Structured Review

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Comparison of the ASFV proteins expressed in WSL-HP, <t>HEK</t> 293 and <t>Vero</t> cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.
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1) Product Images from "The intracellular proteome of African swine fever virus"

Article Title: The intracellular proteome of African swine fever virus

Journal: Scientific Reports

doi: 10.1038/s41598-018-32985-z

Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.
Figure Legend Snippet: Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.

Techniques Used: Mass Spectrometry

Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.
Figure Legend Snippet: Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.

Techniques Used: Expressing

ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.
Figure Legend Snippet: ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.

Techniques Used: In Vitro

2) Product Images from "Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis"

Article Title: Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005455

Characteristics of rsMB generated using reverse genetics. (A) Growth kinetics of MB and rsMB in L929 and Vero cells. Cells were infected with recombinant viruses at an MOI of 0.1 PFU/cell and incubated for the intervals shown. Viral titers were determined using a plaque assay. (B) Syncytium formation in Vero cells infected with MB and rsMB. The cells were infected with the viruses and incubated for 12 h. The fixed cells were stained with Giemsa solution to visualize syncytium formation. (C) The electropherotype of dsRNA of MB and rsMB. The viral dsRNA was extracted from purified virions, separated using SDS-polyacrylamide gel electrophoresis, and visualized by ethidium bromide staining. Classes of gene segments based on their sizes (L, M, and S gene segments) are indicated.
Figure Legend Snippet: Characteristics of rsMB generated using reverse genetics. (A) Growth kinetics of MB and rsMB in L929 and Vero cells. Cells were infected with recombinant viruses at an MOI of 0.1 PFU/cell and incubated for the intervals shown. Viral titers were determined using a plaque assay. (B) Syncytium formation in Vero cells infected with MB and rsMB. The cells were infected with the viruses and incubated for 12 h. The fixed cells were stained with Giemsa solution to visualize syncytium formation. (C) The electropherotype of dsRNA of MB and rsMB. The viral dsRNA was extracted from purified virions, separated using SDS-polyacrylamide gel electrophoresis, and visualized by ethidium bromide staining. Classes of gene segments based on their sizes (L, M, and S gene segments) are indicated.

Techniques Used: Generated, Infection, Recombinant, Incubation, Plaque Assay, Staining, Purification, Polyacrylamide Gel Electrophoresis

Recovery of a recombinant virus expressing a fluorescence protein. (A) Schematic presentation of the S1 gene segment used for the recovery of rsMB/σC-ZsY (pT7-S1MB-σC-ZsY). The nucleotide sequence of 1001–1516 within the σC gene was replaced with the ZsYellow gene. (B) RT-PCR analysis of rsMB and rsMB/σC-ZsY. The S1 gene fragment was amplified by RT-PCR using viral dsRNA extracted from virions and primers specific for the MB S1 and ZsYellow sequences. The numbers show the S1 nucleotide position corresponding to the 5′ end of the S1-specific primers. (C) Growth kinetics of rsMB and rsMB/σC-ZsY in L929 cells. The cells were infected with the viruses at an MOI of 0.01 PFU/cell and incubated for various intervals. After freeze-thawing, the viral titer was determined by a plaque assay. (D) Expression of ZsYellow in cells infected with rsMB/σC-ZsY. Vero cells were infected with rsMB/σC-ZsY at an MOI of 0.05 PFU/cell and incubated for 24 h. Expression of ZsYellow in the syncytia of Vero cells was observed by confocal microscopy (yellow). Infected cells were fixed and stained using NBV-specific antiserum, followed by Alexa Fluor 633 Goat Anti-Mouse IgG second antibody (red). Cells were stained with DAPI to label nuclei (blue).
Figure Legend Snippet: Recovery of a recombinant virus expressing a fluorescence protein. (A) Schematic presentation of the S1 gene segment used for the recovery of rsMB/σC-ZsY (pT7-S1MB-σC-ZsY). The nucleotide sequence of 1001–1516 within the σC gene was replaced with the ZsYellow gene. (B) RT-PCR analysis of rsMB and rsMB/σC-ZsY. The S1 gene fragment was amplified by RT-PCR using viral dsRNA extracted from virions and primers specific for the MB S1 and ZsYellow sequences. The numbers show the S1 nucleotide position corresponding to the 5′ end of the S1-specific primers. (C) Growth kinetics of rsMB and rsMB/σC-ZsY in L929 cells. The cells were infected with the viruses at an MOI of 0.01 PFU/cell and incubated for various intervals. After freeze-thawing, the viral titer was determined by a plaque assay. (D) Expression of ZsYellow in cells infected with rsMB/σC-ZsY. Vero cells were infected with rsMB/σC-ZsY at an MOI of 0.05 PFU/cell and incubated for 24 h. Expression of ZsYellow in the syncytia of Vero cells was observed by confocal microscopy (yellow). Infected cells were fixed and stained using NBV-specific antiserum, followed by Alexa Fluor 633 Goat Anti-Mouse IgG second antibody (red). Cells were stained with DAPI to label nuclei (blue).

Techniques Used: Recombinant, Expressing, Fluorescence, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Incubation, Plaque Assay, Confocal Microscopy, Staining

3) Product Images from "Cytotoxic and Genotoxic Effects of Fluconazole on African Green Monkey Kidney (Vero) Cell Line"

Article Title: Cytotoxic and Genotoxic Effects of Fluconazole on African Green Monkey Kidney (Vero) Cell Line

Journal: BioMed Research International

doi: 10.1155/2018/6271547

Effects of fluconazole in Vero cell line analyzed by comet assay. ∗ P
Figure Legend Snippet: Effects of fluconazole in Vero cell line analyzed by comet assay. ∗ P

Techniques Used: Single Cell Gel Electrophoresis

Effects of fluconazole in Vero cells analyzed by MTT assay. ∗ P
Figure Legend Snippet: Effects of fluconazole in Vero cells analyzed by MTT assay. ∗ P

Techniques Used: MTT Assay

Effects of fluconazole in Vero cell line analyzed by differential fluorescent staining. ∗ P
Figure Legend Snippet: Effects of fluconazole in Vero cell line analyzed by differential fluorescent staining. ∗ P

Techniques Used: Staining

ROS generation induced by fluconazole in Vero cell line. ∗ P
Figure Legend Snippet: ROS generation induced by fluconazole in Vero cell line. ∗ P

Techniques Used:

4) Product Images from "Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection"

Article Title: Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection

Journal: Viruses

doi: 10.3390/v10110580

Effect of the gel formulation on HSV-2 replication in Vero cells and its spreading. Monolayer of Vero cells in 24-well culture plate was infected with HSV-2 (100 PFU/well) followed by 1% low melting point agarose overlay medium containing varying concentration of the gel formulation and acyclovir as described in Materials and Methods section. ( Panel a ) represents the efficacy of the gel formulation as well as gel base at different concentrations to inhibit formation of plaques by HSV-2. ( Panel b ) represents the efficacy of acyclovir to inhibit HSV-2 replication. The Y-axis represents percent inhibition in the number of plaques with respect to the untreated virus control group and X-axis the concentration of the gel formulation/gel base/acyclovir. Each bar in ( Panels a and b ) represent mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005, respectively, between treated and untreated control at tested concentration of the acyclovir/gel formulation.
Figure Legend Snippet: Effect of the gel formulation on HSV-2 replication in Vero cells and its spreading. Monolayer of Vero cells in 24-well culture plate was infected with HSV-2 (100 PFU/well) followed by 1% low melting point agarose overlay medium containing varying concentration of the gel formulation and acyclovir as described in Materials and Methods section. ( Panel a ) represents the efficacy of the gel formulation as well as gel base at different concentrations to inhibit formation of plaques by HSV-2. ( Panel b ) represents the efficacy of acyclovir to inhibit HSV-2 replication. The Y-axis represents percent inhibition in the number of plaques with respect to the untreated virus control group and X-axis the concentration of the gel formulation/gel base/acyclovir. Each bar in ( Panels a and b ) represent mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005, respectively, between treated and untreated control at tested concentration of the acyclovir/gel formulation.

