vero cells  (ATCC)


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  • 99
    Name:
    Vero
    Description:

    Catalog Number:
    ccl-81
    Price:
    None
    Applications:
    This cell line can be used for the detection of verotoxin.This cell line can be used for efficacy testing.This cell line can be used to study malaria biology.This cell line can be used for media testing.This cell line can be used for mycoplasma testing.This cell line is a suitable transfection host.This cell line can be used for the detection of virus in ground beef.
    Host:
    Cercopithecus aethiops
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    Structured Review

    ATCC vero cells
    The presence of DCs and RCs assessed in the TL and BL of the renografin purified E . <t>chaffeensis</t> fractions. ( A ) Western blot analysis performed with ClpB polyclonal antibody which revealed higher levels of the protein expression in the BL derived bacterial fraction proteins; ( B ) cell-free E . chaffeensis recovered from TL and BL assessed for reinfection of naïve <t>Vero</t> cells; infection was detected only with the fraction derived from the TL ( B1 ), but not in the BL ( B2 ) ( C ). Infectivity of fractionated Ehrlichia organisms from the TL and BL was further confirmed by measuring the numbers of infected cells following incubation for three days following inoculation into naïve HL60 cultures. Infectivity with TL-derived Ehrlichia was significantly more than BL derived bacteria.

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    Images

    1) Product Images from "Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium"

    Article Title: Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27574-z

    The presence of DCs and RCs assessed in the TL and BL of the renografin purified E . chaffeensis fractions. ( A ) Western blot analysis performed with ClpB polyclonal antibody which revealed higher levels of the protein expression in the BL derived bacterial fraction proteins; ( B ) cell-free E . chaffeensis recovered from TL and BL assessed for reinfection of naïve Vero cells; infection was detected only with the fraction derived from the TL ( B1 ), but not in the BL ( B2 ) ( C ). Infectivity of fractionated Ehrlichia organisms from the TL and BL was further confirmed by measuring the numbers of infected cells following incubation for three days following inoculation into naïve HL60 cultures. Infectivity with TL-derived Ehrlichia was significantly more than BL derived bacteria.
    Figure Legend Snippet: The presence of DCs and RCs assessed in the TL and BL of the renografin purified E . chaffeensis fractions. ( A ) Western blot analysis performed with ClpB polyclonal antibody which revealed higher levels of the protein expression in the BL derived bacterial fraction proteins; ( B ) cell-free E . chaffeensis recovered from TL and BL assessed for reinfection of naïve Vero cells; infection was detected only with the fraction derived from the TL ( B1 ), but not in the BL ( B2 ) ( C ). Infectivity of fractionated Ehrlichia organisms from the TL and BL was further confirmed by measuring the numbers of infected cells following incubation for three days following inoculation into naïve HL60 cultures. Infectivity with TL-derived Ehrlichia was significantly more than BL derived bacteria.

    Techniques Used: Purification, Western Blot, Expressing, Derivative Assay, Infection, Incubation

    Incorporation of 35 S Cys-Met into E . chaffeensis organisms recovered from renografin fractionated bacteria in the axenic medium. ( A ) E . chaffeensis organisms isolated from Vero cells used in the axenic media assessment; autoradiography image assessing the incorporation of 35 S Cys-Met in E . chaffeensis recovered from renografin purified top (TL) and bottom (BL) layered fractions incubation in the axenic media with or without chloramphenicol (CHL) and with G6P as an energy source and incubated for 24 h (G6P) ( B ). As in panel A, but quantitation of radiolabel incorporation by scintillation count analysis data ( C ). As in panel A except that the organisms were recovered from infected HL60 cells; this experiment also included a fraction of the purified organisms incubated in the axenic media with ATP as the energy source (ATP) ( D ). As in panel C, but the scintillation counting data were presented.
    Figure Legend Snippet: Incorporation of 35 S Cys-Met into E . chaffeensis organisms recovered from renografin fractionated bacteria in the axenic medium. ( A ) E . chaffeensis organisms isolated from Vero cells used in the axenic media assessment; autoradiography image assessing the incorporation of 35 S Cys-Met in E . chaffeensis recovered from renografin purified top (TL) and bottom (BL) layered fractions incubation in the axenic media with or without chloramphenicol (CHL) and with G6P as an energy source and incubated for 24 h (G6P) ( B ). As in panel A, but quantitation of radiolabel incorporation by scintillation count analysis data ( C ). As in panel A except that the organisms were recovered from infected HL60 cells; this experiment also included a fraction of the purified organisms incubated in the axenic media with ATP as the energy source (ATP) ( D ). As in panel C, but the scintillation counting data were presented.

    Techniques Used: Isolation, Autoradiography, Purification, Incubation, Quantitation Assay, Infection

    2) 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

    3) 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

    4) Product Images from "Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis"

    Article Title: Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis

    Journal: Journal of Traditional and Complementary Medicine

    doi: 10.1016/j.jtcme.2014.11.017

    Microscopic images of Vero cells. (A) Untreated Vero cells. (B) Cells treated with Cassia spectabilis leaf extract at the concentration 50 μg/mL. (C) Cells treated with C. spectabilis leaf extract at the concentration 200 μg/mL.
    Figure Legend Snippet: Microscopic images of Vero cells. (A) Untreated Vero cells. (B) Cells treated with Cassia spectabilis leaf extract at the concentration 50 μg/mL. (C) Cells treated with C. spectabilis leaf extract at the concentration 200 μg/mL.

    Techniques Used: Concentration Assay

    5) Product Images from "TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus"

    Article Title: TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.00093-18

    LASV pseudovirion entry is TIM-1 dependent in Vero cells. (A) Cell surface detection of endogenous DG expression on Vero or HEK 293T cells. Live cells were stained with polyclonal DG antisera (top) or the mannosylation-dependent anti-αDG mAb IIH6 (bottom). Filled histograms represent cells stained with isotype control antisera or mAb; unfilled histograms represent cells stained with anti-DG polyclonal antisera or IIH6. (B) Cell surface expression of endogenous TIM-1, Axl, Mer, Tyro3, or TIM-4 on WT Vero cells. Also shown are TIM-1 and Axl staining of Axl-KO, TIM-KO 1 , TIM-KO 2 , or Axl/TIM-KO (DKO) Vero cells. Filled histograms represent cells stained with the isotype control; unfilled histograms represent cells stained with 2 μg/ml anti-TIM-1, anti-Axl, anti-Mer, anti-Tyro3, or anti-TIM-4 polyclonal antibody. (C and D) LASV-VSV (C) or adenovirus-eGFP (D) transduction of WT, TIM-1-KO, Axl-KO, or DKO Vero cells. Serial dilutions of virus were applied to each cell type in duplicate. Transduction is represented as the percentage of cells that are eGFP positive, as assessed by flow cytometry. Data points are representative of results from three independent experiments with the indicated standard errors of the means (SEM). (E) LASV-LCMV transduction (MOI = ∼0.2) of WT, TIM-1-KO, Axl-KO, or DKO Vero cell lines. Data points are representative of results from three independent experiments with the indicated SEM. (F and G) Ability of adenovirus vector (Ad)-delivered TIM-1 to enhance transduction of LASV-VSV in WT, TIM-1-KO, or TIM-1- and Axl-KO (DKO) Vero cells. Forty-eight hours following Ad transduction, cells were divided and assessed independently for TIM-1 expression (F) or LASV-VSV transduction (G). Bars in each panel represent results from one of three independent experiments. (F) Cell surface expression of TIM-1 48 h following transduction of WT or KO Vero cells with Ad-TIM-1 (MOI of 2, 4, or 8). Cells were surface stained with 2 μg/ml anti-TIM-1 polyclonal antibodies. TIM-1 expression is represented as geometric mean fluorescence index (MFI) values. (G) LASV-VSV transductions of empty (E) or TIM-1-expressing (T) adenovirus vector-transduced WT or KO Vero cells. Equivalent volumes of LASV-VSV were applied to cells in duplicate. Transduction is represented as the percentage of eGFP-positive cells, as assessed by flow cytometry.
    Figure Legend Snippet: LASV pseudovirion entry is TIM-1 dependent in Vero cells. (A) Cell surface detection of endogenous DG expression on Vero or HEK 293T cells. Live cells were stained with polyclonal DG antisera (top) or the mannosylation-dependent anti-αDG mAb IIH6 (bottom). Filled histograms represent cells stained with isotype control antisera or mAb; unfilled histograms represent cells stained with anti-DG polyclonal antisera or IIH6. (B) Cell surface expression of endogenous TIM-1, Axl, Mer, Tyro3, or TIM-4 on WT Vero cells. Also shown are TIM-1 and Axl staining of Axl-KO, TIM-KO 1 , TIM-KO 2 , or Axl/TIM-KO (DKO) Vero cells. Filled histograms represent cells stained with the isotype control; unfilled histograms represent cells stained with 2 μg/ml anti-TIM-1, anti-Axl, anti-Mer, anti-Tyro3, or anti-TIM-4 polyclonal antibody. (C and D) LASV-VSV (C) or adenovirus-eGFP (D) transduction of WT, TIM-1-KO, Axl-KO, or DKO Vero cells. Serial dilutions of virus were applied to each cell type in duplicate. Transduction is represented as the percentage of cells that are eGFP positive, as assessed by flow cytometry. Data points are representative of results from three independent experiments with the indicated standard errors of the means (SEM). (E) LASV-LCMV transduction (MOI = ∼0.2) of WT, TIM-1-KO, Axl-KO, or DKO Vero cell lines. Data points are representative of results from three independent experiments with the indicated SEM. (F and G) Ability of adenovirus vector (Ad)-delivered TIM-1 to enhance transduction of LASV-VSV in WT, TIM-1-KO, or TIM-1- and Axl-KO (DKO) Vero cells. Forty-eight hours following Ad transduction, cells were divided and assessed independently for TIM-1 expression (F) or LASV-VSV transduction (G). Bars in each panel represent results from one of three independent experiments. (F) Cell surface expression of TIM-1 48 h following transduction of WT or KO Vero cells with Ad-TIM-1 (MOI of 2, 4, or 8). Cells were surface stained with 2 μg/ml anti-TIM-1 polyclonal antibodies. TIM-1 expression is represented as geometric mean fluorescence index (MFI) values. (G) LASV-VSV transductions of empty (E) or TIM-1-expressing (T) adenovirus vector-transduced WT or KO Vero cells. Equivalent volumes of LASV-VSV were applied to cells in duplicate. Transduction is represented as the percentage of eGFP-positive cells, as assessed by flow cytometry.

    Techniques Used: Expressing, Staining, Transduction, Flow Cytometry, Cytometry, Plasmid Preparation, Fluorescence

    TIM-1 mediates LASV pseudovirion transduction through a PtdSer-dependent mechanism. (A and B) Anti-human TIM-1 antibody ARD5 inhibits TIM-1-dependent LASV pseudovirion transduction in HEK 293T cells in a dose-dependent manner. (A) WT or DG-KO HEK 293T cells transfected with a TIM-1-expressing plasmid were incubated with serial dilutions of ARD5 prior to the addition of LASV-VSV. Relative transduction is represented as the percentage of eGFP-positive cells relative to cells incubated without ARD5, as assessed by flow cytometry. (B) WT or Axl-KO Vero cells were incubated with serial dilutions of ARD5 prior to the addition of LASV-LCMV, and data are shown as relative transduction, as described above. Data points are representative of results from three independent experiments with the indicated SEM. (C to F) Ability of two human TIM-1 mutants to support LASV-VSV transduction in DG-KO HEK 293T cells. Plasmids expressing a PtdSer pocket mutant, TIM-1 (ND115DN), or a chimeric TIM-1 that has the MLD replaced with the proline-rich region (PRR) of murine leukemia virus glycoprotein were transfected into WT or DG-KO cells. Cells were transfected with 0, 0.25, 0.5, or 1 μg ND115DN or PRR plasmid (increase in DNA is indicated with unfilled triangles). After 48 h, cells were divided and assessed independently for TIM-1 expression (C and E) or LASV-VSV transduction (D and F). Equivalent volumes of LASV-VSV were applied to cells in duplicate in transduction studies. Shown are fold change values relative to transduction levels of untransfected cells of the same type, as assessed by flow cytometry. Shown in panels A and B are means and standard errors of the means of data from three independent experiments. Shown in panels C to F are means and standard errors of the means of data from 3 to 5 independent experiments. *, P
    Figure Legend Snippet: TIM-1 mediates LASV pseudovirion transduction through a PtdSer-dependent mechanism. (A and B) Anti-human TIM-1 antibody ARD5 inhibits TIM-1-dependent LASV pseudovirion transduction in HEK 293T cells in a dose-dependent manner. (A) WT or DG-KO HEK 293T cells transfected with a TIM-1-expressing plasmid were incubated with serial dilutions of ARD5 prior to the addition of LASV-VSV. Relative transduction is represented as the percentage of eGFP-positive cells relative to cells incubated without ARD5, as assessed by flow cytometry. (B) WT or Axl-KO Vero cells were incubated with serial dilutions of ARD5 prior to the addition of LASV-LCMV, and data are shown as relative transduction, as described above. Data points are representative of results from three independent experiments with the indicated SEM. (C to F) Ability of two human TIM-1 mutants to support LASV-VSV transduction in DG-KO HEK 293T cells. Plasmids expressing a PtdSer pocket mutant, TIM-1 (ND115DN), or a chimeric TIM-1 that has the MLD replaced with the proline-rich region (PRR) of murine leukemia virus glycoprotein were transfected into WT or DG-KO cells. Cells were transfected with 0, 0.25, 0.5, or 1 μg ND115DN or PRR plasmid (increase in DNA is indicated with unfilled triangles). After 48 h, cells were divided and assessed independently for TIM-1 expression (C and E) or LASV-VSV transduction (D and F). Equivalent volumes of LASV-VSV were applied to cells in duplicate in transduction studies. Shown are fold change values relative to transduction levels of untransfected cells of the same type, as assessed by flow cytometry. Shown in panels A and B are means and standard errors of the means of data from three independent experiments. Shown in panels C to F are means and standard errors of the means of data from 3 to 5 independent experiments. *, P

