Journal: bioRxiv

Article Title: A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine

doi: 10.1101/2020.07.11.198291

Figure Lengend Snippet: Expression of SARS-CoV-2 S protein in Vero and 293T cells. Photographic depiction of fusion activity in Vero or 293T cells 48 h after transfection with 1 µg of SARS-CoV-2 S expression plasmid of control DNA. One representative out of three independent experiments is shown. Scale bar represents 100 mm.

Article Snippet: Cells Vero (African green monkey kidney) (ATCC# CCL-81), Vero clone E6 (ATCC# CRL-1586), 293T (ATCC CRL-3216) and EL-4 (ATCC TIB-39) cell lines were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and 2 mM L-glutamine (L-Gln; Biochrom).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Human dengue virus serotype 2 neutralizing antibodies target two distinct quaternary epitopes

doi: 10.1371/journal.ppat.1006934

Figure Lengend Snippet: HMAbs 2D22 and 1L12 use EDIII in their epitopes. (A) rDENV4/2-EDIII is DENV4 virus containing entire EDIII from DENV2. rDENV4/2-EDIII R323G is rDENV4/2-EDIII virus with single point mutation at residue 323. 2D22 and 1L12 were assessed for their ability to bind (B and D) and neutralize (C and E) recombinant DENVs in ELISA binding assays and Vero-81 Focus Reduction Neutralization Tests (FRNT). Dotted line in ELISA represents the background signal. FRNT 50 represents the concentration of antibody required to neutralize 50% of infection. # = virus was not neutralized at highest concentration of hMAb tested (5ng/μl).

Article Snippet: Vero-81 cells (ATCC CCL-81) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C.

Techniques: Mutagenesis, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization, Concentration Assay, Infection

Journal: PLoS Pathogens

Article Title: Human dengue virus serotype 2 neutralizing antibodies target two distinct quaternary epitopes

doi: 10.1371/journal.ppat.1006934

Figure Lengend Snippet: HMAb 3F9 use an epitope contained within EDI. (A) rDENV2/4-EDI/II is DENV2 virus containing EDI/II hinge region residues from DENV4. rDENV2/4-EDI is DENV2 virus containing EDI residues from DENV4. 3F9 was assessed for its ability to bind (B and E) and neutralize (C and F) recombinant DENVs in ELISA binding assays and Focus Reduction Neutralization Tests (FRNT) in Vero-81 cells (C) or C6/36 cells (F). Dotted line in ELISA represents the background signal. (D) rDENV4/2-EDI is DENV4 virus containing EDI residues from DENV2.

Article Snippet: Vero-81 cells (ATCC CCL-81) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization

Journal: PLoS Pathogens

Article Title: Human dengue virus serotype 2 neutralizing antibodies target two distinct quaternary epitopes

doi: 10.1371/journal.ppat.1006934

Figure Lengend Snippet: HMAbs 2D22 and 1L12 use different critical residues in their epitopes. (A) rDENV4/2-EDIII 5aa is a DENV4 virus with five EDIII residues from DENV2. 2D22 and 1L12 were assessed for their ability to bind (B and D) and neutralize (C and E) recombinant DENV in ELISA binding assays and Vero-81 Focus Reduction Neutralization Tests (FRNT). Dotted line in ELISA represents the background signal, determined as the OD value of wells containing all reagents except for viral antigen.

Article Snippet: Vero-81 cells (ATCC CCL-81) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization

Journal: Veterinary Sciences

Article Title: Stability of Porcine Epidemic Diarrhea Virus on Fomite Materials at Different Temperatures

doi: 10.3390/vetsci5010021

Figure Lengend Snippet: Light microscopic images showing distribution and characteristics of PEDV plaques in immunoplaque assay. ( A ) Virus-positive well showing plaques on Vero 76 cells infected with PEDV; ( B ) un-infected control well; ( C ) high magnification of a typical plaque showing viral-induced syncytium (Scale bar: 100 μm); ( D ) comparison of immunoplaque assay with and without agarose overlay. Data presented are average ± SEM from two independent experiments.

Article Snippet: Virus titration was performed on Vero 76 cells’ monolayers contained in 96-well microtiter plates using serial 5-fold dilutions of samples.

Techniques: Infection

Journal: Infection and Immunity

Article Title: Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

doi: 10.1128/IAI.02316-14

Figure Lengend Snippet: Cytotoxicity of wild-type and mutant TcdB. Vero cells in 96-well plates were exposed to 1 or 10 ng/ml of TcdB, TcdB-L543A (LA), or TcdB-W102A D288N (aTcdB) for 1 to 4 days. Supernatants from each well were harvested, and LDH activity in supernatants was

Article Snippet: Cytotoxicity of Vero cells was measured by a lactate dehydrogenase (LDH) cytotoxicity kit (Pierce) according to the manufacturer's instructions.