Techniques Used: Infection, Concentration Assay, Inhibition

Effect of the gel formulation on the attachment and penetration of HSV-2 to the Vero cells. The effect of gel formulation on HSV-2 attachment and penetration to the Vero cells was assessed as described in Materials and Methods . ( Panel a ) represents the effect of gel formulation on the attachment of HSV-2 to the Vero cells. Pre-chilled monolayer of the Vero cells was incubated with HSV-2 (100 PFU) along with various concentrations of the gel formulation/gel base/acyclovir for 3 h followed by processing for determination of the plaque formation as described in Materials and Methods . The Y-axis represents percent inhibition of HSV-2 infection and X-axis represents concentration of gel formulation/gel base/acyclovir. ( Panel b ) represents the effect of gel formulation on the penetration of HSV-2 to the Vero cell. Pre-chilled confluent monolayer of the Vero cells was incubated with HSV-2 (100 PFU) for 3 h at 4 °C followed by incubation with the varying concentrations of the gel formulation/gel base/acyclovir and subsequently processed for the assessment of plaque formation. Values in ( Panels a and b ) are expressed as mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005 between treated and untreated control at respective concentration of the gel formulation.
Figure Legend Snippet: Effect of the gel formulation on the attachment and penetration of HSV-2 to the Vero cells. The effect of gel formulation on HSV-2 attachment and penetration to the Vero cells was assessed as described in Materials and Methods . ( Panel a ) represents the effect of gel formulation on the attachment of HSV-2 to the Vero cells. Pre-chilled monolayer of the Vero cells was incubated with HSV-2 (100 PFU) along with various concentrations of the gel formulation/gel base/acyclovir for 3 h followed by processing for determination of the plaque formation as described in Materials and Methods . The Y-axis represents percent inhibition of HSV-2 infection and X-axis represents concentration of gel formulation/gel base/acyclovir. ( Panel b ) represents the effect of gel formulation on the penetration of HSV-2 to the Vero cell. Pre-chilled confluent monolayer of the Vero cells was incubated with HSV-2 (100 PFU) for 3 h at 4 °C followed by incubation with the varying concentrations of the gel formulation/gel base/acyclovir and subsequently processed for the assessment of plaque formation. Values in ( Panels a and b ) are expressed as mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005 between treated and untreated control at respective concentration of the gel formulation.

Techniques Used: Incubation, Inhibition, Infection, Concentration Assay

5) Product Images from "Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus"

Article Title: Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus

Journal: Journal of Virology

doi: 10.1128/JVI.00344-19

Inhibitory effect of the glycans against MuV entry. The GFP-recombinant MuV (1.2 × 10 2 PFU) was preincubated with the serially diluted 3′-SL, 6′-SL, LnNT, GM2-GS, or SLe X pentaose. Subsequently, Vero cells were infected with one of the viral combinations for 1 h at 37°C. After the aspiration of the viral solution, the infected cells were washed with phosphate-buffered saline and overlaid with 0.75% agar for the plaque assay. The EGFP-expressing recombinant MeV containing the Edmonston H protein was also preincubated with 3′-SL and then used in a similar fashion to infect Vero cells as a negative control. The viral entry was evaluated by counting GFP-positive plaques using fluorescence microscopy at 3 dpi for MuV and at 2 dpi for MeV (plaque sizes of MeV under the condition used were too large to count accurately at 3 dpi). The plaque numbers obtained from MuV or MeV preincubated with glycan-free medium are set to 1. Data are the means ± standard deviations of three samples. Data shown in this figure are representative of three independently performed experiments.
Figure Legend Snippet: Inhibitory effect of the glycans against MuV entry. The GFP-recombinant MuV (1.2 × 10 2 PFU) was preincubated with the serially diluted 3′-SL, 6′-SL, LnNT, GM2-GS, or SLe X pentaose. Subsequently, Vero cells were infected with one of the viral combinations for 1 h at 37°C. After the aspiration of the viral solution, the infected cells were washed with phosphate-buffered saline and overlaid with 0.75% agar for the plaque assay. The EGFP-expressing recombinant MeV containing the Edmonston H protein was also preincubated with 3′-SL and then used in a similar fashion to infect Vero cells as a negative control. The viral entry was evaluated by counting GFP-positive plaques using fluorescence microscopy at 3 dpi for MuV and at 2 dpi for MeV (plaque sizes of MeV under the condition used were too large to count accurately at 3 dpi). The plaque numbers obtained from MuV or MeV preincubated with glycan-free medium are set to 1. Data are the means ± standard deviations of three samples. Data shown in this figure are representative of three independently performed experiments.

Techniques Used: Recombinant, Infection, Plaque Assay, Expressing, Negative Control, Fluorescence, Microscopy

6) Product Images from "Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling"

Article Title: Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

Journal: Journal of Virology

doi: 10.1128/JVI.02199-05

Wild-type and dsRNA-binding mutant VP35s inhibit IFN-β gene expression induced by SeV infection. (A) Increasing concentrations (25, 250, or 2,500 ng, indicated by wedges) of plasmids expressing VP35 and the K309A and R312A mutants were transfected into 293T cells together with 300 ng each of an IFN-β-CAT reporter and a constitutive pCAGGS-firefly luciferase reporter. Twenty-four h posttransfection, cells were infected with Sendai virus (MOI of 8), and 12 h postinfection, CAT and luciferase activities were determined. Values are expressed as induction (fold) over an empty-plasmid mock-infected control (not shown). Virus-induced CAT activity was normalized to firefly luciferase activity. Error bars indicate standard deviations of at least three independent experiments. Expression levels of the different VP35 constructs were determined by Western blotting (inset). Blots were probed with a monoclonal antibody to VP35 (6C5). Two exposures of the same blot are shown; expression levels in the 25-ng samples were detected only when the film was overexposed (inset, lower panel). (B) After UV irradiation, a series of twofold dilutions of conditioned media from the experiment described in panel A was overlaid onto Vero cells in a 96-well plate. Twenty-four h after treatment, cells were infected with NDV-GFP (MOI of 6), and 24 h postinfection, virus replication was examined by fluorescence microscopy. Shown are the results obtained when the conditioned media from 293T cells transfected with 25, 250, or 2,500 ng of VP35 and R312A and K309A mutant plasmids were used. Right column: in the empty-vector mock-infected panel, NDV-GFP replication is readily detected by the presence of green fluorescence. The empty-vector SeV-infected panel lacks GFP expression, demonstrating the antiviral state created by IFN. IFN is present in the conditioned media due to the Sendai virus infection of the transfected 293T cells. This is demonstrated in the empty-vector SeV-infected plus anti-IFN-β panel, where neutralizing anti-IFN-β antibody rescues NDV-GFP replication. All panels are from cells treated with a 16-fold dilution of the conditioned medium.
Figure Legend Snippet: Wild-type and dsRNA-binding mutant VP35s inhibit IFN-β gene expression induced by SeV infection. (A) Increasing concentrations (25, 250, or 2,500 ng, indicated by wedges) of plasmids expressing VP35 and the K309A and R312A mutants were transfected into 293T cells together with 300 ng each of an IFN-β-CAT reporter and a constitutive pCAGGS-firefly luciferase reporter. Twenty-four h posttransfection, cells were infected with Sendai virus (MOI of 8), and 12 h postinfection, CAT and luciferase activities were determined. Values are expressed as induction (fold) over an empty-plasmid mock-infected control (not shown). Virus-induced CAT activity was normalized to firefly luciferase activity. Error bars indicate standard deviations of at least three independent experiments. Expression levels of the different VP35 constructs were determined by Western blotting (inset). Blots were probed with a monoclonal antibody to VP35 (6C5). Two exposures of the same blot are shown; expression levels in the 25-ng samples were detected only when the film was overexposed (inset, lower panel). (B) After UV irradiation, a series of twofold dilutions of conditioned media from the experiment described in panel A was overlaid onto Vero cells in a 96-well plate. Twenty-four h after treatment, cells were infected with NDV-GFP (MOI of 6), and 24 h postinfection, virus replication was examined by fluorescence microscopy. Shown are the results obtained when the conditioned media from 293T cells transfected with 25, 250, or 2,500 ng of VP35 and R312A and K309A mutant plasmids were used. Right column: in the empty-vector mock-infected panel, NDV-GFP replication is readily detected by the presence of green fluorescence. The empty-vector SeV-infected panel lacks GFP expression, demonstrating the antiviral state created by IFN. IFN is present in the conditioned media due to the Sendai virus infection of the transfected 293T cells. This is demonstrated in the empty-vector SeV-infected plus anti-IFN-β panel, where neutralizing anti-IFN-β antibody rescues NDV-GFP replication. All panels are from cells treated with a 16-fold dilution of the conditioned medium.