    Techniques Used: Transduction, Transfection, Expressing, Plasmid Preparation, Incubation, Flow Cytometry, Cytometry, Mutagenesis

    6) 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

    7) Product Images from "Attenuation and Protective Efficacy of Rift Valley Fever Phlebovirus rMP12-GM50 Strain"

    Article Title: Attenuation and Protective Efficacy of Rift Valley Fever Phlebovirus rMP12-GM50 Strain

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2017.10.036

    Generation of the rMP12-GM50 strain of Rift Valley fever phlebovirus ] at each codon within the ORFs for N, NSs, M, and L were individually plotted onto graphs. The X-axis represents the nucleotide position numbers. Blue square = rMP-12; red triangle = rMP12-GM50. (C) Replication kinetics of rMP-12 and rMP12-GM50 in Vero cells (left panel) and MRC-5 cells (right panel) at 0.01 MOI. Graph represents the means +/− standard deviations of three independent experiments. Blue square = rMP-12; red triangle = rMP12-GM50.
    Figure Legend Snippet: Generation of the rMP12-GM50 strain of Rift Valley fever phlebovirus ] at each codon within the ORFs for N, NSs, M, and L were individually plotted onto graphs. The X-axis represents the nucleotide position numbers. Blue square = rMP-12; red triangle = rMP12-GM50. (C) Replication kinetics of rMP-12 and rMP12-GM50 in Vero cells (left panel) and MRC-5 cells (right panel) at 0.01 MOI. Graph represents the means +/− standard deviations of three independent experiments. Blue square = rMP-12; red triangle = rMP12-GM50.

    Techniques Used:

    8) Product Images from "A Herpes Simplex Virus Type 1 ?34.5 Second-Site Suppressor Mutant That Exhibits Enhanced Growth in Cultured Glioblastoma Cells Is Severely Attenuated in Animals"

    Article Title: A Herpes Simplex Virus Type 1 ?34.5 Second-Site Suppressor Mutant That Exhibits Enhanced Growth in Cultured Glioblastoma Cells Is Severely Attenuated in Animals

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.11.5189-5196.2001

    Enrichment for recombinant viruses with repaired γ34.5 alleles and physical analysis of the purified recombinant genomes. (A) Vero cells were transfected with HSV-1 viral DNA (genotype Δ34.5ΔSUP) and the wild-type (WT) Bam HI SP fragment. Cell-free lysates from the transfection plate were passaged once in nonpermissive U373 cells as described in the text. Prior to plaque purification, the population was used to infect Vero cells at high multiplicity and viral DNA was isolated. This DNA, along with DNA from wild-type HSV-1 and SUP1, was digested with Bam HI, fractionated by electrophoresis on 1% agarose gels, transferred to a nylon membrane, and hybridized to the 32 P-labeled Bam HI- Bst XI segment (nucleotides 123459 to 124679) from the Bam HI S fragment. This probe identifies sequences in the authentic γ34.5 loci. Heterogeneity at the genomic Bam HI S and Bam HI SP loci is due to natural variations in a repetitive sequence component. The slower-migrating SP and S fragments observed in the SUP virus reflect the fact that all γ34.5 coding sequences have been replaced with sequences encoding β-glucuronidase. (B) Following two rounds of plaque purification on Vero cells, viral DNA was isolated from a 34.5RΔSUP isolate. DNA from 34.5RΔSUP, SUP1 (genotype Δ34.5ΔSUP), and the wild-type HSV-1 Patton strain was digested, fractionated, and hybridized as described for panel A. (C) Same as in panel B except that the membrane was hybridized to a 32 P-labeled Bam HI- Bst EII probe (nucleotides 144875 to 145316) from the Bam HI Z fragment. This probe detects sequences near the Us-TR junction in the SUP locus. Slower-migrating forms of the Bam HI Z fragment are due to natural variations in a repetitive sequence component.
    Figure Legend Snippet: Enrichment for recombinant viruses with repaired γ34.5 alleles and physical analysis of the purified recombinant genomes. (A) Vero cells were transfected with HSV-1 viral DNA (genotype Δ34.5ΔSUP) and the wild-type (WT) Bam HI SP fragment. Cell-free lysates from the transfection plate were passaged once in nonpermissive U373 cells as described in the text. Prior to plaque purification, the population was used to infect Vero cells at high multiplicity and viral DNA was isolated. This DNA, along with DNA from wild-type HSV-1 and SUP1, was digested with Bam HI, fractionated by electrophoresis on 1% agarose gels, transferred to a nylon membrane, and hybridized to the 32 P-labeled Bam HI- Bst XI segment (nucleotides 123459 to 124679) from the Bam HI S fragment. This probe identifies sequences in the authentic γ34.5 loci. Heterogeneity at the genomic Bam HI S and Bam HI SP loci is due to natural variations in a repetitive sequence component. The slower-migrating SP and S fragments observed in the SUP virus reflect the fact that all γ34.5 coding sequences have been replaced with sequences encoding β-glucuronidase. (B) Following two rounds of plaque purification on Vero cells, viral DNA was isolated from a 34.5RΔSUP isolate. DNA from 34.5RΔSUP, SUP1 (genotype Δ34.5ΔSUP), and the wild-type HSV-1 Patton strain was digested, fractionated, and hybridized as described for panel A. (C) Same as in panel B except that the membrane was hybridized to a 32 P-labeled Bam HI- Bst EII probe (nucleotides 144875 to 145316) from the Bam HI Z fragment. This probe detects sequences near the Us-TR junction in the SUP locus. Slower-migrating forms of the Bam HI Z fragment are due to natural variations in a repetitive sequence component.

    Techniques Used: Recombinant, Purification, Transfection, Isolation, Electrophoresis, Labeling, Sequencing

    9) 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

    10) Product Images from "B Virus ( Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies"

    Article Title: B Virus ( Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies

    Journal: Journal of Virology

    doi: 10.1128/JVI.00799-16

    Infection of cells with a gD-negative recombinant B virus (BV-ΔgDZ). Nectin-2-expressing, nectin-1-expressing (negative control), and Vero (positive control) cell monolayers were infected with BV-ΔgDZ. Cells were fixed at 48 hpi and then
    Figure Legend Snippet: Infection of cells with a gD-negative recombinant B virus (BV-ΔgDZ). Nectin-2-expressing, nectin-1-expressing (negative control), and Vero (positive control) cell monolayers were infected with BV-ΔgDZ. Cells were fixed at 48 hpi and then

    Techniques Used: Infection, Recombinant, Expressing, Negative Control, Positive Control

    Susceptibility of murine B78H1 cells expressing HSV entry receptors to B virus infection. B virus E2490 was titrated on confluent monolayers of Vero (A), B78-nectin-1 (B), B78-nectin-2 (C), B78-HVEM (D), and B78c (E) cells. At 48 hpi, the cells were fixed
    Figure Legend Snippet: Susceptibility of murine B78H1 cells expressing HSV entry receptors to B virus infection. B virus E2490 was titrated on confluent monolayers of Vero (A), B78-nectin-1 (B), B78-nectin-2 (C), B78-HVEM (D), and B78c (E) cells. At 48 hpi, the cells were fixed

    Techniques Used: Expressing, Infection

    11) Product Images from "Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro"

    Article Title: Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.066084-0

    Antiviral siRNAs inhibit replication of DENV-2, DENV-3 and DENV-4 serotypes. Vero cells were transfected with siRNAs (si-1 and si-3), siSCRM and a positive control siRNA (DC-3) using a Lipofectamine reagent for 48 h. Vero cells were then infected with
    Figure Legend Snippet: Antiviral siRNAs inhibit replication of DENV-2, DENV-3 and DENV-4 serotypes. Vero cells were transfected with siRNAs (si-1 and si-3), siSCRM and a positive control siRNA (DC-3) using a Lipofectamine reagent for 48 h. Vero cells were then infected with

    Techniques Used: Transfection, Positive Control, Infection

    Cationic AuNP–siRNA complexes are not toxic to Vero cells. Vero cells were cultured with or without AuNP–si-1 complexes (80 nM) under similar experimental conditions as for the pre-treatment assays. (a) Cell images were taken under a confocal
    Figure Legend Snippet: Cationic AuNP–siRNA complexes are not toxic to Vero cells. Vero cells were cultured with or without AuNP–si-1 complexes (80 nM) under similar experimental conditions as for the pre-treatment assays. (a) Cell images were taken under a confocal

    Techniques Used: Cell Culture

    Cationic AuNP–siRNA complexes inhibit DENV-2 replication and viral propagation in vitro. Cationic AuNP–siRNA complexes (20 and 80 nM siRNA) were added to Vero cell culture for 48 h and infected with DENV-2 (m.o.i. 0.1) for an additional
    Figure Legend Snippet: Cationic AuNP–siRNA complexes inhibit DENV-2 replication and viral propagation in vitro. Cationic AuNP–siRNA complexes (20 and 80 nM siRNA) were added to Vero cell culture for 48 h and infected with DENV-2 (m.o.i. 0.1) for an additional

    Techniques Used: In Vitro, Cell Culture, Infection

    12) Product Images from "Development of high-yield influenza A virus vaccine viruses"

    Article Title: Development of high-yield influenza A virus vaccine viruses

    Journal: Nature Communications

    doi: 10.1038/ncomms9148

    Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. ( a ) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 °C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. ( b ) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1+C4U_Indo09). Experiments were carried out as described in a . The values presented are the average of three independent experiments±s.d.
    Figure Legend Snippet: Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. ( a ) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 °C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. ( b ) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1+C4U_Indo09). Experiments were carried out as described in a . The values presented are the average of three independent experiments±s.d.

    Techniques Used: Infection, Incubation, Mutagenesis

    13) Product Images from "Development of high-yield influenza A virus vaccine viruses"

    Article Title: Development of high-yield influenza A virus vaccine viruses

    Journal: Nature Communications

    doi: 10.1038/ncomms9148

    Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. ( a ) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 °C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. ( b ) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1+C4U_Indo09). Experiments were carried out as described in a . The values presented are the average of three independent experiments±s.d.
    Figure Legend Snippet: Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells. ( a ) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 °C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. ( b ) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1+C4U_Indo09). Experiments were carried out as described in a . The values presented are the average of three independent experiments±s.d.

    Techniques Used: Infection, Incubation, Mutagenesis

    Evaluation of PR8-HY backbone vaccine candidates propagated in Vero cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild type, or wild-type and chimeric HA and NA segments of the A/Vietnam/1203/2004 (VN04, H5N1) ( a ), A/Hubei/1/2010 (Hubei10, H5N1) ( b ), A/Egypt/N03072/2010 (Egypt10, H5N1) ( c ), A/Indonesia/5/2005 (Indo05, H5N1) ( d ) or A/Anhui/1/2013 (Anhui13, H7N9) ( e ) viruses. Panels ( f ) and ( g ) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Experiments were carried out as described in the legend to Fig. 2 , with the exception of those involving X-181 and X-223A viruses, which were inoculated at an MOI of 0.1. The values presented are the average of three independent experiments±s.d.
    Figure Legend Snippet: Evaluation of PR8-HY backbone vaccine candidates propagated in Vero cells. Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild type, or wild-type and chimeric HA and NA segments of the A/Vietnam/1203/2004 (VN04, H5N1) ( a ), A/Hubei/1/2010 (Hubei10, H5N1) ( b ), A/Egypt/N03072/2010 (Egypt10, H5N1) ( c ), A/Indonesia/5/2005 (Indo05, H5N1) ( d ) or A/Anhui/1/2013 (Anhui13, H7N9) ( e ) viruses. Panels ( f ) and ( g ) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Experiments were carried out as described in the legend to Fig. 2 , with the exception of those involving X-181 and X-223A viruses, which were inoculated at an MOI of 0.1. The values presented are the average of three independent experiments±s.d.

