Journal: Mikrochimica Acta

Article Title: Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

doi: 10.1007/s00604-023-05747-6

Figure Lengend Snippet: Evaluation of specificity of the RCA-enabled fluorometric ( a ) and colorimetric assay ( b ). The signal intensity generated from S protein RBD is compared with that from N protein, MERS protein RBD, SARS-Cov-2 B.1.617.2 spike protein RBD, and a mixture of the three proteins. The total protein concentration in each sample was 50 ng/mL

Article Snippet: Recombinant SARS-CoV-2 receptor-binding domain (RBD, source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541), with a 6x-His tag at C-terminal), recombinant MERS-CoV Spike RBD (Chinese Hamster Ovary-derived MERS-CoV Spike RBD protein (Glu367-Tyr606), with a 6x-His tag at C-terminal), recombinant SARS-CoV-2 B.1.617.2 RBD (source: HEK293-derived SARS-CoV-2 Spike RBD protein (Arg319-Phe541 (Leu452Arg, Thr478Lys), with a 6x-His tag at C-terminal), and recombinant SARS-CoV-2 nucleocapsid (source: Spodoptera frugiperda , Sf 21 (baculovirus)-derived SARS-CoV-2 nucleocapsid (Met1-Ala419), with a 6x-His tag at C-terminal) were purchased from Bio-Techne (Minneapolis, USA).

Techniques: Colorimetric Assay, Generated, Protein Concentration

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 2. Mutations at S1/S2, S2′ positions were needed for SMERS to be expressed on the surface NDV particles. (A) The genomic arrangement of NDV-SMERS-S1/S2. Chicken-codon optimized SMERS with S1/S2 cleavage site mutation, 2P mutation, tPA secretion peptide, transmembrane domain (TMF) and cytoplasmic tail (CTF) of NDV F protein was inserted between P and M genes of NDV genome. (B) Allantoic fluid (ALF) harvested from embryonated chicken eggs inoculated with NDV-SMERS-S1/S2, and the ultracentrifuged virus samples (UC-virus) were subjected to a Western blot using anti-MERS-CoV

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Virus, Western Blot

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 3. PCM1 and PCM2 did not improve SMERS expression. (A) A diagram of different versions of SMERS containing combinations of S1/S2, S2’, 2P, PCM1, PCM2 mutations. (B) HEK-293T/17 cells were transfected with 1 µg pCAGGS containing each version of SMERS gene. Cell lysates harvested at 72 h post transfection were subjected to a Western blot analysis using anti-MERS-CoV S2 and anti-β-actin antibodies. (C) The volumes of the ultracentrifuged virus samples were adjusted to equal TCID50/mL. The samples were then subjected to a Western blot analysis using anti-MERS-CoV S2 and anti-NDV N antibodies.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Expressing, Transfection, Western Blot, Virus

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 4. 2P mutation improved surface SMERS expression. (A) A diagram comparing the gene insert of NDV-SMERS-S1/S2, NDV-SMERS-S2’, and NDV-SMERS-S2’ no2P. (B) The ultracentrifuged virus samples were subjected to Western blot analysis using anti-MERS-CoV S2 and anti-NDV N antibodies, and (C) an MERS-CoV S1-sandwich ELISA. **** indicates significant difference (p < 0.0001). (D) The ultracentrifuged samples were stained with rabbit anti MERS-CoV S1 antibody, and then the 10-nm gold-conjugated goat anti-rabbit IgGs. Images were recorded under a TEM at 50,000× magnification.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Expressing, Virus, Western Blot, Sandwich ELISA, Staining

Journal: Vaccines

Article Title: Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

doi: 10.3390/vaccines13010002

Figure Lengend Snippet: Figure 6. SMERS gene insert was stable after 5 egg-passages. (A) Viral RNAs of NDV-mCh and NDV- SMERS-S2’ from egg-passages 2–5 were purified and subjected to RT-PCRs using SMERS- and NNDV- specific primers. pCI.NDV-SMERS plasmid was used as positive controls. (B) The ultracentrifuged NDV-SMERS-S2’ samples from egg-passage 3–5 (E3, E4, and E5) were stained with rabbit anti MERS- CoV S1 antibody, followed by 10-nm gold-conjugated goat anti-rabbit IgGs. Images were recorded under a TEM at 50,000× magnification.

Article Snippet: Uninfected and NDV-infected DF-1 cells were fixed with 80% ice-cold acetone or 4% formaldehyde for 20 min. After 3 washes with PBS, samples were incubated with a blocking buffer (1% BSA and 10% FBS in PBS) for 1 h, followed by an incubation with mouse anti-MERS-CoV S1 monoclonal antibody (MAB10707, R&D system’s, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Purification, Plasmid Preparation, Staining

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Precision, Accuracy of all 9 CoV SeroAssay Capture Antigens. a

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Concentration Assay

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Identifying Information for Nine Human Coronavirus Spike Antigens Represented on the CoV SeroAssay.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Expressing, Binding Assay

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Sensitivity, Linear Dynamic Range of all 9 CoV SeroAssay Capture Antigens.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques:

Journal: Journal of Virological Methods

Article Title: Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses

doi: 10.1016/j.jviromet.2021.114111

Figure Lengend Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens.