Techniques Used: Binding Assay, Mutagenesis, Expressing, Infection, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct, Western Blot, Irradiation, Fluorescence, Microscopy

7) Product Images from "Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus"

Article Title: Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus

Journal: AMB Express

doi: 10.1186/s13568-019-0916-0

The 50% cytotoxicity concentration of crude extract derived from Bacillus licheniformis (BLFP) in Vero cells. Different concentrations of BLFP crude extract were added to Vero cells and incubated for 48 h. BLFP crude extract showed no cytotoxicity to Vero cells when evaluated using the AlamarBlue™ assay. Data are presented as the mean ± SD out of three test replicates
Figure Legend Snippet: The 50% cytotoxicity concentration of crude extract derived from Bacillus licheniformis (BLFP) in Vero cells. Different concentrations of BLFP crude extract were added to Vero cells and incubated for 48 h. BLFP crude extract showed no cytotoxicity to Vero cells when evaluated using the AlamarBlue™ assay. Data are presented as the mean ± SD out of three test replicates

Techniques Used: Concentration Assay, Derivative Assay, Incubation, Alamar Blue Assay

Replication kinetics of PEDV in Vero cells treated with or without BLFP crude extract. a Extracellular viral titers in the supernatants of PEDV-infected Vero cells treated with and without BLFP crude extract were determined by viral titration in Vero cells using the Reed-Müench method and expressed as the 50% TCID 50 /ml. b Extracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time reverse transcription (RT)-PCR. c Intracellular viral titers in PEDV-infected Vero cells treated with or without BLFP crude extract were determined by viral titration in Vero cells. d Intracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time RT-PCR. Statistical analysis was performed using Student’s t-test, and statistically significant differences were labeled with *( p
Figure Legend Snippet: Replication kinetics of PEDV in Vero cells treated with or without BLFP crude extract. a Extracellular viral titers in the supernatants of PEDV-infected Vero cells treated with and without BLFP crude extract were determined by viral titration in Vero cells using the Reed-Müench method and expressed as the 50% TCID 50 /ml. b Extracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time reverse transcription (RT)-PCR. c Intracellular viral titers in PEDV-infected Vero cells treated with or without BLFP crude extract were determined by viral titration in Vero cells. d Intracellular viral RNA levels in the supernatants of PEDV-infected Vero cells treated with or without BLFP crude extract were determined by real-time RT-PCR. Statistical analysis was performed using Student’s t-test, and statistically significant differences were labeled with *( p

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

Antiviral activity of crude extract derived from Bacillus licheniformis (BLFP) against PEDV infection, as well as the IC 50 , was determined using the AlamarBlue™ assay. a PEDV-infected Vero cells treated with (left) and without (middle) 150 ppm BLFP crude extract. The right panel shows Vero cells without any treatment. Brown coloration indicates a positive PEDV S protein signal located in the cytoplasm of PEDV-infected cells. b Dose-dependent effect of BLFP crude extract against PEDV. The half-maximal inhibitory concentration (IC 50 ) of BLFP crude extract against PEDV in Vero cells is 0.07 ± 0.45 ppm. Data are presented as the mean ± SD out of three test replicates
Figure Legend Snippet: Antiviral activity of crude extract derived from Bacillus licheniformis (BLFP) against PEDV infection, as well as the IC 50 , was determined using the AlamarBlue™ assay. a PEDV-infected Vero cells treated with (left) and without (middle) 150 ppm BLFP crude extract. The right panel shows Vero cells without any treatment. Brown coloration indicates a positive PEDV S protein signal located in the cytoplasm of PEDV-infected cells. b Dose-dependent effect of BLFP crude extract against PEDV. The half-maximal inhibitory concentration (IC 50 ) of BLFP crude extract against PEDV in Vero cells is 0.07 ± 0.45 ppm. Data are presented as the mean ± SD out of three test replicates

Techniques Used: Activity Assay, Derivative Assay, Infection, Alamar Blue Assay, Concentration Assay

8) Product Images from "Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection"

Article Title: Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection

Journal: Viruses

doi: 10.3390/v10110580

Effect of the gel formulation on HSV-2 replication in Vero cells and its spreading. Monolayer of Vero cells in 24-well culture plate was infected with HSV-2 (100 PFU/well) followed by 1% low melting point agarose overlay medium containing varying concentration of the gel formulation and acyclovir as described in Materials and Methods section. ( Panel a ) represents the efficacy of the gel formulation as well as gel base at different concentrations to inhibit formation of plaques by HSV-2. ( Panel b ) represents the efficacy of acyclovir to inhibit HSV-2 replication. The Y-axis represents percent inhibition in the number of plaques with respect to the untreated virus control group and X-axis the concentration of the gel formulation/gel base/acyclovir. Each bar in ( Panels a and b ) represent mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005, respectively, between treated and untreated control at tested concentration of the acyclovir/gel formulation.
Figure Legend Snippet: Effect of the gel formulation on HSV-2 replication in Vero cells and its spreading. Monolayer of Vero cells in 24-well culture plate was infected with HSV-2 (100 PFU/well) followed by 1% low melting point agarose overlay medium containing varying concentration of the gel formulation and acyclovir as described in Materials and Methods section. ( Panel a ) represents the efficacy of the gel formulation as well as gel base at different concentrations to inhibit formation of plaques by HSV-2. ( Panel b ) represents the efficacy of acyclovir to inhibit HSV-2 replication. The Y-axis represents percent inhibition in the number of plaques with respect to the untreated virus control group and X-axis the concentration of the gel formulation/gel base/acyclovir. Each bar in ( Panels a and b ) represent mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005, respectively, between treated and untreated control at tested concentration of the acyclovir/gel formulation.