    Techniques Used: Derivative Assay

    14) Product Images from "Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule"

    Article Title: Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165088

    Comparison of BHK-SLAM cells and Vero cells. (A) BHK-SLAM cells and Vero cells were grown to 80%–90% confluence in 24-well plates. Then, they were infected with rPPRV/GFP at an MOI of 0.01. At various time points after infection (every 24 h), images of GFP expression were taken. (B) Viruses at different time points were collected and stored at −70°C, and viral titers (TCID 50 ) were determined in Vero cells. (C) BHK-SLAM and Vero cells were fixed at the same time after infection with rPPRV/GFP at an MOI = 0.1. The CPE (black arrow) and GFP expression were observed. (D) After fixing with cold 4% paraformaldehyde for 30 min, the BHK-SLAM and Vero cells were used for the IPMA, and AEC staining results were observed (black arrow).
    Figure Legend Snippet: Comparison of BHK-SLAM cells and Vero cells. (A) BHK-SLAM cells and Vero cells were grown to 80%–90% confluence in 24-well plates. Then, they were infected with rPPRV/GFP at an MOI of 0.01. At various time points after infection (every 24 h), images of GFP expression were taken. (B) Viruses at different time points were collected and stored at −70°C, and viral titers (TCID 50 ) were determined in Vero cells. (C) BHK-SLAM and Vero cells were fixed at the same time after infection with rPPRV/GFP at an MOI = 0.1. The CPE (black arrow) and GFP expression were observed. (D) After fixing with cold 4% paraformaldehyde for 30 min, the BHK-SLAM and Vero cells were used for the IPMA, and AEC staining results were observed (black arrow).

    Techniques Used: Infection, Expressing, Staining

    15) Product Images from "Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule"

    Article Title: Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165088

    Comparison of BHK-SLAM cells and Vero cells. (A) BHK-SLAM cells and Vero cells were grown to 80%–90% confluence in 24-well plates. Then, they were infected with rPPRV/GFP at an MOI of 0.01. At various time points after infection (every 24 h), images of GFP expression were taken. (B) Viruses at different time points were collected and stored at −70°C, and viral titers (TCID 50 ) were determined in Vero cells. (C) BHK-SLAM and Vero cells were fixed at the same time after infection with rPPRV/GFP at an MOI = 0.1. The CPE (black arrow) and GFP expression were observed. (D) After fixing with cold 4% paraformaldehyde for 30 min, the BHK-SLAM and Vero cells were used for the IPMA, and AEC staining results were observed (black arrow).
    Figure Legend Snippet: Comparison of BHK-SLAM cells and Vero cells. (A) BHK-SLAM cells and Vero cells were grown to 80%–90% confluence in 24-well plates. Then, they were infected with rPPRV/GFP at an MOI of 0.01. At various time points after infection (every 24 h), images of GFP expression were taken. (B) Viruses at different time points were collected and stored at −70°C, and viral titers (TCID 50 ) were determined in Vero cells. (C) BHK-SLAM and Vero cells were fixed at the same time after infection with rPPRV/GFP at an MOI = 0.1. The CPE (black arrow) and GFP expression were observed. (D) After fixing with cold 4% paraformaldehyde for 30 min, the BHK-SLAM and Vero cells were used for the IPMA, and AEC staining results were observed (black arrow).

    Techniques Used: Infection, Expressing, Staining

    16) Product Images from "A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells"

    Article Title: A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00067-17

    Genome organization of R-213 and generation and properties of the Vero-GCN4R cell line. (A) Genome organization of R-213. The sequence arrangement of the R-213 genome shows the inverted repeat sequences as rectangular boxes. The scFv-HER2 sequence (V L -linker-V H ), bracketed by upstream and downstream linkers, is inserted in place of the deletion of aa 6 to 38 in gD. LOX-P-bracketed pBelo-BAC and EGFP sequences are inserted in the UL3-UL4 intergenic region. The sequence encoding the GCN4 peptide is engineered in gH, between aa 23 and 24. (B) Amino acid sequence of the GCN4 peptide. The epitope residues are in red, two residues flanking the epitope in wt GCN4 transcription factor are in blue, and upstream and downstream Gly-Ser linkers are in black. (C) Schematic drawing of the GCN4R chimeric receptor. The receptor is formed by an N-terminal signal peptide (purple box), an HA tag sequence (yellow box) from pDisplay (Thermo Fisher Scientific), the scFv to GCN4 (red box) bracketed by two short linkers, GA and GSGA, optimized for human codon usage, the human nectin-1 residues from Met 143 to Val 517 , which includes the nectin-1 extracellular domains 2 and 3, the transmembrane (TM) segment (green box), and the cytoplasmic tail. (D to G) Expression of GCN4R and stability of the Vero-GCN4R cell line. The expression of the scFv GCN4-nectin receptor was analyzed by indirect immunofluorescence (D) or by fluorescence-activated cell sorting (E to G) by using a MAb to the HA tag. Diagrams show the percentages of positive cells in cultures of wt-Vero (E) or Vero-GCN4R cells at passages 10 (F) and 40 (G). SSC, side scatter; FITC, fluorescein isothiocyanate. The illustration in panel D was adjusted as follows: increase in brightness of 10%, increase in contrast of 20%.
    Figure Legend Snippet: Genome organization of R-213 and generation and properties of the Vero-GCN4R cell line. (A) Genome organization of R-213. The sequence arrangement of the R-213 genome shows the inverted repeat sequences as rectangular boxes. The scFv-HER2 sequence (V L -linker-V H ), bracketed by upstream and downstream linkers, is inserted in place of the deletion of aa 6 to 38 in gD. LOX-P-bracketed pBelo-BAC and EGFP sequences are inserted in the UL3-UL4 intergenic region. The sequence encoding the GCN4 peptide is engineered in gH, between aa 23 and 24. (B) Amino acid sequence of the GCN4 peptide. The epitope residues are in red, two residues flanking the epitope in wt GCN4 transcription factor are in blue, and upstream and downstream Gly-Ser linkers are in black. (C) Schematic drawing of the GCN4R chimeric receptor. The receptor is formed by an N-terminal signal peptide (purple box), an HA tag sequence (yellow box) from pDisplay (Thermo Fisher Scientific), the scFv to GCN4 (red box) bracketed by two short linkers, GA and GSGA, optimized for human codon usage, the human nectin-1 residues from Met 143 to Val 517 , which includes the nectin-1 extracellular domains 2 and 3, the transmembrane (TM) segment (green box), and the cytoplasmic tail. (D to G) Expression of GCN4R and stability of the Vero-GCN4R cell line. The expression of the scFv GCN4-nectin receptor was analyzed by indirect immunofluorescence (D) or by fluorescence-activated cell sorting (E to G) by using a MAb to the HA tag. Diagrams show the percentages of positive cells in cultures of wt-Vero (E) or Vero-GCN4R cells at passages 10 (F) and 40 (G). SSC, side scatter; FITC, fluorescein isothiocyanate. The illustration in panel D was adjusted as follows: increase in brightness of 10%, increase in contrast of 20%.

    Techniques Used: Sequencing, BAC Assay, Expressing, Immunofluorescence, Fluorescence, FACS

    Double tropism of R-213 for Vero-GCN4R and HER2-positive cells. The indicated cells were infected with R-213 (A) or R-LM113 (B) at an MOI of 1 PFU/cell and monitored for EGFP expression by fluorescence microscopy 24 h postinfection. J-cells express no receptor for wt HSV; J-HER2, J-nectin-1, and J-HVEM express the indicated receptor. Cells in panels e to h and p to s were infected in the presence of the humanized anti-HER2 MAb trastuzumab at a concentration of 28 μg/ml. Some of the panels were adjusted as follows: 90% increase in brightness in panels a, b, c, e, f, g, l, m, n, p, q, and r.
    Figure Legend Snippet: Double tropism of R-213 for Vero-GCN4R and HER2-positive cells. The indicated cells were infected with R-213 (A) or R-LM113 (B) at an MOI of 1 PFU/cell and monitored for EGFP expression by fluorescence microscopy 24 h postinfection. J-cells express no receptor for wt HSV; J-HER2, J-nectin-1, and J-HVEM express the indicated receptor. Cells in panels e to h and p to s were infected in the presence of the humanized anti-HER2 MAb trastuzumab at a concentration of 28 μg/ml. Some of the panels were adjusted as follows: 90% increase in brightness in panels a, b, c, e, f, g, l, m, n, p, q, and r.

    Techniques Used: Infection, Expressing, Fluorescence, Microscopy, Concentration Assay

    17) Product Images from "The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro"

    Article Title: The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.10776-10784.2002

    Immunoprecipitation of RSV-infected proteins infected with wild-type or phosphorylation mutants. Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0 and incubated at 35°C. At 18 h of postinfection, proteins were radiolabeled with [ 35 S]Cys and [ 35 S]Met (100 μCi/ml) in DMEM deficient in cysteine and methionine or 33 P i (100 μCi/ml) in DMEM deficient in phosphate for 4 h, immunoprecipitated either by anti-RSV polyclonal or by anti-P protein monoclonal antibodies, separated by SDS-PAGE (15% polyacrylamide), and autoradiographed. P indicates the mature form of the P protein, and P′ represents the immature form of P protein.
    Figure Legend Snippet: Immunoprecipitation of RSV-infected proteins infected with wild-type or phosphorylation mutants. Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0 and incubated at 35°C. At 18 h of postinfection, proteins were radiolabeled with [ 35 S]Cys and [ 35 S]Met (100 μCi/ml) in DMEM deficient in cysteine and methionine or 33 P i (100 μCi/ml) in DMEM deficient in phosphate for 4 h, immunoprecipitated either by anti-RSV polyclonal or by anti-P protein monoclonal antibodies, separated by SDS-PAGE (15% polyacrylamide), and autoradiographed. P indicates the mature form of the P protein, and P′ represents the immature form of P protein.

    Techniques Used: Immunoprecipitation, Infection, Incubation, SDS Page

    Growth kinetics of rA2-PP2 and rA2-PP5 in Vero and HEp-2 cells. Vero or HEp-2 cells were infected with virus at an MOI of 1.0 or 0.01 and incubated at 35°C. Aliquots of culture supernatant (200 μl) were harvested at 24-h intervals for 96 h. The virus titers are an average of two experiments.
    Figure Legend Snippet: Growth kinetics of rA2-PP2 and rA2-PP5 in Vero and HEp-2 cells. Vero or HEp-2 cells were infected with virus at an MOI of 1.0 or 0.01 and incubated at 35°C. Aliquots of culture supernatant (200 μl) were harvested at 24-h intervals for 96 h. The virus titers are an average of two experiments.

    Techniques Used: Infection, Incubation

    18) Product Images from "The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro"

    Article Title: The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.10776-10784.2002

    Immunoprecipitation of RSV-infected proteins infected with wild-type or phosphorylation mutants. Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0 and incubated at 35°C. At 18 h of postinfection, proteins were radiolabeled with [ 35 S]Cys and [ 35 S]Met (100 μCi/ml) in DMEM deficient in cysteine and methionine or 33 P i (100 μCi/ml) in DMEM deficient in phosphate for 4 h, immunoprecipitated either by anti-RSV polyclonal or by anti-P protein monoclonal antibodies, separated by SDS-PAGE (15% polyacrylamide), and autoradiographed. P indicates the mature form of the P protein, and P′ represents the immature form of P protein.
    Figure Legend Snippet: Immunoprecipitation of RSV-infected proteins infected with wild-type or phosphorylation mutants. Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0 and incubated at 35°C. At 18 h of postinfection, proteins were radiolabeled with [ 35 S]Cys and [ 35 S]Met (100 μCi/ml) in DMEM deficient in cysteine and methionine or 33 P i (100 μCi/ml) in DMEM deficient in phosphate for 4 h, immunoprecipitated either by anti-RSV polyclonal or by anti-P protein monoclonal antibodies, separated by SDS-PAGE (15% polyacrylamide), and autoradiographed. P indicates the mature form of the P protein, and P′ represents the immature form of P protein.

    Techniques Used: Immunoprecipitation, Infection, Incubation, SDS Page

    Growth kinetics of rA2-PP2 and rA2-PP5 in Vero and HEp-2 cells. Vero or HEp-2 cells were infected with virus at an MOI of 1.0 or 0.01 and incubated at 35°C. Aliquots of culture supernatant (200 μl) were harvested at 24-h intervals for 96 h. The virus titers are an average of two experiments.
    Figure Legend Snippet: Growth kinetics of rA2-PP2 and rA2-PP5 in Vero and HEp-2 cells. Vero or HEp-2 cells were infected with virus at an MOI of 1.0 or 0.01 and incubated at 35°C. Aliquots of culture supernatant (200 μl) were harvested at 24-h intervals for 96 h. The virus titers are an average of two experiments.

    Techniques Used: Infection, Incubation

    Cell association of rA2-PP5 in HEp-2 cells. HEp-2 or Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0, and at 24 and 48 h postinfection, the amounts of virus released in the culture media or in the infected cells were determined by plaque assay. The percentages of virus that remained associated with the cells are represented.
    Figure Legend Snippet: Cell association of rA2-PP5 in HEp-2 cells. HEp-2 or Vero cells were infected with rA2, rA2-PP2, or rA2-PP5 at an MOI of 1.0, and at 24 and 48 h postinfection, the amounts of virus released in the culture media or in the infected cells were determined by plaque assay. The percentages of virus that remained associated with the cells are represented.