Article Snippet: Fluorescence microarray images illustrating binding of monoclonal antibodies to the CoV SeroAssay. (a) CR3022 SARS-CoV-1 antibody from Creative Biolabs binding to the nCoV(ii) and SARS antigens, (b) 40021-MM07 HKU1 antibody from Sino Biological binding to HKU1 antigen, (c) 40069-MM23 MERS antibody from Sino Biological binding to the MERS antigen, and (d) GTX632604 SARS-CoV-2 antibody from Genetex binding to the nCoV(i) and nCoV(iii) antigens. (a) Schematic illustration of microarray layout, and representative fluorescence images of the VaxArray CoV SeroAssay microarray in (b) through (l).

Techniques: Fluorescence, Microarray, Binding Assay

Journal: The Lancet. Infectious Diseases

Article Title: T-cell responses to MERS coronavirus infection in people with occupational exposure to dromedary camels in Nigeria: an observational cohort study

doi: 10.1016/S1473-3099(20)30599-5

Figure Lengend Snippet: MERS-CoV-specific CD4 + and CD8 + T-cell responses in camel workers and controls (A) Frequencies of MERS-CoV-specific CD4+ T cells. (B) Frequencies of MERS-CoV-specific CD8+ T cells. (C) Summary of aggregate CD4+ T-cell responses to all structural peptide pools in different study groups. (D) Summary of aggregate CD8+ T-cell responses to all structural peptide pools in different study groups. (E) CD4+ T-cell responses to MERS-CoV accessory protein-specific peptide pools. (F) Phenotypes of virus-specific CD4+ T cells. (G,H) Phenotypes of virus-specific CD8+ T cells. Abattoir workers with exposure to dromedaries are represented by red symbols, those without exposure to dromedaries by green symbols, non-abattoir workers by light blue symbols, MERS-positive controls by dark blue symbols (open shapes represent asymptomatic patients), and negative controls from Guangzhou by purple symbols. Symbol shape identifies the same individual. IFN=interferon. MERS-CoV= Middle East respiratory syndrome coronavirus. TNF=tumour necrosis factor. **=p<0·01. ***=p<0·001.

Article Snippet: Anti-MERS-CoV antibody titres were determined using plaque reduction neutralisation tests., A set of 20-mer peptides overlapping by ten amino acids encompassing the four MERS-CoV (HCoV-EMC/2012) structural proteins (peptides S1, S2, N, and ME encompassing the N-terminal and C-terminal portions of the spike [S] glycoprotein, the nucleocapsid [N] protein, and the transmembrane [M] and envelope [E] proteins) and five accessory proteins (ORF3, ORF4a, ORF4b, ORF5 and ORF8b) were synthesised by Sino Biological (Shanghai, China), and used for stimulation of PBMCs.

Techniques:

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: In vitro characterization of purified equine immunoglobulin against SARS-CoV-2. (A) The neutralizing titers of hyperimmune serum, purified IgG, and F(ab’) 2 derived from equine No. 15 and No. 16 were tested with wild type SARS-CoV-2 Wuhan 01. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (B) The titers of purified SARS-CoV-2-specific IgG in equine sera were examined via RBD-capture ELISA. Two repeated tests were performed on each sample.

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: In Vitro, Purification, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Broad-spectrum neutralizing activity test against SARS-CoV-2 VOC and VOI. The neutralizing antibody titers were calculated as the highest dilution of sera that completely inhibited virus-caused CPE. The serum neutralizing antibody titer was defined as the reciprocal of the highest dilution showing a 100% CPE reduction compared to the virus control. (A) Neutralizing antibody titers of purified IgG and F(ab’) 2 of equine No.15 against SARS-CoV-2 VOC; (B) Neutralizing antibody titers of purified IgG and F(ab’) 2 of equine No.16 against SARS-CoV-2 VOC; (C) Neutralizing antibody titers of purified equine immunoglobulin of equine No.15 against SARS-CoV-2 VOI; (D) Neutralizing antibody titers of purified equine immunoglobulin of equine No.16 against SARS-CoV-2 VOI. Comparison to the wild type SARS-CoV-2 Wuhan01, the number above the column represented the fold by which the neutralizing titer of the IgG or F(ab’) 2 was weakened by the SARS-CoV-2 VOC and VOI. Samples were processed in triplicate, and error bars indicate standard error. Data are presented as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Activity Assay, Purification

