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    Structured Review

    ATCC vero cells
    Mutant <t>UL42</t> resistant to extraction by detergent. <t>Vero</t> cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed
    Vero 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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    Images

    1) Product Images from "Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity ▿"

    Article Title: Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity ▿

    Journal:

    doi: 10.1128/JVI.00193-09

    Mutant UL42 resistant to extraction by detergent. Vero cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed
    Figure Legend Snippet: Mutant UL42 resistant to extraction by detergent. Vero cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed

    Techniques Used: Mutagenesis, Infection

    Viral yields and DNA synthesized by UL42 mutants. (A) Single-cycle growth curve assays were performed as described in Materials and Methods, and the yields of progeny viruses were determined by plaque assay on Vero cells. Since recombinants A and B of
    Figure Legend Snippet: Viral yields and DNA synthesized by UL42 mutants. (A) Single-cycle growth curve assays were performed as described in Materials and Methods, and the yields of progeny viruses were determined by plaque assay on Vero cells. Since recombinants A and B of

    Techniques Used: Synthesized, Plaque Assay

    2) Product Images from "African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast"

    Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200086

    Comparative growth curve analyses of three AF and three AF ZIKV strains in ESCd, JAr, and Vero cells. Cells were infected with the six ZIKV strains at a MOI of 0.1. Cell supernatants were harvested at the indicated time points for titration by plaque assay in Vero cells. Growth curve analyses were performed in triplicate and plotted as SEM. Growth curves representing the AF strains are highlighted in shades of blue and those representing the AS strains are shown in shades of red. (A) In ESCd, significantly higher virus concentrations were observed by 48 h PI for the AF Uganda and the AF Senegal strains when compared to all other strains ( p
    Figure Legend Snippet: Comparative growth curve analyses of three AF and three AF ZIKV strains in ESCd, JAr, and Vero cells. Cells were infected with the six ZIKV strains at a MOI of 0.1. Cell supernatants were harvested at the indicated time points for titration by plaque assay in Vero cells. Growth curve analyses were performed in triplicate and plotted as SEM. Growth curves representing the AF strains are highlighted in shades of blue and those representing the AS strains are shown in shades of red. (A) In ESCd, significantly higher virus concentrations were observed by 48 h PI for the AF Uganda and the AF Senegal strains when compared to all other strains ( p

    Techniques Used: Infection, Titration, Plaque Assay

    Cytopathic effects (CPE) caused by three AF and three AS ZIKV strains in Vero cells. Vero cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Similar CPE are evident after infection with all AF and AS ZIKV strains, although the AF Nigeria and AS Panama strains elicited weaker CPE when compared to the other strains. Scale bar is 1 mm.
    Figure Legend Snippet: Cytopathic effects (CPE) caused by three AF and three AS ZIKV strains in Vero cells. Vero cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Similar CPE are evident after infection with all AF and AS ZIKV strains, although the AF Nigeria and AS Panama strains elicited weaker CPE when compared to the other strains. Scale bar is 1 mm.

    Techniques Used: Infection

    3) Product Images from "Herpes Simplex Virus Type 1 Engages Toll Like Receptor 2 to Recruit Macrophages During Infection of Enteric Neurons"

    Article Title: Herpes Simplex Virus Type 1 Engages Toll Like Receptor 2 to Recruit Macrophages During Infection of Enteric Neurons

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02148

    HSV-1 infection of the ENS triggers TLR2 activation. (A) One, two, and three weeks (wks) post IG inoculum of HSV-1, total RNA was purified from the LMMP of WT and TLR2 ko mice. Quantitative PCR was performed to evaluate the expression of HSV-1 latency-associated transcripts (LATs), infected cell protein (ICP0, ICP4), VP16, and thymidine kinase (tk) mRNA transcripts. Data were normalized to Rn18S and are reported as mRNA fold-change over WT mice. Sham: mice IG inoculated with Vero cell lysate. n = 6 mice per group. ∗ denotes P
    Figure Legend Snippet: HSV-1 infection of the ENS triggers TLR2 activation. (A) One, two, and three weeks (wks) post IG inoculum of HSV-1, total RNA was purified from the LMMP of WT and TLR2 ko mice. Quantitative PCR was performed to evaluate the expression of HSV-1 latency-associated transcripts (LATs), infected cell protein (ICP0, ICP4), VP16, and thymidine kinase (tk) mRNA transcripts. Data were normalized to Rn18S and are reported as mRNA fold-change over WT mice. Sham: mice IG inoculated with Vero cell lysate. n = 6 mice per group. ∗ denotes P

    Techniques Used: Infection, Activation Assay, Purification, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    4) Product Images from "Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures"

    Article Title: Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    Journal: Journal of Virology

    doi: 10.1128/JVI.02351-15

    Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product
    Figure Legend Snippet: Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product

    Techniques Used: Expressing, Activity Assay, Infection, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM
    Figure Legend Snippet: Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM

    Techniques Used: Purification

    Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected
    Figure Legend Snippet: Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected

    Techniques Used: Infection, Derivative Assay, Produced, Purification

    Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (
    Figure Legend Snippet: Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (

    Techniques Used: Infection, Purification, Produced

    5) Product Images from "Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic"

    Article Title: Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic

    Journal: Journal of Virology

    doi: 10.1128/JVI.02615-15

    Schematic representation of the genome of rMACV/Cd#1-GPC and the virus growth curves. (A) For rMACV/Cd#1-GPC, the entire MACV GPC gene was replaced with the Cd#1 GPC gene. The plaque morphology is shown. (B) The growth of rMACV/Cd#1-GPC was characterized in Vero cells (MOI = 0.01). The titer of rMACV was significantly higher than that of rCd#1 at 48 hpi ( n = 5; **, P
    Figure Legend Snippet: Schematic representation of the genome of rMACV/Cd#1-GPC and the virus growth curves. (A) For rMACV/Cd#1-GPC, the entire MACV GPC gene was replaced with the Cd#1 GPC gene. The plaque morphology is shown. (B) The growth of rMACV/Cd#1-GPC was characterized in Vero cells (MOI = 0.01). The titer of rMACV was significantly higher than that of rCd#1 at 48 hpi ( n = 5; **, P

    Techniques Used: Gel Permeation Chromatography

    6) Product Images from "Inhibitory Effects of Norwogonin, Oroxylin A, and Mosloflavone on Enterovirus 71"

    Article Title: Inhibitory Effects of Norwogonin, Oroxylin A, and Mosloflavone on Enterovirus 71

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2015.200

    Antiviral activity of norwogonin, oroxylin A, mosloflavone, and ribavirin against Enterovirus 71 (EV71). (A) Antiviral activity and (B) cytotoxicity of norwogonin, oroxylin A, mosloflavone and ribavirin were measured in Vero cells. Vero cells were infected with EV71 and then treated with the indicated concentrations (0.4, 2, 10, and 50 μg/mL) of norwogonin, oroxylin A, mosloflavone for 48 h. The antiviral activity was investigated using a CPE reduction assay. Data are presented as mean ± SD. from three independent experiments, each carried out in triplicate. * p
    Figure Legend Snippet: Antiviral activity of norwogonin, oroxylin A, mosloflavone, and ribavirin against Enterovirus 71 (EV71). (A) Antiviral activity and (B) cytotoxicity of norwogonin, oroxylin A, mosloflavone and ribavirin were measured in Vero cells. Vero cells were infected with EV71 and then treated with the indicated concentrations (0.4, 2, 10, and 50 μg/mL) of norwogonin, oroxylin A, mosloflavone for 48 h. The antiviral activity was investigated using a CPE reduction assay. Data are presented as mean ± SD. from three independent experiments, each carried out in triplicate. * p

    Techniques Used: Activity Assay, Infection

    The effect of norwogonin, oroxylin and mosloflavone on EV71 replication. (A) Real-time PCR analyses were performed to determine the effect of norwogonin, oroxylin A and mosloflavone on EV71 NCR gene expression levels. Replication of EV71 from Vero cells at 48 h after infection by EV71 in the presence of 50 μg/mL norwogonin, oroxylin A and mosloflavone was determined by real-time PCR. Vehicle (0.1% DMSO)-treated cells without EV71 infection was used as control. (B) Western blot analyses were performed to determine the effect of norwogonin, oroxylin A, mosloflavone, and ribavirin on the production of EV71 VP2 proteins. The reduction in protein expression of EV71 VP2 was identified after treatment with 50 μg/mL norwogonin, oroxylin A, mosloflavone, and ribavirin for 48 h. α-tubulin was used as a loading control for each set of samples. ** p
    Figure Legend Snippet: The effect of norwogonin, oroxylin and mosloflavone on EV71 replication. (A) Real-time PCR analyses were performed to determine the effect of norwogonin, oroxylin A and mosloflavone on EV71 NCR gene expression levels. Replication of EV71 from Vero cells at 48 h after infection by EV71 in the presence of 50 μg/mL norwogonin, oroxylin A and mosloflavone was determined by real-time PCR. Vehicle (0.1% DMSO)-treated cells without EV71 infection was used as control. (B) Western blot analyses were performed to determine the effect of norwogonin, oroxylin A, mosloflavone, and ribavirin on the production of EV71 VP2 proteins. The reduction in protein expression of EV71 VP2 was identified after treatment with 50 μg/mL norwogonin, oroxylin A, mosloflavone, and ribavirin for 48 h. α-tubulin was used as a loading control for each set of samples. ** p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot

    Time-of-addition effect of norwogonin, oroxylin A, and mosloflavone on EV71 replication in Vero cells. Each compound (50 μg/mL) was added either before (−1 h), during (0 h), or after (1, 2, 4, 6, and 8 h) virus infection. After 2 days, the inhibition was evaluated by SRB method and expressed as the inhibition rate. Each value is the result of mean ± SD. of three independent experiments. ** p
    Figure Legend Snippet: Time-of-addition effect of norwogonin, oroxylin A, and mosloflavone on EV71 replication in Vero cells. Each compound (50 μg/mL) was added either before (−1 h), during (0 h), or after (1, 2, 4, 6, and 8 h) virus infection. After 2 days, the inhibition was evaluated by SRB method and expressed as the inhibition rate. Each value is the result of mean ± SD. of three independent experiments. ** p

    Techniques Used: Infection, Inhibition, Sulforhodamine B Assay

    7) Product Images from "Reverse Transcriptase PCR Amplification of Rickettsia typhi from Infected Mammalian Cells and Insect Vectors"

    Article Title: Reverse Transcriptase PCR Amplification of Rickettsia typhi from Infected Mammalian Cells and Insect Vectors

    Journal: Journal of Clinical Microbiology

    doi:

    RT-PCR of R. typhi 120-kDa antigen gene fragment from infected Vero cells. Lane 1, RT-PCR kit control (523 bp); lane 2, PCR contamination control; lane 3, no-RT control, infected Vero cells; lane 4, uninfected Vero cells; lane 5, infected Vero cells (833 bp). ML, low-DNA-mass ladder; the 800-bp rung is indicated.
    Figure Legend Snippet: RT-PCR of R. typhi 120-kDa antigen gene fragment from infected Vero cells. Lane 1, RT-PCR kit control (523 bp); lane 2, PCR contamination control; lane 3, no-RT control, infected Vero cells; lane 4, uninfected Vero cells; lane 5, infected Vero cells (833 bp). ML, low-DNA-mass ladder; the 800-bp rung is indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Polymerase Chain Reaction

    8) Product Images from "Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice"

    Article Title: Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.19.8899-8908.2001

    Immunofluorescence analysis. Vero cells were infected with recombinant PR8/NS-Nef (A), PR8 wt (B), and Aichi/NS-Nef (C) viruses with an MOI of 0.05. Twenty-four hours after infection, cells were collected, fixed on cover slides with acetone, and incubated with anti HIV-1 IIIB Nef mouse MAb followed by incubation with fluorescein isothiocyanate conjugated to goat anti-mouse IgG, as described in Materials and Methods. An analogous immunofluorescence signal was observed when infected cells were incubated with anti-HIV-1 IR-CSF Nef mouse MAb (data not shown). (D) The intracellular localization of the Nef antigen is visible at a higher magnification.
    Figure Legend Snippet: Immunofluorescence analysis. Vero cells were infected with recombinant PR8/NS-Nef (A), PR8 wt (B), and Aichi/NS-Nef (C) viruses with an MOI of 0.05. Twenty-four hours after infection, cells were collected, fixed on cover slides with acetone, and incubated with anti HIV-1 IIIB Nef mouse MAb followed by incubation with fluorescein isothiocyanate conjugated to goat anti-mouse IgG, as described in Materials and Methods. An analogous immunofluorescence signal was observed when infected cells were incubated with anti-HIV-1 IR-CSF Nef mouse MAb (data not shown). (D) The intracellular localization of the Nef antigen is visible at a higher magnification.

    Techniques Used: Immunofluorescence, Infection, Recombinant, Incubation

    Sizes of the wt and recombinant NS1 proteins. (A) Confluent monolayers of Vero cells were infected with recombinant PR8/NS-Nef and PR8 wt viruses at an MOI of 5. Six hours postinfection, the cells were labeled with [ 35 S]methionine and [ 35 S]cysteine (Amersham). Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels containing 5 M urea. Lanes 1 and 2, mock-infected Vero cell extracts; lane 3, PR8 wt-infected Vero cell extract; lane 4, PR8/NS-Nef-infected Vero cell extract. (B and C)Viral proteins derived from the infected Vero cells 24 h postinfection were separated on 16% polyacrylamide gels, transferred to a nitrocellulose membrane, and detected by Western blot analysis with anti HIV-1 IR-CSF Nef mouse MAb (B) and rabbit anti-NS hyperimmune serum (C). Lanes 1 and 4, PR8/NS-Nef; lanes 2 and 3, PR8 wt.
    Figure Legend Snippet: Sizes of the wt and recombinant NS1 proteins. (A) Confluent monolayers of Vero cells were infected with recombinant PR8/NS-Nef and PR8 wt viruses at an MOI of 5. Six hours postinfection, the cells were labeled with [ 35 S]methionine and [ 35 S]cysteine (Amersham). Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels containing 5 M urea. Lanes 1 and 2, mock-infected Vero cell extracts; lane 3, PR8 wt-infected Vero cell extract; lane 4, PR8/NS-Nef-infected Vero cell extract. (B and C)Viral proteins derived from the infected Vero cells 24 h postinfection were separated on 16% polyacrylamide gels, transferred to a nitrocellulose membrane, and detected by Western blot analysis with anti HIV-1 IR-CSF Nef mouse MAb (B) and rabbit anti-NS hyperimmune serum (C). Lanes 1 and 4, PR8/NS-Nef; lanes 2 and 3, PR8 wt.

    Techniques Used: Recombinant, Infection, Labeling, Polyacrylamide Gel Electrophoresis, Derivative Assay, Western Blot

    Virus load in respiratory tract tissue of immunized mice. BALB/c mice were immunized i.n. under ether anesthesia with 10 6 PFU of influenza PR8/NS-Nef, Aichi/NS-Nef, or PR8/NS-124 virus or 10 3 PFU of the PR8 wt virus/mouse. Three mice of each group were sacrificed at days 2, 4, and 6 postinfection. The pooled lungs were homogenized, and the tissue extracts were assayed for infectious virus particles in plaque assays using Vero cells. The results are presented as PFU per milliliter of 10% (wt/vol) tissue extracts.
    Figure Legend Snippet: Virus load in respiratory tract tissue of immunized mice. BALB/c mice were immunized i.n. under ether anesthesia with 10 6 PFU of influenza PR8/NS-Nef, Aichi/NS-Nef, or PR8/NS-124 virus or 10 3 PFU of the PR8 wt virus/mouse. Three mice of each group were sacrificed at days 2, 4, and 6 postinfection. The pooled lungs were homogenized, and the tissue extracts were assayed for infectious virus particles in plaque assays using Vero cells. The results are presented as PFU per milliliter of 10% (wt/vol) tissue extracts.