    Techniques Used: Infection, Plaque Assay

    19) Product Images from "Mutagenic Analysis of the 3? cis-Acting Elements of the Rubella Virus Genome"

    Article Title: Mutagenic Analysis of the 3? cis-Acting Elements of the Rubella Virus Genome

    Journal: Journal of Virology

    doi:

    (A) Mutagenesis of SL2. Because SL2 is located in the E1 coding region, analysis was done by creating point mutations that resulted in silent changes (436) or synonymous codons (435 and 461). However, to eliminate the U-U bulge, we created two missense mutations (444 and 430-AAG/GAG) which led to changes in coding as indicated. Mutation 419 was made to create an Nsi I site for cloning, and 330 was made to conform to HPV77 sequence. The bold characters are the stop codon ending the E1 gene; the nucleotide variations found in other RUB strains are in lowercase. Transfection efficiencies of mutant transcripts are shown as +++ (equivalent to that of Robo302); ++ (about 30 to 50% of that of Robo302) +, (about 5 to 10% of that of Robo302), and N.D., not determinable, but transfected cells inoculated with medium showed CPE. The plaque morphology described is that produced following transfection. Following amplification of 430-AAG from transfection medium, the sequence was found to have changed to CAG. (B) Growth curves of SL2 mutants. Vero cells were infected with viruses amplified from transfection plaques at an MOI of 0.01 PFU/cell. At indicated times postinfection, aliquots of the infected culture media were harvested and titered on Vero cells.
    Figure Legend Snippet: (A) Mutagenesis of SL2. Because SL2 is located in the E1 coding region, analysis was done by creating point mutations that resulted in silent changes (436) or synonymous codons (435 and 461). However, to eliminate the U-U bulge, we created two missense mutations (444 and 430-AAG/GAG) which led to changes in coding as indicated. Mutation 419 was made to create an Nsi I site for cloning, and 330 was made to conform to HPV77 sequence. The bold characters are the stop codon ending the E1 gene; the nucleotide variations found in other RUB strains are in lowercase. Transfection efficiencies of mutant transcripts are shown as +++ (equivalent to that of Robo302); ++ (about 30 to 50% of that of Robo302) +, (about 5 to 10% of that of Robo302), and N.D., not determinable, but transfected cells inoculated with medium showed CPE. The plaque morphology described is that produced following transfection. Following amplification of 430-AAG from transfection medium, the sequence was found to have changed to CAG. (B) Growth curves of SL2 mutants. Vero cells were infected with viruses amplified from transfection plaques at an MOI of 0.01 PFU/cell. At indicated times postinfection, aliquots of the infected culture media were harvested and titered on Vero cells.

    Techniques Used: Mutagenesis, Clone Assay, Sequencing, Transfection, Produced, Amplification, Infection

    Characterization of viruses with mutations in SL4, the leader, and the poly(A) tract. Sequences determined from 15 viable mutants are given (a and b indicate results of independent transfections); altered nucleotides not present in the original transcripts are in lowercase. For comparison, sequences of six nonviable mutants are also given at the bottom. To determine replication ability, mutant viruses isolated from transfection plaques (p) or medium (m) were amplified by one passage in Vero cells, and the titers of the amplified stocks were determined. These stocks were then used for infection of Vero cells (in the first experiment [superscript “1”], which compared the replication of Robo302 and eight mutants, the multiplicity of infection [MOI] was 5; in the second experiment [superscript “2”], which compared Robo302 and six additional mutants, the MOI was 0.01). The infected culture media were harvested at 2 (MOI = 5) or 3 (MOI = 0.01) days postinfection, and the titer (PFU per milliliter) was determined. The relative titer produced by each mutant is shown in comparison with the Robo302 titer (set at 100%) produced in the same experiment (N.D., not determined). The proposed critical stretch of UG(U/C) triplets is underlined.
    Figure Legend Snippet: Characterization of viruses with mutations in SL4, the leader, and the poly(A) tract. Sequences determined from 15 viable mutants are given (a and b indicate results of independent transfections); altered nucleotides not present in the original transcripts are in lowercase. For comparison, sequences of six nonviable mutants are also given at the bottom. To determine replication ability, mutant viruses isolated from transfection plaques (p) or medium (m) were amplified by one passage in Vero cells, and the titers of the amplified stocks were determined. These stocks were then used for infection of Vero cells (in the first experiment [superscript “1”], which compared the replication of Robo302 and eight mutants, the multiplicity of infection [MOI] was 5; in the second experiment [superscript “2”], which compared Robo302 and six additional mutants, the MOI was 0.01). The infected culture media were harvested at 2 (MOI = 5) or 3 (MOI = 0.01) days postinfection, and the titer (PFU per milliliter) was determined. The relative titer produced by each mutant is shown in comparison with the Robo302 titer (set at 100%) produced in the same experiment (N.D., not determined). The proposed critical stretch of UG(U/C) triplets is underlined.

    Techniques Used: Transfection, Mutagenesis, Isolation, Amplification, Infection, Produced

    20) Product Images from "The Major Neutralizing Antigenic Site on Herpes Simplex Virus Glycoprotein D Overlaps a Receptor-Binding Domain"

    Article Title: The Major Neutralizing Antigenic Site on Herpes Simplex Virus Glycoprotein D Overlaps a Receptor-Binding Domain

    Journal: Journal of Virology

    doi:

    Analysis of gD-1(Δ222–224, 306t) for blocking of HSV entry. Cells in 96-well tissue culture plates were incubated with increasing concentrations (shown on the x axis) of gD-1(306t), gD-1(250t), or gD-1(Δ222–224, 306t) prior to infection with HSV-1(KOS) carrying a β-galactosidase reporter gene ( hr R3). At 5 h postinfection, cells were lysed and assayed for β-galactosidase activity. (A) HeLa cells. (B) Vero cells. Symbols: □, gD-1(306t); ○, gD-1(250t); ▵, gD-1(Δ222–224, 306t); , bovine serum albumin.
    Figure Legend Snippet: Analysis of gD-1(Δ222–224, 306t) for blocking of HSV entry. Cells in 96-well tissue culture plates were incubated with increasing concentrations (shown on the x axis) of gD-1(306t), gD-1(250t), or gD-1(Δ222–224, 306t) prior to infection with HSV-1(KOS) carrying a β-galactosidase reporter gene ( hr R3). At 5 h postinfection, cells were lysed and assayed for β-galactosidase activity. (A) HeLa cells. (B) Vero cells. Symbols: □, gD-1(306t); ○, gD-1(250t); ▵, gD-1(Δ222–224, 306t); , bovine serum albumin.

    Techniques Used: Blocking Assay, Incubation, Infection, Activity Assay

    Analysis of gD truncation mutants for blocking of HSV entry. Cells in 96-well tissue culture plates were incubated with increasing concentrations (shown on the x axis) of various forms of gD prior to infection with HSV-1(KOS) carrying a β-galactosidase reporter gene ( hr R3). At 5 h postinfection, cells were lysed and assayed for β-galactosidase activity. (A) HeLa cells. (B) Vero cells. Symbols: □, gD-1(306t); ■, gD-1(285t); ▴, gD-1(260t); ○, gD-1(250t); ●, gD-1(240t); ✖, gD-1(234t); , BSA.
    Figure Legend Snippet: Analysis of gD truncation mutants for blocking of HSV entry. Cells in 96-well tissue culture plates were incubated with increasing concentrations (shown on the x axis) of various forms of gD prior to infection with HSV-1(KOS) carrying a β-galactosidase reporter gene ( hr R3). At 5 h postinfection, cells were lysed and assayed for β-galactosidase activity. (A) HeLa cells. (B) Vero cells. Symbols: □, gD-1(306t); ■, gD-1(285t); ▴, gD-1(260t); ○, gD-1(250t); ●, gD-1(240t); ✖, gD-1(234t); , BSA.

    Techniques Used: Blocking Assay, Incubation, Infection, Activity Assay

    21) 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

    22) 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

    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

    23) Product Images from "Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico"

    Article Title: Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18682-3

    Cytopathic effect in infected and uninfected cell cultures. Cytopathic effects consisting of cell rounding, detachment and culture degeneration were observed in C6/36 and Vero cell lines at day 4 post-ZIKV inoculation, under an inverted microscope (original magnification ×20 in C6/36 and ×10 in Vero cells. Scale bars, 100 μm). ( A ) Uninfected C6/36 cells, ( B ) C6/36 cells inoculated with salivary glands homogenates showing CPE, ( C ) Uninfected Vero cells, ( D ) Vero cells inoculated with salivary glands homogenates showing CPE.
    Figure Legend Snippet: Cytopathic effect in infected and uninfected cell cultures. Cytopathic effects consisting of cell rounding, detachment and culture degeneration were observed in C6/36 and Vero cell lines at day 4 post-ZIKV inoculation, under an inverted microscope (original magnification ×20 in C6/36 and ×10 in Vero cells. Scale bars, 100 μm). ( A ) Uninfected C6/36 cells, ( B ) C6/36 cells inoculated with salivary glands homogenates showing CPE, ( C ) Uninfected Vero cells, ( D ) Vero cells inoculated with salivary glands homogenates showing CPE.

    Techniques Used: Infection, Inverted Microscopy

    24) Product Images from "Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014"

    Article Title: Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2018.19.1.71

    Cytopathic effects of (A–C), and immunofluorescence results for (D–F), porcine epidemic diarrhea virus isolates in infected Vero cells at 200×. Vero cells were infected with the QIAP1401 or QIAP1402 isolates and maintained in DMEM containing 1 µg/mL trypsin
    Figure Legend Snippet: Cytopathic effects of (A–C), and immunofluorescence results for (D–F), porcine epidemic diarrhea virus isolates in infected Vero cells at 200×. Vero cells were infected with the QIAP1401 or QIAP1402 isolates and maintained in DMEM containing 1 µg/mL trypsin

    Techniques Used: Immunofluorescence, Infection

    Virus titers according to number of the porcine epidemic diarrhea virus (PEDV) passages (A) and growth curves of the QIAP1401-P10, -P40, and -P70 strains according to time of harvesting in Vero cells (B). TCID, tissue culture infectious dose.
    Figure Legend Snippet: Virus titers according to number of the porcine epidemic diarrhea virus (PEDV) passages (A) and growth curves of the QIAP1401-P10, -P40, and -P70 strains according to time of harvesting in Vero cells (B). TCID, tissue culture infectious dose.

    Techniques Used:

    Virus particles from the QIAP1401 strain propagated in Vero cells. Negatively stained porcine epidemic diarrhea virus particles of 80 to 100 nm in diameter are visible (100,000×). Scale bar = 100 nm.
    Figure Legend Snippet: Virus particles from the QIAP1401 strain propagated in Vero cells. Negatively stained porcine epidemic diarrhea virus particles of 80 to 100 nm in diameter are visible (100,000×). Scale bar = 100 nm.

    Techniques Used: Staining

    25) Product Images from "Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014"

    Article Title: Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2018.19.1.71

    Cytopathic effects of (A–C), and immunofluorescence results for (D–F), porcine epidemic diarrhea virus isolates in infected Vero cells at 200×. Vero cells were infected with the QIAP1401 or QIAP1402 isolates and maintained in DMEM containing 1 µg/mL trypsin
    Figure Legend Snippet: Cytopathic effects of (A–C), and immunofluorescence results for (D–F), porcine epidemic diarrhea virus isolates in infected Vero cells at 200×. Vero cells were infected with the QIAP1401 or QIAP1402 isolates and maintained in DMEM containing 1 µg/mL trypsin

    Techniques Used: Immunofluorescence, Infection

    Virus titers according to number of the porcine epidemic diarrhea virus (PEDV) passages (A) and growth curves of the QIAP1401-P10, -P40, and -P70 strains according to time of harvesting in Vero cells (B). TCID, tissue culture infectious dose.
    Figure Legend Snippet: Virus titers according to number of the porcine epidemic diarrhea virus (PEDV) passages (A) and growth curves of the QIAP1401-P10, -P40, and -P70 strains according to time of harvesting in Vero cells (B). TCID, tissue culture infectious dose.

    Techniques Used:

    Virus particles from the QIAP1401 strain propagated in Vero cells. Negatively stained porcine epidemic diarrhea virus particles of 80 to 100 nm in diameter are visible (100,000×). Scale bar = 100 nm.
    Figure Legend Snippet: Virus particles from the QIAP1401 strain propagated in Vero cells. Negatively stained porcine epidemic diarrhea virus particles of 80 to 100 nm in diameter are visible (100,000×). Scale bar = 100 nm.

    Techniques Used: Staining

    26) Product Images from "Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus"

    Article Title: Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01816-15

    GRFT interference with infection as a measure of activity against HIV (A), HSV (B), and HPV (C). We determined antiviral activity using the TZM-bl cell assay for anti-HIV-1 activity and the luciferase assay (HeLa cells) for anti-HPV16 PsV activity. The dye uptake assay (with PrestoBlue) was used to test the in vitro susceptibility of HSV-2 in Vero cells. The graphs show the percent virus replication or reporter gene expression (mean ± SD) relative to that for the virus control (triplicate assays were performed per condition). The dotted and shaded vertical lines represent the EC 50 s with their 95% confidence intervals.
    Figure Legend Snippet: GRFT interference with infection as a measure of activity against HIV (A), HSV (B), and HPV (C). We determined antiviral activity using the TZM-bl cell assay for anti-HIV-1 activity and the luciferase assay (HeLa cells) for anti-HPV16 PsV activity. The dye uptake assay (with PrestoBlue) was used to test the in vitro susceptibility of HSV-2 in Vero cells. The graphs show the percent virus replication or reporter gene expression (mean ± SD) relative to that for the virus control (triplicate assays were performed per condition). The dotted and shaded vertical lines represent the EC 50 s with their 95% confidence intervals.

