human rsv  (ATCC)


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    Name:
    Human respiratory syncytial virus Long
    Description:

    Catalog Number:
    vr-26
    Price:
    None
    Applications:
    Testing virucidesRespiratory research
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    Structured Review

    ATCC human rsv

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    Average 85 stars, based on 14 article reviews
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    Related Articles

    Plaque Assay:

    Article Title: Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state
    Article Snippet: .. RSV A2, an A subtype of RSV (ATCC, VR-1540, Rockville), RSV B49, a B subtype of RSV (BE/5649/08 clinical strain , source described in ref. , obtained from Prof Marc Van Ranst, KU Leuven, Leuven, Belgium), RSV A Long (ATCC, VR-26, kind gift from Dr Rik De Swart, Erasmus MC, Rotterdam, The Netherlands), MAD/GM2_2/12, MAD/GM2_12/12, MAD/GM2_13/12, MAD/GM2_14/12, MAD/GM3_10/14, MON/9/92 (primary RSV A strains, isolated in Madrid (MAD) or Montevideo (MON)) and MAD/GM3_7/13 (a primary RSV B strain isolated in Madrid) were propagated in HEp-2 cells and quantified on Vero cells by plaque assay using goat anti-RSV serum (AB1128, Chemicon International). ..

    Isolation:

    Article Title: Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state
    Article Snippet: .. RSV A2, an A subtype of RSV (ATCC, VR-1540, Rockville), RSV B49, a B subtype of RSV (BE/5649/08 clinical strain , source described in ref. , obtained from Prof Marc Van Ranst, KU Leuven, Leuven, Belgium), RSV A Long (ATCC, VR-26, kind gift from Dr Rik De Swart, Erasmus MC, Rotterdam, The Netherlands), MAD/GM2_2/12, MAD/GM2_12/12, MAD/GM2_13/12, MAD/GM2_14/12, MAD/GM3_10/14, MON/9/92 (primary RSV A strains, isolated in Madrid (MAD) or Montevideo (MON)) and MAD/GM3_7/13 (a primary RSV B strain isolated in Madrid) were propagated in HEp-2 cells and quantified on Vero cells by plaque assay using goat anti-RSV serum (AB1128, Chemicon International). ..

    Article Title: The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents
    Article Snippet: .. Viruses, cells, viral ELISA antigens RSV-A Long strain (ATCC VR-26, American Type Culture Collection, Rockville, MD), and strain BT2a, (derived from an infant hospitalised with bronchiolitis and isolated in Dr. Power's laboratory) as well as RSV-B strain 8/60 (kindly provided by Dr. G. Taylor, Institute of Animal Health, Compton, UK) and strain CH18537 (kindly provided by Prof. Geoff Toms, University of Newcastle-upon-Tyne, UK) were propagated in HEp-2 cells (ECACC 86030501, European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) as previously described , except that DMEM was supplemented with 1% fetal calf serum. ..

    Infection:

    Article Title: RSV Infection in Human Macrophages Promotes CXCL10/IP-10 Expression during Bacterial Co-Infection
    Article Snippet: .. These human MDMs were then exposed to RSV (long strain, ATCC VR-26) at different multiplicity of infection (MOI) to investigate the kinetics of infection by monitoring of genome copy number using a specific RT-qPCR on cell lysates harvested at different time points ( A). .. As a comparison, immortalized monocyte-like THP-1 cells were infected in parallel with the same protocol.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein
    Article Snippet: .. Propagation of RSV-A (Long strain; ATCC VR-26 [American Type Culture Collection, Rockville, Md.]) in HEp-2 cells (ECACC 86030501; European Collection of Animal Cell Cultures, Porton Down, Salisbury, United Kingdom), as well as the production of viral protein and uninfected-cell enzyme-linked immunosorbent assay (ELISA) antigens, was performed as previously described ( ). .. Recombinant protein G2ΔCa was produced by expressing and purifying BBG2ΔCa as previously described , cleaving BBG2ΔCa by cyanogen bromide hydrolysis into BB and G2ΔCa components, and purifying the products by reverse-phase high-performance liquid chromatography (RP-HPLC).

