Structured Review

BEI Resources vero e6 cells
Positive control data collected on <t>Vero</t> <t>E6</t> cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.
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1) Product Images from "Validation of the Filovirus Plaque Assay for Use in Preclinical Studies"

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

Journal: Viruses

doi: 10.3390/v8040113

Positive control data collected on Vero E6 cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.
Figure Legend Snippet: Positive control data collected on Vero E6 cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.

Techniques Used: Positive Control

2) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

3) Product Images from "Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L"

Article Title: Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L

Journal: bioRxiv

doi: 10.1101/2020.07.27.223727

Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).
Figure Legend Snippet: Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).

Techniques Used: Activity Assay, Immunofluorescence, Infection, Staining, Fluorescence, Plaque Assay

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

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

Journal: Viruses

doi: 10.3390/v4123511

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

Techniques Used: Produced

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

Techniques Used: Quantitation Assay, Plaque Assay

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

Techniques Used:

5) Product Images from "A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease"

Article Title: A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

Journal: mSphere

doi: 10.1128/mSphere.00401-17

Changes in MARV particle/PFU ratio after cell culture passage in Vero E6 cells. Passage 2 MARV was serially passaged 10 times in Vero E6 cells at an MOI of 0.001. (A) After each passage, the number of virus particles per milliliter was determined via TEM (singlicate). (B) After each passage, the viral titer (PFU per milliliter) was determined via plaque assay (singlicate). (C) After each passage, the particle/PFU ratio was determined based on the titer and number of particles.
Figure Legend Snippet: Changes in MARV particle/PFU ratio after cell culture passage in Vero E6 cells. Passage 2 MARV was serially passaged 10 times in Vero E6 cells at an MOI of 0.001. (A) After each passage, the number of virus particles per milliliter was determined via TEM (singlicate). (B) After each passage, the viral titer (PFU per milliliter) was determined via plaque assay (singlicate). (C) After each passage, the particle/PFU ratio was determined based on the titer and number of particles.

Techniques Used: Cell Culture, Transmission Electron Microscopy, Plaque Assay

Percentage of abundance of reads containing SNP in the MARV GP gene in populations over passage in Vero E6 cell culture. MARV was serially passaged in Vero E6 cells at three different MOI (0.001, 0.01, and 0.1). Deep sequencing was used to investigate genotypic changes that occurred after cell culture passaging. Graphs display the relative abundance of reads containing cytosine (C) at the SNP locus site, after each passage. (A) MARV was passaged 10 times at an MOI of 0.001. (B) The first five passages were repeated at the same MOI (0.001). (C) The first five passages were repeated at two different higher MOI (0.01 and 0.1).
Figure Legend Snippet: Percentage of abundance of reads containing SNP in the MARV GP gene in populations over passage in Vero E6 cell culture. MARV was serially passaged in Vero E6 cells at three different MOI (0.001, 0.01, and 0.1). Deep sequencing was used to investigate genotypic changes that occurred after cell culture passaging. Graphs display the relative abundance of reads containing cytosine (C) at the SNP locus site, after each passage. (A) MARV was passaged 10 times at an MOI of 0.001. (B) The first five passages were repeated at the same MOI (0.001). (C) The first five passages were repeated at two different higher MOI (0.01 and 0.1).

Techniques Used: Cell Culture, Sequencing, Passaging

6) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

7) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

8) Product Images from "Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome"