    Techniques Used: Infection, Activity Assay, Luciferase, In Vitro, Expressing

    GRFT prevents HSV-2 and HPV postadsorption events. (A) HSV-2 G or HPV16 PsV and different concentrations of GRFT or medium (virus control) were preincubated for 0 h, 0.5 h, and 2 h at 37°C before being added to prechilled Vero cells (for HSV-2) or HeLa cells (for HPV16 PsV) and kept at 4°C for 2 h. The cells were washed 3 times before addition of the overlay and incubation at 48 h and 37°C in a 5% CO 2 atmosphere with 98% humidity. Finally, the cells were fixed and stained prior to counting the numbers of PFU. (B) The same as in panel A for HSV-2, but after 2 h of incubation at 4°C, the cells were switched to 37°C for an additional 2 h, followed by a 2-min treatment with citric acid buffer (pH 3.0). (C) Different concentrations of CG or GRFT were added at the indicated time points, with time zero representing the time of initial HPV16 PsV inoculation. The graph shows the percent reporter gene expression (mean ± SD) relative to that for the virus control (triplicate assays were performed per condition). (D) Western blot of purified HSV-2 lysate. Membranes were incubated overnight at 4°C with mouse anti-gD MAb DL11 (lane 1) or 1 μg/ml GRFT (lane 2). The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated anti-mouse Ig (lane 1) or were incubated overnight at 4°C with rabbit anti-GRFT antibody (lane 2), before being washed and incubated with HRP-conjugated anti-rabbit immunoglobulin antibody (1 h at room temperature). (E) HSV-2-infected Vero cells were lysed, preincubated with GRFT, and immunoprecipitated with anti-GRFT antibody. Lanes 1, cell lysate without immunoprecipitation; lanes 2, HeLa cell lysate 24 h after HSV-2 infection with immunoprecipitation in the presence of GRFT and anti-GRFT antibody; lanes 3, same as lanes 2 but 48 h after HSV-2 infection; lanes 4 and 5, immunoprecipitation controls without and with GRFT, respectively. In the experiments described in the legends to panels D and E, the membranes were processed using an ECL Western blotting detection system to capture the bands on X-ray films. A representative blot of three repeats is shown. (F) Soluble GRFT, N-glycosylated (N-glyco), nonglycosylated gD, or a mixture of N-glycosylated and nonglycosylated gD-GRFT was preincubated before addition to Vero cells. HSV-2 G was then added, and infection was detected by an immunohistochemical assay after overnight incubation. Statistical analysis was performed using the Mann-Whitney U test ( P
    Figure Legend Snippet: GRFT prevents HSV-2 and HPV postadsorption events. (A) HSV-2 G or HPV16 PsV and different concentrations of GRFT or medium (virus control) were preincubated for 0 h, 0.5 h, and 2 h at 37°C before being added to prechilled Vero cells (for HSV-2) or HeLa cells (for HPV16 PsV) and kept at 4°C for 2 h. The cells were washed 3 times before addition of the overlay and incubation at 48 h and 37°C in a 5% CO 2 atmosphere with 98% humidity. Finally, the cells were fixed and stained prior to counting the numbers of PFU. (B) The same as in panel A for HSV-2, but after 2 h of incubation at 4°C, the cells were switched to 37°C for an additional 2 h, followed by a 2-min treatment with citric acid buffer (pH 3.0). (C) Different concentrations of CG or GRFT were added at the indicated time points, with time zero representing the time of initial HPV16 PsV inoculation. The graph shows the percent reporter gene expression (mean ± SD) relative to that for the virus control (triplicate assays were performed per condition). (D) Western blot of purified HSV-2 lysate. Membranes were incubated overnight at 4°C with mouse anti-gD MAb DL11 (lane 1) or 1 μg/ml GRFT (lane 2). The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated anti-mouse Ig (lane 1) or were incubated overnight at 4°C with rabbit anti-GRFT antibody (lane 2), before being washed and incubated with HRP-conjugated anti-rabbit immunoglobulin antibody (1 h at room temperature). (E) HSV-2-infected Vero cells were lysed, preincubated with GRFT, and immunoprecipitated with anti-GRFT antibody. Lanes 1, cell lysate without immunoprecipitation; lanes 2, HeLa cell lysate 24 h after HSV-2 infection with immunoprecipitation in the presence of GRFT and anti-GRFT antibody; lanes 3, same as lanes 2 but 48 h after HSV-2 infection; lanes 4 and 5, immunoprecipitation controls without and with GRFT, respectively. In the experiments described in the legends to panels D and E, the membranes were processed using an ECL Western blotting detection system to capture the bands on X-ray films. A representative blot of three repeats is shown. (F) Soluble GRFT, N-glycosylated (N-glyco), nonglycosylated gD, or a mixture of N-glycosylated and nonglycosylated gD-GRFT was preincubated before addition to Vero cells. HSV-2 G was then added, and infection was detected by an immunohistochemical assay after overnight incubation. Statistical analysis was performed using the Mann-Whitney U test ( P

    Techniques Used: Incubation, Staining, Expressing, Western Blot, Purification, Infection, Immunoprecipitation, Immunohistochemistry, MANN-WHITNEY

    27) Product Images from "dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA"

    Article Title: dsRNA-Seq: Identification of Viral Infection by Purifying and Sequencing dsRNA

    Journal: Viruses

    doi: 10.3390/v11100943

    dsRNA-Seq detects viral infections of cultured mammalian cells. ( A ) Outline of the dsRNA purification method; ( B ) number of dsRNA contigs assembled from dsRNA-Seq reads from infected or mock infected Vero cell samples and their classification based on mapping to host nuclear or mitochondrial chromosomes or BLASTn analysis against NCBI nt; ( C ) percentage of dsRNA-Seq reads that align to the host nuclear or mitochondrial chromosomes, influenza A viral genome, dengue virus type 2 genome, or did not align (unknown). For ( D ) and ( E ), viral genomes are illustrated with protein coding regions indicated by colored boxes. Arrows indicate the alignment of contigs to viral genomes or genome segments. Contigs representing the positive strand are in red; negative strand in blue; ( D ) alignment of contigs assembled from Vero 2 sample to dengue virus type 2 genome; ( E ) alignment of contigs assembled from Vero 3 sample to influenza A viral segments.
    Figure Legend Snippet: dsRNA-Seq detects viral infections of cultured mammalian cells. ( A ) Outline of the dsRNA purification method; ( B ) number of dsRNA contigs assembled from dsRNA-Seq reads from infected or mock infected Vero cell samples and their classification based on mapping to host nuclear or mitochondrial chromosomes or BLASTn analysis against NCBI nt; ( C ) percentage of dsRNA-Seq reads that align to the host nuclear or mitochondrial chromosomes, influenza A viral genome, dengue virus type 2 genome, or did not align (unknown). For ( D ) and ( E ), viral genomes are illustrated with protein coding regions indicated by colored boxes. Arrows indicate the alignment of contigs to viral genomes or genome segments. Contigs representing the positive strand are in red; negative strand in blue; ( D ) alignment of contigs assembled from Vero 2 sample to dengue virus type 2 genome; ( E ) alignment of contigs assembled from Vero 3 sample to influenza A viral segments.

    Techniques Used: Cell Culture, Purification, Infection

    28) Product Images from "Rapid detection of feline morbillivirus by a reverse transcription loop-mediated isothermal amplification"

    Article Title: Rapid detection of feline morbillivirus by a reverse transcription loop-mediated isothermal amplification

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.15-0239

    Specificity and sensitivity of the RT-LAMP products and clinical application. (A) Specificity of the FmoPV RT-LAMP assay. RT-LAMP products amplified from RNAs extracted from CRFK infected with FmoPV strains, SS1, SS2 and SS3 (lanes 2, 4 and 6, respectively), and RT-LAMP products digested with Ava II (lanes 3, 5 and 7, respectively) are shown. An RT-LAMP product amplified from RNA extracted from uninfected CRFK cells is shown as a negative control (lane 1). Lane M: 100 bp DNA ladder. (B) Potential cross-reactions of the FmoPV RT-LAMP assay with CDV. RT-LAMP products amplified from RNA extracted from CRFK cells infected with FmoPV strain SS1 and uninfected Vero cells are shown as positive (lane 1) and negative (lane 2) controls, respectively. An RT-LAMP product amplified from RNA extracted from Vero cells infected with CDV strain Snyder Hill is shown in lane 3. Lane M: 100 bp DNA ladder. (C) Sensitivity comparison of the RT-LAMP assay with conventional RT-PCR. RT-LAMP (upper panel) and RT-PCR products (lower panel) from 10-fold serial dilutions of stock virus of FmoPV strain SS1 were electrophoresed. Amplicons from 10 −1 (lane 1), 10 −2 (lane 2), 10 −3 (lane 3), 10 −4 (lane 4), 10 −5 (lane 5), 10 −6 (lane 6), 10 −7 (lane 7) and 10 −8 dilutions (lane 8) are shown. RT-LAMP and RT-PCR products amplified from RNA extracted from uninfected CRFK cells are shown as a negative control (lane 9). Lane M: 100 bp DNA ladder. (D) Detection of FmoPV RNAs in clinical urine samples. RT-LAMP products amplified from RNAs extracted from urine samples of cats infected with FmoPV strains, SS1, SS2 and SS3 (lanes 2, 4 and 6, respectively), and the RT-LAMP products digested with Ava II (lanes 3, 5 and 7, respectively) are shown. An RT-LAMP product amplified from RNA extracted from an uninfected cat is shown as a negative control (lane 1). Lane M: 100 bp DNA ladder.
    Figure Legend Snippet: Specificity and sensitivity of the RT-LAMP products and clinical application. (A) Specificity of the FmoPV RT-LAMP assay. RT-LAMP products amplified from RNAs extracted from CRFK infected with FmoPV strains, SS1, SS2 and SS3 (lanes 2, 4 and 6, respectively), and RT-LAMP products digested with Ava II (lanes 3, 5 and 7, respectively) are shown. An RT-LAMP product amplified from RNA extracted from uninfected CRFK cells is shown as a negative control (lane 1). Lane M: 100 bp DNA ladder. (B) Potential cross-reactions of the FmoPV RT-LAMP assay with CDV. RT-LAMP products amplified from RNA extracted from CRFK cells infected with FmoPV strain SS1 and uninfected Vero cells are shown as positive (lane 1) and negative (lane 2) controls, respectively. An RT-LAMP product amplified from RNA extracted from Vero cells infected with CDV strain Snyder Hill is shown in lane 3. Lane M: 100 bp DNA ladder. (C) Sensitivity comparison of the RT-LAMP assay with conventional RT-PCR. RT-LAMP (upper panel) and RT-PCR products (lower panel) from 10-fold serial dilutions of stock virus of FmoPV strain SS1 were electrophoresed. Amplicons from 10 −1 (lane 1), 10 −2 (lane 2), 10 −3 (lane 3), 10 −4 (lane 4), 10 −5 (lane 5), 10 −6 (lane 6), 10 −7 (lane 7) and 10 −8 dilutions (lane 8) are shown. RT-LAMP and RT-PCR products amplified from RNA extracted from uninfected CRFK cells are shown as a negative control (lane 9). Lane M: 100 bp DNA ladder. (D) Detection of FmoPV RNAs in clinical urine samples. RT-LAMP products amplified from RNAs extracted from urine samples of cats infected with FmoPV strains, SS1, SS2 and SS3 (lanes 2, 4 and 6, respectively), and the RT-LAMP products digested with Ava II (lanes 3, 5 and 7, respectively) are shown. An RT-LAMP product amplified from RNA extracted from an uninfected cat is shown as a negative control (lane 1). Lane M: 100 bp DNA ladder.

    Techniques Used: RT Lamp Assay, Amplification, Infection, Antiviral Assay, Negative Control, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2"

    Article Title: Drug repurposing screens reveal FDA approved drugs active against SARS-Cov-2

    Journal: bioRxiv

    doi: 10.1101/2020.06.19.161042

    High-throughput screening in Vero cells to identify antivirals against SARS-CoV-2. A. Schematic of the screening strategy. Vero cells were plated in 384 well plates, drugs were added and the cells were infected with SARS-CoV-2 (MOI=1). 30 hpi cells were stained for viral infection (dsRNA, Spike) and imaged using automated microscopy to define cell number and percent infection. Antivirals show little impact on cell number and block viral infection. B. Dose-response analysis of Vero cells treated with hydroxychloroquine or remdesvir. C. Z-scores of the Vero drug screen performed at 1μM. 6 drugs had > 60% reduction in infection with > 80% cell viability. D. Dose-response analysis of three candidates identified in the screen.
    Figure Legend Snippet: High-throughput screening in Vero cells to identify antivirals against SARS-CoV-2. A. Schematic of the screening strategy. Vero cells were plated in 384 well plates, drugs were added and the cells were infected with SARS-CoV-2 (MOI=1). 30 hpi cells were stained for viral infection (dsRNA, Spike) and imaged using automated microscopy to define cell number and percent infection. Antivirals show little impact on cell number and block viral infection. B. Dose-response analysis of Vero cells treated with hydroxychloroquine or remdesvir. C. Z-scores of the Vero drug screen performed at 1μM. 6 drugs had > 60% reduction in infection with > 80% cell viability. D. Dose-response analysis of three candidates identified in the screen.