    Techniques Used: Mouse Assay

    9) Product Images from "An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters"

    Article Title: An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.16.10678-10689.2005

    Relative fusion of Vero cells infected with hMPV viruses. Confluent monolayers of Vero cells were inoculated with the indicated hMPV viruses at an MOI of 3 PFU/cell with or without 0.2 μg/ml trypsin and incubated under 1% methyl cellulose. The
    Figure Legend Snippet: Relative fusion of Vero cells infected with hMPV viruses. Confluent monolayers of Vero cells were inoculated with the indicated hMPV viruses at an MOI of 3 PFU/cell with or without 0.2 μg/ml trypsin and incubated under 1% methyl cellulose. The

    Techniques Used: Infection, Incubation

    Titers and plaques of four subtypes of wt hMPV and recombinant hMPV. Subconfluent monolayers of Vero cells were inoculated with each of the indicated biologically derived viruses, rhMPV/NL/1/00/101P or rhMPV/NL/1/00/101S, at an MOI of 0.1 PFU/cell with
    Figure Legend Snippet: Titers and plaques of four subtypes of wt hMPV and recombinant hMPV. Subconfluent monolayers of Vero cells were inoculated with each of the indicated biologically derived viruses, rhMPV/NL/1/00/101P or rhMPV/NL/1/00/101S, at an MOI of 0.1 PFU/cell with

    Techniques Used: Recombinant, Derivative Assay

    Expression of hMPV F vectored in b/h PIV3 as detected by Western blot analysis. Subconfluent monolayers of Vero cells were inoculated with wt hMPV/NL/1/00, b/h PIV3/hMPV F/101P, b/h PIV3/hMPV F/101S, or b/h PIV3 with or without 0.2 μg/ml trypsin.
    Figure Legend Snippet: Expression of hMPV F vectored in b/h PIV3 as detected by Western blot analysis. Subconfluent monolayers of Vero cells were inoculated with wt hMPV/NL/1/00, b/h PIV3/hMPV F/101P, b/h PIV3/hMPV F/101S, or b/h PIV3 with or without 0.2 μg/ml trypsin.

    Techniques Used: Expressing, Western Blot

    10) Product Images from "Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)"

    Article Title: Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

    Journal: Veterinary Microbiology

    doi: 10.1016/j.vetmic.2016.02.002

    Mouse serum antibody neutralization activity against STb toxin. A : Normal Vero cells grown in cell culture medium. B : Vero cells (in 700 μl culture medium) incubated with 300 μl 8020 (STb) overnight grown culture filtrates, showing over 50% cell death and detachment. C : Vero cells incubated 300 μl 8020 (STb) filtrates pre-mixed with 150 μl pooled serum of the control mice. D : Vero cells incubated with 300 μl 8020 (STb) filtrates pre-mixed with 150 μl pooled serum of the mice immunized with ‘LT R192G -STb-Stx2e-3xSTa P12F ’ MEFA. E : Vero cells incubated with 300 μl 8020 (STb) filtrates premixed with 50 μl pooled serum of the immunized mice. F : Vero cells incubated with 300 μl 8020 (STb) filtrates and 25 μl pooled serum of the immunized mice.
    Figure Legend Snippet: Mouse serum antibody neutralization activity against STb toxin. A : Normal Vero cells grown in cell culture medium. B : Vero cells (in 700 μl culture medium) incubated with 300 μl 8020 (STb) overnight grown culture filtrates, showing over 50% cell death and detachment. C : Vero cells incubated 300 μl 8020 (STb) filtrates pre-mixed with 150 μl pooled serum of the control mice. D : Vero cells incubated with 300 μl 8020 (STb) filtrates pre-mixed with 150 μl pooled serum of the mice immunized with ‘LT R192G -STb-Stx2e-3xSTa P12F ’ MEFA. E : Vero cells incubated with 300 μl 8020 (STb) filtrates premixed with 50 μl pooled serum of the immunized mice. F : Vero cells incubated with 300 μl 8020 (STb) filtrates and 25 μl pooled serum of the immunized mice.

    Techniques Used: Neutralization, Activity Assay, Cell Culture, Incubation, Mouse Assay

    11) Product Images from "Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation"

    Article Title: Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02502-15

    Examining type III IFN signaling in Vero cells using Western blotting. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection, Vero cells were stimulated with recombinant IL-29/-28A/-28B (1 μg/ml)
    Figure Legend Snippet: Examining type III IFN signaling in Vero cells using Western blotting. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection, Vero cells were stimulated with recombinant IL-29/-28A/-28B (1 μg/ml)

    Techniques Used: Western Blot, Infection, Recombinant

    Examining type III IFN signaling in BEAS-2B cells using Western blotting. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection, Vero cells were stimulated with recombinant IL-29/-28A/-28B (1 μg/ml)
    Figure Legend Snippet: Examining type III IFN signaling in BEAS-2B cells using Western blotting. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection, Vero cells were stimulated with recombinant IL-29/-28A/-28B (1 μg/ml)

    Techniques Used: Western Blot, Infection, Recombinant

    Real-time PCR of antiviral molecules downstream of the IFN receptor in Vero cells stimulated with recombinant type III IFNs after PIV-3 infection. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection,
    Figure Legend Snippet: Real-time PCR of antiviral molecules downstream of the IFN receptor in Vero cells stimulated with recombinant type III IFNs after PIV-3 infection. Vero cells were infected with dolphin (T), bovine (B), or human (H) PIV-3 for 24 h. At 24 h postinfection,

    Techniques Used: Real-time Polymerase Chain Reaction, Recombinant, Infection

    12) Product Images from "A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics"

    Article Title: A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics

    Journal: Viruses

    doi: 10.3390/v10050246

    In Nedel-infected cells, mNeonGreen co-localizes with HSV antigen, and Nedel plaques are similar to wild-type. ( a ) Representative confocal image of a Nedel plaque on Vero cells. Nuclei ( upper left ) are stained with DAPI, mNeonGreen is detected ( upper right ) by fluorescence, and HSV-2 antigens are detected by immunofluorescent staining using a polyclonal HSV-2 antibody ( lower left ). Merged image is shown at lower right . Red bar = 100 µm. Enlarged images taken from the plaque shown in panel ( a ) illustrate fluorescence in the ( b ) nucleus, ( c ) cytoplasm, ( d ) and cell membrane, in some cases ( e ) obscuring the entire cell. Yellow arrow indicates location referenced in text; yellow bar = 15 µm. ( f ) Plaque size comparison, mean of 20 plaques, Error bars reflect SEM, Unpaired two-tailed t test p = 0.82.
    Figure Legend Snippet: In Nedel-infected cells, mNeonGreen co-localizes with HSV antigen, and Nedel plaques are similar to wild-type. ( a ) Representative confocal image of a Nedel plaque on Vero cells. Nuclei ( upper left ) are stained with DAPI, mNeonGreen is detected ( upper right ) by fluorescence, and HSV-2 antigens are detected by immunofluorescent staining using a polyclonal HSV-2 antibody ( lower left ). Merged image is shown at lower right . Red bar = 100 µm. Enlarged images taken from the plaque shown in panel ( a ) illustrate fluorescence in the ( b ) nucleus, ( c ) cytoplasm, ( d ) and cell membrane, in some cases ( e ) obscuring the entire cell. Yellow arrow indicates location referenced in text; yellow bar = 15 µm. ( f ) Plaque size comparison, mean of 20 plaques, Error bars reflect SEM, Unpaired two-tailed t test p = 0.82.

    Techniques Used: Infection, Staining, Fluorescence, Two Tailed Test

    Western blot, visualized with anti-VP26 antibody, indicating that Nedel expresses VP26-mNeonGreen fusion protein. Vero cells were harvested 24 h after infection at an MOI of 10 with either Strain 333 or Nedel. Magic Marker protein standards (M), uninfected Vero cells, HSV-2 Strain 333-infected Vero cells, and Nedel-infected Vero cells are in the lanes left to right.
    Figure Legend Snippet: Western blot, visualized with anti-VP26 antibody, indicating that Nedel expresses VP26-mNeonGreen fusion protein. Vero cells were harvested 24 h after infection at an MOI of 10 with either Strain 333 or Nedel. Magic Marker protein standards (M), uninfected Vero cells, HSV-2 Strain 333-infected Vero cells, and Nedel-infected Vero cells are in the lanes left to right.

    Techniques Used: Western Blot, Infection, Marker

    13) Product Images from "Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence"

    Article Title: Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

    Journal: Journal of Virology

    doi: 10.1128/JVI.02861-15

    Plaque morphology and particle stability of chimeric viruses. (A) After 72 h of incubation, the plaque morphologies of WNV/KOUV-EIII and WNV/BAGV-EIII in Vero cells were significantly smaller than those of WNV NY99, whereas those of WNV/JEV-EIII and WNV/SLEV-EIII
    Figure Legend Snippet: Plaque morphology and particle stability of chimeric viruses. (A) After 72 h of incubation, the plaque morphologies of WNV/KOUV-EIII and WNV/BAGV-EIII in Vero cells were significantly smaller than those of WNV NY99, whereas those of WNV/JEV-EIII and WNV/SLEV-EIII

    Techniques Used: Incubation

    In vitro growth kinetics for chimeric viruses. Growth kinetics were determined for WNV NY99 and each of the four chimeras following infection of Vero (A) and C6/36 (B) cells in triplicate flasks at a low MOI of 0.0005. Statistical significance was determined
    Figure Legend Snippet: In vitro growth kinetics for chimeric viruses. Growth kinetics were determined for WNV NY99 and each of the four chimeras following infection of Vero (A) and C6/36 (B) cells in triplicate flasks at a low MOI of 0.0005. Statistical significance was determined

    Techniques Used: In Vitro, Infection

    14) Product Images from "The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis"

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0589-8

    Bacterial adherence and invasion assays. Strains CH3 and RA1062 were tested on Vero cells. A Adherence assay; B Invasion assay. The data represent the number of bacteria bound to or invaded into Vero cells in each well of a 24-well plate. The error bars represent mean ± standard deviations from three independent experiments (** p
    Figure Legend Snippet: Bacterial adherence and invasion assays. Strains CH3 and RA1062 were tested on Vero cells. A Adherence assay; B Invasion assay. The data represent the number of bacteria bound to or invaded into Vero cells in each well of a 24-well plate. The error bars represent mean ± standard deviations from three independent experiments (** p

    Techniques Used: Invasion Assay

    15) Product Images from "Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies"

    Article Title: Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

    Journal: Viruses

    doi: 10.3390/v4123511

    Filovirus plaques produced on Vero E6 cells from two sources are similar in appearance and titer. ( A ) EBOV plaques on ATCC Vero E6 cells plated at (1) 24 hours before assay, and (2) 72 hours before assay. EBOV plaques on BEI Vero E6 cells plated at (3) 24 hours before assay, and (4) 72 hours before assay. (5) MARV plaques on Vero cells from (5) ATCC and (6) BEI plated 24 hours before assay. ( B ) EBOV titers are similar in Vero E6 cells from ATCC and BEI when measured independently by two operators. This experiment was performed twice with up to 3 operators (data not shown), and one representative graph is shown. Each bar represents an average of 7 replicates.
    Figure Legend Snippet: Filovirus plaques produced on Vero E6 cells from two sources are similar in appearance and titer. ( A ) EBOV plaques on ATCC Vero E6 cells plated at (1) 24 hours before assay, and (2) 72 hours before assay. EBOV plaques on BEI Vero E6 cells plated at (3) 24 hours before assay, and (4) 72 hours before assay. (5) MARV plaques on Vero cells from (5) ATCC and (6) BEI plated 24 hours before assay. ( B ) EBOV titers are similar in Vero E6 cells from ATCC and BEI when measured independently by two operators. This experiment was performed twice with up to 3 operators (data not shown), and one representative graph is shown. Each bar represents an average of 7 replicates.

    Techniques Used: Produced

    Vero E6 cells are suitable for quantitation of EBOV plaques. ( A ) Vero and Vero E6 cells produce EBOV plaques. ( B ) EBOV titers are similar in ATCC Vero E6 cells plated 24, 48 or 72 hours prior to plaque assay. This experiment was performed twice, and one representative graph is shown. Each bar represents an average of 5 replicates. The * indicates p = 0.006 between 24 and 72 hour samples for passage 29.
    Figure Legend Snippet: Vero E6 cells are suitable for quantitation of EBOV plaques. ( A ) Vero and Vero E6 cells produce EBOV plaques. ( B ) EBOV titers are similar in ATCC Vero E6 cells plated 24, 48 or 72 hours prior to plaque assay. This experiment was performed twice, and one representative graph is shown. Each bar represents an average of 5 replicates. The * indicates p = 0.006 between 24 and 72 hour samples for passage 29.

    Techniques Used: Quantitation Assay, Plaque Assay

    ( A ) EBOV titers in ATCC or BEI Vero E6 cells of various passage ages. This experiment was performed by three independent investigators, four replicates per cell type. The combined data are presented, where each bar represents 12 replicates. * indicates a significant difference in value between BEI passage 40 and passage 27 ( p = 0.0003), ** a difference between ATCC passage 54 and 28 ( p = 0.007), and *** a difference between BEI passage 40 and ATCC passage 28 ( p = 0.000002). For these experiments, p value cutoff was ≤0.008). ( B ) Analysis of EBOV titer changes in cells of various passages. The arrows point out data from passages plotted in (A).
    Figure Legend Snippet: ( A ) EBOV titers in ATCC or BEI Vero E6 cells of various passage ages. This experiment was performed by three independent investigators, four replicates per cell type. The combined data are presented, where each bar represents 12 replicates. * indicates a significant difference in value between BEI passage 40 and passage 27 ( p = 0.0003), ** a difference between ATCC passage 54 and 28 ( p = 0.007), and *** a difference between BEI passage 40 and ATCC passage 28 ( p = 0.000002). For these experiments, p value cutoff was ≤0.008). ( B ) Analysis of EBOV titer changes in cells of various passages. The arrows point out data from passages plotted in (A).