Techniques: Mutagenesis, Activity Assay

Journal: bioRxiv

Article Title: Establishment of a Replicon System for Bourbon Virus and Identification of Small Molecules that Efficiently Inhibit Virus Replication

doi: 10.1101/2020.04.24.058693

Figure Lengend Snippet: Determination of the CC 50 and EC 50 of compound #9 in cell cultures. (A B) EC 50 of compound #9. HEK293T cells (A) or Vero cells (B) were infected with BRBV at an MOI of 10, followed by application of compound #9 at various concentrations as indicated. At 3 days post treatment, RT-qPCR was performed to quantify the viral genome copy (vgc) numbers in the cell culture media. Values are the means with standard deviation and are normalized to a mock group, and were obtained from three experiments, each performed in duplicate. The EC 50 was calculated using GraphPad Prism software. (C D) CC 50 of compound #9 . A cytotoxicity assay was used to measure the viability of HEK293T cells (C) or Vero cells (D) affected by compound #9 at various concentrations as indicated. The percentage of viable cells were determined using a CytoTox-Glo cytotoxicity assay kit (Promega). The results are shown as relative values to the mock control cells. Values are the means with standard deviation obtained from three experiments performed in duplicate. The CC 50 was calculated using GraphPad Prism software.

Article Snippet: BRBV-KS infection of HEK293T cells We propagated BRBV-KS, obtained from BEI Resources ( www.beiresources.org/ ), in Vero cells.

Techniques: Infection, Quantitative RT-PCR, Cell Culture, Standard Deviation, Software, Cytotoxicity Assay

Journal: bioRxiv

Article Title: Establishment of a Replicon System for Bourbon Virus and Identification of Small Molecules that Efficiently Inhibit Virus Replication

doi: 10.1101/2020.04.24.058693

Figure Lengend Snippet: Comparisons of BRBV-KS infection between Vero and HEK293T cells (A) BRBV growth kinetics. Cells were infected with BRBV-KS at an MOI=10 pfu/cell. Cell culture media (supernatants) were collected daily for 6 days. Viral RNA was extracted from the samples of supernatants and then titrated by a RT-qPCR assay. The data presented the means with standard deviations, which are obtained from three independent experiments, with each experiment analyzing samples in duplicate. (B) Western blotting. The M protein in BRBV-infected Vero and HEK293T cells was detected at ~30 kDa as indicated, but not in mock-infected cells. β-action was probed as a loading control.

Article Snippet: BRBV-KS infection of HEK293T cells We propagated BRBV-KS, obtained from BEI Resources ( www.beiresources.org/ ), in Vero cells.

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Establishment of a Replicon System for Bourbon Virus and Identification of Small Molecules that Efficiently Inhibit Virus Replication

doi: 10.1101/2020.04.24.058693

Figure Lengend Snippet: Titration of BRBV stock. (A) Plaque assay. Vero cell monolayers on a 6-well plate were infected with 200 μl of serial 10-fold diluted BRBV-KS as indicated. Plaques were visualized by staining with crystal violet after 5 days of infection on Vero cells. Arrows indicate plaques. (B) Comparison between plaque assay and RT-qPCR. The same virus stock was diluted by serial 10-fold dilution. RT-qPCR was used to quantify the viral genome copy (vgc) number. The linear regression has a R 2 of 0.9875, indicating 1 plaque forming unit (pfu) equals ~100 vgc. The data presented are averages and standard deviations from three independent experiments, with each experiment analyzing samples in duplicate.

Article Snippet: BRBV-KS infection of HEK293T cells We propagated BRBV-KS, obtained from BEI Resources ( www.beiresources.org/ ), in Vero cells.

Techniques: Titration, Plaque Assay, Infection, Staining, Quantitative RT-PCR

Journal: mBio

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

doi: 10.1128/mBio.02168-20

Figure Lengend 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.

Article Snippet: The SARS-CoV-2 USA-WA1/2020 natural isolate was obtained from BEI Resources (NR-52281) and amplified on Vero E6 cells.

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

Journal: mBio

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

doi: 10.1128/mBio.02168-20

Figure Lengend 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.

Article Snippet: The SARS-CoV-2 USA-WA1/2020 natural isolate was obtained from BEI Resources (NR-52281) and amplified on Vero E6 cells.

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

Journal: PLoS Pathogens

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

doi: 10.1371/journal.ppat.1005455

Figure Lengend 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.

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.

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

Journal: PLoS Pathogens

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

doi: 10.1371/journal.ppat.1005455

Figure Lengend 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).

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.

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