    Techniques Used: High Throughput Screening Assay, Infection, Staining, Microscopy, Blocking Assay

    Cell type specific dependencies of entry inhibitors. A. The human lung epithelial Calu-3 cells were infected with SARS-CoV-2 (MOI=0.5) and 48 hpi processed for microscopy. B. Dose response analysis of Calu-3 cells treated with quinolines or remdesvir. C. IC50, CC50 and SI for Vero, Huh7.5 and Calu-3 cells treated with a panel of quinolines or remdesivir. D. Dose response analysis of Calu-3 cells treated with cathepsin inhibitor Z-FA-FMK. E. Dose response analysis of Calu-3, Vero and Huh7.5 cells treated with camostat. F. IC50, CC50 and SI for camostat across cell types.
    Figure Legend Snippet: Cell type specific dependencies of entry inhibitors. A. The human lung epithelial Calu-3 cells were infected with SARS-CoV-2 (MOI=0.5) and 48 hpi processed for microscopy. B. Dose response analysis of Calu-3 cells treated with quinolines or remdesvir. C. IC50, CC50 and SI for Vero, Huh7.5 and Calu-3 cells treated with a panel of quinolines or remdesivir. D. Dose response analysis of Calu-3 cells treated with cathepsin inhibitor Z-FA-FMK. E. Dose response analysis of Calu-3, Vero and Huh7.5 cells treated with camostat. F. IC50, CC50 and SI for camostat across cell types.

    Techniques Used: Infection, Microscopy

    30) Product Images from "Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay"

    Article Title: Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay

    Journal: BioMed Research International

    doi: 10.1155/2018/8431243

    Characterization of rRSV-EGFP. (a) Identification of the rescued rRSV-EGFP by RT-PCR. M, DNA Ladder DL2000; control of HEp-2 cells (1), wt RSV Long infected HEp-2 cells (2), or rRSV-EGFP infected HEp-2 cells (3). (b) The replication titers of rRSV-EGFP during serial blind passages from p1 to p9 by immunoplaque assay. (c) The growth kinetic of rRSV-EGFP. The growth curve for rRSV-EGFP was compared with those for wt RSV Long, rRSV-RFP, and rRSV-Luc. Each virus was harvested every other 24 h postinfection and titers were assayed by immunoplaque assay. (d) The replication capacity of the rRSV-EGFP in HEp-2, Vero, or 293T cells. Viruses were harvested at 48 h postinfection and titers were determined by assays of immunoplaque and RT-qPCR. Data were shown as mean ± SD. ∗ P
    Figure Legend Snippet: Characterization of rRSV-EGFP. (a) Identification of the rescued rRSV-EGFP by RT-PCR. M, DNA Ladder DL2000; control of HEp-2 cells (1), wt RSV Long infected HEp-2 cells (2), or rRSV-EGFP infected HEp-2 cells (3). (b) The replication titers of rRSV-EGFP during serial blind passages from p1 to p9 by immunoplaque assay. (c) The growth kinetic of rRSV-EGFP. The growth curve for rRSV-EGFP was compared with those for wt RSV Long, rRSV-RFP, and rRSV-Luc. Each virus was harvested every other 24 h postinfection and titers were assayed by immunoplaque assay. (d) The replication capacity of the rRSV-EGFP in HEp-2, Vero, or 293T cells. Viruses were harvested at 48 h postinfection and titers were determined by assays of immunoplaque and RT-qPCR. Data were shown as mean ± SD. ∗ P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Quantitative RT-PCR

    31) Product Images from "Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1"

    Article Title: Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080712

    Neutralization of Stx1 holotoxin by the plantibody. Stx1 holotoxin was pre-treated with an extract of dimer Tg plants, an extract of a wild-type plants (WT), or control IgA (TEPC 15) for 1 h, and then the mixture was added to Stx1-sensitive cells. A. Cell viability assay. Vero cells were cultured for 48 h in the presence of 20 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA at various concentrations (abscissa). The IgA concentration is effective for Tg and TEPC 15, and TSP for Tg and WT. Cell viability was exhibited as a percentage of the control level (without toxin exposure). Data are expressed as means ± SD of triplicate determinations. Error bars underneath the symbols are not visible. B. DNA fragmentation. Vero cells were cultured for 48 h in the presence of 10 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA. DNA ladder formation was observed on agarose gel electrophoresis. Lane 1, untreated; lane 2, Stx1 only; lane 3, Stx1 + TEPC 15 (300 ng/ml IgA); lane 4, Stx1 + Tg (30 ng/ml IgA); lane 5, Stx1 + Tg (300 ng/ml IgA; 270 µg/ml TSP); lane 6, Stx1 + WT (270 µg/ml TSP). C. Caspase 3 activation. Ramos cells were cultured for 5 h in the presence of 10 pg/ml of Stx1 that had been pre-treated as indicated. Activated caspase 3 was probed with FITC-DEVD-FMK (abscissa), and analyzed with a flow cytometer. The number in each graph indicates the percentage of cells with activated caspase 3. D. Annexin V binding. Ramos cells were cultured as described for panel C. The binding of FITC-annexin V (abscissa) revealed cell surface exposure of phosphatidylserine. The number in each graph indicates the percentage of cells labeled with annexin V. The results are representative of three experiments.
    Figure Legend Snippet: Neutralization of Stx1 holotoxin by the plantibody. Stx1 holotoxin was pre-treated with an extract of dimer Tg plants, an extract of a wild-type plants (WT), or control IgA (TEPC 15) for 1 h, and then the mixture was added to Stx1-sensitive cells. A. Cell viability assay. Vero cells were cultured for 48 h in the presence of 20 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA at various concentrations (abscissa). The IgA concentration is effective for Tg and TEPC 15, and TSP for Tg and WT. Cell viability was exhibited as a percentage of the control level (without toxin exposure). Data are expressed as means ± SD of triplicate determinations. Error bars underneath the symbols are not visible. B. DNA fragmentation. Vero cells were cultured for 48 h in the presence of 10 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA. DNA ladder formation was observed on agarose gel electrophoresis. Lane 1, untreated; lane 2, Stx1 only; lane 3, Stx1 + TEPC 15 (300 ng/ml IgA); lane 4, Stx1 + Tg (30 ng/ml IgA); lane 5, Stx1 + Tg (300 ng/ml IgA; 270 µg/ml TSP); lane 6, Stx1 + WT (270 µg/ml TSP). C. Caspase 3 activation. Ramos cells were cultured for 5 h in the presence of 10 pg/ml of Stx1 that had been pre-treated as indicated. Activated caspase 3 was probed with FITC-DEVD-FMK (abscissa), and analyzed with a flow cytometer. The number in each graph indicates the percentage of cells with activated caspase 3. D. Annexin V binding. Ramos cells were cultured as described for panel C. The binding of FITC-annexin V (abscissa) revealed cell surface exposure of phosphatidylserine. The number in each graph indicates the percentage of cells labeled with annexin V. The results are representative of three experiments.

    Techniques Used: Neutralization, Viability Assay, Cell Culture, Incubation, Concentration Assay, Agarose Gel Electrophoresis, Activation Assay, Flow Cytometry, Cytometry, Binding Assay, Labeling

    32) Product Images from "Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response"

    Article Title: Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002783

    Activation of the UPR and WP1130 treatment show broad antiviral effects. (A–D) Cells were treated with DMSO, 3 µM thapsigargin (Thapsi), 5 µM WP1130, 2.5 µM Irestatin (Ires.), or both 2.5 µM Irestatin and 5 µM WP1130 (WP1130 Ires.) prior to infection. (A) La Crosse virus infection of Be2-c cells is inhibited by WP1130 or thapsigargin. Treated Be2-c cells were infected with La Crosse virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (B) Encephalomyocarditis virus (EMCV) infection of Vero cells is inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with EMCV virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (C) Vesicular stomatitis virus (VSV) infection of Vero cells is not inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with VSV virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (D) Sindbis virus infection of Vero cells is inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with Sindbis virus (MOI 5) for 12 hours, and viral titers determined by plaque assay on Vero cells. In all cases, data from at least three independent experiments with two experimental replicates per condition are presented as means +/− S.E.M. * P
    Figure Legend Snippet: Activation of the UPR and WP1130 treatment show broad antiviral effects. (A–D) Cells were treated with DMSO, 3 µM thapsigargin (Thapsi), 5 µM WP1130, 2.5 µM Irestatin (Ires.), or both 2.5 µM Irestatin and 5 µM WP1130 (WP1130 Ires.) prior to infection. (A) La Crosse virus infection of Be2-c cells is inhibited by WP1130 or thapsigargin. Treated Be2-c cells were infected with La Crosse virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (B) Encephalomyocarditis virus (EMCV) infection of Vero cells is inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with EMCV virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (C) Vesicular stomatitis virus (VSV) infection of Vero cells is not inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with VSV virus (MOI 5) for 12 hours and viral titers determined by plaque assay on Vero cells. (D) Sindbis virus infection of Vero cells is inhibited by WP1130 or thapsigargin. Treated Vero cells were infected with Sindbis virus (MOI 5) for 12 hours, and viral titers determined by plaque assay on Vero cells. In all cases, data from at least three independent experiments with two experimental replicates per condition are presented as means +/− S.E.M. * P

    Techniques Used: Activation Assay, Infection, Plaque Assay

    33) Product Images from "Role of Nuclear Factor Y in Stress-Induced Activation of the Herpes Simplex Virus Type 1 ICP0 Promoter ▿Role of Nuclear Factor Y in Stress-Induced Activation of the Herpes Simplex Virus Type 1 ICP0 Promoter ▿ †"

    Article Title: Role of Nuclear Factor Y in Stress-Induced Activation of the Herpes Simplex Virus Type 1 ICP0 Promoter ▿Role of Nuclear Factor Y in Stress-Induced Activation of the Herpes Simplex Virus Type 1 ICP0 Promoter ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01377-09

    Effect of heat shock on global HSV-1 transcription during lytic infection. Vero cells were infected with HSV-1 KOS at an MOI of 10 PFU/cell for 30 min, heat shocked for 1 h at 43°C or maintained at 37°C (mock heat shocked), and allowed to recover at 37°C for 1 h. Total RNA was then harvested, labeled, and hybridized to HSV-1 microarrays. Arrays were visualized by chemiluminescence, and images were captured with a charge-coupled device camera and processed with GEArray Expression Analysis Suite 2.0 (SABiosciences). The microarrays were standardized by using the interquartile method. The upper line represents a 1.5-fold increase, the middle line represents no change in expression, and the lower line represents a 1.5-fold decrease relative to the mock-treated control. Red stars indicate data points upregulated by > 1.5-fold with treatment, black stars indicate data points not significantly affected by treatment, and green stars indicate data points downregulated by > 1.5-fold compared to control. Upregulated genes of interest are indicated by circles and arrows. The figure represents three independent experiments.
    Figure Legend Snippet: Effect of heat shock on global HSV-1 transcription during lytic infection. Vero cells were infected with HSV-1 KOS at an MOI of 10 PFU/cell for 30 min, heat shocked for 1 h at 43°C or maintained at 37°C (mock heat shocked), and allowed to recover at 37°C for 1 h. Total RNA was then harvested, labeled, and hybridized to HSV-1 microarrays. Arrays were visualized by chemiluminescence, and images were captured with a charge-coupled device camera and processed with GEArray Expression Analysis Suite 2.0 (SABiosciences). The microarrays were standardized by using the interquartile method. The upper line represents a 1.5-fold increase, the middle line represents no change in expression, and the lower line represents a 1.5-fold decrease relative to the mock-treated control. Red stars indicate data points upregulated by > 1.5-fold with treatment, black stars indicate data points not significantly affected by treatment, and green stars indicate data points downregulated by > 1.5-fold compared to control. Upregulated genes of interest are indicated by circles and arrows. The figure represents three independent experiments.