Techniques Used: Infection, Concentration Assay, Inhibition

Effect of the gel formulation on the attachment and penetration of HSV-2 to the Vero cells. The effect of gel formulation on HSV-2 attachment and penetration to the Vero cells was assessed as described in Materials and Methods . ( Panel a ) represents the effect of gel formulation on the attachment of HSV-2 to the Vero cells. Pre-chilled monolayer of the Vero cells was incubated with HSV-2 (100 PFU) along with various concentrations of the gel formulation/gel base/acyclovir for 3 h followed by processing for determination of the plaque formation as described in Materials and Methods . The Y-axis represents percent inhibition of HSV-2 infection and X-axis represents concentration of gel formulation/gel base/acyclovir. ( Panel b ) represents the effect of gel formulation on the penetration of HSV-2 to the Vero cell. Pre-chilled confluent monolayer of the Vero cells was incubated with HSV-2 (100 PFU) for 3 h at 4 °C followed by incubation with the varying concentrations of the gel formulation/gel base/acyclovir and subsequently processed for the assessment of plaque formation. Values in ( Panels a and b ) are expressed as mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005 between treated and untreated control at respective concentration of the gel formulation.
Figure Legend Snippet: Effect of the gel formulation on the attachment and penetration of HSV-2 to the Vero cells. The effect of gel formulation on HSV-2 attachment and penetration to the Vero cells was assessed as described in Materials and Methods . ( Panel a ) represents the effect of gel formulation on the attachment of HSV-2 to the Vero cells. Pre-chilled monolayer of the Vero cells was incubated with HSV-2 (100 PFU) along with various concentrations of the gel formulation/gel base/acyclovir for 3 h followed by processing for determination of the plaque formation as described in Materials and Methods . The Y-axis represents percent inhibition of HSV-2 infection and X-axis represents concentration of gel formulation/gel base/acyclovir. ( Panel b ) represents the effect of gel formulation on the penetration of HSV-2 to the Vero cell. Pre-chilled confluent monolayer of the Vero cells was incubated with HSV-2 (100 PFU) for 3 h at 4 °C followed by incubation with the varying concentrations of the gel formulation/gel base/acyclovir and subsequently processed for the assessment of plaque formation. Values in ( Panels a and b ) are expressed as mean ± SEM of the three independent experiments performed in duplicate. * p ≤ 0.05, ** p ≤ 0.005 between treated and untreated control at respective concentration of the gel formulation.

Techniques Used: Incubation, Inhibition, Infection, Concentration Assay

9) Product Images from "Viral RNA is a target for Wolbachia-mediated pathogen blocking"

Article Title: Viral RNA is a target for Wolbachia-mediated pathogen blocking

Journal: bioRxiv

doi: 10.1101/2020.04.03.023556

Presence of Wolbachia in mosquito cells reduces progeny virus infectivity in mammalian cells. RML12 mosquito cells with and without Wolbachia ( w Mel) were infected with alphaviruses Sindbis (SINV), Chikungunya (CHIKV) or flavivirus Zika (ZIKV) at MOI of 10. Viral supernatants were harvested at 48 hours post infection after which infectious virus titer was quantified by performing plaque assays on BHK-21 (SINV, CHIKV) and Vero (ZIKV) cells. Total virus particles were determined by quantifying viral genome copies present in the supernatant using qRT-PCR. Reported specific infectivity (SI) ratios were calculated as total infectious virus titer divided by total particles produced per mL of viral supernatant. (A) Percentage of total SINV particles produced from cells with and without Wolbachia that are infectious on BHK-21 cells. (B) Specific Infectivity Ratios of progeny SINV, CHIKV and ZIKV viruses. (C) Maximum-likelihood tree representing the phylogenetic relationship between the Wolbachia strains used in the study was generated using MEGA X, using a MUSCLE alignment of concatenated sequences of multi-locus typing (MLST) genes ( coxA, gatB, ftsZ, hcpA, fbpA) . Sequences from Wolbachia strain w Bm, native to the filarial nematode Brugia malayi , was used as a distant outgroup. Scale bar represent branch lengths. (D) Specific Infectivity of progeny SINV derived from Aedes albopictus cells colonized with non-native ( w Mel and w Stri) and native ( w AlbB) Wolbachia strains. Cells were infected with virus at MOI=0.1 and infectious virus titer produced after 48 hours was quantified via plaque assays on BHK-21 cells. Error bars represent standard error of mean (SEM) of biological replicates (n=3-6). Student’s t-test performed on log-transformed values. *P
Figure Legend Snippet: Presence of Wolbachia in mosquito cells reduces progeny virus infectivity in mammalian cells. RML12 mosquito cells with and without Wolbachia ( w Mel) were infected with alphaviruses Sindbis (SINV), Chikungunya (CHIKV) or flavivirus Zika (ZIKV) at MOI of 10. Viral supernatants were harvested at 48 hours post infection after which infectious virus titer was quantified by performing plaque assays on BHK-21 (SINV, CHIKV) and Vero (ZIKV) cells. Total virus particles were determined by quantifying viral genome copies present in the supernatant using qRT-PCR. Reported specific infectivity (SI) ratios were calculated as total infectious virus titer divided by total particles produced per mL of viral supernatant. (A) Percentage of total SINV particles produced from cells with and without Wolbachia that are infectious on BHK-21 cells. (B) Specific Infectivity Ratios of progeny SINV, CHIKV and ZIKV viruses. (C) Maximum-likelihood tree representing the phylogenetic relationship between the Wolbachia strains used in the study was generated using MEGA X, using a MUSCLE alignment of concatenated sequences of multi-locus typing (MLST) genes ( coxA, gatB, ftsZ, hcpA, fbpA) . Sequences from Wolbachia strain w Bm, native to the filarial nematode Brugia malayi , was used as a distant outgroup. Scale bar represent branch lengths. (D) Specific Infectivity of progeny SINV derived from Aedes albopictus cells colonized with non-native ( w Mel and w Stri) and native ( w AlbB) Wolbachia strains. Cells were infected with virus at MOI=0.1 and infectious virus titer produced after 48 hours was quantified via plaque assays on BHK-21 cells. Error bars represent standard error of mean (SEM) of biological replicates (n=3-6). Student’s t-test performed on log-transformed values. *P

Techniques Used: Infection, Quantitative RT-PCR, Produced, Generated, Derivative Assay, Transformation Assay

10) Product Images from "Simkania negevensis may produce long-lasting infections in human pneumocytes and endometrial cells"

Article Title: Simkania negevensis may produce long-lasting infections in human pneumocytes and endometrial cells

Journal: Pathogens and Disease

doi: 10.1093/femspd/ftw115

Assessment of the interactions of the SnCV with different host organelles. ( A) ER and mitochondria as observed in Vero cells. Bacteria ( S. negevensis or W. chondrophila ) were detected with a species-specific polyclonal antibody (green), PDI was used to label the ER (red) and MitoTracker was used to stain mitochondria (red); nucleic acids were stained with DAPI (blue). ( B) Golgi apparatus as observed in Vero cells. Simkania negevensis particles were detected with a specific polyclonal antibody (green); C. trachomatis was either detected with a specific polyclonal antibody (green) or DAPI to stain the nucleic acids (blue); for clarity, dashed lines highlight the DAPI-positive C. trachomatis inclusion. Gm130 and RCAS1 specific markers were used to mark the Golgi apparatus (red); nucleic acids in all experiments were stained with DAPI (blue).
Figure Legend Snippet: Assessment of the interactions of the SnCV with different host organelles. ( A) ER and mitochondria as observed in Vero cells. Bacteria ( S. negevensis or W. chondrophila ) were detected with a species-specific polyclonal antibody (green), PDI was used to label the ER (red) and MitoTracker was used to stain mitochondria (red); nucleic acids were stained with DAPI (blue). ( B) Golgi apparatus as observed in Vero cells. Simkania negevensis particles were detected with a specific polyclonal antibody (green); C. trachomatis was either detected with a specific polyclonal antibody (green) or DAPI to stain the nucleic acids (blue); for clarity, dashed lines highlight the DAPI-positive C. trachomatis inclusion. Gm130 and RCAS1 specific markers were used to mark the Golgi apparatus (red); nucleic acids in all experiments were stained with DAPI (blue).

Techniques Used: Staining

Infectivity of bacterial particles released upon Vero cells infection. Two milliliters of the supernatant of a Vero cells co-culture were taken at various time points to inoculate fresh Vero cells. Cells were harvested for DNA extraction at 0 h and day 6 post-inoculation with the supernatant to quantify S. negevensis DNA by qPCR and monitor the amount of bacteria present in the supernatant and their ability to produce an efficient infection, as assessed at day 6. Dashed lines at day 14 represent the results of a similar experiment performed with a 1/1000 dilution of the supernatant prior to inoculation of fresh Vero cells. Results are representative of one experiment. In total, two independent experiments were performed. Abbreviation: D, day.
Figure Legend Snippet: Infectivity of bacterial particles released upon Vero cells infection. Two milliliters of the supernatant of a Vero cells co-culture were taken at various time points to inoculate fresh Vero cells. Cells were harvested for DNA extraction at 0 h and day 6 post-inoculation with the supernatant to quantify S. negevensis DNA by qPCR and monitor the amount of bacteria present in the supernatant and their ability to produce an efficient infection, as assessed at day 6. Dashed lines at day 14 represent the results of a similar experiment performed with a 1/1000 dilution of the supernatant prior to inoculation of fresh Vero cells. Results are representative of one experiment. In total, two independent experiments were performed. Abbreviation: D, day.