Article Title: Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

Journal: mBio

doi: 10.1128/mBio.02168-20

Rescue of rSARS-CoV-2. (A) Schematic representation of the approach followed to rescue rSARS-CoV-2. Vero E6 cells were transiently transfected with the SARS-CoV-2 BAC at day 1. After 24 h, transfection medium was changed to postinfection medium. At day 4, cells were split into T75 flasks and the tissue culture supernatant was used to infect fresh Vero E6 cells. At 48 h postinfection, Vero E6 cells were fixed for detection of rSARS-CoV-2 by immunofluorescence, and the tissue culture supernatant of the scaled-up Vero E6 cells was collected at 72 h. As an internal control for this experiment, Vero E6 cells were transfected with the empty BAC. (B) CPE. Images of empty or SARS-CoV-2 BAC-transfected Vero E6 cells at 72 h posttransfection are shown. Scale bars, 100 μm. (C) Viral titers. Tissue culture supernatant from mock-infected (empty BAC) or transfected Vero E6 cells in T75 flasks was collected and titrated by immunofluorescence. Data are presented as means ± SDs. LOD, limit of detection. (D) IFA. Vero E6 cells infected with the tissue culture supernatants from transfected Vero E6 cells were fixed at 48 h postinfection, and viral detection was carried out by using a SARS-CoV cross-reactive monoclonal antibody (1C7) against the N protein (green). Cellular nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 μm.
Figure Legend Snippet: Rescue of rSARS-CoV-2. (A) Schematic representation of the approach followed to rescue rSARS-CoV-2. Vero E6 cells were transiently transfected with the SARS-CoV-2 BAC at day 1. After 24 h, transfection medium was changed to postinfection medium. At day 4, cells were split into T75 flasks and the tissue culture supernatant was used to infect fresh Vero E6 cells. At 48 h postinfection, Vero E6 cells were fixed for detection of rSARS-CoV-2 by immunofluorescence, and the tissue culture supernatant of the scaled-up Vero E6 cells was collected at 72 h. As an internal control for this experiment, Vero E6 cells were transfected with the empty BAC. (B) CPE. Images of empty or SARS-CoV-2 BAC-transfected Vero E6 cells at 72 h posttransfection are shown. Scale bars, 100 μm. (C) Viral titers. Tissue culture supernatant from mock-infected (empty BAC) or transfected Vero E6 cells in T75 flasks was collected and titrated by immunofluorescence. Data are presented as means ± SDs. LOD, limit of detection. (D) IFA. Vero E6 cells infected with the tissue culture supernatants from transfected Vero E6 cells were fixed at 48 h postinfection, and viral detection was carried out by using a SARS-CoV cross-reactive monoclonal antibody (1C7) against the N protein (green). Cellular nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 100 μm.

Techniques Used: Transfection, BAC Assay, Immunofluorescence, Infection, Staining

Characterization of rSARS2-CoV-2 in vitro . (A and B) Genotypic characterization. Vero E6 cells were mock infected or infected (MOI, 0.01) with rSARS-CoV-2 or the SARS-CoV-2 USA-WA1/2020 natural isolate. At 24 h postinfection, total RNA from Vero E6 cells was extracted and a 1,297-bp region of the M gene (nt 26488 to 27784) was amplified by RT-PCR. Amplified DNA was subjected to MluI digestion ( Fig. 1 ). (A) Undigested (top) and digested (bottom) samples were separated in a 0.7% agarose gel. The RT-PCR-amplified DNA product was also sequenced to verify the presence of the silent mutation in the MluI restriction site introduced in the viral genome of the rSARS-CoV-2 ( Fig. 1 ). (B) The MluI restriction site is underlined in red, and the silent mutation introduced to remove the MluI restriction site (T to A) is shown in the black box. (C) Verification of BAC and rSARS-CoV-2 sequences. The SARS-CoV-2 non-reference allele frequency was calculated by comparing short reads to the reference genome of the USA-WA1/2020 reference. All variants were at low frequency in the P6 natural isolate (top), the BAC (bottom), and rSARS-CoV-2 (middle), with the exception of introduced variants at positions 21895 and 26843, which were fixed in the BAC and in rSARS-CoV-2. Non-reference alleles present in less than 1% of reads are not shown. (D) Plaque phenotype. Vero E6 cells were infected with ∼20 PFU of rSARS-CoV-2 (left) or the natural SARS-CoV-2 isolate (right). After 72 h of incubation at 37°C, cells were fixed and immunostained with the N protein 1C7 monoclonal antibody. (E) Growth kinetics. Vero E6 cells were infected (MOI, 0.01) with rSARS-CoV-2 or the natural SARS-CoV-2 isolate. At the indicated times postinfection, tissue culture supernatants were collected and viral titers were assessed by plaque assay (PFU/ml). Data are presented as means ± SDs. LOD, limit of detection.
Figure Legend Snippet: Characterization of rSARS2-CoV-2 in vitro . (A and B) Genotypic characterization. Vero E6 cells were mock infected or infected (MOI, 0.01) with rSARS-CoV-2 or the SARS-CoV-2 USA-WA1/2020 natural isolate. At 24 h postinfection, total RNA from Vero E6 cells was extracted and a 1,297-bp region of the M gene (nt 26488 to 27784) was amplified by RT-PCR. Amplified DNA was subjected to MluI digestion ( Fig. 1 ). (A) Undigested (top) and digested (bottom) samples were separated in a 0.7% agarose gel. The RT-PCR-amplified DNA product was also sequenced to verify the presence of the silent mutation in the MluI restriction site introduced in the viral genome of the rSARS-CoV-2 ( Fig. 1 ). (B) The MluI restriction site is underlined in red, and the silent mutation introduced to remove the MluI restriction site (T to A) is shown in the black box. (C) Verification of BAC and rSARS-CoV-2 sequences. The SARS-CoV-2 non-reference allele frequency was calculated by comparing short reads to the reference genome of the USA-WA1/2020 reference. All variants were at low frequency in the P6 natural isolate (top), the BAC (bottom), and rSARS-CoV-2 (middle), with the exception of introduced variants at positions 21895 and 26843, which were fixed in the BAC and in rSARS-CoV-2. Non-reference alleles present in less than 1% of reads are not shown. (D) Plaque phenotype. Vero E6 cells were infected with ∼20 PFU of rSARS-CoV-2 (left) or the natural SARS-CoV-2 isolate (right). After 72 h of incubation at 37°C, cells were fixed and immunostained with the N protein 1C7 monoclonal antibody. (E) Growth kinetics. Vero E6 cells were infected (MOI, 0.01) with rSARS-CoV-2 or the natural SARS-CoV-2 isolate. At the indicated times postinfection, tissue culture supernatants were collected and viral titers were assessed by plaque assay (PFU/ml). Data are presented as means ± SDs. LOD, limit of detection.