    Techniques Used: Infection, Labeling, Expressing

    NF-Y activity is necessary for induction of the ICP0 promoter after heat shock during lytic infection. Vero cells were infected with wild-type, DN NF-YA, or GFP adenoviruses at an MOI of 25 PFU/cell and superinfected with HSV-1 KOS at an MOI of 1 PFU/cell for 30 min, heat shocked for 1 h at 43°C, and allowed to recover for 1 h at 37°C, and the RNA was harvested for RT-PCR. PCRs were performed in triplicate with primers specific for ICP0, ICP22, and β-actin. Reactions were standardized to β-actin. ICP0 (A) and ICP22 (B) transcript levels. The results are presented as the fold induction of transcription in response to heat shock (43 or 37°C) ± the SEM ( n = 3).
    Figure Legend Snippet: NF-Y activity is necessary for induction of the ICP0 promoter after heat shock during lytic infection. Vero cells were infected with wild-type, DN NF-YA, or GFP adenoviruses at an MOI of 25 PFU/cell and superinfected with HSV-1 KOS at an MOI of 1 PFU/cell for 30 min, heat shocked for 1 h at 43°C, and allowed to recover for 1 h at 37°C, and the RNA was harvested for RT-PCR. PCRs were performed in triplicate with primers specific for ICP0, ICP22, and β-actin. Reactions were standardized to β-actin. ICP0 (A) and ICP22 (B) transcript levels. The results are presented as the fold induction of transcription in response to heat shock (43 or 37°C) ± the SEM ( n = 3).

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

    Response of selected HSV-1 promoters to heat shock. (A) Viral promoter- and control-luciferase constructs used in these studies. Vero cells were transfected with the indicated HSV-1 promoter-firefly luciferase constructs in duplicate. At 24 h posttransfection, cells were heat shocked for 3 h at 43°C or maintained at 37°C (mock heat shocked). After a 4-h recovery period at 37°C, cells were harvested and subjected to the luciferase reporter assay (Promega). (B) Basal promoter activity (mock heat shocked) presented as relative light units (RLU). (C) Fold induction following heat shock. The data are presented as the fold change versus non-heat-shocked control (43°C/37°C) ± the standard error of the mean (SEM). IE genes are represented by hatched bars, E genes are represented by cross-hatched bars, DE genes are represented by small cross-hatched bars, and L genes are represented by a striped bar ( n ≥ 3). The luciferase activity of all samples was at least threefold higher than the background level (mock-transfected cells).
    Figure Legend Snippet: Response of selected HSV-1 promoters to heat shock. (A) Viral promoter- and control-luciferase constructs used in these studies. Vero cells were transfected with the indicated HSV-1 promoter-firefly luciferase constructs in duplicate. At 24 h posttransfection, cells were heat shocked for 3 h at 43°C or maintained at 37°C (mock heat shocked). After a 4-h recovery period at 37°C, cells were harvested and subjected to the luciferase reporter assay (Promega). (B) Basal promoter activity (mock heat shocked) presented as relative light units (RLU). (C) Fold induction following heat shock. The data are presented as the fold change versus non-heat-shocked control (43°C/37°C) ± the standard error of the mean (SEM). IE genes are represented by hatched bars, E genes are represented by cross-hatched bars, DE genes are represented by small cross-hatched bars, and L genes are represented by a striped bar ( n ≥ 3). The luciferase activity of all samples was at least threefold higher than the background level (mock-transfected cells).

    Techniques Used: Luciferase, Construct, Transfection, Reporter Assay, Activity Assay

    34) Product Images from "Cation-Independent Mannose 6-Phosphate Receptor Blocks Apoptosis Induced by Herpes Simplex Virus 1 Mutants Lacking Glycoprotein D and Is Likely the Target of Antiapoptotic Activity of the Glycoprotein"

    Article Title: Cation-Independent Mannose 6-Phosphate Receptor Blocks Apoptosis Induced by Herpes Simplex Virus 1 Mutants Lacking Glycoprotein D and Is Likely the Target of Antiapoptotic Activity of the Glycoprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.12.6197-6204.2002

    gD −/− and gD −/+ stocks of gD null mutants of HSV-1 induce cellular DNA fragmentation apoptosis in HEp-2, HeLa, SK-N-SH, and Vero cells. Replicate cultures of SK-N-SH, Vero, HEp-2, and HeLa cells were infected with gD −/− (A) or gD −/+ (B) stocks as shown. The cell were harvested at 18 h after infection and processed for the DNA fragmentation assay as described in Materials and Methods.
    Figure Legend Snippet: gD −/− and gD −/+ stocks of gD null mutants of HSV-1 induce cellular DNA fragmentation apoptosis in HEp-2, HeLa, SK-N-SH, and Vero cells. Replicate cultures of SK-N-SH, Vero, HEp-2, and HeLa cells were infected with gD −/− (A) or gD −/+ (B) stocks as shown. The cell were harvested at 18 h after infection and processed for the DNA fragmentation assay as described in Materials and Methods.

    Techniques Used: Infection, DNA Fragmentation Assay

    35) Product Images from "Cellular and Viral Requirements for Rapid Endocytic Entry of Herpes Simplex Virus"

    Article Title: Cellular and Viral Requirements for Rapid Endocytic Entry of Herpes Simplex Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.14.7508-7517.2004

    HSV glycoprotein requirements for infection of cells that support distinct entry pathways. HeLa, CHO-nectin-1, or Vero cells were infected with HSV-1 strain KOS mutants devoid of gB, gC, gD, or gL for 10 h. Total cell number and HSV antigen-positive cells were quantitated by immunofluorescence microscopy. Four-hundred to 500 cells per sample were evaluated. One-hundred percent represents the infectivity of an equivalent number of particles of the corresponding complemented virus.
    Figure Legend Snippet: HSV glycoprotein requirements for infection of cells that support distinct entry pathways. HeLa, CHO-nectin-1, or Vero cells were infected with HSV-1 strain KOS mutants devoid of gB, gC, gD, or gL for 10 h. Total cell number and HSV antigen-positive cells were quantitated by immunofluorescence microscopy. Four-hundred to 500 cells per sample were evaluated. One-hundred percent represents the infectivity of an equivalent number of particles of the corresponding complemented virus.

    Techniques Used: Infection, Immunofluorescence, Microscopy

    Intracellular trafficking of HSV. HSV-1 KOS was bound to CHO-nectin-1 cells (A) or CHO cells (B) for 2 h at 4°C. Cultures were shifted to 37°C in the presence or absence of 200 nM BFLA. At the indicated time p.i., following inactivation of extracellular virus, cells were lysed by two cycles of freezing and thawing. The numbers of infectious, intracellular particles were assayed by plaque titration on Vero cells. Data shown are representative of at least three independent experiments. For ultrastructural analysis, HSV-1 KOS (MOI of 50) was bound to cells for 2 h at 4°C followed by a shift to 37°C for 1 h, and then samples were processed for EM. (C) Naked, empty viral capsid docked at the nuclear pore complex of a CHO-nectin-1 cell. (D) Accumulation of degraded HSV particles in CHO cells. Enveloped virions are 150 to 200 nm, and capsids are ∼100 nm in diameter. Magnification, 90,000×. n, nucleus; ne, nuclear envelope.
    Figure Legend Snippet: Intracellular trafficking of HSV. HSV-1 KOS was bound to CHO-nectin-1 cells (A) or CHO cells (B) for 2 h at 4°C. Cultures were shifted to 37°C in the presence or absence of 200 nM BFLA. At the indicated time p.i., following inactivation of extracellular virus, cells were lysed by two cycles of freezing and thawing. The numbers of infectious, intracellular particles were assayed by plaque titration on Vero cells. Data shown are representative of at least three independent experiments. For ultrastructural analysis, HSV-1 KOS (MOI of 50) was bound to cells for 2 h at 4°C followed by a shift to 37°C for 1 h, and then samples were processed for EM. (C) Naked, empty viral capsid docked at the nuclear pore complex of a CHO-nectin-1 cell. (D) Accumulation of degraded HSV particles in CHO cells. Enveloped virions are 150 to 200 nm, and capsids are ∼100 nm in diameter. Magnification, 90,000×. n, nucleus; ne, nuclear envelope.

    Techniques Used: Titration

    36) 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 Genotype Viruses Differ in Growth and Foci Morphology (A and B) Viruses were analyzed for their ability to replicate in (A) C6/36 and (B) Vero cells at multiplicities of infection (MOI) of either 0.01 or 0.5 (mean ± SD of biological triplicates). (C–F) Viral foci were immunostained on both (C) C6/36 and (E) Vero cells, and average foci area for (D) C6/36 and (F) Vero cells were calculated using CTL ImmunoSpot analyzer software (mean ± SD of biological triplicates).
    Figure Legend Snippet: DENV4 Genotype Viruses Differ in Growth and Foci Morphology (A and B) Viruses were analyzed for their ability to replicate in (A) C6/36 and (B) Vero cells at multiplicities of infection (MOI) of either 0.01 or 0.5 (mean ± SD of biological triplicates). (C–F) Viral foci were immunostained on both (C) C6/36 and (E) Vero cells, and average foci area for (D) C6/36 and (F) Vero cells were calculated using CTL ImmunoSpot analyzer software (mean ± SD of biological triplicates).

    Techniques Used: Infection, CTL Assay, Software

    37) 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

    38) Product Images from "Effect of natural products on the production and activity of Clostridium difficile toxins in vitro"

    Article Title: Effect of natural products on the production and activity of Clostridium difficile toxins in vitro

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33954-2

    Protection from cytopathic effect on Vero cells using microscopy ( C . difficile NCTC 13366 culture filtrate and zingerone were incubated at 37 °C for 2 h prior to being added to Vero cell monolayers). ( A ) No culture filtrate; ( B ) culture filtrate only; ( C ) zingerone (1.2 mg/ml); ( D ) zingerone (0.6 mg/ml); ( E ) zingerone (0.3 mg/ml); Light microscopy ×40, Scale: 50 µm.
    Figure Legend Snippet: Protection from cytopathic effect on Vero cells using microscopy ( C . difficile NCTC 13366 culture filtrate and zingerone were incubated at 37 °C for 2 h prior to being added to Vero cell monolayers). ( A ) No culture filtrate; ( B ) culture filtrate only; ( C ) zingerone (1.2 mg/ml); ( D ) zingerone (0.6 mg/ml); ( E ) zingerone (0.3 mg/ml); Light microscopy ×40, Scale: 50 µm.

    Techniques Used: Microscopy, Incubation, Light Microscopy

    39) Product Images from "Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses"

    Article Title: Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses

    Journal: Viruses

    doi: 10.3390/v10040184

    ( A ) Structure of arbidol. ( B ) Cytotoxicities of arbidol with Huh-7, PS, UKF-NB-4, HBCA, and Vero cells within the compound concentration ranges 0–100 μM, 48 h post infection. ( C ) Antiviral effects of arbidol against ZIKV, WNV and TBEV infection in different cell lines. Given differential arbidol cytotoxicities with respect to different cell lines, indicated cell lines were treated with different maximum concentrations of arbidol (12.5 µM for Huh-7, 25 µM for HBCA and PS, 30 µM for UKF-NB-4, and 50 µM for Vero) 24 h prior to virus infection. Culture supernatants were then collected 48 h post infection and individual viral titers were determined by plaque assay. ( D ) Dose-dependent effects of arbidol on virus titers 48 h post infection in Vero cells. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log 10 PFU/mL. ( E ) Inhibition of indicated flaviviruses in the presence of a serial dilution of arbidol. Data from two ( C ) or three ( B – E ) independent experiments done in triplicates. ** p
    Figure Legend Snippet: ( A ) Structure of arbidol. ( B ) Cytotoxicities of arbidol with Huh-7, PS, UKF-NB-4, HBCA, and Vero cells within the compound concentration ranges 0–100 μM, 48 h post infection. ( C ) Antiviral effects of arbidol against ZIKV, WNV and TBEV infection in different cell lines. Given differential arbidol cytotoxicities with respect to different cell lines, indicated cell lines were treated with different maximum concentrations of arbidol (12.5 µM for Huh-7, 25 µM for HBCA and PS, 30 µM for UKF-NB-4, and 50 µM for Vero) 24 h prior to virus infection. Culture supernatants were then collected 48 h post infection and individual viral titers were determined by plaque assay. ( D ) Dose-dependent effects of arbidol on virus titers 48 h post infection in Vero cells. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log 10 PFU/mL. ( E ) Inhibition of indicated flaviviruses in the presence of a serial dilution of arbidol. Data from two ( C ) or three ( B – E ) independent experiments done in triplicates. ** p

    Techniques Used: Concentration Assay, Infection, Plaque Assay, Inhibition, Serial Dilution

    40) Product Images from "Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses"

    Article Title: Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses

    Journal: Viruses

    doi: 10.3390/v10040184

    ( A ) Structure of arbidol. ( B ) Cytotoxicities of arbidol with Huh-7, PS, UKF-NB-4, HBCA, and Vero cells within the compound concentration ranges 0–100 μM, 48 h post infection. ( C ) Antiviral effects of arbidol against ZIKV, WNV and TBEV infection in different cell lines. Given differential arbidol cytotoxicities with respect to different cell lines, indicated cell lines were treated with different maximum concentrations of arbidol (12.5 µM for Huh-7, 25 µM for HBCA and PS, 30 µM for UKF-NB-4, and 50 µM for Vero) 24 h prior to virus infection. Culture supernatants were then collected 48 h post infection and individual viral titers were determined by plaque assay. ( D ) Dose-dependent effects of arbidol on virus titers 48 h post infection in Vero cells. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log 10 PFU/mL. ( E ) Inhibition of indicated flaviviruses in the presence of a serial dilution of arbidol. Data from two ( C ) or three ( B – E ) independent experiments done in triplicates. ** p
    Figure Legend Snippet: ( A ) Structure of arbidol. ( B ) Cytotoxicities of arbidol with Huh-7, PS, UKF-NB-4, HBCA, and Vero cells within the compound concentration ranges 0–100 μM, 48 h post infection. ( C ) Antiviral effects of arbidol against ZIKV, WNV and TBEV infection in different cell lines. Given differential arbidol cytotoxicities with respect to different cell lines, indicated cell lines were treated with different maximum concentrations of arbidol (12.5 µM for Huh-7, 25 µM for HBCA and PS, 30 µM for UKF-NB-4, and 50 µM for Vero) 24 h prior to virus infection. Culture supernatants were then collected 48 h post infection and individual viral titers were determined by plaque assay. ( D ) Dose-dependent effects of arbidol on virus titers 48 h post infection in Vero cells. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log 10 PFU/mL. ( E ) Inhibition of indicated flaviviruses in the presence of a serial dilution of arbidol. Data from two ( C ) or three ( B – E ) independent experiments done in triplicates. ** p

    Techniques Used: Concentration Assay, Infection, Plaque Assay, Inhibition, Serial Dilution

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    Article Snippet: .. Effect of interferon treatment on JUNV multiplication in murine cells To understand if IFN has direct impact on JUNV infection, we characterized the effects of IFN treatment on JUNV multiplication in primary murine embryonic fibroblast cells (MEF) derived from C57BL/6 mice, Vero cells or human lung epithelial A549 cells. .. MEF cells were treated with mouse IFN-β at 1, 10, 50 or 100 U/ml for 16 hrs before and after virus infection, meanwhile Vero cells and A549 cells were treated with human IFN-α, IFN-β or IFN-γ for 16 hrs before and after infection at 125, 250, 500 or 1000 U/ml ( ).