Techniques Used: Infection, Co-Culture Assay, DNA Extraction, Real-time Polymerase Chain Reaction

Growth of S. negevensis within mammalian cells, insect cells and amoebae. ( A) Replication in Vero cells—inoculum 25 bacteria per cell, endometrial cells (Ishikawa cell line)—inoculum 25 bacteria per cell, pneumocytes (A549 cell line)—inoculum 5 bacteria per cell, Spodoptera frugiperda ovarian epithelial cells (Sf9 cells)—inoculum 25 bacteria per cell, and amoebae ( A. castellanii )—inoculum 25 bacteria per cell, measured by quantitative PCR. Results are the mean of one representative experiment performed in duplicate. In total, three independent experiments in duplicate were performed for each cell line. ( B) Growth of S. negevensis observed by immunofluorescence— S. negevensis particles are detected with a rabbit polyclonal anti- S. negevensis antibody (1:2500), followed by a secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), mammalian cells (red) are stained with texas red-conjugated Concanavalin A (1:50) and nucleic acids (blue) are stained with DAPI (1:1000). Abbreviation: D, day.
Figure Legend Snippet: Growth of S. negevensis within mammalian cells, insect cells and amoebae. ( A) Replication in Vero cells—inoculum 25 bacteria per cell, endometrial cells (Ishikawa cell line)—inoculum 25 bacteria per cell, pneumocytes (A549 cell line)—inoculum 5 bacteria per cell, Spodoptera frugiperda ovarian epithelial cells (Sf9 cells)—inoculum 25 bacteria per cell, and amoebae ( A. castellanii )—inoculum 25 bacteria per cell, measured by quantitative PCR. Results are the mean of one representative experiment performed in duplicate. In total, three independent experiments in duplicate were performed for each cell line. ( B) Growth of S. negevensis observed by immunofluorescence— S. negevensis particles are detected with a rabbit polyclonal anti- S. negevensis antibody (1:2500), followed by a secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), mammalian cells (red) are stained with texas red-conjugated Concanavalin A (1:50) and nucleic acids (blue) are stained with DAPI (1:1000). Abbreviation: D, day.

Techniques Used: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

11) Product Images from "Robust three-dimensional expansion of human adult alveolar stem cells and SARS-CoV-2 infection"

Article Title: Robust three-dimensional expansion of human adult alveolar stem cells and SARS-CoV-2 infection

Journal: bioRxiv

doi: 10.1101/2020.07.10.194498

Establishment of long-term and three-dimensional human alveolar type 2 cell culture and SARS-CoV-2 infection in the three-dimensional model system a, Schematic diagram outlining the method for dissociation and processing of human adult lung parenchymal tissues for in vitro three-dimensional cultures (top). Schematic illustration of SARS-CoV-2 infection experiments in this study (bottom). b, Representative bright-field images of hAOs derived from HTII-280 + hAT2 cells at day 28 in culture. Insets (top left) show high-power view of cystic-like (top right) and budding-like (bottom left) alveolar organoids. Scale bar, 2000 μm. Of note, the majority of hAOs exhibited uptake of Lysotracker (red), indicative of mature AT2 cells (bottom right). Scale bar, 1000 μm. c, Immunofluorescent staining of hAOs expressing AT2 markers. Left and right; HTII-280 (for hAT2, green), pro-SFTPC (for hAT2, red). Middle; ABCA3 (for hAT2, white). DAPI (blue). Scale bar, 50 μm. d, Representative bright-field images of hBOs derived from HTII-280 − non-hAT2 cells at day 14 in culture. Insets (left) show high-power view (right). Scale bar, 2000 μm. e, Immunofluorescent staining of hBOs expressing airway lineage markers. Left; SCGB1A1 (for secretory, green), pro-SFTPC (for hAT2, red). Middle; KRT5 (basal, white), TP63 (basal, green), HTII-280 (for hAT2, red). Right; ABCA3 (for hAT2, green), HOPX (for hAT1, white). DAPI (blue). Scale bar, 50 μm. f, Immunofluorescent staining of ACE2 (green) and HTII-280 (red) in hAOs. HTII-280 is stained in the apical membrane of hAT2 cells. Scale bar, 100 μm. g, Immunofluorescent staining of TMPRSS2 (green) in hAOs. Scale bar, 100 μm. h, Representative images for plaque assay at 3 dpi. Dilution factors are shown in numbers on the right upper corner. Scale bar, 1 cm. i, Plaque assay showing that SARS-CoV-2 actively replicates in hAOs at 1 dpi. SARS-CoV-2 can infect hBOs, but the viral amplification is much lower than in hAOs. Given the different culture and viral infection techniques between 2D Vero cells and 3D organoids, direct comparison is not applicable between Vero cells and hAOs. Error bars represent SEM. n=2. j, Quantitative PCR (qPCR) analysis for measuring the viral RNA levels in lysed hAOs. Error bars represent SEM. n=3. k, qPCR analysis for measuring the viral RNA levels in hAO media. Error bars represent SEM. n=3.
Figure Legend Snippet: Establishment of long-term and three-dimensional human alveolar type 2 cell culture and SARS-CoV-2 infection in the three-dimensional model system a, Schematic diagram outlining the method for dissociation and processing of human adult lung parenchymal tissues for in vitro three-dimensional cultures (top). Schematic illustration of SARS-CoV-2 infection experiments in this study (bottom). b, Representative bright-field images of hAOs derived from HTII-280 + hAT2 cells at day 28 in culture. Insets (top left) show high-power view of cystic-like (top right) and budding-like (bottom left) alveolar organoids. Scale bar, 2000 μm. Of note, the majority of hAOs exhibited uptake of Lysotracker (red), indicative of mature AT2 cells (bottom right). Scale bar, 1000 μm. c, Immunofluorescent staining of hAOs expressing AT2 markers. Left and right; HTII-280 (for hAT2, green), pro-SFTPC (for hAT2, red). Middle; ABCA3 (for hAT2, white). DAPI (blue). Scale bar, 50 μm. d, Representative bright-field images of hBOs derived from HTII-280 − non-hAT2 cells at day 14 in culture. Insets (left) show high-power view (right). Scale bar, 2000 μm. e, Immunofluorescent staining of hBOs expressing airway lineage markers. Left; SCGB1A1 (for secretory, green), pro-SFTPC (for hAT2, red). Middle; KRT5 (basal, white), TP63 (basal, green), HTII-280 (for hAT2, red). Right; ABCA3 (for hAT2, green), HOPX (for hAT1, white). DAPI (blue). Scale bar, 50 μm. f, Immunofluorescent staining of ACE2 (green) and HTII-280 (red) in hAOs. HTII-280 is stained in the apical membrane of hAT2 cells. Scale bar, 100 μm. g, Immunofluorescent staining of TMPRSS2 (green) in hAOs. Scale bar, 100 μm. h, Representative images for plaque assay at 3 dpi. Dilution factors are shown in numbers on the right upper corner. Scale bar, 1 cm. i, Plaque assay showing that SARS-CoV-2 actively replicates in hAOs at 1 dpi. SARS-CoV-2 can infect hBOs, but the viral amplification is much lower than in hAOs. Given the different culture and viral infection techniques between 2D Vero cells and 3D organoids, direct comparison is not applicable between Vero cells and hAOs. Error bars represent SEM. n=2. j, Quantitative PCR (qPCR) analysis for measuring the viral RNA levels in lysed hAOs. Error bars represent SEM. n=3. k, qPCR analysis for measuring the viral RNA levels in hAO media. Error bars represent SEM. n=3.

