) and Western blot, using CHIKV hyperimmune mouse ascitic fluid (HMAF) VR-1241AF (ATCC).

    Techniques: Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Western Blot, Plaque Assay, Derivative Assay