    Cell Culture:

    Article Title: Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium
    Article Snippet: .. Similarly, E . chaffeensis in Vero cells (ATCC, Manassas, VA) was cultured in the complete MEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 7% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 2 mM L-glutamine (Mediatech, Manassas, VA). .. Cultivation of E . chaffeensis in HL60 cells (ATCC, Manassas, VA) was in complete RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 2 mM L-glutamine (Mediatech, Manassas, VA) by following the protocols described elsewhere for Anaplasma phagocytophilum .

    Mouse Assay:

    Article Title: Potent Inhibition of Jun?n Virus Infection by Interferon in Murine Cells
    Article Snippet: .. Effect of interferon treatment on JUNV multiplication in murine cells To understand if IFN has direct impact on JUNV infection, we characterized the effects of IFN treatment on JUNV multiplication in primary murine embryonic fibroblast cells (MEF) derived from C57BL/6 mice, Vero cells or human lung epithelial A549 cells. .. MEF cells were treated with mouse IFN-β at 1, 10, 50 or 100 U/ml for 16 hrs before and after virus infection, meanwhile Vero cells and A549 cells were treated with human IFN-α, IFN-β or IFN-γ for 16 hrs before and after infection at 125, 250, 500 or 1000 U/ml ( ).

    other:

    Article Title: A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics
    Article Snippet: The fluorescent capsid proteins in Nedel exhibited cellular distribution similar to that of HSV-2 glycoproteins, as assessed by immunolabelling in Vero cells ( a).

    Article Title: Exploration of the anticandidal mechanism of Cassia spectabilis in debilitating candidiasis
    Article Snippet: Nevertheless, it was surprising that the toxic effect of C. spectabilis leaf extract was exhibited on C. albicans rather than on Vero cells, suggesting that the extract displayed selective toxicity towards yeast cells.

    Infection:

    Article Title: SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses
    Article Snippet: .. For each of these four competition experiments, three wells of a 6-well culture plate containing confluent Vero ATCC cells were inoculated with a mix of both viruses (TCID50 ratio of 50/50; global multiplicity of infection of 0.5) for 2 h at 37 °C with 5% CO2 . .. Then, the plates were washed (HBSS) and incubated for 48 h after the addition of 4 ml of fresh medium.

    Article Title: Potent Inhibition of Jun?n Virus Infection by Interferon in Murine Cells
    Article Snippet: .. Effect of interferon treatment on JUNV multiplication in murine cells To understand if IFN has direct impact on JUNV infection, we characterized the effects of IFN treatment on JUNV multiplication in primary murine embryonic fibroblast cells (MEF) derived from C57BL/6 mice, Vero cells or human lung epithelial A549 cells. .. MEF cells were treated with mouse IFN-β at 1, 10, 50 or 100 U/ml for 16 hrs before and after virus infection, meanwhile Vero cells and A549 cells were treated with human IFN-α, IFN-β or IFN-γ for 16 hrs before and after infection at 125, 250, 500 or 1000 U/ml ( ).

    Expressing:

    Article Title: TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus
    Article Snippet: .. LASV GP expressed in the pCAGG vector was used to generate the LASV-LCMV pseudovirions from constitutively expressing Vero cells ( ). .. VSV pseudovirions were produced by transfecting HEK 293T cells with plasmids containing either the LASV GP (Josiah; a kind gift from Robert Mandell, NewLink Genetics), VSV G (Indiana; a kind gift from Paul McCray, University of Iowa), or full-length EBOV GP (Mayinga; a kind gift Robert Davey, Texas Biomedical Research Institute) gene 18 to 24 h prior to transduction with LASV GPC-pseudotyped VSVΔG-eGFP at a multiplicity of infection (MOI) of ∼1.

    Plasmid Preparation:

    Article Title: TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus
    Article Snippet: .. LASV GP expressed in the pCAGG vector was used to generate the LASV-LCMV pseudovirions from constitutively expressing Vero cells ( ). .. VSV pseudovirions were produced by transfecting HEK 293T cells with plasmids containing either the LASV GP (Josiah; a kind gift from Robert Mandell, NewLink Genetics), VSV G (Indiana; a kind gift from Paul McCray, University of Iowa), or full-length EBOV GP (Mayinga; a kind gift Robert Davey, Texas Biomedical Research Institute) gene 18 to 24 h prior to transduction with LASV GPC-pseudotyped VSVΔG-eGFP at a multiplicity of infection (MOI) of ∼1.

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    ATCC vero e6 cells
    siRNA No. 14 inhibition of SARS-CoV-2 cytopathicity in <t>Vero</t> E6 cells (CPE assay and EC 50 ). Vero E6 cells were infected with SARS-CoV-2 and incubated for 2 days. (a) Mock-siRNA (100 nM). (b) 5 nM. (c) 10 nM. (d) 20 nM. (e) 30 nM. (f) 40 nM. (g) 50 nM. (h) 60 nM. (i) 70 nM. (j) 80 nM. (k) 90 nM. (l) 100 nM. (m) EC 50 of siRNA No. 14 using qRT-PCR.
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    siRNA No. 14 inhibition of SARS-CoV-2 cytopathicity in Vero E6 cells (CPE assay and EC 50 ). Vero E6 cells were infected with SARS-CoV-2 and incubated for 2 days. (a) Mock-siRNA (100 nM). (b) 5 nM. (c) 10 nM. (d) 20 nM. (e) 30 nM. (f) 40 nM. (g) 50 nM. (h) 60 nM. (i) 70 nM. (j) 80 nM. (k) 90 nM. (l) 100 nM. (m) EC 50 of siRNA No. 14 using qRT-PCR.

    Journal: bioRxiv

    Article Title: A Small interfering RNA lead targeting RNA-dependent RNA-polymerase effectively inhibit the SARS-CoV-2 infection in Golden Syrian hamster and Rhesus macaque

    doi: 10.1101/2020.07.07.190967

    Figure Lengend Snippet: siRNA No. 14 inhibition of SARS-CoV-2 cytopathicity in Vero E6 cells (CPE assay and EC 50 ). Vero E6 cells were infected with SARS-CoV-2 and incubated for 2 days. (a) Mock-siRNA (100 nM). (b) 5 nM. (c) 10 nM. (d) 20 nM. (e) 30 nM. (f) 40 nM. (g) 50 nM. (h) 60 nM. (i) 70 nM. (j) 80 nM. (k) 90 nM. (l) 100 nM. (m) EC 50 of siRNA No. 14 using qRT-PCR.

    Article Snippet: In vitro efficacy test using Vero E6 cells (plaque assay) To assess viral titers, a plaque assay was performed using Vero E6 cells in 6-well culture plates.

    Techniques: Inhibition, Infection, Incubation, Quantitative RT-PCR

    Radio-uptake of 99m Tc-pertechnetate by planar scintigraphy. (a) Experimental overview of in vitro evaluation of the rMERS-CoV/ hNIS virus. Vero E6 cells were infected with rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 or 0.04. At various time points postinfection, the cells were incubated with 99m Tc-pertechnetate, and images of the plates were acquired. (b) Plate layout for hNIS functional assays. (c) Representative images of the plates acquired at 24 h postinfection at an MOI of 0.01 (top plates) or 0.04 (bottom plates) after incubation with 99m Tc-pertechnetate.

    Journal: mSphere

    Article Title: The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis

    doi: 10.1128/mSphere.00540-18

    Figure Lengend Snippet: Radio-uptake of 99m Tc-pertechnetate by planar scintigraphy. (a) Experimental overview of in vitro evaluation of the rMERS-CoV/ hNIS virus. Vero E6 cells were infected with rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 or 0.04. At various time points postinfection, the cells were incubated with 99m Tc-pertechnetate, and images of the plates were acquired. (b) Plate layout for hNIS functional assays. (c) Representative images of the plates acquired at 24 h postinfection at an MOI of 0.01 (top plates) or 0.04 (bottom plates) after incubation with 99m Tc-pertechnetate.

    Article Snippet: Vero E6 cells (ATCC CRL-1586) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Lonza) supplemented with 5% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2 without antibiotics or antimycotics.

    Techniques: In Vitro, Infection, Incubation, Functional Assay

    Retention of hNIS transgene following viral kinetics analysis and serial passage. (a and b) Vero E6 cells were infected with rMERS-CoV/hNIS (a) or parental rMERS-CoV (b) at an MOI of 0.01 or 3 and then collected at 96 h postinfection for RT-PCR. (c) Retention of the hNIS gene following serial passage. RNA was extracted from cells 72 h postinfection followed by RT-PCR at passage 6. A positive-control virus (C+) and uninfected negative-control cells (C−) were used as controls.

    Journal: mSphere

    Article Title: The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis

    doi: 10.1128/mSphere.00540-18

    Figure Lengend Snippet: Retention of hNIS transgene following viral kinetics analysis and serial passage. (a and b) Vero E6 cells were infected with rMERS-CoV/hNIS (a) or parental rMERS-CoV (b) at an MOI of 0.01 or 3 and then collected at 96 h postinfection for RT-PCR. (c) Retention of the hNIS gene following serial passage. RNA was extracted from cells 72 h postinfection followed by RT-PCR at passage 6. A positive-control virus (C+) and uninfected negative-control cells (C−) were used as controls.

    Article Snippet: Vero E6 cells (ATCC CRL-1586) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Lonza) supplemented with 5% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2 without antibiotics or antimycotics.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

    Kinetics of rMERS-CoV/ hNIS and parental rMERS-CoV replication in Vero E6 cells. (a and b) Multistep (a) and one-step (b) growth curves of Vero E6 cells infected with rMERS-CoV (Parental) and rMERS-CoV/ hNIS ( hNIS ). Quantification of the release of infectious virus at the indicated time points (hours postexposure) was determined by plaque assays. Each data point represents the mean ± standard deviation (SD) (error bar) averaged from three independent experiments. (c and d) Cytopathology of rMERS-CoV and rMERS-CoV/ hNIS in Vero E6 cells. The cells were infected with either rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 (c) or 3 (d) and analyzed by light microscopy at the indicated time points. Photomicrographs were taken using a 40× objective.

    Journal: mSphere

    Article Title: The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis

    doi: 10.1128/mSphere.00540-18

    Figure Lengend Snippet: Kinetics of rMERS-CoV/ hNIS and parental rMERS-CoV replication in Vero E6 cells. (a and b) Multistep (a) and one-step (b) growth curves of Vero E6 cells infected with rMERS-CoV (Parental) and rMERS-CoV/ hNIS ( hNIS ). Quantification of the release of infectious virus at the indicated time points (hours postexposure) was determined by plaque assays. Each data point represents the mean ± standard deviation (SD) (error bar) averaged from three independent experiments. (c and d) Cytopathology of rMERS-CoV and rMERS-CoV/ hNIS in Vero E6 cells. The cells were infected with either rMERS-CoV or rMERS-CoV/ hNIS at an MOI of 0.01 (c) or 3 (d) and analyzed by light microscopy at the indicated time points. Photomicrographs were taken using a 40× objective.

    Article Snippet: Vero E6 cells (ATCC CRL-1586) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Lonza) supplemented with 5% fetal bovine serum (FBS) and incubated at 37°C and 5% CO2 without antibiotics or antimycotics.

    Techniques: Infection, Standard Deviation, Light Microscopy