    Techniques Used:

    16) Product Images from "Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry"

    Article Title: Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01130-17

    Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P
    Figure Legend Snippet: Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P

    Techniques Used: Luciferase, Sequencing, Synthesized, In Vitro, Transfection, Incubation, Activity Assay

    Effects of Ca 2+ , SMase, and MβCD on RuV binding to mammalian cells. (A and B) Vero, RK13, 293T, LLC-MK2, HeLa, Jurkat, and Raji cells were incubated with RuV antigens at 4°C in DMEM containing 2 mM CaCl 2 or in Ca 2+ -free DMEM for 1 h. The bound RuV antigens were detected by flow cytometry using a MAb specific for the RuV E1 protein and a PE-conjugated secondary antibody. (A) Representative histograms of three independent experiments. Black and red lines indicate cells incubated with RuV in DMEM containing 2 mM CaCl 2 and in Ca 2+ -free DMEM, respectively. Shaded areas indicate cells incubated without RuV antigens. (B) The geometric mean fluorescence intensity (MFI) and standard deviation of triplicate samples. (C and D) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with 150 mU/ml SMase for 1 h or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (C). Black lines indicate cells with or without SMase pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (D). (E and F) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with MβCD (10 mM and 5 mM, respectively) for 15 min or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (E). Black lines indicate cells with or without MβCD pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (F).
    Figure Legend Snippet: Effects of Ca 2+ , SMase, and MβCD on RuV binding to mammalian cells. (A and B) Vero, RK13, 293T, LLC-MK2, HeLa, Jurkat, and Raji cells were incubated with RuV antigens at 4°C in DMEM containing 2 mM CaCl 2 or in Ca 2+ -free DMEM for 1 h. The bound RuV antigens were detected by flow cytometry using a MAb specific for the RuV E1 protein and a PE-conjugated secondary antibody. (A) Representative histograms of three independent experiments. Black and red lines indicate cells incubated with RuV in DMEM containing 2 mM CaCl 2 and in Ca 2+ -free DMEM, respectively. Shaded areas indicate cells incubated without RuV antigens. (B) The geometric mean fluorescence intensity (MFI) and standard deviation of triplicate samples. (C and D) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with 150 mU/ml SMase for 1 h or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (C). Black lines indicate cells with or without SMase pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (D). (E and F) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with MβCD (10 mM and 5 mM, respectively) for 15 min or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (E). Black lines indicate cells with or without MβCD pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (F).

    Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation, Cell Culture

    Effects of sphingomyelinase (SMase), myriocin, and HPA-12 on rubella virus (RuV) infection. (A) RK13 cells, untreated or treated with various doses of SMase for 1 h, were infected with RuV (HS wild-type or TO-336 vaccine strain) and Sindbis virus (SINV), and then standard plaque assays were performed. (B) Similar experiments were performed using Vero cells and green fluorescent protein (GFP)-expressing recombinant RuV (RuV-rHS/p150-AG1), measles virus, (MeV-IC323/Ed-H-EGFP), and human metapneumovirus (HMPV-rJPS02-76EGFP). Plaque numbers of GFP-expressing recombinant viruses were counted under a fluorescence microscope. (C) Vero cells, untreated or treated with various doses of myriocin for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. (D) Vero cells, untreated or treated with various doses of HPA-12 for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. For panels A to D, the average infectious titer of each virus in untreated cells was set to 100%. Asterisks indicate significant differences based on a t test ( P
    Figure Legend Snippet: Effects of sphingomyelinase (SMase), myriocin, and HPA-12 on rubella virus (RuV) infection. (A) RK13 cells, untreated or treated with various doses of SMase for 1 h, were infected with RuV (HS wild-type or TO-336 vaccine strain) and Sindbis virus (SINV), and then standard plaque assays were performed. (B) Similar experiments were performed using Vero cells and green fluorescent protein (GFP)-expressing recombinant RuV (RuV-rHS/p150-AG1), measles virus, (MeV-IC323/Ed-H-EGFP), and human metapneumovirus (HMPV-rJPS02-76EGFP). Plaque numbers of GFP-expressing recombinant viruses were counted under a fluorescence microscope. (C) Vero cells, untreated or treated with various doses of myriocin for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. (D) Vero cells, untreated or treated with various doses of HPA-12 for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. For panels A to D, the average infectious titer of each virus in untreated cells was set to 100%. Asterisks indicate significant differences based on a t test ( P

    Techniques Used: Infection, Expressing, Recombinant, Fluorescence, Microscopy

    Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P
    Figure Legend Snippet: Effects of SMase, myriocin, HPA-12, and IFN on RuV replicon systems. (A) Structure of the subgenomic replicon RuV-Luc genome (HS-Rep-C-P2R). P2R, a reporter fusion protein composed of puromycin N -acetyl-transferase (Puro), the foot-and-mouth disease virus 2A self-cleavage domain, and Renilla luciferase (RLuc); IRES, internal ribosome entry site sequence of encephalomyocarditis virus; P150 and P90, RuV nonstructural proteins; C, RuV capsid protein; AG1, monomeric Aami-Green1. (B) The replicon RuV-Luc genome was synthesized in vitro , and Vero cells were transfected with the in vitro -synthesized replicon RuV-Luc genome. At 4 h posttransfection, the cells were left untreated or treated with SMase (150 mU/ml) and incubated for 72 h at 35°C. The luciferase activity in the cells was then measured. (C) RuV-RNA replicon cells (Vero-HS-Rep-C-P2R cells) were left untreated or treated with SMase (150 mU/ml), myriocin (100 nM), HPA-12 (5 μM), or IFN (100 units/ml) and incubated for 2 days at 35°C. The Renilla luciferase activity in the cells was then measured. For panels B and C, the average luciferase activity in untreated cells (−) was set to 100%. The asterisk indicates a significant difference based on a t test ( P

    Techniques Used: Luciferase, Sequencing, Synthesized, In Vitro, Transfection, Incubation, Activity Assay

    Effects of Ca 2+ , SMase, and MβCD on RuV binding to mammalian cells. (A and B) Vero, RK13, 293T, LLC-MK2, HeLa, Jurkat, and Raji cells were incubated with RuV antigens at 4°C in DMEM containing 2 mM CaCl 2 or in Ca 2+ -free DMEM for 1 h. The bound RuV antigens were detected by flow cytometry using a MAb specific for the RuV E1 protein and a PE-conjugated secondary antibody. (A) Representative histograms of three independent experiments. Black and red lines indicate cells incubated with RuV in DMEM containing 2 mM CaCl 2 and in Ca 2+ -free DMEM, respectively. Shaded areas indicate cells incubated without RuV antigens. (B) The geometric mean fluorescence intensity (MFI) and standard deviation of triplicate samples. (C and D) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with 150 mU/ml SMase for 1 h or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (C). Black lines indicate cells with or without SMase pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (D). (E and F) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with MβCD (10 mM and 5 mM, respectively) for 15 min or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (E). Black lines indicate cells with or without MβCD pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (F).
    Figure Legend Snippet: Effects of Ca 2+ , SMase, and MβCD on RuV binding to mammalian cells. (A and B) Vero, RK13, 293T, LLC-MK2, HeLa, Jurkat, and Raji cells were incubated with RuV antigens at 4°C in DMEM containing 2 mM CaCl 2 or in Ca 2+ -free DMEM for 1 h. The bound RuV antigens were detected by flow cytometry using a MAb specific for the RuV E1 protein and a PE-conjugated secondary antibody. (A) Representative histograms of three independent experiments. Black and red lines indicate cells incubated with RuV in DMEM containing 2 mM CaCl 2 and in Ca 2+ -free DMEM, respectively. Shaded areas indicate cells incubated without RuV antigens. (B) The geometric mean fluorescence intensity (MFI) and standard deviation of triplicate samples. (C and D) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with 150 mU/ml SMase for 1 h or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (C). Black lines indicate cells with or without SMase pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (D). (E and F) Vero and Jurkat cells cultured in medium supplemented with 2 mM CaCl 2 were treated with MβCD (10 mM and 5 mM, respectively) for 15 min or remained untreated. The cells were then incubated with RuV antigens at 4°C for 1 h. The bound RuV antigens were detected by flow cytometry. Representative histograms of three independent experiments are shown (E). Black lines indicate cells with or without MβCD pretreatment and then incubated with RuV. Shaded areas indicate cells incubated without RuV antigens. The geometric MFIs and standard deviations of triplicate samples were determined (F).

    Techniques Used: Binding Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Standard Deviation, Cell Culture

    Effects of sphingomyelinase (SMase), myriocin, and HPA-12 on rubella virus (RuV) infection. (A) RK13 cells, untreated or treated with various doses of SMase for 1 h, were infected with RuV (HS wild-type or TO-336 vaccine strain) and Sindbis virus (SINV), and then standard plaque assays were performed. (B) Similar experiments were performed using Vero cells and green fluorescent protein (GFP)-expressing recombinant RuV (RuV-rHS/p150-AG1), measles virus, (MeV-IC323/Ed-H-EGFP), and human metapneumovirus (HMPV-rJPS02-76EGFP). Plaque numbers of GFP-expressing recombinant viruses were counted under a fluorescence microscope. (C) Vero cells, untreated or treated with various doses of myriocin for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. (D) Vero cells, untreated or treated with various doses of HPA-12 for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. For panels A to D, the average infectious titer of each virus in untreated cells was set to 100%. Asterisks indicate significant differences based on a t test ( P
    Figure Legend Snippet: Effects of sphingomyelinase (SMase), myriocin, and HPA-12 on rubella virus (RuV) infection. (A) RK13 cells, untreated or treated with various doses of SMase for 1 h, were infected with RuV (HS wild-type or TO-336 vaccine strain) and Sindbis virus (SINV), and then standard plaque assays were performed. (B) Similar experiments were performed using Vero cells and green fluorescent protein (GFP)-expressing recombinant RuV (RuV-rHS/p150-AG1), measles virus, (MeV-IC323/Ed-H-EGFP), and human metapneumovirus (HMPV-rJPS02-76EGFP). Plaque numbers of GFP-expressing recombinant viruses were counted under a fluorescence microscope. (C) Vero cells, untreated or treated with various doses of myriocin for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. (D) Vero cells, untreated or treated with various doses of HPA-12 for 48 h, were infected with RuV-rHS/p150-AG1 or MeV-IC323/Ed-H-EGFP, and standard plaque assays were performed. For panels A to D, the average infectious titer of each virus in untreated cells was set to 100%. Asterisks indicate significant differences based on a t test ( P

    Techniques Used: Infection, Expressing, Recombinant, Fluorescence, Microscopy

    17) Product Images from "Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures"

    Article Title: Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    Journal: Journal of Virology

    doi: 10.1128/JVI.02351-15

    Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product
    Figure Legend Snippet: Cathepsin L and B expression and activity in HeLa and Vero cells. (A) Cathepsin L mRNA from mock-treated or rgRSV-infected HeLa and Vero cells was reverse transcribed and amplified by reverse transcription-PCR (RT-PCR), and the specific 294-bp product

    Techniques Used: Expressing, Activity Assay, Infection, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM
    Figure Legend Snippet: Cathepsin L treatment of purified virions that had been grown in the presence of a cathepsin L inhibitor (inh). Vero cells (A) or HeLa cells (B) were inoculated with rgRSV and treated with medium containing vehicle (first and third lanes) or 0.5 μM

    Techniques Used: Purification

    Protease inhibition of G-protein cleavage in Vero cells. Immunoblotting was used to detect biotinylated cell surface RSV G protein (55 or 90 kDa) produced in rgRSV-inoculated Vero cells treated with the following protease inhibitors: 2-fold dilutions
    Figure Legend Snippet: Protease inhibition of G-protein cleavage in Vero cells. Immunoblotting was used to detect biotinylated cell surface RSV G protein (55 or 90 kDa) produced in rgRSV-inoculated Vero cells treated with the following protease inhibitors: 2-fold dilutions

    Techniques Used: Inhibition, Produced

    Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected
    Figure Legend Snippet: Comparison of rgRSV and rgRSV-L208A infection of HAE cells. HeLa cell-derived (H-D) and Vero cell-derived (V-D) rgRSV and rgRSV-L208A were produced in HeLa or Vero cells. (A) Virus was purified by use of a sucrose gradient, and G protein was detected

    Techniques Used: Infection, Derivative Assay, Produced, Purification

    Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (
    Figure Legend Snippet: Ability of rgRSV grown in the presence of a cathepsin L inhibitor to infect HAE cells. The infectivity for HAE cells of partially purified rgRSV virions that were produced in HeLa or Vero cells in the presence of a cathepsin L inhibitor or DMSO (

    Techniques Used: Infection, Purification, Produced

    18) Product Images from "Imaging of the Alphavirus Capsid Protein during Virus Replication"

    Article Title: Imaging of the Alphavirus Capsid Protein during Virus Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01299-13

    Examples of the three types of capsid foci. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. Cells were then fixed, permeabilized, and stained with a MAb against the SINV E2 protein. The images illustrate three groups of capsid foci. (A) Small internal capsid puncta that colocalize with the E2 protein (arrows point to representative examples). (B) Irregular internal capsid structures that did not colocalize with the E2 protein (arrows point to representative examples). (C) PM-proximal capsid puncta that colocalize with E2 protein. The inset shows a zoomed view of the boxed region (3× magnification). All images are single z sections, where panels A and B are internal sections and C is a PM-proximal section. All are representative examples from three experiments. Bar, 10 μM.
    Figure Legend Snippet: Examples of the three types of capsid foci. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. Cells were then fixed, permeabilized, and stained with a MAb against the SINV E2 protein. The images illustrate three groups of capsid foci. (A) Small internal capsid puncta that colocalize with the E2 protein (arrows point to representative examples). (B) Irregular internal capsid structures that did not colocalize with the E2 protein (arrows point to representative examples). (C) PM-proximal capsid puncta that colocalize with E2 protein. The inset shows a zoomed view of the boxed region (3× magnification). All images are single z sections, where panels A and B are internal sections and C is a PM-proximal section. All are representative examples from three experiments. Bar, 10 μM.

    Techniques Used: Infection, Labeling, Staining

    Properties of PM capsid puncta. (A) Generation of PM capsid puncta does not require cytoplasmic NC formation. Vero cells infected with TC-LL for 7 h were labeled with ReAsH and then fixed, permeabilized, and stained with E2 MAb R6. (B) PM capsid puncta do not colocalize with dsRNA. Vero cells were infected with Q94-TC for 7 h, labeled with ReAsH, and then fixed, permeabilized, and stained with MAb against dsRNA. (C) Newly synthesized capsid protein was delivered to preexisting PM capsid puncta. Vero cells were infected with Q94-TC for 7 h and stained with FlAsH to label the existing capsid protein pool (second column). At the indicated chase time, the cells were labeled with ReAsH (first column). After 1 h of chase, ReAsH-labeled capsid protein was detected in PM capsid puncta containing the FlAsH signal. The inset is a zoomed view of the boxed region (2.5× magnification). Images are representative of two independent experiments. Bar, 10 μM.
    Figure Legend Snippet: Properties of PM capsid puncta. (A) Generation of PM capsid puncta does not require cytoplasmic NC formation. Vero cells infected with TC-LL for 7 h were labeled with ReAsH and then fixed, permeabilized, and stained with E2 MAb R6. (B) PM capsid puncta do not colocalize with dsRNA. Vero cells were infected with Q94-TC for 7 h, labeled with ReAsH, and then fixed, permeabilized, and stained with MAb against dsRNA. (C) Newly synthesized capsid protein was delivered to preexisting PM capsid puncta. Vero cells were infected with Q94-TC for 7 h and stained with FlAsH to label the existing capsid protein pool (second column). At the indicated chase time, the cells were labeled with ReAsH (first column). After 1 h of chase, ReAsH-labeled capsid protein was detected in PM capsid puncta containing the FlAsH signal. The inset is a zoomed view of the boxed region (2.5× magnification). Images are representative of two independent experiments. Bar, 10 μM.