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Evaluation of the protective efficacy of purified equine immunoglobulin in a mouse model. Groups of 13 BALB/c mice were administered with IgG or F(ab’) 2 at 1 day before mouse-adapted SARS-CoV-2 (BMA8) infection or 1 dpi with BMA8. Each mouse was given 250 µg of antibody at a dose of 10 mg/kg. BALB/c mice were challenged intranasally with a lethal dose 50 LD 50 of BMA8 before treatment or after administration. The survival rate, weight change, body temperature and clinical scores of BALB/c mice were monitored daily after SARS-CoV-2 BMA8 infection. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure; (B) Survival rate. (C) Percent weight change. (D) Body temperature change. Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Purification, Infection

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Blood counts in SARS-CoV-2-infected mice. The hematological values of BALB/c mice were analysed, including lymphocyte (LYM), neutrophil percentage (Neu%), monocytes (Mon), platelet count (PLT) and white blood cell count (WBC), at 3 dpi after SARS-CoV-2 BMA8 infection. Four infected mice were sacrificed at 3 dpi to collect the whole blood for blood counts test. (A) White blood cell (WBC) count; (B) neutrophil (Neu) percentage; (C) lymphocyte (LYM) percentage; (D) platelet (PLT) (E) Monocyte(Mno). Data are presented as the mean ± SEM (n=4). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Infection, Cell Counting

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Histopathological and immunohistochemistry findings in SARS-CoV-2-infected mice. The lungs and spleens were collected from the control mice infected with SARS-CoV-2 without equine immunoglobulin drug injection at 3dpi, and the lungs, spleens, livers and kidneys were harvested from recovered mice. After each tissue was embedded in paraffin, the sections were sectioned for HE staining. (A, B, E, F) Lung tissue changes of control mice were characterized by more necrotic epithelial cells (blue arrow), a small amount of neutrophil infiltration, and perivascular edema with a small amount of inflammatory cell infiltration in the local alveolar cavity (yellow arrow). (C, D, G, H) Spleen tissue changes of control mice were characterized with spotted apoptosis of lymphocytes, nuclear pyknosis and deep staining or fragmentation in the spleen nodules (black arrows), and the expansion of germinal centers (yellow arrow), scattered neutrophils mostly seen in the red pulp granulocyte infiltration (red arrow), and more brown‒yellow particles in the red pulp (blue arrow). (I-L) The basically normal structure of the lung, spleen liver, and kidney tissues were found in administration groups given equine IgG or F(ab’) 2 . The figure showed immunohistochemistry (IHC) labeling against SARS-CoV-2 N. (M) Viral antigen was not detectable in prevention group given purified IgG; (N) Viral antigen was not detectable in prevention group given purified F(ab’) 2 ; (O) Viral antigen was detected for positive in prevention control group; (P) Viral antigen was not detectable in treatment group given purified IgG; (Q) Viral antigen was not detectable in treatment group given purified IgG F(ab’) 2 ; (R) Viral antigen was detected for positive in treatment control group. (scale bar = 100 μm).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Immunohistochemistry, Infection, Injection, Staining, Labeling, Purification

Journal: Frontiers in Immunology

Article Title: Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1066730

Figure Lengend Snippet: Evaluation of the protective efficacy of purified equine immunoglobulin in the golden hamster model. Each golden hamster was given 500 µg of antibody at a dose of 10 mg/kg. Groups of golden hamsters were infected intranasally with 1,000 TCID 50 of wild-type SARS-CoV-2 Wuhan 01 before treatment and or after administration. The survival rate and weight change of BALB/c mice were monitored daily after SARS-CoV-2 Wuhan01 infection. Four infected golden hamsters in each group were sacrificed at 3 dpi, and the turbinate and lung samples were collected to analyze the viral RNA loads by RT‒qPCR and TCID 50 , respectively. (A) Schematic diagram of the administration of equine immunoglobulin drugs and virus challenge procedure. (B) Survival rate. (C) Percent weight change; Body weight change of mice in a with comparison to isotype control was measured by repeated measurements two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Data are mean ± s.e.m. of each experimental group. (D) The viral loads of turbinate were quantified by RT‒qPCR at 3 dpi in each group; (E) The viral loads of lung were quantified by RT‒qPCR at 3 dpi in each group; (F) The viral loads of turbinate were determined by TCID 50 at 3 dpi in each group; (G) The viral loads of lung were determined by TCID 50 at 3 dpi in each group. Data are presented as the mean ± SEM (n=5). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN).

Techniques: Purification, Infection

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b – f Detection of CoVs S protein in cells lysate by western blot. Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g – j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa. Experiments were done twice and one is shown. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Western Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a , b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Transduction, Luciferase, Stable Transfection, Expressing, Binding Assay, Incubation, Flow Cytometry, Inhibition

Journal: Nature Communications

Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

doi: 10.1038/s41467-020-15562-9

Figure Lengend Snippet: a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d , e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod ( d ), YM201636 ( e ), or tetrandrine ( f ), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Inhibition, Transduction, Luciferase, Activity Assay, Infection, MTT Assay