Techniques Used: In Vitro, Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis, BAC Assay, Incubation, Plaque Assay

9) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

10) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

11) Product Images from "A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease"

Article Title: A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

Journal: mSphere

doi: 10.1128/mSphere.00401-17

Percentage of abundance of reads containing SNP in the MARV GP gene in populations over passage in Vero E6 cell culture. MARV was serially passaged in Vero E6 cells at three different MOI (0.001, 0.01, and 0.1). Deep sequencing was used to investigate genotypic changes that occurred after cell culture passaging. Graphs display the relative abundance of reads containing cytosine (C) at the SNP locus site, after each passage. (A) MARV was passaged 10 times at an MOI of 0.001. (B) The first five passages were repeated at the same MOI (0.001). (C) The first five passages were repeated at two different higher MOI (0.01 and 0.1).
Figure Legend Snippet: Percentage of abundance of reads containing SNP in the MARV GP gene in populations over passage in Vero E6 cell culture. MARV was serially passaged in Vero E6 cells at three different MOI (0.001, 0.01, and 0.1). Deep sequencing was used to investigate genotypic changes that occurred after cell culture passaging. Graphs display the relative abundance of reads containing cytosine (C) at the SNP locus site, after each passage. (A) MARV was passaged 10 times at an MOI of 0.001. (B) The first five passages were repeated at the same MOI (0.001). (C) The first five passages were repeated at two different higher MOI (0.01 and 0.1).

Techniques Used: Cell Culture, Sequencing, Passaging

12) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

13) Product Images from "Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice"

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

Journal: Viruses

doi: 10.3390/v11020161

10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.
Figure Legend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

Techniques Used: Cell Culture

14) Product Images from "Rescue of SARS-CoV-2 from a single bacterial artificial chromosome"