    Techniques Used: Infection, Labeling, Staining, Synthesized

    Growth properties of Q94-TC SINV. (A) Growth kinetics of Q94-TC versus WT virus. BHK-21 cells were electroporated with WT or mutant virus RNA and incubated at 37°C for the indicated times. Media were collected, and titers of progeny viruses were determined by plaque assay. (B) The effect of ReAsH labeling on Q94-TC virus production. Vero cells were infected with Q94-TC at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and mock treated or treated with ReAsH using the conditions described in Materials and Methods. The incubation was continued at 37°C for the indicated times, and progeny virus production was quantitated by infectious center assays on Vero cells. Data are averages from two independent experiments with ranges indicated.
    Figure Legend Snippet: Growth properties of Q94-TC SINV. (A) Growth kinetics of Q94-TC versus WT virus. BHK-21 cells were electroporated with WT or mutant virus RNA and incubated at 37°C for the indicated times. Media were collected, and titers of progeny viruses were determined by plaque assay. (B) The effect of ReAsH labeling on Q94-TC virus production. Vero cells were infected with Q94-TC at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and mock treated or treated with ReAsH using the conditions described in Materials and Methods. The incubation was continued at 37°C for the indicated times, and progeny virus production was quantitated by infectious center assays on Vero cells. Data are averages from two independent experiments with ranges indicated.

    Techniques Used: Mutagenesis, Incubation, Plaque Assay, Labeling, Infection, Cell Culture

    Localization and dynamics of the PM-proximal capsid puncta. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. (A) Localization of the PM-proximal capsid puncta. Following ReAsH labeling, cells were immunolabeled on ice to detect the cell surface E2 protein as described in Materials and Methods. A middle section in the z direction is shown. (B) Time series of PM capsid puncta. The positions of specific puncta (indicated by arrows) were tracked by images acquired every second during a 37°C incubation. Images of a z section at the PM collected at 0, 20, and 40 s are shown, with a zoomed view of the boxed region on the upper-right side, documenting the relative immobility of this puncta type. Images are representative examples from two independent experiments. Bar, 10 μM.
    Figure Legend Snippet: Localization and dynamics of the PM-proximal capsid puncta. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. (A) Localization of the PM-proximal capsid puncta. Following ReAsH labeling, cells were immunolabeled on ice to detect the cell surface E2 protein as described in Materials and Methods. A middle section in the z direction is shown. (B) Time series of PM capsid puncta. The positions of specific puncta (indicated by arrows) were tracked by images acquired every second during a 37°C incubation. Images of a z section at the PM collected at 0, 20, and 40 s are shown, with a zoomed view of the boxed region on the upper-right side, documenting the relative immobility of this puncta type. Images are representative examples from two independent experiments. Bar, 10 μM.

    Techniques Used: Infection, Labeling, Immunolabeling, Incubation

    Dynamics of irregular internal capsid structures. (A) Time series of internal capsid structures. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. The positions of specific foci (indicated by arrows) were tracked by images acquired every second. Images collected at 0, 20, and 40 s are shown, documenting the relative immobility of these structures. (B) Recruitment of newly synthesized capsid protein to preexisting irregular internal capsid foci. Vero cells were infected with Q94-TC and labeled with FlAsH at 7 h postinfection (right column). At the indicated chase time, the cells were labeled with ReAsH (left column). Images all were acquired at the same gain and are representative of two independent experiments. Bar, 10 μM.
    Figure Legend Snippet: Dynamics of irregular internal capsid structures. (A) Time series of internal capsid structures. Vero cells were infected with Q94-TC and labeled with ReAsH at 7 h postinfection. The positions of specific foci (indicated by arrows) were tracked by images acquired every second. Images collected at 0, 20, and 40 s are shown, documenting the relative immobility of these structures. (B) Recruitment of newly synthesized capsid protein to preexisting irregular internal capsid foci. Vero cells were infected with Q94-TC and labeled with FlAsH at 7 h postinfection (right column). At the indicated chase time, the cells were labeled with ReAsH (left column). Images all were acquired at the same gain and are representative of two independent experiments. Bar, 10 μM.

    Techniques Used: Infection, Labeling, Synthesized

    Specificity of biarsenical dye labeling. Vero cells were infected with Q94-TC or WT SINV at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and labeled with ReAsH as described in Materials and Methods. Cells were then fixed, permeabilized, and stained with antibody against the capsid protein. Images are single internal z sections and are representative of two independent experiments. Bar, 10 μM.
    Figure Legend Snippet: Specificity of biarsenical dye labeling. Vero cells were infected with Q94-TC or WT SINV at a multiplicity of 0.5 IC/cell, cultured for 7 h at 37°C, and labeled with ReAsH as described in Materials and Methods. Cells were then fixed, permeabilized, and stained with antibody against the capsid protein. Images are single internal z sections and are representative of two independent experiments. Bar, 10 μM.

    Techniques Used: Labeling, Infection, Cell Culture, Staining

    Irregular internal capsid structures colocalize with G3BP and nsP3. (A) Vero cells were infected with Q94-TC and labeled with ReAsH (left) at 7 h postinfection. Cells were then fixed, permeabilized, and stained with antibodies against G3BP, dsRNA, or eIF3 (middle panels). (B) Vero cells were infected with Q94-TC virus, incubated for 7 h, fixed, and costained with MAb recognizing the capsid protein and rabbit antibody against nsP3. (C) Vero cells were infected with either Q94-TC or Q94-TC plus L108A/L110A (indicated as TC-LL). Alternatively, cells were transfected with RNA for the budding-defective mutant Q94-TC plus E2Y400K (indicated as TC-Y400K). Cells were incubated for 7 h, fixed, and costained with MAb to capsid and rabbit antibody against G3BP. Images are representative of two independent experiments. Scale bar, 10 μM.
    Figure Legend Snippet: Irregular internal capsid structures colocalize with G3BP and nsP3. (A) Vero cells were infected with Q94-TC and labeled with ReAsH (left) at 7 h postinfection. Cells were then fixed, permeabilized, and stained with antibodies against G3BP, dsRNA, or eIF3 (middle panels). (B) Vero cells were infected with Q94-TC virus, incubated for 7 h, fixed, and costained with MAb recognizing the capsid protein and rabbit antibody against nsP3. (C) Vero cells were infected with either Q94-TC or Q94-TC plus L108A/L110A (indicated as TC-LL). Alternatively, cells were transfected with RNA for the budding-defective mutant Q94-TC plus E2Y400K (indicated as TC-Y400K). Cells were incubated for 7 h, fixed, and costained with MAb to capsid and rabbit antibody against G3BP. Images are representative of two independent experiments. Scale bar, 10 μM.

    Techniques Used: Infection, Labeling, Staining, Incubation, Transfection, Mutagenesis

    19) Product Images from "Genetic Variation between Dengue Virus Type 4 Strains Impacts Human Antibody Binding and Neutralization"

    Article Title: Genetic Variation between Dengue Virus Type 4 Strains Impacts Human Antibody Binding and Neutralization

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.10.006

    DENV4 Polyclonal Immune Sera Have a Range of Neutralization Titers against DENV4 Variants Driven by Serotype-Specific Antibodies (A and B) Using Vero cell focus reduction neutralization test (FRNT), (A) pooled polyclonal immune sera from DENV4-infected non-human primates (NHPs) or from naturally infected individuals, or from (B) individuals who received the NIH DENV4 monovalent vaccine were evaluated for their ability to neutralize DENV4 genotype viruses. (C–F) DENV4 natural infection sera (C), DENV4 monovalent vaccine sera (D), and NIH DENV tetravalent vaccine sera were control depleted with BSA (E), or depleted of cross-reactive antibodies (F) and evaluated for their ability to neutralize DENV4 genotype viruses. The y axis represents the dilution factor of immune sera required to neutralize 50% of infectious virus (mean ± SD of technical duplicates). The dashed line represents one-half the assay limit of detection.
    Figure Legend Snippet: DENV4 Polyclonal Immune Sera Have a Range of Neutralization Titers against DENV4 Variants Driven by Serotype-Specific Antibodies (A and B) Using Vero cell focus reduction neutralization test (FRNT), (A) pooled polyclonal immune sera from DENV4-infected non-human primates (NHPs) or from naturally infected individuals, or from (B) individuals who received the NIH DENV4 monovalent vaccine were evaluated for their ability to neutralize DENV4 genotype viruses. (C–F) DENV4 natural infection sera (C), DENV4 monovalent vaccine sera (D), and NIH DENV tetravalent vaccine sera were control depleted with BSA (E), or depleted of cross-reactive antibodies (F) and evaluated for their ability to neutralize DENV4 genotype viruses. The y axis represents the dilution factor of immune sera required to neutralize 50% of infectious virus (mean ± SD of technical duplicates). The dashed line represents one-half the assay limit of detection.

    Techniques Used: Neutralization, Infection

    DENV4 Genotypic Variants Are Differentially Neutralized by Monoclonal Antibodies (A and B) DENV4 serotype-specific antibodies D4-126, D4-131, D4-141, and 5H2 and DENV cross-reactive antibodies C10 and B7 were evaluated for their ability to neutralize DENV4 genotype viruses in (A) Vero cell focus reduction neutralization test (FRNT) and (B) flow cytometry-based neutralization assay (Neut) (mean ± SD of technical duplicates). The y-axes represent the concentration of antibody required to neutralize 50% of infectious virus. The dashed line represents assay limit of detection.
    Figure Legend Snippet: DENV4 Genotypic Variants Are Differentially Neutralized by Monoclonal Antibodies (A and B) DENV4 serotype-specific antibodies D4-126, D4-131, D4-141, and 5H2 and DENV cross-reactive antibodies C10 and B7 were evaluated for their ability to neutralize DENV4 genotype viruses in (A) Vero cell focus reduction neutralization test (FRNT) and (B) flow cytometry-based neutralization assay (Neut) (mean ± SD of technical duplicates). The y-axes represent the concentration of antibody required to neutralize 50% of infectious virus. The dashed line represents assay limit of detection.

    Techniques Used: Neutralization, Flow Cytometry, Cytometry, Concentration Assay

    20) Product Images from "Cellular Proteasome Activity Facilitates Herpes Simplex Virus Entry at a Postpenetration Step "

    Article Title: Cellular Proteasome Activity Facilitates Herpes Simplex Virus Entry at a Postpenetration Step

    Journal: Journal of Virology

    doi: 10.1128/JVI.02296-07

    Incoming HSV relies on proteasome activity at a postpenetration step. (A and B) Citrate treatment of the cell surface has no effect on the infectivity of MG132-arrested virions. (A) Vero cells were chilled on ice, and HSV-1 KOS (100 PFU/well) was added for 1 h at 4°C. Cells were treated with warmed PBS or citrate buffer (pH 3.0). Plates were shifted to 37°C, and plaques were quantified at 24 h p.i. (B) Vero cells were mock treated (no inhibitor) or treated with 25 μM MG132 for 30 min at 37°C. Cells were chilled, and then HSV-1 KOS (100 PFU/well) was added for 1 h at 4°C to synchronize the infection. Cultures were shifted to 37°C, and MG132 concentrations were maintained for 2.5 h at 37°C. Cells were then treated with PBS or citrate buffer (Citrate) and then extensively washed with culture medium to reverse the effect of MG132. Plaques were quantified at 18 h p.i. The data are the means of triplicate samples with the standard deviations. (C to G) Permeabilization of the plasma membrane followed by treatment with protease allows the removal of MG132-arrested virions. HSV-1 KOS K26GFP (MOI of 10) was bound to prechilled Vero cells for 1 h at 4°C (C), and then 100 μg of proteinase K/ml was added for 2.5 min (D) prior to fixation and confocal microscopy. Bar, 10 μm. (E to G) HSV-1 KOS K26GFP (MOI of 10) was added to cells in the presence of 25 μM MG132 for 2.5 h p.i. at 37°C. MG132-treated cells received no additional treatment (E), were treated with proteinase K (F), or were permeabilized with digitonin followed by proteinase K treatment (G). Cells were fixed and analyzed by confocal microscopy. Bar, 10 μm. (H to K) MG132 has no effect on virion-induced fusion in a fusion-from-without assay. Vero cells were mock treated (H and I) or treated with 50 μM MG132 (J) for 30 min at 37°C. Cells were left uninfected (H), or HSV-1 ANG path (MOI of 50) was added (I and J) for 3 h in the presence of cycloheximide. Cells were fixed and stained with Giemsa. (K) Fusion was quantitated from photomicrographs of random fields as described in Materials and Methods. The data are means of triplicate determinations with the standard errors.
    Figure Legend Snippet: Incoming HSV relies on proteasome activity at a postpenetration step. (A and B) Citrate treatment of the cell surface has no effect on the infectivity of MG132-arrested virions. (A) Vero cells were chilled on ice, and HSV-1 KOS (100 PFU/well) was added for 1 h at 4°C. Cells were treated with warmed PBS or citrate buffer (pH 3.0). Plates were shifted to 37°C, and plaques were quantified at 24 h p.i. (B) Vero cells were mock treated (no inhibitor) or treated with 25 μM MG132 for 30 min at 37°C. Cells were chilled, and then HSV-1 KOS (100 PFU/well) was added for 1 h at 4°C to synchronize the infection. Cultures were shifted to 37°C, and MG132 concentrations were maintained for 2.5 h at 37°C. Cells were then treated with PBS or citrate buffer (Citrate) and then extensively washed with culture medium to reverse the effect of MG132. Plaques were quantified at 18 h p.i. The data are the means of triplicate samples with the standard deviations. (C to G) Permeabilization of the plasma membrane followed by treatment with protease allows the removal of MG132-arrested virions. HSV-1 KOS K26GFP (MOI of 10) was bound to prechilled Vero cells for 1 h at 4°C (C), and then 100 μg of proteinase K/ml was added for 2.5 min (D) prior to fixation and confocal microscopy. Bar, 10 μm. (E to G) HSV-1 KOS K26GFP (MOI of 10) was added to cells in the presence of 25 μM MG132 for 2.5 h p.i. at 37°C. MG132-treated cells received no additional treatment (E), were treated with proteinase K (F), or were permeabilized with digitonin followed by proteinase K treatment (G). Cells were fixed and analyzed by confocal microscopy. Bar, 10 μm. (H to K) MG132 has no effect on virion-induced fusion in a fusion-from-without assay. Vero cells were mock treated (H and I) or treated with 50 μM MG132 (J) for 30 min at 37°C. Cells were left uninfected (H), or HSV-1 ANG path (MOI of 50) was added (I and J) for 3 h in the presence of cycloheximide. Cells were fixed and stained with Giemsa. (K) Fusion was quantitated from photomicrographs of random fields as described in Materials and Methods. The data are means of triplicate determinations with the standard errors.

    Techniques Used: Activity Assay, Infection, Confocal Microscopy, Staining

    The block to entry imposed by proteasome inhibitors is partially reversible. Vero cells were mock treated (A) or treated with 25 μΜ MG132 (B and C) for 15 min at 37°C. HSV-1 KOS K26GFP was allowed to infect cells for 2 h in the absence (A) or presence of MG132 (B and C). Cells were fixed (B) or were washed four times for 5 min each time with culture medium and returned to 37°C for an additional hour in normal culture medium prior to fixation (A and C). Bar, 10 μm.
    Figure Legend Snippet: The block to entry imposed by proteasome inhibitors is partially reversible. Vero cells were mock treated (A) or treated with 25 μΜ MG132 (B and C) for 15 min at 37°C. HSV-1 KOS K26GFP was allowed to infect cells for 2 h in the absence (A) or presence of MG132 (B and C). Cells were fixed (B) or were washed four times for 5 min each time with culture medium and returned to 37°C for an additional hour in normal culture medium prior to fixation (A and C). Bar, 10 μm.

