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

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: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Sequence and model of MERS-CoV S protein fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FPs). FP1 and FP2 designate the two different domains in the FP. Sequences illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E; in green are the A substitutions. (B) Modeling of the MERS-CoV S protein monomer with an emphasis on the FP. Negatively charges D and E are depicted as atomic bonds in red. The S2’ site is orange, and the FP1 and FP2 domains are labeled blue and pink, respectively.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Sequencing, Mutagenesis, Labeling

Journal: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S protein WT and mutants. (A) Plasmid DNA encoding MERS-CoV S protein WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B and C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection, and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full-length S proteins are visible at approximately 250 kDa. S1/S2-cleaved S proteins are visible at approximately 115 kDa.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

Journal: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Immunofluorescence assay of MERS-CoV S protein WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding the respective MERS-CoV S protein variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleave MERS-CoV S proteins for fusion, no further protease treatment was needed to induce syncytium formation. WT + furin inhibitor (FI) indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 μM was added at the time of transfection to block fusion. Syncytia were visualized using immunofluorescence microscopy by staining the MERS-CoV S protein with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI; in blue). Images were taken at a magnification of ×25. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted, and the average number of nuclei per syncytium was calculated. Error bars represent standard deviations (n = 9). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against each of the respective mutant *, P > 0.5; **, P > 0.05; ***, P > 0.005.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Mutagenesis

Journal: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Western blot analysis of S proteins incorporated into PPs. A total of 1 ml of DMEM containing PPs per each tested S protein was ultracentrifuged, washed in PBS, and resuspended in SDS Laemmli buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S protein antibodies.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Western Blot, SDS Page

Journal: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Pseudoparticle assays of MERS-CoV S protein WT and mutants. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S protein WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays. (C) Impact of intracellular Ca2+ on MERS-CoV fusion. Cells were pretreated with growth medium containing either 50 μM calcium chelator BAPTA-AM or dimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PPs in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessment for luciferase activity. (D) Impact of extracellular Ca2+ on MERS-CoV fusion. Cells were pretreated with growth medium either with or without 1.8 mM Ca2+ for 1 h. The infection protocol is as described above except PPs were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviations (n = 3). Statistical analysis was performed using an unpaired Student’s t test, as indicated. *, P > 0.5; **, P > 0.05; ***, P > 0.005.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Infection, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Ca 2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity

doi: 10.1128/JVI.00426-20

Figure Lengend Snippet: Pseudoparticle assays of MERS-CoV S protein WT and E891A/D896A. Huh-7 cells were infected with MLV-based pseudoparticles (PPs) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to the WT sample. Error bars represent standard deviations (n = 3). Statistical analysis was performed using an unpaired Student’s t test comparing the WT against the respective mutant (for B and C, the untreated WT was compared to each sample). *, P > 0.5; **, P > 0.05; ***, P > 0.005. (A) Infectivity of PPs without pretreatment of cells. (B) Impact of intracellular Ca2+ on MERS-CoV fusion. Cells and PPs were treated as described for Fig. 5C. (C) Impact of extracellular Ca2+ on MERS-CoV fusion. Cells and PPs were treated as described for Fig. 5D.

Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (catalog no. 40069-RP01; Sino Biological) as the primary antibody and Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

Techniques: Infection, Mutagenesis

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal antibody (pAb) to MERS-CoV S2 antibody , Sino Biological , Cat:40070-T62 , WB (1:1000).

Techniques: Purification, In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Plasmid Preparation, Sequencing