Article Title: Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

Journal: bioRxiv

doi: 10.1101/2020.07.22.216358

Characterization of rSARS2-CoV-2 in vitro . A-B) Genotypic characterization: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were mock-infected or infected (MOI 0.01) with rSARS-CoV-2 or the SARS-CoV-2 USA-WA1/2020 natural isolate. After 1 h viral adsorption at 37°C, Vero E6 cells were washed 3 times with PBS and incubated in post-infection media at 37°C. At 24 h post-infection, total RNA from Vero E6 cells was extracted and a 1,297 bp region of the M gene (nt 26,488 to 27,784) was amplified by RT-PCR. Amplified DNA was subjected to MluI digestion ( Figure 1 ). Undigested (top) and digested (bottom) samples were separated in a 0.7% agarose gel ( A ). The RT-PCR amplified DNA product was also sequenced to verify the presence of the silent mutation in the MluI restriction site introduced in the viral genome of the rSARS-CoV-2 ( Figure 1 ). The MluI restriction site is underlined in red and the silent mutation introduced to remove the MluI restriction site (T to A) is shown in the black box ( B ). C ) Verification of BAC and rSARS-CoV-2 sequences: The SARS-CoV-2 non-reference allele frequency was calculated by comparing short reads to the reference genome of USA-WA1/2020 reference. All variants were at low frequency in P6 natural isolate (top), BAC (bottom), and rSARS-CoV-2 (middle) with the exception of introduced variants at positions 21,895 and 26,843, which were fixed in the BAC and rSARS-CoV-2 virus. Non-reference alleles present in less than 1% of reads are not shown. D ) Plaque phenotype: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were infected with ∼20 PFU of rSARS-CoV-2 (left) or the natural SARS-CoV-2 isolate (right) for 1h at 37°C. After viral adsorption, cells were washed 3 times with PBS and overlaid with 2 ml of post-infection media containing 1% agar. After 72 h incubation at 37°C, cells were fixed and immunostained with the N protein 1C7 monoclonal antibody. D ) Growth kinetics: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were infected (MOI 0.01) with rSARS-CoV-2 or the natural SARS-CoV-2 isolate for 1h at 37°C. After viral adsorption, cells were washed 3 times with PBS and incubated in 2 ml of post-infection media. At the indicated times post-infection, tissue culture supernatants were collected and viral titers were assessed by plaque assay (PFU/ml). Data were presented in mean ± SD. LOD: limit of detection.
Figure Legend Snippet: Characterization of rSARS2-CoV-2 in vitro . A-B) Genotypic characterization: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were mock-infected or infected (MOI 0.01) with rSARS-CoV-2 or the SARS-CoV-2 USA-WA1/2020 natural isolate. After 1 h viral adsorption at 37°C, Vero E6 cells were washed 3 times with PBS and incubated in post-infection media at 37°C. At 24 h post-infection, total RNA from Vero E6 cells was extracted and a 1,297 bp region of the M gene (nt 26,488 to 27,784) was amplified by RT-PCR. Amplified DNA was subjected to MluI digestion ( Figure 1 ). Undigested (top) and digested (bottom) samples were separated in a 0.7% agarose gel ( A ). The RT-PCR amplified DNA product was also sequenced to verify the presence of the silent mutation in the MluI restriction site introduced in the viral genome of the rSARS-CoV-2 ( Figure 1 ). The MluI restriction site is underlined in red and the silent mutation introduced to remove the MluI restriction site (T to A) is shown in the black box ( B ). C ) Verification of BAC and rSARS-CoV-2 sequences: The SARS-CoV-2 non-reference allele frequency was calculated by comparing short reads to the reference genome of USA-WA1/2020 reference. All variants were at low frequency in P6 natural isolate (top), BAC (bottom), and rSARS-CoV-2 (middle) with the exception of introduced variants at positions 21,895 and 26,843, which were fixed in the BAC and rSARS-CoV-2 virus. Non-reference alleles present in less than 1% of reads are not shown. D ) Plaque phenotype: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were infected with ∼20 PFU of rSARS-CoV-2 (left) or the natural SARS-CoV-2 isolate (right) for 1h at 37°C. After viral adsorption, cells were washed 3 times with PBS and overlaid with 2 ml of post-infection media containing 1% agar. After 72 h incubation at 37°C, cells were fixed and immunostained with the N protein 1C7 monoclonal antibody. D ) Growth kinetics: Vero E6 cells (6-well plate, 10 6 cells/well, triplicates) were infected (MOI 0.01) with rSARS-CoV-2 or the natural SARS-CoV-2 isolate for 1h at 37°C. After viral adsorption, cells were washed 3 times with PBS and incubated in 2 ml of post-infection media. At the indicated times post-infection, tissue culture supernatants were collected and viral titers were assessed by plaque assay (PFU/ml). Data were presented in mean ± SD. LOD: limit of detection.