    Techniques Used: Blocking Assay

    Effect of proteasome inhibitors on HSV infection. (A) Effect of MG132 on HSV plaque formation. HSV-1 KOS (100 PFU/well) was added to Vero cells in the presence of MG132. At 3 h p.i., medium was removed, extracellular virus was acid inactivated, and plates were incubated for 24 h. Plaques were detected by immunoperoxidase staining and quantified. The data are means of quadruplicate determinations with the standard error. Vero cells (B and C) or CHO cells expressing nectin-1 (D and E) were treated with the indicated concentrations of inhibitor for 15 min. HSV-1 KOS-tk12 was added for 7.5 h in the continued presence of inhibitor. The percent β-galactosidase activity relative to that obtained in the absence of agent is indicated. The data are means of quadruplicate determinations with the standard errors. The effect of inhibitor on cell viability was measured by trypan blue exclusion. Viability in the absence of inhibitor was set to 100%. The mean viabilities and the standard errors of quadruplicate samples is shown.
    Figure Legend Snippet: Effect of proteasome inhibitors on HSV infection. (A) Effect of MG132 on HSV plaque formation. HSV-1 KOS (100 PFU/well) was added to Vero cells in the presence of MG132. At 3 h p.i., medium was removed, extracellular virus was acid inactivated, and plates were incubated for 24 h. Plaques were detected by immunoperoxidase staining and quantified. The data are means of quadruplicate determinations with the standard error. Vero cells (B and C) or CHO cells expressing nectin-1 (D and E) were treated with the indicated concentrations of inhibitor for 15 min. HSV-1 KOS-tk12 was added for 7.5 h in the continued presence of inhibitor. The percent β-galactosidase activity relative to that obtained in the absence of agent is indicated. The data are means of quadruplicate determinations with the standard errors. The effect of inhibitor on cell viability was measured by trypan blue exclusion. Viability in the absence of inhibitor was set to 100%. The mean viabilities and the standard errors of quadruplicate samples is shown.

    Techniques Used: Infection, Incubation, Immunoperoxidase Staining, Expressing, Activity Assay

    MG132 affects an entry event that occurs after surface binding, but prior to incoming capsid arrival at the nucleus. (A to D) Effect of MG132 on virus binding to cells. HSV-1 KOS K26GFP (MOI of 30) was added to prechilled Vero cells for 1 h at 4°C in the presence of either no inhibitor (A), 25 μΜ MG132 (B), or 1 μg of heparin/ml (C). Cells were fixed, and confocal images were obtained. Bar, 100 μm. (D) The number of bound GFP-tagged virions was quantified by three observers blinded to experimental conditions (Virions). The data are means with the standard errors. In parallel, mean GFP intensity was measured by the histogram function of Adobe Photoshop. Binding in the absence of inhibitor was set to 100%. (E to H) Effect of proteasome inhibitors on incoming capsid transport to the nuclear periphery. Vero cells were mock treated (E) or treated with 25 μΜ MG132 (F), 10 μΜ lactacystin (G), or 10 μΜ epoxomicin (H) for 15 min at 37°C. HSV-1 KOS K26GFP (MOI of 10) was added for 2.5 h in the constant presence of agent and 0.5 mM cycloheximide. Cells were fixed, and confocal images were obtained. Images are representative of cell population. Bar, 10 μm.
    Figure Legend Snippet: MG132 affects an entry event that occurs after surface binding, but prior to incoming capsid arrival at the nucleus. (A to D) Effect of MG132 on virus binding to cells. HSV-1 KOS K26GFP (MOI of 30) was added to prechilled Vero cells for 1 h at 4°C in the presence of either no inhibitor (A), 25 μΜ MG132 (B), or 1 μg of heparin/ml (C). Cells were fixed, and confocal images were obtained. Bar, 100 μm. (D) The number of bound GFP-tagged virions was quantified by three observers blinded to experimental conditions (Virions). The data are means with the standard errors. In parallel, mean GFP intensity was measured by the histogram function of Adobe Photoshop. Binding in the absence of inhibitor was set to 100%. (E to H) Effect of proteasome inhibitors on incoming capsid transport to the nuclear periphery. Vero cells were mock treated (E) or treated with 25 μΜ MG132 (F), 10 μΜ lactacystin (G), or 10 μΜ epoxomicin (H) for 15 min at 37°C. HSV-1 KOS K26GFP (MOI of 10) was added for 2.5 h in the constant presence of agent and 0.5 mM cycloheximide. Cells were fixed, and confocal images were obtained. Images are representative of cell population. Bar, 10 μm.

    Techniques Used: Binding Assay

    21) Product Images from "The effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines"

    Article Title: The effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines

    Journal: Antiviral Chemistry & Chemotherapy

    doi: 10.1177/2040206618807580

    Antiviral effect of favipiravir (FAV) against chikungunya virus (CHIKV) in Vero (a) HUH-7 (b), and A549 (c) cells. Vero cells were inoculated with CHIKV at a multiplicity of infection of 0.0001 and HUH-7 and A549 cells were inoculated at a MOI of 0.1. Extracellular infectious CHIKV was quantified by plaque assay on Vero cells and reported as Log 10 plaque forming units per ml (PFU/ml). Data points represent the mean of three independent samples and error bars correspond to one standard deviation. The dashed line signifies the assay limit of detection.
    Figure Legend Snippet: Antiviral effect of favipiravir (FAV) against chikungunya virus (CHIKV) in Vero (a) HUH-7 (b), and A549 (c) cells. Vero cells were inoculated with CHIKV at a multiplicity of infection of 0.0001 and HUH-7 and A549 cells were inoculated at a MOI of 0.1. Extracellular infectious CHIKV was quantified by plaque assay on Vero cells and reported as Log 10 plaque forming units per ml (PFU/ml). Data points represent the mean of three independent samples and error bars correspond to one standard deviation. The dashed line signifies the assay limit of detection.

    Techniques Used: Infection, Plaque Assay, Standard Deviation

    Antiviral effect of interferon-alpha (IFN-α) against chikungunya virus (CHIKV) in Vero (a) HUH-7 (b), and A549 (c) cells. Vero cells were inoculated with CHIKV at a multiplicity of infection of 0.0001 and HUH-7 and A549 cells were inoculated at a MOI of 0.1. Infectious CHIKV, reported as Log 10 plaque forming units per ml (PFU/ml), was quantified by plaque assay on Vero cells. Data points represent the mean of three independent samples and error bars correspond to one standard deviation. The dashed line signifies the assay limit of detection.
    Figure Legend Snippet: Antiviral effect of interferon-alpha (IFN-α) against chikungunya virus (CHIKV) in Vero (a) HUH-7 (b), and A549 (c) cells. Vero cells were inoculated with CHIKV at a multiplicity of infection of 0.0001 and HUH-7 and A549 cells were inoculated at a MOI of 0.1. Infectious CHIKV, reported as Log 10 plaque forming units per ml (PFU/ml), was quantified by plaque assay on Vero cells. Data points represent the mean of three independent samples and error bars correspond to one standard deviation. The dashed line signifies the assay limit of detection.

    Techniques Used: Infection, Plaque Assay, Standard Deviation

    22) Product Images from "Zika Virus Replication Is Substantially Inhibited by Novel Favipiravir and Interferon Alpha Combination Regimens"

    Article Title: Zika Virus Replication Is Substantially Inhibited by Novel Favipiravir and Interferon Alpha Combination Regimens

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01983-17

    Antiviral activities of favipiravir (FAV), interferon alpha (IFN), and ribavirin (RBV) as monotherapy and combination therapy against Zika virus (ZIKV). (A to C) Vero cells were inoculated with ZIKV at a multiplicity of infection of 0.01 PFU/cell; different concentrations of FAV (A), IFN (B), or RBV (C) were added after infection for single-agent evaluations; and cell culture supernatants were harvested daily. (D to F) For combination evaluations, different concentrations of FAV plus IFN (D), FAV plus RBV (E), or RBV plus IFN (F) were added after inoculation, and cell culture supernatants were sampled on day 3 posttreatment. (G to I) 3D plots illustrating viral burdens for each combination of concentrations are shown for the combinations of FAV plus IFN (G), FAV plus RBV (H), and RBV plus IFN (I). The infectious virus burden, reported as log 10 PFU per milliliter, was quantified from cell culture supernatants by a plaque assay on Vero cells. The data are representative of results from one experiment. Data points represent the mean observed viral burdens for three independent samples, and error bars correspond to one standard deviation from three independent samples. Lines through the data points signify the predicted viral burden as determined by the mathematical model. The dashed horizontal line represents the assay limit of detection of 100 PFU/ml.
    Figure Legend Snippet: Antiviral activities of favipiravir (FAV), interferon alpha (IFN), and ribavirin (RBV) as monotherapy and combination therapy against Zika virus (ZIKV). (A to C) Vero cells were inoculated with ZIKV at a multiplicity of infection of 0.01 PFU/cell; different concentrations of FAV (A), IFN (B), or RBV (C) were added after infection for single-agent evaluations; and cell culture supernatants were harvested daily. (D to F) For combination evaluations, different concentrations of FAV plus IFN (D), FAV plus RBV (E), or RBV plus IFN (F) were added after inoculation, and cell culture supernatants were sampled on day 3 posttreatment. (G to I) 3D plots illustrating viral burdens for each combination of concentrations are shown for the combinations of FAV plus IFN (G), FAV plus RBV (H), and RBV plus IFN (I). The infectious virus burden, reported as log 10 PFU per milliliter, was quantified from cell culture supernatants by a plaque assay on Vero cells. The data are representative of results from one experiment. Data points represent the mean observed viral burdens for three independent samples, and error bars correspond to one standard deviation from three independent samples. Lines through the data points signify the predicted viral burden as determined by the mathematical model. The dashed horizontal line represents the assay limit of detection of 100 PFU/ml.

    Techniques Used: Infection, Cell Culture, Plaque Assay, Standard Deviation

    23) Product Images from "Herpes Simplex Virus Type 1 Primary Envelopment: UL34 Protein Modification and the US3-UL34 Catalytic Relationship"

    Article Title: Herpes Simplex Virus Type 1 Primary Envelopment: UL34 Protein Modification and the US3-UL34 Catalytic Relationship

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.1.399-412.2004

    Expression and phosphorylation of UL34 protein in recombinant virus-infected cells. HEp-2 cells (A and B) or Vero cells (C and D) were infected at an MOI of 5 with vRR1202rep (lane 1), HSV-1(F) (lane 2), vRR1202 (lane 3), vRR1204 #1 (lane 4), vRR1204 #2 (lane 5), vRR1205 #1 (lane 6), vRR1205 #2 (lane 7), or vRR1072 (lane 8) or mock infected (lane 9) and were then labeled with 32 P as inorganic phosphate. Equal amounts of total infected cell proteins were either separated by SDS-PAGE (12%), transferred to a nitrocellulose membrane, and probed with an anti-UL34 antibody (conjugated with alkaline phosphatase) (A and C) or immunoprecipitated with an anti-UL34 antibody, separated by SDS-PAGE, and analyzed by autoradiography (B and D).
    Figure Legend Snippet: Expression and phosphorylation of UL34 protein in recombinant virus-infected cells. HEp-2 cells (A and B) or Vero cells (C and D) were infected at an MOI of 5 with vRR1202rep (lane 1), HSV-1(F) (lane 2), vRR1202 (lane 3), vRR1204 #1 (lane 4), vRR1204 #2 (lane 5), vRR1205 #1 (lane 6), vRR1205 #2 (lane 7), or vRR1072 (lane 8) or mock infected (lane 9) and were then labeled with 32 P as inorganic phosphate. Equal amounts of total infected cell proteins were either separated by SDS-PAGE (12%), transferred to a nitrocellulose membrane, and probed with an anti-UL34 antibody (conjugated with alkaline phosphatase) (A and C) or immunoprecipitated with an anti-UL34 antibody, separated by SDS-PAGE, and analyzed by autoradiography (B and D).

    Techniques Used: Expressing, Recombinant, Infection, Labeling, SDS Page, Immunoprecipitation, Autoradiography

    Proper localization of the UL34 and US3 proteins requires US3-directed phosphorylation of an infected cell protein other than UL34. Vero cells were infected at an MOI of 5 with HSV-1(F) (A to C), vRR1202 (D to F), vRR1202rep (G to I), vRR1204 #1 (J to L), vRR1204 #2 (M to O), vRR1205 #1 (P to R), vRR1205 #2 (S to U), or vRR1072 (V to X). At 12 h.p.i., infected cells were formaldehyde fixed, immunostained with anti-UL34 (conjugated with fluorescein isothiocyanate) and anti-US3 (conjugated with Texas red), and analyzed by confocal microscopy. Representative confocal Z sections are shown. Original magnification, ×1,000.
    Figure Legend Snippet: Proper localization of the UL34 and US3 proteins requires US3-directed phosphorylation of an infected cell protein other than UL34. Vero cells were infected at an MOI of 5 with HSV-1(F) (A to C), vRR1202 (D to F), vRR1202rep (G to I), vRR1204 #1 (J to L), vRR1204 #2 (M to O), vRR1205 #1 (P to R), vRR1205 #2 (S to U), or vRR1072 (V to X). At 12 h.p.i., infected cells were formaldehyde fixed, immunostained with anti-UL34 (conjugated with fluorescein isothiocyanate) and anti-US3 (conjugated with Texas red), and analyzed by confocal microscopy. Representative confocal Z sections are shown. Original magnification, ×1,000.

    Techniques Used: Infection, Confocal Microscopy

    Expression of US3 protein in recombinant virus-infected cells. HEp-2 cells (A) or Vero cells (B) were infected at an MOI of 5 for 12 h with HSV-1(F) (lane 1), vRR1202 (lane 2), vRR1202rep (lane 3), vRR1204 #1 (lane 4), vRR1204 #2 (lane5), vRR1205 #1 (lane 6), or vRR1205 #2 (lane 7). Equal amounts of total infected cell proteins were separated by SDS-PAGE (8%), transferred to a nitrocellulose membrane, and probed with an anti-US3 antibody (conjugated with alkaline phosphatase).
    Figure Legend Snippet: Expression of US3 protein in recombinant virus-infected cells. HEp-2 cells (A) or Vero cells (B) were infected at an MOI of 5 for 12 h with HSV-1(F) (lane 1), vRR1202 (lane 2), vRR1202rep (lane 3), vRR1204 #1 (lane 4), vRR1204 #2 (lane5), vRR1205 #1 (lane 6), or vRR1205 #2 (lane 7). Equal amounts of total infected cell proteins were separated by SDS-PAGE (8%), transferred to a nitrocellulose membrane, and probed with an anti-US3 antibody (conjugated with alkaline phosphatase).

    Techniques Used: Expressing, Recombinant, Infection, SDS Page

    The significance of the US3-UL34 catalytic relationship to virus replication is cell type dependent. Replicate cultures of HEp-2 cells (A and B) or Vero cells (C and D) were infected an at MOI of 5 with HSV-1(F) (A to D), vRR1202 (A to D), vRR1202rep (A and C), vRR1204 #1 (A and C), vRR1204 #2 (A and C), vRR1205 #1 (B and D), or vRR1205 #2 (B and D). Residual virus was removed or inactivated with a low-pH wash, and at the indicated times total culture virus was titrated on 143/1099E cells. Virus yields are expressed as PFU per milliliter. Each datum point represents the mean of three experiments. Error bars indicate the sample standard deviations. For ease of comparison with recombinant viruses, the growth data presented in panels A and C for HSV-1(F) and vRR1202 are duplicated in panels B and D.
    Figure Legend Snippet: The significance of the US3-UL34 catalytic relationship to virus replication is cell type dependent. Replicate cultures of HEp-2 cells (A and B) or Vero cells (C and D) were infected an at MOI of 5 with HSV-1(F) (A to D), vRR1202 (A to D), vRR1202rep (A and C), vRR1204 #1 (A and C), vRR1204 #2 (A and C), vRR1205 #1 (B and D), or vRR1205 #2 (B and D). Residual virus was removed or inactivated with a low-pH wash, and at the indicated times total culture virus was titrated on 143/1099E cells. Virus yields are expressed as PFU per milliliter. Each datum point represents the mean of three experiments. Error bars indicate the sample standard deviations. For ease of comparison with recombinant viruses, the growth data presented in panels A and C for HSV-1(F) and vRR1202 are duplicated in panels B and D.