Techniques Used: Cell Culture, Infection, In Vitro, Derivative Assay, Staining, Expressing, Plaque Assay, Amplification, Real-time Polymerase Chain Reaction

12) Product Images from "The intracellular proteome of African swine fever virus"

Article Title: The intracellular proteome of African swine fever virus

Journal: Scientific Reports

doi: 10.1038/s41598-018-32985-z

Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.
Figure Legend Snippet: Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.

Techniques Used: Mass Spectrometry

Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.
Figure Legend Snippet: Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.

Techniques Used: Expressing

ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.
Figure Legend Snippet: ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.

Techniques Used: In Vitro

13) Product Images from "Identification and characterization of linear B cell epitopes on the nucleocapsid protein of porcine epidemic diarrhea virus using monoclonal antibodies"

Article Title: Identification and characterization of linear B cell epitopes on the nucleocapsid protein of porcine epidemic diarrhea virus using monoclonal antibodies

Journal: Virus Research

doi: 10.1016/j.virusres.2020.197912

Reactivity of McAb with the epidemic PEDV strains by IFA and Western blot. Vero cells inoculated with PEDV strain YZ, SH and MS were incubated with 1C9, 6A11 and 3F10, and then stained with FITC labeled goat anti-mouse IgG. Vero cells inoculated with YZ and MS showed specific fluorescence, but Vero cells inoculated with SH 2016 only reacted with the McAb 3F10 and 6A11, not with 1C9. To further verify reactivity of McAb with the epidemic PEDV strains, Vero cells inoculated with PEDV strain YZ, SH2016 and MS were separated by 12 % agarose gel and transferred onto a nitrocellulose (NC) membranes. Non-specific antibody binding sites were blocked with PBST containing 10 % skim milk at room temperature for 2 h. The membranes were successively incubated with McAbs and HRP-labeled Goat Anti-Mouse IgG (H + L) antibody (Beyotime, China). Then the reaction was visualized by using ECL Western blot substrate (Thermo). The result of Western blot was consistent with that of IFA. Vero cells inoculated with PEDV strain CV777 were used as positive control.
Figure Legend Snippet: Reactivity of McAb with the epidemic PEDV strains by IFA and Western blot. Vero cells inoculated with PEDV strain YZ, SH and MS were incubated with 1C9, 6A11 and 3F10, and then stained with FITC labeled goat anti-mouse IgG. Vero cells inoculated with YZ and MS showed specific fluorescence, but Vero cells inoculated with SH 2016 only reacted with the McAb 3F10 and 6A11, not with 1C9. To further verify reactivity of McAb with the epidemic PEDV strains, Vero cells inoculated with PEDV strain YZ, SH2016 and MS were separated by 12 % agarose gel and transferred onto a nitrocellulose (NC) membranes. Non-specific antibody binding sites were blocked with PBST containing 10 % skim milk at room temperature for 2 h. The membranes were successively incubated with McAbs and HRP-labeled Goat Anti-Mouse IgG (H + L) antibody (Beyotime, China). Then the reaction was visualized by using ECL Western blot substrate (Thermo). The result of Western blot was consistent with that of IFA. Vero cells inoculated with PEDV strain CV777 were used as positive control.

Techniques Used: Immunofluorescence, Western Blot, Incubation, Staining, Labeling, Fluorescence, Agarose Gel Electrophoresis, Binding Assay, Positive Control

Identification of monoclonal antibodies by IFA(A) and Western-blot(B). A: After inoculated with PEDV CV777 strain, the Vero cells were fixed and analyzed by IFA with the five McAb. Non-inoculated cells were used as control. To confirm the specificity of McAb, positive serum and the RPMI-1640 containing 20 % FCS (N) were used as antibody to incubated the cells as control. The Vero cells inoculated with CV777 could reacted with the five McAb and positive serum, and showed obvious green fluorescence on the surface. The non-inoculated cells and N control showed no any fluorescence. B: The purified PEDV and N protein expressed in Baculovirus were separated by 10 % agarose gel and transferred onto a nitrocellulose (NC) membranes. After blocking, the membranes were incubated with McAb overnight at 4 °C. After 3 times washes with PBST, the membranes were incubated with HRP-labeled Goat Anti-Mouse IgG (H + L) antibody. The reaction was visualized by using ECL Western blot substrate. Vero cells and Sf9 cells were used as control. Line 1: Purified PEDV; Line 2: Vero cell control; Line 3: N protein expressed in Baculovirus; Line 4: Sf9 cell control.
Figure Legend Snippet: Identification of monoclonal antibodies by IFA(A) and Western-blot(B). A: After inoculated with PEDV CV777 strain, the Vero cells were fixed and analyzed by IFA with the five McAb. Non-inoculated cells were used as control. To confirm the specificity of McAb, positive serum and the RPMI-1640 containing 20 % FCS (N) were used as antibody to incubated the cells as control. The Vero cells inoculated with CV777 could reacted with the five McAb and positive serum, and showed obvious green fluorescence on the surface. The non-inoculated cells and N control showed no any fluorescence. B: The purified PEDV and N protein expressed in Baculovirus were separated by 10 % agarose gel and transferred onto a nitrocellulose (NC) membranes. After blocking, the membranes were incubated with McAb overnight at 4 °C. After 3 times washes with PBST, the membranes were incubated with HRP-labeled Goat Anti-Mouse IgG (H + L) antibody. The reaction was visualized by using ECL Western blot substrate. Vero cells and Sf9 cells were used as control. Line 1: Purified PEDV; Line 2: Vero cell control; Line 3: N protein expressed in Baculovirus; Line 4: Sf9 cell control.

Techniques Used: Immunofluorescence, Western Blot, Incubation, Fluorescence, Purification, Agarose Gel Electrophoresis, Blocking Assay, Labeling

Related Articles

In Vitro:

Article Title: Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus
Article Snippet: .. In vitro cytotoxicity assay of BLFP crude extract To evaluate the cytotoxicity of BLFP crude extract in vitro, Vero cells were first grown in a 96-well microplate (Corning Life Sciences, Corning, NY, USA) at a density of 20,000 cells per well 1 day prior to the experiment. .. After removing the culture supernatant, ten-fold serially diluted BLFP crude extract in PI medium (DMEM supplemented with 0.3% tryptose phosphate broth (Sigma-Aldrich, Mo, USA), 0.02% yeast extract (Acumedia, Michigan, USA), and 10 μg/ml of trypsin (Sigma-Aldrich, Mo, USA) was added to the cell monolayer.

Infection:

Article Title: Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis
Article Snippet: .. Growth kinetics of recombinant viruses Monolayers of L929 or Vero cells (2 × 105 cells) in 24-well plates (Corning) were infected with viruses at an MOI of 0.1 PFU/cell. .. After 1 h of incubation, the cells were washed using phosphate-buffered saline (PBS) twice and incubated with maintenance medium.

Article Title: Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus
Article Snippet: .. GFP-expressing recombinant MuV of the Hoshino strain was preincubated with the medium containing the serially diluted 3′-SL, SLeX pentaose, GM2-GS, 6′-SL, or LnNT at 37°C for 60 min. Vero cells cultured in a 96-well dish (Corning) were infected with the viruses (multiplicity of infection [MOI] of 0.01) for 60 min at 37°C. .. After the virus-containing medium was removed, cells were overlaid with 0.75% (w/vol) Sea Kem ME Agar (Invitrogen)-DMEM supplemented with 5% (vol/vol) FBS, 0.16% (w/vol) NaHCO3 , and penicillin-streptomycin at 37°C and 5% CO2 .