Techniques Used: In Vitro, Infection, Adsorption, Incubation, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis, BAC Assay, Plaque Assay

Rescue of rSARS-CoV-2. A ) Schematic representation of the approach followed to rescue rSARS-CoV-2: Vero E6 cells (6-well plates, 10 6 cells/well, triplicates) were transiently transfected with the SARS-CoV-2 BAC at day 1. After 24 h, transfection media was changed by post-infection media. At day 4, cells were split into T75 flask and the tissue culture supernatant was used to infect fresh Vero E6 cells (6-well plates, 10 6 cells/well, triplicates). At 48 h post-infection, Vero E6 cells were fixed for detection of rSARS-CoV-2 by immunofluorescence and the tissue culture supernatant of the scaled-up Vero E6 cells was collected at 72 h. As internal control for this experiment, Vero E6 cells were transfected with the empty BAC. B) CPE: Images of empty or SARS-CoV-2 BACs transfected Vero E6 cells at 72 h post-transfection. Scale bars, 100 μm. C) Viral titers: Tissue culture supernatant from mock (empty BAC) or transfected Vero E6 cells in T75 flask was collected and titrated by immunofluorescence. Data were presented in mean ± SD. LOD: limit of detection. D) IFA: Vero E6 cells (6-well plate format, 10 6 cells/well) infected with the tissue culture supernatants from transfected Vero E6 cells were fixed at 48 h post-infection and viral detection was carried out by using a SARS-CoV cross-reactive monoclonal antibody (1C7) against the N protein (green). Cellular nuclei were stained by DAPI (blue). Scale bars, 100 μm.
Figure Legend Snippet: Rescue of rSARS-CoV-2. A ) Schematic representation of the approach followed to rescue rSARS-CoV-2: Vero E6 cells (6-well plates, 10 6 cells/well, triplicates) were transiently transfected with the SARS-CoV-2 BAC at day 1. After 24 h, transfection media was changed by post-infection media. At day 4, cells were split into T75 flask and the tissue culture supernatant was used to infect fresh Vero E6 cells (6-well plates, 10 6 cells/well, triplicates). At 48 h post-infection, Vero E6 cells were fixed for detection of rSARS-CoV-2 by immunofluorescence and the tissue culture supernatant of the scaled-up Vero E6 cells was collected at 72 h. As internal control for this experiment, Vero E6 cells were transfected with the empty BAC. B) CPE: Images of empty or SARS-CoV-2 BACs transfected Vero E6 cells at 72 h post-transfection. Scale bars, 100 μm. C) Viral titers: Tissue culture supernatant from mock (empty BAC) or transfected Vero E6 cells in T75 flask was collected and titrated by immunofluorescence. Data were presented in mean ± SD. LOD: limit of detection. D) IFA: Vero E6 cells (6-well plate format, 10 6 cells/well) infected with the tissue culture supernatants from transfected Vero E6 cells were fixed at 48 h post-infection and viral detection was carried out by using a SARS-CoV cross-reactive monoclonal antibody (1C7) against the N protein (green). Cellular nuclei were stained by DAPI (blue). Scale bars, 100 μm.