    Techniques Used: Infection, Recombinant

    24) Product Images from "Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice"

    Article Title: Hyperattenuated Recombinant Influenza A Virus Nonstructural-Protein-Encoding Vectors Induce Human Immunodeficiency Virus Type 1 Nef-Specific Systemic and Mucosal Immune Responses in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.19.8899-8908.2001

    Immunofluorescence analysis. Vero cells were infected with recombinant PR8/NS-Nef (A), PR8 wt (B), and Aichi/NS-Nef (C) viruses with an MOI of 0.05. Twenty-four hours after infection, cells were collected, fixed on cover slides with acetone, and incubated with anti HIV-1 IIIB Nef mouse MAb followed by incubation with fluorescein isothiocyanate conjugated to goat anti-mouse IgG, as described in Materials and Methods. An analogous immunofluorescence signal was observed when infected cells were incubated with anti-HIV-1 IR-CSF Nef mouse MAb (data not shown). (D) The intracellular localization of the Nef antigen is visible at a higher magnification.
    Figure Legend Snippet: Immunofluorescence analysis. Vero cells were infected with recombinant PR8/NS-Nef (A), PR8 wt (B), and Aichi/NS-Nef (C) viruses with an MOI of 0.05. Twenty-four hours after infection, cells were collected, fixed on cover slides with acetone, and incubated with anti HIV-1 IIIB Nef mouse MAb followed by incubation with fluorescein isothiocyanate conjugated to goat anti-mouse IgG, as described in Materials and Methods. An analogous immunofluorescence signal was observed when infected cells were incubated with anti-HIV-1 IR-CSF Nef mouse MAb (data not shown). (D) The intracellular localization of the Nef antigen is visible at a higher magnification.

    Techniques Used: Immunofluorescence, Infection, Recombinant, Incubation

    Sizes of the wt and recombinant NS1 proteins. (A) Confluent monolayers of Vero cells were infected with recombinant PR8/NS-Nef and PR8 wt viruses at an MOI of 5. Six hours postinfection, the cells were labeled with [ 35 S]methionine and [ 35 S]cysteine (Amersham). Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels containing 5 M urea. Lanes 1 and 2, mock-infected Vero cell extracts; lane 3, PR8 wt-infected Vero cell extract; lane 4, PR8/NS-Nef-infected Vero cell extract. (B and C)Viral proteins derived from the infected Vero cells 24 h postinfection were separated on 16% polyacrylamide gels, transferred to a nitrocellulose membrane, and detected by Western blot analysis with anti HIV-1 IR-CSF Nef mouse MAb (B) and rabbit anti-NS hyperimmune serum (C). Lanes 1 and 4, PR8/NS-Nef; lanes 2 and 3, PR8 wt.
    Figure Legend Snippet: Sizes of the wt and recombinant NS1 proteins. (A) Confluent monolayers of Vero cells were infected with recombinant PR8/NS-Nef and PR8 wt viruses at an MOI of 5. Six hours postinfection, the cells were labeled with [ 35 S]methionine and [ 35 S]cysteine (Amersham). Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels containing 5 M urea. Lanes 1 and 2, mock-infected Vero cell extracts; lane 3, PR8 wt-infected Vero cell extract; lane 4, PR8/NS-Nef-infected Vero cell extract. (B and C)Viral proteins derived from the infected Vero cells 24 h postinfection were separated on 16% polyacrylamide gels, transferred to a nitrocellulose membrane, and detected by Western blot analysis with anti HIV-1 IR-CSF Nef mouse MAb (B) and rabbit anti-NS hyperimmune serum (C). Lanes 1 and 4, PR8/NS-Nef; lanes 2 and 3, PR8 wt.

    Techniques Used: Recombinant, Infection, Labeling, Polyacrylamide Gel Electrophoresis, Derivative Assay, Western Blot

    Virus load in respiratory tract tissue of immunized mice. BALB/c mice were immunized i.n. under ether anesthesia with 10 6 PFU of influenza PR8/NS-Nef, Aichi/NS-Nef, or PR8/NS-124 virus or 10 3 PFU of the PR8 wt virus/mouse. Three mice of each group were sacrificed at days 2, 4, and 6 postinfection. The pooled lungs were homogenized, and the tissue extracts were assayed for infectious virus particles in plaque assays using Vero cells. The results are presented as PFU per milliliter of 10% (wt/vol) tissue extracts.
    Figure Legend Snippet: Virus load in respiratory tract tissue of immunized mice. BALB/c mice were immunized i.n. under ether anesthesia with 10 6 PFU of influenza PR8/NS-Nef, Aichi/NS-Nef, or PR8/NS-124 virus or 10 3 PFU of the PR8 wt virus/mouse. Three mice of each group were sacrificed at days 2, 4, and 6 postinfection. The pooled lungs were homogenized, and the tissue extracts were assayed for infectious virus particles in plaque assays using Vero cells. The results are presented as PFU per milliliter of 10% (wt/vol) tissue extracts.

    Techniques Used: Mouse Assay

    25) Product Images from "Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry"

    Article Title: Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00318-18

    Determination of the kinetics of PDCoV replication, propagation, and release in Vero-pAPN or control LLC-PK1 cells. (A) The amounts of extracellular and intracellular viral RNA in Vero-pAPN, Vero, and LLC-PK1 cells were assessed in triplicate by qRT-PCR, respectively. (B) Virus titers of PDCoV or TGEV released into the supernatant of inoculated Vero-pAPN, Vero, or LLC-PK1 cells were determined in triplicate on fresh LLC-PK1 cells. Samples of supernatants and cells were collected at intervals between 2 and 72 h postinoculation. Error bars indicate standard deviations. (C) Infection of fresh LLC-PK1 cells with progeny PDCoV collected from Vero-pAPN cells inoculated with PDCoV. IFA was performed at 36 hpi. The expression of PDCoV N protein was detected by staining with anti-PDCoV-N MAb and Alexa Fluor 488-conjugated goat anti-mouse IgG Ab (middle panel).
    Figure Legend Snippet: Determination of the kinetics of PDCoV replication, propagation, and release in Vero-pAPN or control LLC-PK1 cells. (A) The amounts of extracellular and intracellular viral RNA in Vero-pAPN, Vero, and LLC-PK1 cells were assessed in triplicate by qRT-PCR, respectively. (B) Virus titers of PDCoV or TGEV released into the supernatant of inoculated Vero-pAPN, Vero, or LLC-PK1 cells were determined in triplicate on fresh LLC-PK1 cells. Samples of supernatants and cells were collected at intervals between 2 and 72 h postinoculation. Error bars indicate standard deviations. (C) Infection of fresh LLC-PK1 cells with progeny PDCoV collected from Vero-pAPN cells inoculated with PDCoV. IFA was performed at 36 hpi. The expression of PDCoV N protein was detected by staining with anti-PDCoV-N MAb and Alexa Fluor 488-conjugated goat anti-mouse IgG Ab (middle panel).

    Techniques Used: Quantitative RT-PCR, Infection, Immunofluorescence, Expressing, Staining

    Vero or BHK-21 cells stably expressing pAPN confer susceptibility to PDCoV and TGEV infection. (A) Vero, Vero-pAPN, BHK-21, and BHK-pAPN cells were challenged with TGEV or PDCoV. At 36 h postchallenge, TGEV-challenged cells were costained with a rabbit anti-TGEV-N pAb and a mouse anti-Myc MAb, whereas PDCoV-challenged cells were costained with a mouse anti-PDCoV-N MAb and a rabbit anti-APN pAb. Alexa Fluor 488- or 594-conjugated anti-rabbit or anti-mouse IgG was costained for secondary antibody detection, followed by DAPI incubation. Magnification, ×200×. (B) Evidence of PDCoV infection in Vero-pAPN cells showing cytopathic effects (CPEs) with cell rounding and aggregation (indicated by red arrows) at 36 h postinfection.
    Figure Legend Snippet: Vero or BHK-21 cells stably expressing pAPN confer susceptibility to PDCoV and TGEV infection. (A) Vero, Vero-pAPN, BHK-21, and BHK-pAPN cells were challenged with TGEV or PDCoV. At 36 h postchallenge, TGEV-challenged cells were costained with a rabbit anti-TGEV-N pAb and a mouse anti-Myc MAb, whereas PDCoV-challenged cells were costained with a mouse anti-PDCoV-N MAb and a rabbit anti-APN pAb. Alexa Fluor 488- or 594-conjugated anti-rabbit or anti-mouse IgG was costained for secondary antibody detection, followed by DAPI incubation. Magnification, ×200×. (B) Evidence of PDCoV infection in Vero-pAPN cells showing cytopathic effects (CPEs) with cell rounding and aggregation (indicated by red arrows) at 36 h postinfection.

    Techniques Used: Stable Transfection, Expressing, Infection, Incubation

    Soluble TGEV-S1 and PDCoV-S1 bind to porcine permissive cells endogenously expressing pAPN. (A) Binding of soluble TGEV S1 protein to the cell surface as determined by flow cytometry analysis. Equal amounts (10 μg/ml) of TGEV-S1-hFc (filled histogram) or hFc (dashed line) were incubated with susceptible LLC-PK1 or ST cells or with nonsusceptible Vero or BHK-21 cells. Cell surface binding was detected by an FITC-conjugated anti-human IgG Fc. (B) Binding of soluble PDCoV S1 to LLC-PK1, ST, Vero, or BHK-21 cells. Equal amounts (10 μg/ml) of PDCoV-S1-hFc (filled histogram) or Fc only (dashed line) were incubated with four cell lines, followed by FITC-conjugated anti-human IgG Fc detection. (C) Detection of endogenous expression of pAPN on LLC-PK1 or ST cells or of pAPN exogenous expression on Vero-pAPN or BHK-pAPN stable cells by immunoblot analysis using an anti-APN Ab. Max, maximum.
    Figure Legend Snippet: Soluble TGEV-S1 and PDCoV-S1 bind to porcine permissive cells endogenously expressing pAPN. (A) Binding of soluble TGEV S1 protein to the cell surface as determined by flow cytometry analysis. Equal amounts (10 μg/ml) of TGEV-S1-hFc (filled histogram) or hFc (dashed line) were incubated with susceptible LLC-PK1 or ST cells or with nonsusceptible Vero or BHK-21 cells. Cell surface binding was detected by an FITC-conjugated anti-human IgG Fc. (B) Binding of soluble PDCoV S1 to LLC-PK1, ST, Vero, or BHK-21 cells. Equal amounts (10 μg/ml) of PDCoV-S1-hFc (filled histogram) or Fc only (dashed line) were incubated with four cell lines, followed by FITC-conjugated anti-human IgG Fc detection. (C) Detection of endogenous expression of pAPN on LLC-PK1 or ST cells or of pAPN exogenous expression on Vero-pAPN or BHK-pAPN stable cells by immunoblot analysis using an anti-APN Ab. Max, maximum.

    Techniques Used: Expressing, Binding Assay, Flow Cytometry, Incubation

    Infection of PDCoV in LLC-PK1 or Vero-pAPN cells was inhibited by soluble pAPN (at a concentration of 39 or 78 μg/ml) within 24 h. PDCoV RNA titers were measured by one-step quantitative RT-PCR targeting the M gene. *, P
    Figure Legend Snippet: Infection of PDCoV in LLC-PK1 or Vero-pAPN cells was inhibited by soluble pAPN (at a concentration of 39 or 78 μg/ml) within 24 h. PDCoV RNA titers were measured by one-step quantitative RT-PCR targeting the M gene. *, P

    Techniques Used: Infection, Concentration Assay, Quantitative RT-PCR

    26) Product Images from "Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets"

    Article Title: Deletion of a 197-Amino-Acid Region in the N-Terminal Domain of Spike Protein Attenuates Porcine Epidemic Diarrhea Virus in Piglets

    Journal: Journal of Virology

    doi: 10.1128/JVI.00227-17

    Characterization of recombinant viruses. (A) Cytopathic effects (CPE) of recombinant viruses occurred at 2 days posttransfection of the mixture of full-length genomic RNA and N gene transcripts into Vero cells. (B) Sanger sequencing chromatogram of a silent mutation (nucleotide position 16507; C to G) was identified exclusively in the recombinant viruses icPC22A (P1) and icPC22A-S1Δ197 (P1). (C) Multistep growth kinetics of two recombinant viruses and parental PC22A (P4). Vero cells were inoculated with each virus at an MOI of 0.01. Supernatants were sampled at different time points and titrated for TCID 50 ). (D) Plaque assay of two recombinant viruses, parental PC22A (P4), TC-PC177 P10C8, and mock inoculation. Images were taken at 3 days postinoculation.
    Figure Legend Snippet: Characterization of recombinant viruses. (A) Cytopathic effects (CPE) of recombinant viruses occurred at 2 days posttransfection of the mixture of full-length genomic RNA and N gene transcripts into Vero cells. (B) Sanger sequencing chromatogram of a silent mutation (nucleotide position 16507; C to G) was identified exclusively in the recombinant viruses icPC22A (P1) and icPC22A-S1Δ197 (P1). (C) Multistep growth kinetics of two recombinant viruses and parental PC22A (P4). Vero cells were inoculated with each virus at an MOI of 0.01. Supernatants were sampled at different time points and titrated for TCID 50 ). (D) Plaque assay of two recombinant viruses, parental PC22A (P4), TC-PC177 P10C8, and mock inoculation. Images were taken at 3 days postinoculation.