Cytotoxicity Assay:

Article Title: Evaluation of antiviral activity of Bacillus licheniformis-fermented products against porcine epidemic diarrhea virus
Article Snippet: .. In vitro cytotoxicity assay of BLFP crude extract To evaluate the cytotoxicity of BLFP crude extract in vitro, Vero cells were first grown in a 96-well microplate (Corning Life Sciences, Corning, NY, USA) at a density of 20,000 cells per well 1 day prior to the experiment. .. After removing the culture supernatant, ten-fold serially diluted BLFP crude extract in PI medium (DMEM supplemented with 0.3% tryptose phosphate broth (Sigma-Aldrich, Mo, USA), 0.02% yeast extract (Acumedia, Michigan, USA), and 10 μg/ml of trypsin (Sigma-Aldrich, Mo, USA) was added to the cell monolayer.

Concentration Assay:

Article Title: Cytotoxic and Genotoxic Effects of Fluconazole on African Green Monkey Kidney (Vero) Cell Line
Article Snippet: .. Vero cells were treated with fluconazole for 24 h in 25-cm2 sterile flasks (Corning) at a concentration of 1x106 cells/mL. .. After treatment, 3- μ g/mL cytochalasin B (Sigma Chemical Co.) was added for another 24 hours at 37°C.

Activity Assay:

Article Title: Herbal Gel Formulation Developed for Anti-Human Immunodeficiency Virus (HIV)-1 Activity Also Inhibits In Vitro HSV-2 Infection
Article Snippet: .. HSV-2 Virucidal Assay To screen for the HSV-2 virucidal activity (direct anti-HSV-2 activity) of the gel formulation, Vero cells (8 × 104 /well) were seeded in 24-well culture plates (Corning Incorporated Costar, NY, USA) and grown for 24 h. Next day, HSV-2 virus (100 PFU/well) was pre-incubated with serial dilutions of the gel formulation/gel base and acyclovir at 37 °C for 1 h to directly inactivate the virus. .. Subsequently, the pretreated HSV-2 virus was added to the confluent Vero cells monolayer and incubated for 1 h at 37 °C under the humidified 5% CO2 atmosphere.

Cell Culture:

Article Title: The intracellular proteome of African swine fever virus
Article Snippet: .. For the MS analyses, HEK 293, WSL-HP, and Vero cells were cultivated in 6-well cell culture dishes (Corning, #3506) to 90% confluency. .. After infection at a MOI of 2 (HEK 293) or 5 (Vero and WSL-HP) and further incubation for 24 h (HEK 293 and Vero cells) or 48 hours (WSL-HP cells) the complete cell layer was positive for GFP fluorescence.

Article Title: Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus
Article Snippet: .. GFP-expressing recombinant MuV of the Hoshino strain was preincubated with the medium containing the serially diluted 3′-SL, SLeX pentaose, GM2-GS, 6′-SL, or LnNT at 37°C for 60 min. Vero cells cultured in a 96-well dish (Corning) were infected with the viruses (multiplicity of infection [MOI] of 0.01) for 60 min at 37°C. .. After the virus-containing medium was removed, cells were overlaid with 0.75% (w/vol) Sea Kem ME Agar (Invitrogen)-DMEM supplemented with 5% (vol/vol) FBS, 0.16% (w/vol) NaHCO3 , and penicillin-streptomycin at 37°C and 5% CO2 .

Mass Spectrometry:

Article Title: The intracellular proteome of African swine fever virus
Article Snippet: .. For the MS analyses, HEK 293, WSL-HP, and Vero cells were cultivated in 6-well cell culture dishes (Corning, #3506) to 90% confluency. .. After infection at a MOI of 2 (HEK 293) or 5 (Vero and WSL-HP) and further incubation for 24 h (HEK 293 and Vero cells) or 48 hours (WSL-HP cells) the complete cell layer was positive for GFP fluorescence.

Recombinant:

Article Title: Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis
Article Snippet: .. Growth kinetics of recombinant viruses Monolayers of L929 or Vero cells (2 × 105 cells) in 24-well plates (Corning) were infected with viruses at an MOI of 0.1 PFU/cell. .. After 1 h of incubation, the cells were washed using phosphate-buffered saline (PBS) twice and incubated with maintenance medium.

Article Title: Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus
Article Snippet: .. GFP-expressing recombinant MuV of the Hoshino strain was preincubated with the medium containing the serially diluted 3′-SL, SLeX pentaose, GM2-GS, 6′-SL, or LnNT at 37°C for 60 min. Vero cells cultured in a 96-well dish (Corning) were infected with the viruses (multiplicity of infection [MOI] of 0.01) for 60 min at 37°C. .. After the virus-containing medium was removed, cells were overlaid with 0.75% (w/vol) Sea Kem ME Agar (Invitrogen)-DMEM supplemented with 5% (vol/vol) FBS, 0.16% (w/vol) NaHCO3 , and penicillin-streptomycin at 37°C and 5% CO2 .

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    Corning Life Sciences vero cells
    Comparison of the ASFV proteins expressed in WSL-HP, <t>HEK</t> 293 and <t>Vero</t> cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.
    Vero Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences vero e6 cells
    Antiviral activity of T-705 against MARV in cell culture (A) <t>Vero</t> <t>E6</t> cells were treated with a dilution series of T-705 (starting at 50 µg/ml followed by 1:3 dilutions) 1 h prior to infection with wild type MARV at MOI of 0.01. After 72 hours, MARV RNA in the supernatant was quantified by RT-qPCR. The EC50 and EC90 values for T-705 with 95% confidence interval (95% CI) were calculated from the sigmoidal function. (B) Virus titers in Vero E6 cell culture supernatants were determined by TCID50 assay.
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    Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.

    Journal: Scientific Reports

    Article Title: The intracellular proteome of African swine fever virus

    doi: 10.1038/s41598-018-32985-z

    Figure Lengend Snippet: Comparison of the ASFV proteins expressed in WSL-HP, HEK 293 and Vero cells. In total, 94 ASFV proteins were identified by MS, 54 of them in all three cell lines. Of the remaining 40, 23 were shared by Vero (light grey) and WSL-HP (white). Ten, and four proteins were exclusively expressed in WSL-HP and in Vero cells, respectively.

    Article Snippet: For the MS analyses, HEK 293, WSL-HP, and Vero cells were cultivated in 6-well cell culture dishes (Corning, #3506) to 90% confluency.

    Techniques: Mass Spectrometry

    Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.

    Journal: Scientific Reports

    Article Title: The intracellular proteome of African swine fever virus

    doi: 10.1038/s41598-018-32985-z

    Figure Lengend Snippet: Correlation of ASFV protein expression levels in WSL-HP, HEK 293 and Vero cells. Protein abundances determined in the cells indicated at the axes are given in mole % in a logarithmic scale. Non-structural (ns) proteins are marked as full black circles, structural (s) and uncharacterized (u) proteins as open squares and triangles, respectively. Correlation coefficients are given as R, the y-intercepts and slopes of the dotted regression lines are given as Y0 and m, respectively. Dissecting lines are dashed.

    Article Snippet: For the MS analyses, HEK 293, WSL-HP, and Vero cells were cultivated in 6-well cell culture dishes (Corning, #3506) to 90% confluency.

    Techniques: Expressing

    ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.

    Journal: Scientific Reports

    Article Title: The intracellular proteome of African swine fever virus

    doi: 10.1038/s41598-018-32985-z

    Figure Lengend Snippet: ASFV OURT 88/3-ΔTK-GFP productively infects WSL-HP, Vero, and HEK 293 cells. After inoculation of WSL-HP, Vero, and HEK 293 cells with OURT 88/3-ΔTK-GFP, supernatants were collected at the times indicated and titers of the progeny virus were determined on WSL-HP cells. The in-vitro growth kinetics show that ASFV OURT 88/3-dTK-GFP replicates well on all three cell lines, although final titers on Vero and HEK 293 cells are over tenfold lower than on WSL-HP cells.