Techniques Used: Transfection, BAC Assay, Infection, Immunofluorescence, Staining

15) Product Images from "Genetic Changes at the Glycoprotein Editing Site Associated With Serial Passage of Sudan Virus"

Article Title: Genetic Changes at the Glycoprotein Editing Site Associated With Serial Passage of Sudan Virus

Journal: The Journal of Infectious Diseases

doi: 10.1093/infdis/jiv216

Editing site genotype of Sudan virus (SUDV) after serial passage in Vero E6 cells. SUDV was serially passaged in Vero E6 cells at a low multiplicity of infection (MOI) of 0.001 plaque-forming units (PFU)/cell. Ultra-deep sequencing was used to investigate the relative abundance of uridine (U) at the editing site after each passage. A , SUDV was passaged 18 times. B , The first 5 passages were repeated at the same MOI. C , The first 5 passages were repeated at different MOIs (0.01, 0.1, and 1.0 PFU/cell).
Figure Legend Snippet: Editing site genotype of Sudan virus (SUDV) after serial passage in Vero E6 cells. SUDV was serially passaged in Vero E6 cells at a low multiplicity of infection (MOI) of 0.001 plaque-forming units (PFU)/cell. Ultra-deep sequencing was used to investigate the relative abundance of uridine (U) at the editing site after each passage. A , SUDV was passaged 18 times. B , The first 5 passages were repeated at the same MOI. C , The first 5 passages were repeated at different MOIs (0.01, 0.1, and 1.0 PFU/cell).

Techniques Used: Infection, Sequencing

16) Product Images from "SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2"

Article Title: SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

Journal: Cell

doi: 10.1016/j.cell.2020.09.033

Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .
Figure Legend Snippet: Manipulation of Cellular Heparan Sulfate Decreases Infection of Authentic SARS-CoV-2 Virus (A,) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) Vero cells stained with antibodies against SARS-CoV-2 nucleocapsid and spike protein. (B) SARS-CoV-2 infection of Vero cells performed in the absence and presence of HSase, or with incubation with different concentrations of unfractionated heparin (UFH). The extent of infection was analyzed by flow cytometry as in (A). The graph shows a composite of five separate experiments, each performed in triplicate. The MOI was 0.5, but the extent of infection varied. The MOI in the experiment shown in maroon and blue was 0.2. The mean data from the individual experiments are colorized to allow for separate visualization (C) Same data as in (B), but with the experimental data normalized to the mock infection for each respective experiment. (D) SARS-CoV-2 infection of Hep3B mutants altered in HS biosynthesis enzymes. Cells were infected for 1 h and incubated 48 h, allowing for new virus to form. The resulting viral titers in the culture supernatants were determined by plaque assays on Vero E6 cells. Average values with standard error mean are shown, along with the individual data points. The experiment was initially optimized and then performed in triplicate. (E) Flow cytometry analysis of SARS-CoV-2-infected (red) or uninfected (black) human bronchial epithelial cells at an air-liquid interface stained with antibodies against SARS-CoV-2 nucleocapsid. (F) SARS-CoV-2 infection of human bronchial epithelial cells at an air-liquid interface was performed in the absence and presence of HSase, or with incubation UFH. The extent of infection was analyzed by flow cytometry. The graph shows a composite of three separate experiments, each performed in triplicate. The mean data from the individual experiments are colorized to allow for separate visualization. (G) Same data as in (F), but with each experimental dataset normalized to the uninfected control. Statistical analysis by one-way ANOVA (B, C, and G) or unpaired t test (D); ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001). See also Figure S7 .

Techniques Used: Infection, Flow Cytometry, Staining, Incubation

Heparin and Heparin Lyases Have No Effect on ACE2 Expression or Cell Viability, Related to Figure 7 (A) Western blot analysis of ACE2 expression in Vero E6 cells treated with heparin lyases or 100 μg/mL UFH. (B and C) Assessment of cell viability after treatment with heparin lyase or 100 μg/mL UFH for 16 h in Vero (B) and human bronchial epithelial cells (HBEC) (C). Cell viability was measured using CellTiter-Blue. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).
Figure Legend Snippet: Heparin and Heparin Lyases Have No Effect on ACE2 Expression or Cell Viability, Related to Figure 7 (A) Western blot analysis of ACE2 expression in Vero E6 cells treated with heparin lyases or 100 μg/mL UFH. (B and C) Assessment of cell viability after treatment with heparin lyase or 100 μg/mL UFH for 16 h in Vero (B) and human bronchial epithelial cells (HBEC) (C). Cell viability was measured using CellTiter-Blue. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ : p ≤ 0.05, ∗∗ : p ≤ 0.01, ∗∗∗ : p ≤ 0.001, ∗∗∗∗ : p ≤ 0.0001).