    Article Title: The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents
    Article Snippet: .. Viruses, cells, viral ELISA antigens RSV-A Long strain (ATCC VR-26, American Type Culture Collection, Rockville, MD), and strain BT2a, (derived from an infant hospitalised with bronchiolitis and isolated in Dr. Power's laboratory) as well as RSV-B strain 8/60 (kindly provided by Dr. G. Taylor, Institute of Animal Health, Compton, UK) and strain CH18537 (kindly provided by Prof. Geoff Toms, University of Newcastle-upon-Tyne, UK) were propagated in HEp-2 cells (ECACC 86030501, European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) as previously described , except that DMEM was supplemented with 1% fetal calf serum. ..

    other:

    Article Title: Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B
    Article Snippet: Amino acid variability of RSV subgroups The F domains of RSV/A and RSV/B were compared to the historical RSV/A Long strain (ATCC VR-26; RSV/A Long).

    Article Title: Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B
    Article Snippet: To determine the amino acid variability in the F domains of RSV/A and RSV/B, the RSV/A and RSV/B subgroups were compared to the historical RSV/A Long strain (ATCC VR-26; RSV/A Long), a GA1 genotype.

    Article Title: Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B
    Article Snippet: Amino acid variability among RSV genotypes The F antigenic sites of isolates for each genotype were compared to the historical RSV/A Long strain (ATCC VR-26; RSV/A Long).

    Quantitative RT-PCR:

    Article Title: RSV Infection in Human Macrophages Promotes CXCL10/IP-10 Expression during Bacterial Co-Infection
    Article Snippet: .. These human MDMs were then exposed to RSV (long strain, ATCC VR-26) at different multiplicity of infection (MOI) to investigate the kinetics of infection by monitoring of genome copy number using a specific RT-qPCR on cell lysates harvested at different time points ( A). .. As a comparison, immortalized monocyte-like THP-1 cells were infected in parallel with the same protocol.

    Derivative Assay:

    Article Title: The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents
    Article Snippet: .. Viruses, cells, viral ELISA antigens RSV-A Long strain (ATCC VR-26, American Type Culture Collection, Rockville, MD), and strain BT2a, (derived from an infant hospitalised with bronchiolitis and isolated in Dr. Power's laboratory) as well as RSV-B strain 8/60 (kindly provided by Dr. G. Taylor, Institute of Animal Health, Compton, UK) and strain CH18537 (kindly provided by Prof. Geoff Toms, University of Newcastle-upon-Tyne, UK) were propagated in HEp-2 cells (ECACC 86030501, European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) as previously described , except that DMEM was supplemented with 1% fetal calf serum. ..