    Techniques Used: Recombinant, Sequencing, Mutagenesis, Plaque Assay

    27) Product Images from "Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells"

    Article Title: Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.17.11507-11512.2005

    Localization of SARS-CoV N-EGFP in Vero cells. (A, B, and C) Confocal microscopic images of a single cell at 24 h after transfection with pSARS-N-EGFP. (A) EGFP fluorescence; (B) TO-PRO-3 fluorescence; (C) merged image. (D) Image of a single cell transfected
    Figure Legend Snippet: Localization of SARS-CoV N-EGFP in Vero cells. (A, B, and C) Confocal microscopic images of a single cell at 24 h after transfection with pSARS-N-EGFP. (A) EGFP fluorescence; (B) TO-PRO-3 fluorescence; (C) merged image. (D) Image of a single cell transfected

    Techniques Used: Transfection, Fluorescence

    28) Product Images from "Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells"

    Article Title: Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.17.11507-11512.2005

    Localization of SARS-CoV N-EGFP in Vero cells. (A, B, and C) Confocal microscopic images of a single cell at 24 h after transfection with pSARS-N-EGFP. (A) EGFP fluorescence; (B) TO-PRO-3 fluorescence; (C) merged image. (D) Image of a single cell transfected
    Figure Legend Snippet: Localization of SARS-CoV N-EGFP in Vero cells. (A, B, and C) Confocal microscopic images of a single cell at 24 h after transfection with pSARS-N-EGFP. (A) EGFP fluorescence; (B) TO-PRO-3 fluorescence; (C) merged image. (D) Image of a single cell transfected

    Techniques Used: Transfection, Fluorescence

    29) Product Images from "Potent Inhibition of Jun?n Virus Infection by Interferon in Murine Cells"

    Article Title: Potent Inhibition of Jun?n Virus Infection by Interferon in Murine Cells

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002933

    Interferon sensitivity of JUNV in different cell lines. (A) mouse embryonic fibroblast cells (MEF), (B) Vero cells (ATCC) and (C) human lung carcinoma epithelial A549 cells (ATCC) were treated with IFNs at the indicated concentrations for 16 h. Vero cells and A549 cells were treated with human IFN-α2b (Schering), IFN-β (PBL) or IFN-γ (Sigma), while MEF cells were treated with mouse IFN-β (PBL), respectively. Cells were then infected with VSV, Candid#1 JUNV or Romero JUNV at an MOI of 0.1 PFU/cell. IFNs were supplemented after virus infection. During Romero and Candid#1 JUNV infection, supernatants were collected at 3 days p.i. and assayed for virus production by plaque assay. During VSV infection, supernatants were collected at 16 h.p.i.. Dotted lines indicate the limitation of plaque assay. Data represent the mean of three experiments ±SEM.
    Figure Legend Snippet: Interferon sensitivity of JUNV in different cell lines. (A) mouse embryonic fibroblast cells (MEF), (B) Vero cells (ATCC) and (C) human lung carcinoma epithelial A549 cells (ATCC) were treated with IFNs at the indicated concentrations for 16 h. Vero cells and A549 cells were treated with human IFN-α2b (Schering), IFN-β (PBL) or IFN-γ (Sigma), while MEF cells were treated with mouse IFN-β (PBL), respectively. Cells were then infected with VSV, Candid#1 JUNV or Romero JUNV at an MOI of 0.1 PFU/cell. IFNs were supplemented after virus infection. During Romero and Candid#1 JUNV infection, supernatants were collected at 3 days p.i. and assayed for virus production by plaque assay. During VSV infection, supernatants were collected at 16 h.p.i.. Dotted lines indicate the limitation of plaque assay. Data represent the mean of three experiments ±SEM.

    Techniques Used: Infection, Plaque Assay

    Immunoblotting analysis of viral protein expression. MEF cells (A), A549 cells (B) and Vero cells (C) were pretreated with mouse IFN-β (A) or human IFN-β (B and C) at different concentrations as indicated for 16 hr. Cells were then infected with Candid#1 virus at an MOI of 3. Cell lysates were prepared at 1 and 2 days p.i. from MEF cells and A549 cells, or at 2 days p.i. from Vero cells. The viral NP protein was detected with a monoclonal mouse anti-JUNV NP antibody (AG12, BEI) by Western Blotting assay. Equal loading of samples was confirmed by immunoblotting of the same membranes with an antibody to β-actin protein (Santa Cruz). Relative NP protein level in A549 cell samples is shown (B) after densitometry measurement and normalization to the actin protein level.
    Figure Legend Snippet: Immunoblotting analysis of viral protein expression. MEF cells (A), A549 cells (B) and Vero cells (C) were pretreated with mouse IFN-β (A) or human IFN-β (B and C) at different concentrations as indicated for 16 hr. Cells were then infected with Candid#1 virus at an MOI of 3. Cell lysates were prepared at 1 and 2 days p.i. from MEF cells and A549 cells, or at 2 days p.i. from Vero cells. The viral NP protein was detected with a monoclonal mouse anti-JUNV NP antibody (AG12, BEI) by Western Blotting assay. Equal loading of samples was confirmed by immunoblotting of the same membranes with an antibody to β-actin protein (Santa Cruz). Relative NP protein level in A549 cell samples is shown (B) after densitometry measurement and normalization to the actin protein level.

    Techniques Used: Expressing, Infection, Western Blot

    30) Product Images from "Effect of black tea extract on herpes simplex virus-1 infection of cultured cells"

    Article Title: Effect of black tea extract on herpes simplex virus-1 infection of cultured cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-139

    Gel electrophoresis of PCR products. A . PCR products extracted from HSV-1 infected A549 cells either treated with 1.4 mM BTE (columns 2–4) or untreated (columns 5–7). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp). Columns 2 and 5, 3 and 6, 4 and 7 contain DNA amplified with primers for the HSV-1 gD, GFP and pUL46 genes, respectively. B . Gel electrophoresis of HSV-1 GFP PCR products extracted from HSV-1 infected Vero cells either untreated (column 2) or treated with 0.14 μM, 1.4 μM, or 1.4 mM BTE (columns 3 – 5, respectively). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp).
    Figure Legend Snippet: Gel electrophoresis of PCR products. A . PCR products extracted from HSV-1 infected A549 cells either treated with 1.4 mM BTE (columns 2–4) or untreated (columns 5–7). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp). Columns 2 and 5, 3 and 6, 4 and 7 contain DNA amplified with primers for the HSV-1 gD, GFP and pUL46 genes, respectively. B . Gel electrophoresis of HSV-1 GFP PCR products extracted from HSV-1 infected Vero cells either untreated (column 2) or treated with 0.14 μM, 1.4 μM, or 1.4 mM BTE (columns 3 – 5, respectively). Column 1 contains the DNA ladder, with visible bands identified to the left in base pairs (bp).

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Infection, Amplification

    31) Product Images from "The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis"

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0589-8

    Bacterial adherence and invasion assays. Strains CH3 and RA1062 were tested on Vero cells. A Adherence assay; B Invasion assay. The data represent the number of bacteria bound to or invaded into Vero cells in each well of a 24-well plate. The error bars represent mean ± standard deviations from three independent experiments (** p
    Figure Legend Snippet: Bacterial adherence and invasion assays. Strains CH3 and RA1062 were tested on Vero cells. A Adherence assay; B Invasion assay. The data represent the number of bacteria bound to or invaded into Vero cells in each well of a 24-well plate. The error bars represent mean ± standard deviations from three independent experiments (** p

    Techniques Used: Invasion Assay

    32) Product Images from "A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: Biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate"

    Article Title: A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: Biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate

    Journal: Virus Research

    doi: 10.1016/j.virusres.2018.10.012

    Electron micrograph of PEDV virions in cell culture media of infected Vero cells or on the cell surface of infected Vero cells. (A) Images of PEDV virions from cell culture media of Vero cells infected with the PEDV CH/HNPJ/2017 strain, as shown by the arrow. Scale bar = 100 nm; (B) Images of a PEDV-infected Vero cell. PEDV particles (arrow heads) on the cell surface of an infected Vero cell, as shown by the arrow. Scale bar = 200 nm.
    Figure Legend Snippet: Electron micrograph of PEDV virions in cell culture media of infected Vero cells or on the cell surface of infected Vero cells. (A) Images of PEDV virions from cell culture media of Vero cells infected with the PEDV CH/HNPJ/2017 strain, as shown by the arrow. Scale bar = 100 nm; (B) Images of a PEDV-infected Vero cell. PEDV particles (arrow heads) on the cell surface of an infected Vero cell, as shown by the arrow. Scale bar = 200 nm.

    Techniques Used: Cell Culture, Infection

    Viral titers of the PEDV CH/HNPJ/2017 strain propagated in Vero cells after serial passage. All the results of a representative experiment performed with triplicate samples are shown.
    Figure Legend Snippet: Viral titers of the PEDV CH/HNPJ/2017 strain propagated in Vero cells after serial passage. All the results of a representative experiment performed with triplicate samples are shown.

    Techniques Used:

    Isolation and detection of the PEDV CH/HNPJ/2017 strain in Vero cells. The upper and lower panels show light and immunofluorescence images, respectively, of Vero cells infected with the PEDV CH/HNPJ/2017 strain. (A) Cytopathic effects (CPE) caused by the 1st passage of the PEDV CH/HNPJ/2017 strain at 24 h after inoculation (400×). Vero cells were seeded into T-25 flasks and infected at a multiplicity of infection (MOI) of 0.1; (B) CPE at 72 h after inoculation; (C) Control (uninfected) Vero cells; (D) Immunofluorescence detection results for the 50th passage of the PEDV CH/HNPJ/2017 strain in infected Vero cells at 6 h after inoculation (400×). PEDV antigens and nuclei were detected with mouse anti-PEDV N protein monoclonal antibodies (McAbs) and 4′, 6-diamidino-2-phenylindole (DAPI), respectively; (E) Immunofluorescence detection results at 12 h after inoculation (400×); (F) Immunofluorescence detection results at 24 h after inoculation (400×).
    Figure Legend Snippet: Isolation and detection of the PEDV CH/HNPJ/2017 strain in Vero cells. The upper and lower panels show light and immunofluorescence images, respectively, of Vero cells infected with the PEDV CH/HNPJ/2017 strain. (A) Cytopathic effects (CPE) caused by the 1st passage of the PEDV CH/HNPJ/2017 strain at 24 h after inoculation (400×). Vero cells were seeded into T-25 flasks and infected at a multiplicity of infection (MOI) of 0.1; (B) CPE at 72 h after inoculation; (C) Control (uninfected) Vero cells; (D) Immunofluorescence detection results for the 50th passage of the PEDV CH/HNPJ/2017 strain in infected Vero cells at 6 h after inoculation (400×). PEDV antigens and nuclei were detected with mouse anti-PEDV N protein monoclonal antibodies (McAbs) and 4′, 6-diamidino-2-phenylindole (DAPI), respectively; (E) Immunofluorescence detection results at 12 h after inoculation (400×); (F) Immunofluorescence detection results at 24 h after inoculation (400×).

    Techniques Used: Isolation, Immunofluorescence, Infection

    33) Product Images from "Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission"

    Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006061

    Alphavirus infection induces actin-positive intercellular extensions. (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see methods ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P
    Figure Legend Snippet: Alphavirus infection induces actin-positive intercellular extensions. (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see methods ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P

    Techniques Used: Infection, Transfection, Incubation, Staining, Confocal Microscopy, Standard Deviation

    The alphavirus structural proteins induce the formation of intercellular extensions that preferentially target non-expressing cells. (A) Vero cells were transfected with plasmids encoding the SFV structural proteins with or without capsid and with or without the E2 Y399R mutation. At 24 h post-transfection the cells were fixed, permeabilized, immunostained for the SFV E2 protein and stained with phalloidin-Alexa568 to detect F-actin. Images were acquired by confocal microscopy and are representative of three independent experiments. Images from one optical section are shown. Bar = 20 μm. (B) Using the conditions described in (A), the number of intercellular extensions per E2-expressing cell was quantitated based on positive staining for actin and contact with a neighboring cell. The graph represents the mean and standard deviation of three independent experiments. *P
    Figure Legend Snippet: The alphavirus structural proteins induce the formation of intercellular extensions that preferentially target non-expressing cells. (A) Vero cells were transfected with plasmids encoding the SFV structural proteins with or without capsid and with or without the E2 Y399R mutation. At 24 h post-transfection the cells were fixed, permeabilized, immunostained for the SFV E2 protein and stained with phalloidin-Alexa568 to detect F-actin. Images were acquired by confocal microscopy and are representative of three independent experiments. Images from one optical section are shown. Bar = 20 μm. (B) Using the conditions described in (A), the number of intercellular extensions per E2-expressing cell was quantitated based on positive staining for actin and contact with a neighboring cell. The graph represents the mean and standard deviation of three independent experiments. *P

    Techniques Used: Expressing, Transfection, Mutagenesis, Staining, Confocal Microscopy, Standard Deviation

    SINV cell-cell transmission is independent of NRAMP in target or producer cells. (A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.
    Figure Legend Snippet: SINV cell-cell transmission is independent of NRAMP in target or producer cells. (A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.

    Techniques Used: Transmission Assay, Transfection, Incubation, Stable Transfection, Expressing, Marker, Cell Culture, Infection, Staining, Standard Deviation

    Intercellular extensions are induced by various alphaviruses in Vero and primary human cells. (A) Vero cells were mock-infected (Uninf.), transfected with SINV Y400K RNA, or infected with WT SINV, SFV or CHIKV (MOI 20, 10, 10, respectively). Cells were then incubated at 37°C for 11 h, fixed, permeabilized, and stained with antibodies to detect viral envelope proteins (virus GP) and α-tubulin, and with phalloidin to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments. Bar = 20 μm. (B) Quantitation of the number of extensions in Vero cells from experiments as described in (A). Intercellular extensions emanating from infected cells were identified based on positive staining for GP, actin and tubulin and contact with a neighboring cell (see methods ). (C) HUVECs were infected and stained as in (A) and imaged and quantitated as in (B). Graphs in B and C show the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. * P
    Figure Legend Snippet: Intercellular extensions are induced by various alphaviruses in Vero and primary human cells. (A) Vero cells were mock-infected (Uninf.), transfected with SINV Y400K RNA, or infected with WT SINV, SFV or CHIKV (MOI 20, 10, 10, respectively). Cells were then incubated at 37°C for 11 h, fixed, permeabilized, and stained with antibodies to detect viral envelope proteins (virus GP) and α-tubulin, and with phalloidin to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments. Bar = 20 μm. (B) Quantitation of the number of extensions in Vero cells from experiments as described in (A). Intercellular extensions emanating from infected cells were identified based on positive staining for GP, actin and tubulin and contact with a neighboring cell (see methods ). (C) HUVECs were infected and stained as in (A) and imaged and quantitated as in (B). Graphs in B and C show the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. * P

    Techniques Used: Infection, Transfection, Incubation, Staining, Confocal Microscopy, Quantitation Assay, Standard Deviation

    Formation of intercellular extensions does not require virus budding. Vero cells were mock-transfected (Uninf.) or transfected with SFV WT or G91D mutant RNA, incubated at the permissive temperature (28°C) overnight (A) or at the non-permissive temperature (37°C) for 8 h (B), and fixed. Note that different times of incubation at these two temperatures were used to allow virus replication and extension formation. Cells were permeabilized and immunostained to detect the virus glycoproteins (GP) or α-tubulin, and stained with phalloidin-Alexa488 to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (C,D) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for GP, actin and tubulin and their contact with a neighboring cell. (E,F) Vero cells were transfected with WT or G91D SFV RNA and incubated at 37°C for 2h (producer cells). At 4 h post-transfection Vero target cells stably expressing the PM-GFP marker were plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated overnight at 37 or 28°C. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in C-F represent the mean and standard deviation of three independent experiments. * P
    Figure Legend Snippet: Formation of intercellular extensions does not require virus budding. Vero cells were mock-transfected (Uninf.) or transfected with SFV WT or G91D mutant RNA, incubated at the permissive temperature (28°C) overnight (A) or at the non-permissive temperature (37°C) for 8 h (B), and fixed. Note that different times of incubation at these two temperatures were used to allow virus replication and extension formation. Cells were permeabilized and immunostained to detect the virus glycoproteins (GP) or α-tubulin, and stained with phalloidin-Alexa488 to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (C,D) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for GP, actin and tubulin and their contact with a neighboring cell. (E,F) Vero cells were transfected with WT or G91D SFV RNA and incubated at 37°C for 2h (producer cells). At 4 h post-transfection Vero target cells stably expressing the PM-GFP marker were plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated overnight at 37 or 28°C. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in C-F represent the mean and standard deviation of three independent experiments. * P

    Techniques Used: Transfection, Mutagenesis, Incubation, Staining, Confocal Microscopy, Infection, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

    Intercellular extensions do not transfer cytosolic or PM markers. (A) Vero cells were transfected with SINV WT-mCherry RNA (t = 0) and incubated for 6.5 h at 37°C. Cells were labeled for 30 min with CellTracker Green and then uninfected Vero cells were added and the co-cultures were incubated for 2 h at 37°C. Cells were then fixed and imaged by epifluorescence microscopy. Images are representative of three independent experiments, evaluating 10 cell pairs/experiment. Bar = 20 μm. (B) Vero cells expressing the PM-GFP marker were transfected with SINV WT-mCherry RNA and incubated at 37°C for 7 h. Uninfected Vero cells were then added and the co-cultures were incubated for 2 h at 37°C. Cells were fixed, stained with antibodies to detect α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown, and are representative of three independent experiments, 10 cell pairs/experiment. Bar = 20 μm.
    Figure Legend Snippet: Intercellular extensions do not transfer cytosolic or PM markers. (A) Vero cells were transfected with SINV WT-mCherry RNA (t = 0) and incubated for 6.5 h at 37°C. Cells were labeled for 30 min with CellTracker Green and then uninfected Vero cells were added and the co-cultures were incubated for 2 h at 37°C. Cells were then fixed and imaged by epifluorescence microscopy. Images are representative of three independent experiments, evaluating 10 cell pairs/experiment. Bar = 20 μm. (B) Vero cells expressing the PM-GFP marker were transfected with SINV WT-mCherry RNA and incubated at 37°C for 7 h. Uninfected Vero cells were then added and the co-cultures were incubated for 2 h at 37°C. Cells were fixed, stained with antibodies to detect α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown, and are representative of three independent experiments, 10 cell pairs/experiment. Bar = 20 μm.

    Techniques Used: Transfection, Incubation, Labeling, Epifluorescence Microscopy, Expressing, Marker, Staining, Confocal Microscopy

    Intercellular extensions are generated by stable cell-cell contacts and migration of infected cells, and contain budding virus particles. (A) Vero cells stably expressing GFP-actin were infected with WT-mCherry SINV and incubated for 7 h at 37°C. The cells were then imaged using the TIRF microscope in the wide-field mode and both the 561-nm and 488-nm lasers. Images were acquired every 10 s for 60 min (see S1 Movie ). Arrows indicate stable contacts established between an infected cell and a neighboring cell, and marker lines are used to follow cell position during migration. Bar = 20 μm. (B-E) Vero cells were infected with WT SINV, incubated at 37°C for 9 h, fixed, processed for SEM, and imaged using a Zeiss Supra 40 field emission SEM. (B) A representative image of infected cells. Bar = 10 μm. (C) SEM image of the region indicated by the dashed white box in panel B. Bar = 10 μm. (D and E) SEM images of the regions indicated by the dashed white boxes in panel C. Arrowheads indicate virus-sized structures. Bar = 1 μm.
    Figure Legend Snippet: Intercellular extensions are generated by stable cell-cell contacts and migration of infected cells, and contain budding virus particles. (A) Vero cells stably expressing GFP-actin were infected with WT-mCherry SINV and incubated for 7 h at 37°C. The cells were then imaged using the TIRF microscope in the wide-field mode and both the 561-nm and 488-nm lasers. Images were acquired every 10 s for 60 min (see S1 Movie ). Arrows indicate stable contacts established between an infected cell and a neighboring cell, and marker lines are used to follow cell position during migration. Bar = 20 μm. (B-E) Vero cells were infected with WT SINV, incubated at 37°C for 9 h, fixed, processed for SEM, and imaged using a Zeiss Supra 40 field emission SEM. (B) A representative image of infected cells. Bar = 10 μm. (C) SEM image of the region indicated by the dashed white box in panel B. Bar = 10 μm. (D and E) SEM images of the regions indicated by the dashed white boxes in panel C. Arrowheads indicate virus-sized structures. Bar = 1 μm.

    Techniques Used: Generated, Stable Transfection, Migration, Infection, Expressing, Incubation, Microscopy, Marker

    Formation of intercellular extensions and virus cell-cell transmission are independent of NRAMP2. (A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH 4 Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P
    Figure Legend Snippet: Formation of intercellular extensions and virus cell-cell transmission are independent of NRAMP2. (A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH 4 Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P

    Techniques Used: Transmission Assay, Infection, Cell Culture, Staining, Standard Deviation

    Production of fusion-active virus is required for cell-to-cell transmission. (A) Vero cells were transfected with SFV WT or D188K mutant RNA, incubated at 37°C and fixed at 8 hours post transfection. Cells were permeabilized and immunostained to detect the virus E2 glycoprotein or α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2 and tubulin and their contact with a neighboring cell. (C) Vero cells were transfected with WT or D188K SFV RNA and incubated at 37°C for 2 h (producer cells). At 4 hours post transfection Vero target cells stably expressing the PM-GFP marker were plated onto the producer cells at an approximate ratio of 1:1 and the co-cultures incubated at 37°C overnight. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in B and C represent the mean and standard deviation of three independent experiments. *** P
    Figure Legend Snippet: Production of fusion-active virus is required for cell-to-cell transmission. (A) Vero cells were transfected with SFV WT or D188K mutant RNA, incubated at 37°C and fixed at 8 hours post transfection. Cells were permeabilized and immunostained to detect the virus E2 glycoprotein or α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2 and tubulin and their contact with a neighboring cell. (C) Vero cells were transfected with WT or D188K SFV RNA and incubated at 37°C for 2 h (producer cells). At 4 hours post transfection Vero target cells stably expressing the PM-GFP marker were plated onto the producer cells at an approximate ratio of 1:1 and the co-cultures incubated at 37°C overnight. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in B and C represent the mean and standard deviation of three independent experiments. *** P

    Techniques Used: Transmission Assay, Transfection, Mutagenesis, Incubation, Confocal Microscopy, Infection, Staining, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

    34) Product Images from "Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2"

    Article Title: Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.03818-14

    Specific toxicity of R33ExoA for gD2-expressing cells. Dilutions of immunotoxins were added to Vero cells (A) or z4/6 cells (B), and their cytotoxicity was measured using an MTS assay. While R33ExoA demonstrates cytotoxic activity against gD2-expressing cell lines (IC 50 = 0.5 nM) (95% CI, 0.1810 to 1.403), P10ExoA does not. Neither immunotoxin demonstrates activity against Vero cells, which do not express gD2. Dilutions of each protein were added to wells in triplicate, and error bars represent standard deviations.
    Figure Legend Snippet: Specific toxicity of R33ExoA for gD2-expressing cells. Dilutions of immunotoxins were added to Vero cells (A) or z4/6 cells (B), and their cytotoxicity was measured using an MTS assay. While R33ExoA demonstrates cytotoxic activity against gD2-expressing cell lines (IC 50 = 0.5 nM) (95% CI, 0.1810 to 1.403), P10ExoA does not. Neither immunotoxin demonstrates activity against Vero cells, which do not express gD2. Dilutions of each protein were added to wells in triplicate, and error bars represent standard deviations.

    Techniques Used: Expressing, MTS Assay, Activity Assay

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    Plasmid Preparation:

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    Construct:

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    Derivative Assay:

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    Passaging:

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    other:

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    Mutagenesis:

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    ATCC vero e6 cells
    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of <t>Vero</t> <t>E6</t> cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p
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    ATCC vero cells
    Mutant <t>UL42</t> resistant to extraction by detergent. <t>Vero</t> cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed
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    Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p

    Journal: Viruses

    Article Title: Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus

    doi: 10.3390/v11010049

    Figure Lengend Snippet: Pre-treatment of cells with emodin and berberine. Graphic of relative foci number after treatment of Vero E6 cells with berberine or emodin. Cells treated with drug diluent were used as negative control. Results are the means (±SD) from three independent experiments and are expressed as relative values compared to untreated cells. *** p

    Article Snippet: Berberine reduced virus infectivity by almost 80% in Vero E6 cells.

    Techniques: Negative Control

    Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.

    Journal: Viruses

    Article Title: Natural Products Isolated from Oriental Medicinal Herbs Inactivate Zika Virus

    doi: 10.3390/v11010049

    Figure Lengend Snippet: Viability of emodin- and berberine-treated Vero E6 cells. Cells were incubated with different concentrations of emodin ( A ) or berberine ( B ) and cell viability was evaluated after 24 h, 48 h, and 72 h.

    Article Snippet: Berberine reduced virus infectivity by almost 80% in Vero E6 cells.

    Techniques: Incubation

    Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture. (A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture. (A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Cell Culture, Infection, Plaque Assay

    Generation of rMP12-PTNSs and rMP12-SFSNSs. (A) Schematics of MP-12 S-segments encoding mutation or foreign gene in place of MP-12 NSs. The rMP12-C13type (C13type) lacks 69% of the NSs ORF as described previously [44] . The rMP12-PTNSs, and rMP12-SFSNSs encode NSs of Punta Toro virus Adames strain and Sandfly fever Sicilian virus, respectively. The expected phenotype corresponding to each S-segment is also presented. (B) Plaque phenotypes of MP-12, rMP12-PTNSs and rMP12-SFSNSs at 4 dpi. Plaque assay was performed with VeroE6 cells overlaid with 0.6% noble agar and stained with Neutral red.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Generation of rMP12-PTNSs and rMP12-SFSNSs. (A) Schematics of MP-12 S-segments encoding mutation or foreign gene in place of MP-12 NSs. The rMP12-C13type (C13type) lacks 69% of the NSs ORF as described previously [44] . The rMP12-PTNSs, and rMP12-SFSNSs encode NSs of Punta Toro virus Adames strain and Sandfly fever Sicilian virus, respectively. The expected phenotype corresponding to each S-segment is also presented. (B) Plaque phenotypes of MP-12, rMP12-PTNSs and rMP12-SFSNSs at 4 dpi. Plaque assay was performed with VeroE6 cells overlaid with 0.6% noble agar and stained with Neutral red.

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Mutagenesis, Plaque Assay, Staining

    Degradation of PKR in VeroE6 cells infected with rMP12-PTNSs or rMP12-SFSNSs. (A) VeroE6 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and cells were collected at 16 hpi. Western blot was performed with anti-PKR, anti-RVFV and anti-β-actin antibodies. (B) VeroE6 cells were mock-infected or infected with rMP12-NSs-Flag [32] , rMP12-C13type, rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag at a m.o.i of 3, and Western blot was performed as described above. Anti-Flag antibody was used for the detection of NSs-Flag. Representative data from three independent experiments are shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

    doi: 10.1371/journal.pntd.0002181

    Figure Lengend Snippet: Degradation of PKR in VeroE6 cells infected with rMP12-PTNSs or rMP12-SFSNSs. (A) VeroE6 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3, and cells were collected at 16 hpi. Western blot was performed with anti-PKR, anti-RVFV and anti-β-actin antibodies. (B) VeroE6 cells were mock-infected or infected with rMP12-NSs-Flag [32] , rMP12-C13type, rMP12-PTNSs-Flag or rMP12-SFSNSs-Flag at a m.o.i of 3, and Western blot was performed as described above. Anti-Flag antibody was used for the detection of NSs-Flag. Representative data from three independent experiments are shown.

    Article Snippet: Media, cells and viruses VeroE6 cells (ATCC CRL-1586), 293 cells (ATCC CRL-1573), MRC-5 cells (ATCC CCL-171) and MEF cells were maintained in Dulbecco's modified minimum essential medium (DMEM) containing 10% fetal calf serum (FCS).

    Techniques: Infection, Western Blot

    Mutant UL42 resistant to extraction by detergent. Vero cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed

    Journal:

    Article Title: Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity ▿

    doi: 10.1128/JVI.00193-09

    Figure Lengend Snippet: Mutant UL42 resistant to extraction by detergent. Vero cells infected with the virus at an MOI of 3 were harvested at 6.5 h postinfection. Cell lysates were subjected to Triton X-100 extraction as described in Materials and Methods and separately probed

    Article Snippet: The HSV type 1 strains 17 syn+ , the parental strain of CgalΔ42, and two independently constructed control viruses, C-700-A and -B , which are derived from CgalΔ42 and retain the lac Z gene but express wild-type UL42, were propagated in Vero cells.

    Techniques: Mutagenesis, Infection

    Viral yields and DNA synthesized by UL42 mutants. (A) Single-cycle growth curve assays were performed as described in Materials and Methods, and the yields of progeny viruses were determined by plaque assay on Vero cells. Since recombinants A and B of

    Journal:

    Article Title: Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity ▿

    doi: 10.1128/JVI.00193-09

    Figure Lengend Snippet: Viral yields and DNA synthesized by UL42 mutants. (A) Single-cycle growth curve assays were performed as described in Materials and Methods, and the yields of progeny viruses were determined by plaque assay on Vero cells. Since recombinants A and B of

    Article Snippet: The HSV type 1 strains 17 syn+ , the parental strain of CgalΔ42, and two independently constructed control viruses, C-700-A and -B , which are derived from CgalΔ42 and retain the lac Z gene but express wild-type UL42, were propagated in Vero cells.

    Techniques: Synthesized, Plaque Assay

    Comparative growth curve analyses of three AF and three AF ZIKV strains in ESCd, JAr, and Vero cells. Cells were infected with the six ZIKV strains at a MOI of 0.1. Cell supernatants were harvested at the indicated time points for titration by plaque assay in Vero cells. Growth curve analyses were performed in triplicate and plotted as SEM. Growth curves representing the AF strains are highlighted in shades of blue and those representing the AS strains are shown in shades of red. (A) In ESCd, significantly higher virus concentrations were observed by 48 h PI for the AF Uganda and the AF Senegal strains when compared to all other strains ( p

    Journal: PLoS ONE

    Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

    doi: 10.1371/journal.pone.0200086

    Figure Lengend Snippet: Comparative growth curve analyses of three AF and three AF ZIKV strains in ESCd, JAr, and Vero cells. Cells were infected with the six ZIKV strains at a MOI of 0.1. Cell supernatants were harvested at the indicated time points for titration by plaque assay in Vero cells. Growth curve analyses were performed in triplicate and plotted as SEM. Growth curves representing the AF strains are highlighted in shades of blue and those representing the AS strains are shown in shades of red. (A) In ESCd, significantly higher virus concentrations were observed by 48 h PI for the AF Uganda and the AF Senegal strains when compared to all other strains ( p

    Article Snippet: The third host cell type chosen for ZIKV infections was Vero cells, which are commonly used to propagate and titer viruses due to the absence of the type I interferon gene locus [ ].

    Techniques: Infection, Titration, Plaque Assay

    Cytopathic effects (CPE) caused by three AF and three AS ZIKV strains in Vero cells. Vero cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Similar CPE are evident after infection with all AF and AS ZIKV strains, although the AF Nigeria and AS Panama strains elicited weaker CPE when compared to the other strains. Scale bar is 1 mm.

    Journal: PLoS ONE

    Article Title: African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast

    doi: 10.1371/journal.pone.0200086

    Figure Lengend Snippet: Cytopathic effects (CPE) caused by three AF and three AS ZIKV strains in Vero cells. Vero cells were infected with each ZIKV strain at 1 MOI and fixed either at 48 h or 60 h PI. Respective mock infected controls are shown below. Similar CPE are evident after infection with all AF and AS ZIKV strains, although the AF Nigeria and AS Panama strains elicited weaker CPE when compared to the other strains. Scale bar is 1 mm.

    Article Snippet: The third host cell type chosen for ZIKV infections was Vero cells, which are commonly used to propagate and titer viruses due to the absence of the type I interferon gene locus [ ].

    Techniques: Infection