Techniques Used: Expressing, Western Blot

SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .
Figure Legend Snippet: SARS-CoV-2 Pseudovirus Infection Depends on Heparan Sulfate (A) Left, SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells measured by flow cytometry with and without HSase. Right, heparin and split-glycol heparin inhibit SARS-CoV-2 spike protein (20 μg/mL) binding to Vero cells by flow cytometry. Statistical analysis by unpaired t test. (B) Western blot analysis of ACE2 expression in Vero E6 cells compared to A549, H1299, and A375 cells. A representative blot of three extracts is shown for each strain. (C) Infection of Vero E6 cells with SARS-CoV-2 spike protein expressing pseudotyped virus expressing GFP. Infection was done with and without HSase treatment of the cells. Insert shows GFP expression in the infected cells by imaging. Counting was performed by flow cytometry with gating for GFP-positive cells as indicated by “infected.” (D) Quantitative analysis of GFP-positive cells. (E) Infection of Vero E6 cells with SARS-CoV-2 S protein pseudotyped virus expressing luciferase, as measured by the addition of Bright-Glo and detection of luminescence. The figure shows infection experiments done at low and high titer. (F) HSase treatment diminishes infection by SARS-CoV-2 S protein pseudotyped virus (luciferase) at low and high titer. (G) Heparin (0.5 μg/mL) blocks infection with SARS-CoV-2 S protein pseudotyped virus (luciferase). (H) Effect of HSase treatment of Vero E6 cells on the infection of both SARS-CoV-1 S and SARS-CoV-2 S protein pseudotyped virus expressing luciferase. (I) Infection of Hep3B with and without HSase and in Hep3B cells containing mutations in EXT1 , NDST1 , and HS6ST1 / HS6ST2 . Cells were infected with SARS-CoV-2 S protein pseudotyped virus expressing luciferase. All experiments were repeated at least three times. Graphs shows representative experiments performed in technical triplicates. Statistical analysis by unpaired t test. (ns: p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). See also Figure S6 .

Techniques Used: Infection, Binding Assay, Flow Cytometry, Western Blot, Expressing, Imaging, Luciferase

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Amplification:

Article Title: Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome
Article Snippet: .. The SARS-CoV-2 USA-WA1/2020 natural isolate was obtained from BEI Resources (NR-52281) and amplified on Vero E6 cells. .. This strain was selected because it was isolated from an oropharyngeal swab from a patient with respiratory illness in January 2020 in Washington, DC.

Plaque Assay:

Article Title: Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
Article Snippet: .. Cells The specific Vero E6 cells cultured and plated for the plaque assay (C1008) were obtained from BEI Resources at passage Number 24 (Manassas, VA, USA). ..

Mutagenesis:

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice
Article Snippet: .. Virus growth in Huh-7 cells also shows reduced to no mutation frequency as compared to virus growth in Vero E6 cells [ , ]. .. Reduced mutation frequency will be especially important if further characterization of virus found in breast milk and semen is to be completed.

Isolation:

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice
Article Snippet: .. When target titers ranged from 1 to 10 PFU/mL, isolation success decreased to 25% (2/8) in Vero E6 cells, 50% (5/10) in Huh-7 cells, 38% (3/8) in MDM cells, and 50% (2/4) in mice. .. Isolation attempts were unsuccessful for all methods tested when the titer dropped below 1 PFU/mL (0%).

Cell Culture:

Article Title: Validation of the Filovirus Plaque Assay for Use in Preclinical Studies
Article Snippet: .. Cells The specific Vero E6 cells cultured and plated for the plaque assay (C1008) were obtained from BEI Resources at passage Number 24 (Manassas, VA, USA). ..