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  • 93
    ATCC rsv strain b
    Neutralizing antibody titers. Neutralizing antibody titers with time after boosts of <t>RSV-primed</t> (set 1) and VLP-primed (set 2) animals are shown in panels A and B, respectively. Aliquots of sera from each group of animals acquired at each time point after the VLP or RSV boost were pooled. The experiment was accomplished twice for the RSV-primed animals (10 animals/group) and three times for the VLP-primed animals (15 animals/group). Serum from each experiment at each time point was pooled separately and titers of each pool determined separately. The NA titers are the reciprocal of the dilution of sera resulting in a 50% reduction of virus titer in a plaque reduction assay. The results are averages from 4 to 6 separate determinations on each pool. Since the results from each of the pools from equivalent groups were not statistically different, the results were combined. The averages and standard deviations are shown. Note the change in y axis scales to visualize lower titers. (C) Titers in each group of animals in the two sets of mice at week 4 after the VLP boost with statistically significant differences in values for between groups within a set and between groups in the two sets of animals. The significance of results at 17 weeks postboost is indicated in panel A. There were no significant differences at late times in VLP-primed animals. (D) RSV, strain B, neutralization titers using sera from animals primed with either RSV or with pre-F VLPs, boosted with pre-F VLPs, and harvested at 4 weeks postboost. Results are from pools of sera from two separate experiments, and the assay with each pool was accomplished twice. *, P = 0.05; **, P = 0.005; ***, P = 0.0005.
    Rsv Strain B, 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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    99
    ATCC rsv infected human epithelial cell lines
    NLRP3 and ASC are required for caspase-1 activation following <t>RSV</t> infection. ( A ) Wild type (WT), NLRP3 knock-out (KO) and ASC KO BMDMs were infected with RSV (1 MOI) for 12 h. The cell lysate from mock infected and RSV infected cells were subjected to Western blot analysis with mouse caspase-1 p10 subunit specific antibody. ( B ) RT-PCR analysis of <t>pro-IL-1β,</t> ASC and NLRP3 expression in RSV infected WT, NLRP3 KO and ASC KO BMDMs. The gels shown in (A) and (B) is representative of three independent experiments that yielded similar results.
    Rsv Infected Human Epithelial Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rsv  (ATCC)
    95
    ATCC rsv
    Characterization of <t>RSV</t> infection in human monocyte-derived macrophages <t>(MDMs).</t> Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages as previously described (1). RSV (Lon strain, ATCC VR-26) was propagated in HEp2 cells and isolated as described previously [ 40 ]. ( A ) THP-1 or MDMs cells were exposed to RSV Long at different multiplicity of infection (MOI, 0.1, 1 and 10) to investigate the kinetics of infection over time. Uninfected control replicates were exposed to cell culture medium. At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify RSV (F fusion gene). The RT-PCR reaction was performed using the AgPath-ID™ One-Step RT-PCR kit (Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s instructions. Samples with a cycle threshold (C t ) value of ≥40 were recorded as negative. A standard curve was prepared using serially diluted RNA extracts from a known quantity and used to calculate genomic copies/mL; ( B ) In the same experimental condition, cell survival in infected MDMs was monitored using flow cytometry, using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer’s instructions. Percent survival is expressed compared to viable cells measured at T = 0 hpi; ( C ) Immunofluorescence microscopy of MDMs infected by RSV (MOI of 1) at 8 hpi. Magnification ×20. Immunofluorescence protocol was performed as previously published [ 41 ]. RSV F antigen (Green), Nuclei stained by DAPI (Blue). Each experiment was performed in duplicate, from two separate experiments.
    Rsv, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neutralizing antibody titers. Neutralizing antibody titers with time after boosts of RSV-primed (set 1) and VLP-primed (set 2) animals are shown in panels A and B, respectively. Aliquots of sera from each group of animals acquired at each time point after the VLP or RSV boost were pooled. The experiment was accomplished twice for the RSV-primed animals (10 animals/group) and three times for the VLP-primed animals (15 animals/group). Serum from each experiment at each time point was pooled separately and titers of each pool determined separately. The NA titers are the reciprocal of the dilution of sera resulting in a 50% reduction of virus titer in a plaque reduction assay. The results are averages from 4 to 6 separate determinations on each pool. Since the results from each of the pools from equivalent groups were not statistically different, the results were combined. The averages and standard deviations are shown. Note the change in y axis scales to visualize lower titers. (C) Titers in each group of animals in the two sets of mice at week 4 after the VLP boost with statistically significant differences in values for between groups within a set and between groups in the two sets of animals. The significance of results at 17 weeks postboost is indicated in panel A. There were no significant differences at late times in VLP-primed animals. (D) RSV, strain B, neutralization titers using sera from animals primed with either RSV or with pre-F VLPs, boosted with pre-F VLPs, and harvested at 4 weeks postboost. Results are from pools of sera from two separate experiments, and the assay with each pool was accomplished twice. *, P = 0.05; **, P = 0.005; ***, P = 0.0005.

    Journal: Journal of Virology

    Article Title: Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein-Containing Virus-Like Particles

    doi: 10.1128/JVI.00087-19

    Figure Lengend Snippet: Neutralizing antibody titers. Neutralizing antibody titers with time after boosts of RSV-primed (set 1) and VLP-primed (set 2) animals are shown in panels A and B, respectively. Aliquots of sera from each group of animals acquired at each time point after the VLP or RSV boost were pooled. The experiment was accomplished twice for the RSV-primed animals (10 animals/group) and three times for the VLP-primed animals (15 animals/group). Serum from each experiment at each time point was pooled separately and titers of each pool determined separately. The NA titers are the reciprocal of the dilution of sera resulting in a 50% reduction of virus titer in a plaque reduction assay. The results are averages from 4 to 6 separate determinations on each pool. Since the results from each of the pools from equivalent groups were not statistically different, the results were combined. The averages and standard deviations are shown. Note the change in y axis scales to visualize lower titers. (C) Titers in each group of animals in the two sets of mice at week 4 after the VLP boost with statistically significant differences in values for between groups within a set and between groups in the two sets of animals. The significance of results at 17 weeks postboost is indicated in panel A. There were no significant differences at late times in VLP-primed animals. (D) RSV, strain B, neutralization titers using sera from animals primed with either RSV or with pre-F VLPs, boosted with pre-F VLPs, and harvested at 4 weeks postboost. Results are from pools of sera from two separate experiments, and the assay with each pool was accomplished twice. *, P = 0.05; **, P = 0.005; ***, P = 0.0005.

    Article Snippet: To determine if the sera resulting from the A2-derived Pre-F VLP immunization could induce cross-reactive protection to RSV, strain B, sera obtained 4 weeks after Pre-F VLP immunization of RSV (A2)-primed animals or VLP-primed animals were used in a plaque reduction assay using RSV strain B (WV/14617/85; ATCC VR-1400). shows that the RSV (A2)-primed sera neutralize strain B significantly more effectively that the VLP-primed sera.

    Techniques: Mouse Assay, Neutralization

    NLRP3 and ASC are required for caspase-1 activation following RSV infection. ( A ) Wild type (WT), NLRP3 knock-out (KO) and ASC KO BMDMs were infected with RSV (1 MOI) for 12 h. The cell lysate from mock infected and RSV infected cells were subjected to Western blot analysis with mouse caspase-1 p10 subunit specific antibody. ( B ) RT-PCR analysis of pro-IL-1β, ASC and NLRP3 expression in RSV infected WT, NLRP3 KO and ASC KO BMDMs. The gels shown in (A) and (B) is representative of three independent experiments that yielded similar results.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: NLRP3 and ASC are required for caspase-1 activation following RSV infection. ( A ) Wild type (WT), NLRP3 knock-out (KO) and ASC KO BMDMs were infected with RSV (1 MOI) for 12 h. The cell lysate from mock infected and RSV infected cells were subjected to Western blot analysis with mouse caspase-1 p10 subunit specific antibody. ( B ) RT-PCR analysis of pro-IL-1β, ASC and NLRP3 expression in RSV infected WT, NLRP3 KO and ASC KO BMDMs. The gels shown in (A) and (B) is representative of three independent experiments that yielded similar results.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Activation Assay, Infection, Knock-Out, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

    Caspase-1 dependent IL-1β release during RSV infection. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β and/or pro-caspase-1 plasmids. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Human macrophagic U937 cells were infected with RSV (1 MOI) in the presence of either water (vehicle control) or caspase-1 inhibitor (10 µM of Ac-YVAD-CHO). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( C ) Primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of either water (vehicle control) or caspase-1 inhibitor (10 µM of Ac-YVAD-CHO). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( D ) Wild type (WT) or caspase-1 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: Caspase-1 dependent IL-1β release during RSV infection. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β and/or pro-caspase-1 plasmids. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Human macrophagic U937 cells were infected with RSV (1 MOI) in the presence of either water (vehicle control) or caspase-1 inhibitor (10 µM of Ac-YVAD-CHO). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( C ) Primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of either water (vehicle control) or caspase-1 inhibitor (10 µM of Ac-YVAD-CHO). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( D ) Wild type (WT) or caspase-1 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Knock-Out, Standard Deviation

    A schematic model depicting the mechanism of IL-1β secretion during RSV infection. RSV infection activates TLR2/MyD88 pathway that culminates in NF-κB activation. Nuclear translocation of NF-κB results in NF-κB mediated trans-activation of pro-IL-1β and NLRP3 genes. Intracellular ROS generated during RSV infection and potassium efflux due to stimulation of ATP-sensitive ion channel triggers assembly of NLRP3/ASC inflammasome complex. NLRP3/ASC inflammasome activates caspase-1, which subsequently cleaves pro-IL-1β protein into its mature form. Mature IL-1β is secreted from the cells.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: A schematic model depicting the mechanism of IL-1β secretion during RSV infection. RSV infection activates TLR2/MyD88 pathway that culminates in NF-κB activation. Nuclear translocation of NF-κB results in NF-κB mediated trans-activation of pro-IL-1β and NLRP3 genes. Intracellular ROS generated during RSV infection and potassium efflux due to stimulation of ATP-sensitive ion channel triggers assembly of NLRP3/ASC inflammasome complex. NLRP3/ASC inflammasome activates caspase-1, which subsequently cleaves pro-IL-1β protein into its mature form. Mature IL-1β is secreted from the cells.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Activation Assay, Translocation Assay, Generated

    ASC expression is essential for IL-1β production during RSV infection. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β+pro-caspase-1 plasmids and ASC plasmid. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Wild type (WT) or ASC knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: ASC expression is essential for IL-1β production during RSV infection. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β+pro-caspase-1 plasmids and ASC plasmid. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Wild type (WT) or ASC knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Knock-Out, Standard Deviation

    Potassium efflux plays a critical role in IL-1β production during RSV infection. ( A ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of ATP-sensitive potassium channel inhibitor glibenclamide (50 µM). DMSO served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Cells were also treated with LPS and ATP in the presence of either DMSO or glibenclamide. ( B ) U937 cells were infected with RSV (1 MOI) in the presence of buffer containing either NaCl (150 mM) (control) or KCl (150 mM). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( C ) BMDM were infected with RSV (1 MOI) in the presence of buffer containing either NaCl (150 mM) (control) or KCl (150 mM). IL-1β levels in the medium supernatant were assayed by ELISA at 6 h and 12 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: Potassium efflux plays a critical role in IL-1β production during RSV infection. ( A ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of ATP-sensitive potassium channel inhibitor glibenclamide (50 µM). DMSO served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Cells were also treated with LPS and ATP in the presence of either DMSO or glibenclamide. ( B ) U937 cells were infected with RSV (1 MOI) in the presence of buffer containing either NaCl (150 mM) (control) or KCl (150 mM). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h post-infection. ( C ) BMDM were infected with RSV (1 MOI) in the presence of buffer containing either NaCl (150 mM) (control) or KCl (150 mM). IL-1β levels in the medium supernatant were assayed by ELISA at 6 h and 12 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    TLR2/MyD88 pathway is required for IL-1β release during RSV infection. ( A ) Wild type (WT), TLR2 knock-out (KO) and TLR4 KO BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) Wild type (WT) and MyD88 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( C ) RT-PCR analysis of pro-IL-1β expression in RSV infected WT, TLR2 KO and MyD88 KO BMDMs. (D) RT-PCR analysis of caspase-1 and NLRP3 expression in RSV infected WT, TLR2 KO and MyD88 KO BMDMs. The gels shown in (C) and (D) are representative of three independent experiments that yielded similar results. For ELISA assay, each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: TLR2/MyD88 pathway is required for IL-1β release during RSV infection. ( A ) Wild type (WT), TLR2 knock-out (KO) and TLR4 KO BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) Wild type (WT) and MyD88 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( C ) RT-PCR analysis of pro-IL-1β expression in RSV infected WT, TLR2 KO and MyD88 KO BMDMs. (D) RT-PCR analysis of caspase-1 and NLRP3 expression in RSV infected WT, TLR2 KO and MyD88 KO BMDMs. The gels shown in (C) and (D) are representative of three independent experiments that yielded similar results. For ELISA assay, each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation

    ROS produced during RSV infection triggers IL-1β secretion. ( A ) U937 cells were infected with RSV (1 MOI) in the presence of ROS inhibitor APDC (50 µM). Water served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) U937 cells were infected with RSV (1 MOI) in the presence of ROS inhibitor DPI (10 µM). DMSO served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( C ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of ROS inhibitor DPI (2 µM) or DMSO (vehicle control). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: ROS produced during RSV infection triggers IL-1β secretion. ( A ) U937 cells were infected with RSV (1 MOI) in the presence of ROS inhibitor APDC (50 µM). Water served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) U937 cells were infected with RSV (1 MOI) in the presence of ROS inhibitor DPI (10 µM). DMSO served as the vehicle control. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( C ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of ROS inhibitor DPI (2 µM) or DMSO (vehicle control). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Produced, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Standard Deviation

    NF-κB signaling is critical for IL-1β secretion during RSV infection. ( A ) Human U937 cells were infected with RSV (1 MOI) in the presence of either DMSO (control) or NF-κB inhibitor (BAY 11-7082). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) RT-PCR analysis of pro-IL-1β, caspase-1 and NLRP3 expression in U937 cells infected with RSV in the presence of either DMSO (control) or BAY 11-7082. ( C ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of either DMSO (control) or BAY 11-7082. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( D ) RT-PCR analysis of pro-IL-1β, ASC and NLRP3 expression in WT BMDMs infected with RSV in the presence of either DMSO (control) or BAY 11-7082. The gels shown in (B) and (D) are representative of three independent experiments that yielded similar results. For ELISA results, each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: NF-κB signaling is critical for IL-1β secretion during RSV infection. ( A ) Human U937 cells were infected with RSV (1 MOI) in the presence of either DMSO (control) or NF-κB inhibitor (BAY 11-7082). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( B ) RT-PCR analysis of pro-IL-1β, caspase-1 and NLRP3 expression in U937 cells infected with RSV in the presence of either DMSO (control) or BAY 11-7082. ( C ) Wild type primary mouse bone marrow derived macrophages (BMDM) were infected with RSV (1 MOI) in the presence of either DMSO (control) or BAY 11-7082. IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. ( D ) RT-PCR analysis of pro-IL-1β, ASC and NLRP3 expression in WT BMDMs infected with RSV in the presence of either DMSO (control) or BAY 11-7082. The gels shown in (B) and (D) are representative of three independent experiments that yielded similar results. For ELISA results, each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Standard Deviation

    IL-1β secretion during RSV infection requires NLRP3 expression. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β+pro-caspase-1 plasmids and NLRP3 plasmid. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Wild type (WT) or NLRP3 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Journal: PLoS ONE

    Article Title: TLR2/MyD88/NF-?B Pathway, Reactive Oxygen Species, Potassium Efflux Activates NLRP3/ASC Inflammasome during Respiratory Syncytial Virus Infection

    doi: 10.1371/journal.pone.0029695

    Figure Lengend Snippet: IL-1β secretion during RSV infection requires NLRP3 expression. ( A ) 293 cells were transfected either with pcDNA (control), pro-IL-1β+pro-caspase-1 plasmids and NLRP3 plasmid. At 24 h post-transfection, cells were infected with RSV (1 MOI). At 12 h post-infection, medium supernatant was assessed for IL-1β protein by ELISA analysis. ( B ) Wild type (WT) or NLRP3 knock-out (KO) BMDMs were infected with RSV (1 MOI). IL-1β levels in the medium supernatant were assayed by ELISA at 12 h and 24 h post-infection. Each value represents the mean ± standard deviation from three independent experiments.

    Article Snippet: Interestingly, we failed to detect IL-1β secretion from RSV infected human epithelial cell-lines [human lung epithelial A549 (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells and Hela (obtained from American Type Culture Collection (ATCC), Manassas, VA) cells (data not shown).

    Techniques: Infection, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Knock-Out, Standard Deviation

    Characterization of RSV infection in human monocyte-derived macrophages (MDMs). Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages as previously described (1). RSV (Lon strain, ATCC VR-26) was propagated in HEp2 cells and isolated as described previously [ 40 ]. ( A ) THP-1 or MDMs cells were exposed to RSV Long at different multiplicity of infection (MOI, 0.1, 1 and 10) to investigate the kinetics of infection over time. Uninfected control replicates were exposed to cell culture medium. At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify RSV (F fusion gene). The RT-PCR reaction was performed using the AgPath-ID™ One-Step RT-PCR kit (Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s instructions. Samples with a cycle threshold (C t ) value of ≥40 were recorded as negative. A standard curve was prepared using serially diluted RNA extracts from a known quantity and used to calculate genomic copies/mL; ( B ) In the same experimental condition, cell survival in infected MDMs was monitored using flow cytometry, using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer’s instructions. Percent survival is expressed compared to viable cells measured at T = 0 hpi; ( C ) Immunofluorescence microscopy of MDMs infected by RSV (MOI of 1) at 8 hpi. Magnification ×20. Immunofluorescence protocol was performed as previously published [ 41 ]. RSV F antigen (Green), Nuclei stained by DAPI (Blue). Each experiment was performed in duplicate, from two separate experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: RSV Infection in Human Macrophages Promotes CXCL10/IP-10 Expression during Bacterial Co-Infection

    doi: 10.3390/ijms18122654

    Figure Lengend Snippet: Characterization of RSV infection in human monocyte-derived macrophages (MDMs). Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages as previously described (1). RSV (Lon strain, ATCC VR-26) was propagated in HEp2 cells and isolated as described previously [ 40 ]. ( A ) THP-1 or MDMs cells were exposed to RSV Long at different multiplicity of infection (MOI, 0.1, 1 and 10) to investigate the kinetics of infection over time. Uninfected control replicates were exposed to cell culture medium. At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify RSV (F fusion gene). The RT-PCR reaction was performed using the AgPath-ID™ One-Step RT-PCR kit (Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s instructions. Samples with a cycle threshold (C t ) value of ≥40 were recorded as negative. A standard curve was prepared using serially diluted RNA extracts from a known quantity and used to calculate genomic copies/mL; ( B ) In the same experimental condition, cell survival in infected MDMs was monitored using flow cytometry, using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer’s instructions. Percent survival is expressed compared to viable cells measured at T = 0 hpi; ( C ) Immunofluorescence microscopy of MDMs infected by RSV (MOI of 1) at 8 hpi. Magnification ×20. Immunofluorescence protocol was performed as previously published [ 41 ]. RSV F antigen (Green), Nuclei stained by DAPI (Blue). Each experiment was performed in duplicate, from two separate experiments.

    Article Snippet: These human MDMs were then exposed to RSV (long strain, ATCC VR-26) at different multiplicity of infection (MOI) to investigate the kinetics of infection by monitoring of genome copy number using a specific RT-qPCR on cell lysates harvested at different time points ( A).

    Techniques: Infection, Derivative Assay, Isolation, Cell Culture, Multiplex Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Immunofluorescence, Microscopy, Staining

    Characterization of RSV/ Sp mixed infection in human monocyte-derived macrophages (MDMs). Human MDMs were infected by RSV at an MOI of 1. After 4 h of viral infection, cells were mock-infected or infected by Sp at 1 CFU/cell. Encapsulated Streptococcus pneumoniae serotype 14 was obtained from the National Reference Center for Streptococci (Department of Medical Microbiology, Aachen, Germany). Pneumococci were opsonized in anti-pneumococcal immune serum and incubated for 30 min at 37 °C prior to infections for all experiments. MDM cells showed significant loss of viability following overnight incubation with S. pneumoniae , and gentamicin (10 µg/mL) was therefore added to all wells at the 2-h time point to prevent bacterial overgrowth and loss of cell viability. ( A ) At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify RSV; ( B ) RSV viral production in supernatants was performed by quantification of viral titers (TCID 50/mL) with a limit-dilution assay and using the Reed Muench statistical method. Results are expressed as log10 TCID50/mL values; ( C ) In the same experimental condition, cell survival in infected MDMs was monitored using flow cytometry, with the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer’s instructions. Percent survival is expressed compared to viable cells measured at T = 0 hpi; ( D , E ) Co-infection alters the timing and extent of IP-10 expression in human monocyte-derived macrophages (MDMs). Human MDMs were infected by RSV at an MOI of 1. After 4 h of viral infection, cells were mock-infected or infected by Sp at 1 CFU/cell. At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify CXCL10 mRNA levels. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify CXCL10 (F: 5′-GTGGCATTCAAGGAGTACCTC-3′, R: 5′-TGATGGCCTTCGATTCTGGATT-3′) and β-Actin for normalization (F: 5′-CTCTTCCAGCCTTCCTTCCT-3′, R: 5′-AGCACTGTGTTGGCGTACAG-3′); ( E ) Similar strategy with different combinations of RSV and Sp , at 8 hpi. Each experiment was performed in duplicate, from two separate experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: RSV Infection in Human Macrophages Promotes CXCL10/IP-10 Expression during Bacterial Co-Infection

    doi: 10.3390/ijms18122654

    Figure Lengend Snippet: Characterization of RSV/ Sp mixed infection in human monocyte-derived macrophages (MDMs). Human MDMs were infected by RSV at an MOI of 1. After 4 h of viral infection, cells were mock-infected or infected by Sp at 1 CFU/cell. Encapsulated Streptococcus pneumoniae serotype 14 was obtained from the National Reference Center for Streptococci (Department of Medical Microbiology, Aachen, Germany). Pneumococci were opsonized in anti-pneumococcal immune serum and incubated for 30 min at 37 °C prior to infections for all experiments. MDM cells showed significant loss of viability following overnight incubation with S. pneumoniae , and gentamicin (10 µg/mL) was therefore added to all wells at the 2-h time point to prevent bacterial overgrowth and loss of cell viability. ( A ) At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify RSV; ( B ) RSV viral production in supernatants was performed by quantification of viral titers (TCID 50/mL) with a limit-dilution assay and using the Reed Muench statistical method. Results are expressed as log10 TCID50/mL values; ( C ) In the same experimental condition, cell survival in infected MDMs was monitored using flow cytometry, with the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer’s instructions. Percent survival is expressed compared to viable cells measured at T = 0 hpi; ( D , E ) Co-infection alters the timing and extent of IP-10 expression in human monocyte-derived macrophages (MDMs). Human MDMs were infected by RSV at an MOI of 1. After 4 h of viral infection, cells were mock-infected or infected by Sp at 1 CFU/cell. At different time points, total cell extracts were harvested and analyzed by multiplex real-time RT-qPCR to quantify CXCL10 mRNA levels. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify CXCL10 (F: 5′-GTGGCATTCAAGGAGTACCTC-3′, R: 5′-TGATGGCCTTCGATTCTGGATT-3′) and β-Actin for normalization (F: 5′-CTCTTCCAGCCTTCCTTCCT-3′, R: 5′-AGCACTGTGTTGGCGTACAG-3′); ( E ) Similar strategy with different combinations of RSV and Sp , at 8 hpi. Each experiment was performed in duplicate, from two separate experiments.

    Article Snippet: These human MDMs were then exposed to RSV (long strain, ATCC VR-26) at different multiplicity of infection (MOI) to investigate the kinetics of infection by monitoring of genome copy number using a specific RT-qPCR on cell lysates harvested at different time points ( A).

    Techniques: Infection, Derivative Assay, Incubation, Multiplex Assay, Quantitative RT-PCR, Dilution Assay, Flow Cytometry, Cytometry, Expressing, Purification, Transfection