    Article Snippet: For the MS analyses, HEK 293, WSL-HP, and Vero cells were cultivated in 6-well cell culture dishes (Corning, #3506) to 90% confluency.

    Techniques: In Vitro

    Antiviral activity of T-705 against MARV in cell culture (A) Vero E6 cells were treated with a dilution series of T-705 (starting at 50 µg/ml followed by 1:3 dilutions) 1 h prior to infection with wild type MARV at MOI of 0.01. After 72 hours, MARV RNA in the supernatant was quantified by RT-qPCR. The EC50 and EC90 values for T-705 with 95% confidence interval (95% CI) were calculated from the sigmoidal function. (B) Virus titers in Vero E6 cell culture supernatants were determined by TCID50 assay.

    Journal: Antiviral research

    Article Title: Successful treatment of Marburg virus with orally administrated T-705 (Favipiravir) in a mouse model

    doi: 10.1016/j.antiviral.2018.01.011

    Figure Lengend Snippet: Antiviral activity of T-705 against MARV in cell culture (A) Vero E6 cells were treated with a dilution series of T-705 (starting at 50 µg/ml followed by 1:3 dilutions) 1 h prior to infection with wild type MARV at MOI of 0.01. After 72 hours, MARV RNA in the supernatant was quantified by RT-qPCR. The EC50 and EC90 values for T-705 with 95% confidence interval (95% CI) were calculated from the sigmoidal function. (B) Virus titers in Vero E6 cell culture supernatants were determined by TCID50 assay.

    Article Snippet: Vero E6 cells (at 95% confluence in a 96-well plate (Corning)) were then inoculated with 100 µl of each dilution, in triplicate, and cells were incubated at 37°C for 1 h. Following the incubation, the supernatant was removed and replaced with 100 µl of fresh DMEM with 2% FBS.

    Techniques: Activity Assay, Cell Culture, Infection, Quantitative RT-PCR, TCID50 Assay

    Competition evaluation. Dose-dependent binding competitions between sera and IgG#33 were performed. FACS analyses were performed using IgG#33 combined with serial dilutions (1:100 to 1:800) of both neutralizing (NT) serum (A) and nonneutralizing (non-NT) (B) serum to evaluate the possible competitive binding of antibodies to gD1-transfected cells. As shown by the graphs, no competition resulted even with the lower serum dilution tested. NT (C) and PEI (D) assays next were performed on Vero E6 cells with NT serum and a non-NT serum supplemented by IgG#33 against both HSV-1 and -2 infections. In NT assays, IgG#33 was used at its 50% inhibitory concentration (1.092 μg/ml against HSV-1 and 0.731 μg/ml against HSV-2 tested isolates), while in PEI assays it was used at 25 μg/ml and 100 μg/ml against HSV-1 and -2, respectively. Sera were used at 1:100 dilution, and the MAb alone control is indicated by white columns. Mean values ± SD are reported for each experimental condition (*, P

    Journal: Journal of Virology

    Article Title: Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects

    doi: 10.1128/JVI.00070-19

    Figure Lengend Snippet: Competition evaluation. Dose-dependent binding competitions between sera and IgG#33 were performed. FACS analyses were performed using IgG#33 combined with serial dilutions (1:100 to 1:800) of both neutralizing (NT) serum (A) and nonneutralizing (non-NT) (B) serum to evaluate the possible competitive binding of antibodies to gD1-transfected cells. As shown by the graphs, no competition resulted even with the lower serum dilution tested. NT (C) and PEI (D) assays next were performed on Vero E6 cells with NT serum and a non-NT serum supplemented by IgG#33 against both HSV-1 and -2 infections. In NT assays, IgG#33 was used at its 50% inhibitory concentration (1.092 μg/ml against HSV-1 and 0.731 μg/ml against HSV-2 tested isolates), while in PEI assays it was used at 25 μg/ml and 100 μg/ml against HSV-1 and -2, respectively. Sera were used at 1:100 dilution, and the MAb alone control is indicated by white columns. Mean values ± SD are reported for each experimental condition (*, P

    Article Snippet: HSV-1 HF and HSV-2 MS strains were expanded in confluent Vero E6 cells cultured in T-25 flasks (Corning CellBIND).

    Techniques: Binding Assay, FACS, Transfection, Concentration Assay

    Evaluation of purified IgG activity against HSV-1 and -2 infections. PEI assay was performed on Vero E6 cells using lower dilutions of the neutralizing (NT) serum (1:100) control and purified total IgGs obtained from the same serum sample (100 μg/ml). Nonneutralizing (non-NT) serum and its purified total IgG fraction were used as a negative control. Mean values ± SD are reported for each experimental condition (***, P

    Journal: Journal of Virology

    Article Title: Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects

    doi: 10.1128/JVI.00070-19

    Figure Lengend Snippet: Evaluation of purified IgG activity against HSV-1 and -2 infections. PEI assay was performed on Vero E6 cells using lower dilutions of the neutralizing (NT) serum (1:100) control and purified total IgGs obtained from the same serum sample (100 μg/ml). Nonneutralizing (non-NT) serum and its purified total IgG fraction were used as a negative control. Mean values ± SD are reported for each experimental condition (***, P

    Article Snippet: HSV-1 HF and HSV-2 MS strains were expanded in confluent Vero E6 cells cultured in T-25 flasks (Corning CellBIND).

    Techniques: Purification, Activity Assay, Negative Control

    Evaluation of serum activity against HSV-1 and -2 infections. (A) Neutralizing (NT) activity of 44 sera was assessed on Vero E6 cells, and results show that HSV-1 (▲) was more efficiently blocked than HSV-2 (●). (B) When tested in PEI assay on the same cells, no samples (except one) impaired virus replication efficiently, although higher activity again was observed against HSV-1 (▲) than HSV-2 (●). Sera were used at 1:200 dilution. Median values with interquartile ranges are reported for each experiment (****, P

    Journal: Journal of Virology

    Article Title: Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects

    doi: 10.1128/JVI.00070-19

    Figure Lengend Snippet: Evaluation of serum activity against HSV-1 and -2 infections. (A) Neutralizing (NT) activity of 44 sera was assessed on Vero E6 cells, and results show that HSV-1 (▲) was more efficiently blocked than HSV-2 (●). (B) When tested in PEI assay on the same cells, no samples (except one) impaired virus replication efficiently, although higher activity again was observed against HSV-1 (▲) than HSV-2 (●). Sera were used at 1:200 dilution. Median values with interquartile ranges are reported for each experiment (****, P

    Article Snippet: HSV-1 HF and HSV-2 MS strains were expanded in confluent Vero E6 cells cultured in T-25 flasks (Corning CellBIND).

    Techniques: Activity Assay

    Subcellular localization of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 p4b in transfected cells. Plasmids expressing either GFP alone (green; top panel) or a GFP-tagged version of p4b of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 were transfected into Vero E6 cells, which were double labelled using Lysotracker (a lysosomal marker), Mitotracker (a mitochondrial marker) or concanavalin A (an ER marker) (all staining red). Confocal microscopy images were produced for each stained slide with the red and green images merged onto one overlapping image. Bar, 25 µm.

    Journal: The Journal of General Virology

    Article Title: The ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling

    doi: 10.1099/vir.0.062059-0

    Figure Lengend Snippet: Subcellular localization of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 p4b in transfected cells. Plasmids expressing either GFP alone (green; top panel) or a GFP-tagged version of p4b of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 were transfected into Vero E6 cells, which were double labelled using Lysotracker (a lysosomal marker), Mitotracker (a mitochondrial marker) or concanavalin A (an ER marker) (all staining red). Confocal microscopy images were produced for each stained slide with the red and green images merged onto one overlapping image. Bar, 25 µm.

    Article Snippet: Vero E6 cells were seeded into 24-well plates (Corning) containing round coverslips (Fisher) and cultured overnight at 37 °C.

    Techniques: Transfection, Expressing, Marker, Staining, Confocal Microscopy, Produced