Mouse Assay:

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice
Article Snippet: .. When target titers ranged from 1 to 10 PFU/mL, isolation success decreased to 25% (2/8) in Vero E6 cells, 50% (5/10) in Huh-7 cells, 38% (3/8) in MDM cells, and 50% (2/4) in mice. .. Isolation attempts were unsuccessful for all methods tested when the titer dropped below 1 PFU/mL (0%).

other:

Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice
Article Snippet: This stock was passaged three times in Vero E6 cells (obtained through BEI Resources, NIAID, NIH: VERO C1008 (E6), Kidney (African green monkey), Working Cell Bank, NR-596) in MEM-α, L-alanyl-L-glutamine without nucleosides (Gibco, ThermoFisher Scientific) supplemented with 2% US-origin, certified HI-FBS.

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    BEI Resources vero e6 cells
    Positive control data collected on <t>Vero</t> <t>E6</t> cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.
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    Positive control data collected on Vero E6 cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.

    Journal: Viruses

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

    doi: 10.3390/v8040113

    Figure Lengend Snippet: Positive control data collected on Vero E6 cells of various passage ages. Twenty eight measurements of the EBOV Positive Control (PC) collected over pre-, post- and on-validation studies were performed on cell passages ranging from 30 to 41 (filled circles). Averages and standard deviations are presented for multiple results on the same passage. The solid line depicts the nominal PC target titer of 1.09 × 10 5 PFU/mL, and the dotted line denotes the lower PC cutoff limit of 34,469 PFU/mL, below which the assay fails and a repeat is required.

    Article Snippet: Cells The specific Vero E6 cells cultured and plated for the plaque assay (C1008) were obtained from BEI Resources at passage Number 24 (Manassas, VA, USA).

    Techniques: Positive Control

    10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

    Journal: Viruses

    Article Title: Ebola Virus Isolation Using Huh-7 Cells has Methodological Advantages and Similar Sensitivity to Isolation Using Other Cell Types and Suckling BALB/c Laboratory Mice

    doi: 10.3390/v11020161

    Figure Lengend Snippet: 10× images of MDM, Vero E6, and Huh-7 cells taken 10 days following exposure to select volumes of normal semen, breast milk, or cell culture media (control). Breast milk was toxic at 50 and 100 µL/T-25 flask on Vero E6 cells (no cells present after 10 days) and MDM cells, but toxicity was less on these cells than on Huh7 cells.

    Article Snippet: When target titers ranged from 1 to 10 PFU/mL, isolation success decreased to 25% (2/8) in Vero E6 cells, 50% (5/10) in Huh-7 cells, 38% (3/8) in MDM cells, and 50% (2/4) in mice.

    Techniques: Cell Culture

    Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).

    Journal: bioRxiv

    Article Title: Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L

    doi: 10.1101/2020.07.27.223727

    Figure Lengend Snippet: Antiviral activity of GC-376 analogues. a-d Antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay. a GC-376 ; b UAWJ246 ; c UAWJ247 ; d UAWJ248 . Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hpi, the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (Green). The nuclei were counterstained with Hoechst dye (Blue). For each well, fluorescence images of approximately 10K cells were acquired and shown. The images are representatives of two repeats. e-h Antiviral activity of GC-376 analogues against SARS-CoV-2 in the plaque assay. e GC-376 ; f UAWJ246 ; g UAWJ247 ; h UAWJ248 . Vero E6 cells in 6-well plates were infected with approximately 40 PFU/well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 dpi, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogues against SARS-CoV-2 in the immunofluorescence assay ( i ) and the plaque assay ( j ).

    Article Snippet: SARS-CoV-2, isolate USA-WA1/2020 (NR-52281), was obtained through BEI Resources and propagated once on VERO E6 cells before it was used for this study.

    Techniques: Activity Assay, Immunofluorescence, Infection, Staining, Fluorescence, Plaque Assay

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

    Journal: Viruses

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

    doi: 10.3390/v4123511

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

    Article Snippet: Vero E6 cells were obtained from BEI Resources (Manassas, VA, USA).

    Techniques: Produced

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

    Journal: Viruses

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

    doi: 10.3390/v4123511

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

    Article Snippet: Vero E6 cells were obtained from BEI Resources (Manassas, VA, USA).

    Techniques: Quantitation Assay, Plaque Assay

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

    Journal: Viruses

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

    doi: 10.3390/v4123511

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

    Article Snippet: Vero E6 cells were obtained from BEI Resources (Manassas, VA, USA).

    Techniques: