Journal: Journal of Virology

Article Title: Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

doi: 10.1128/JVI.00015-16

Figure Lengend Snippet: Analysis of expression of pFP mutants of MERS-CoV S protein in 293T cells. (A) Western blot analysis of expression of WT or mutant MERS S protein in cell lysate. The MERS S protein was detected by using mouse monoclonal anti-MERS S antibody; β-actin was detected with mouse monoclonal anti-actin antibody. FL, full length. (B) Analysis of surface expression of mutant MERS-CoV S protein by flow cytometry. MERS-CoV S protein-expressing 293T cells were stained with mouse monoclonal anti-MERS S antibody. The amount of wild-type S protein on cell surfaces was set as 100%. All of the experiments shown were repeated at least three times.

Article Snippet: The primary antibodies for SARS-CoV SΔ19, MERS-CoV SΔ16, and MHV S protein were rabbit polyclonal anti-SARS S1 antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), mouse monoclonal anti-MERS S antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), and goat polyclonal anti-MHV S antibody (AO4) (1:200 dilution), respectively.

Techniques: Expressing, Western Blot, Mutagenesis, Flow Cytometry, Staining

Journal: Journal of Virology

Article Title: Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

doi: 10.1128/JVI.00015-16

Figure Lengend Snippet: Receptor binding by mutant MERS S proteins. MERS-CoV S protein-expressing 293T cells were incubated with soluble AVI-tagged hDPP4, followed with polyclonal rabbit anti-AVI antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. The results from the wild type were set as 100%.

Article Snippet: The primary antibodies for SARS-CoV SΔ19, MERS-CoV SΔ16, and MHV S protein were rabbit polyclonal anti-SARS S1 antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), mouse monoclonal anti-MERS S antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), and goat polyclonal anti-MHV S antibody (AO4) (1:200 dilution), respectively.

Techniques: Binding Assay, Mutagenesis, Expressing, Incubation

Journal: Journal of Virology

Article Title: Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

doi: 10.1128/JVI.00015-16

Figure Lengend Snippet: Entry of pseudotype virions with wild-type or mutant MERS S protein. (A) Entry of pseudovirions with wild-type or mutant MERS-CoV S proteins into HeLa/hDPP4 cells. Pseudovirus entry was quantitated by luciferase activity at 40 h postinoculation. A typical transduction by wild-type S protein pseudoviruses resulted in a more than 10,000-fold increase of luciferase activity. The experiments were repeated at least three times, and an average from three experiments is shown. (B) Detection of wild-type or mutant S protein incorporation into pseudovirions by Western blot analysis. MERS S protein was detected using mouse monoclonal anti-MERS S antibody; p24, a gag protein of HIV, was detected using rabbit polyclonal anti-p24 antibodies. FL S, full-length S protein. The experiments were repeated twice and a representative is shown.

Article Snippet: The primary antibodies for SARS-CoV SΔ19, MERS-CoV SΔ16, and MHV S protein were rabbit polyclonal anti-SARS S1 antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), mouse monoclonal anti-MERS S antibody (1:300 dilution) (Sinobiological Inc., Beijing, China), and goat polyclonal anti-MHV S antibody (AO4) (1:200 dilution), respectively.

Techniques: Mutagenesis, Luciferase, Activity Assay, Transduction, Western Blot

Journal: Emerging Microbes & Infections

Article Title: Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection

doi: 10.1080/22221751.2019.1620083

Figure Lengend Snippet: Intranasal immunization with AdC68-S induces a robust antibody and T cell response in BALB/c mice. (A) Timeline for vaccination and characterization of virologic and immunologic responses in two batches of animals. In batch I, a total of 12 groups of mice were immunized and monitored for serum binding and neutralizing activities. Group 1–6 (G1-G6) were negative controls whereas group 7–12 (G7–G12) were vaccinated with a single and varying dose of AdC68-S. In batch II, G8 and G11 mice were immunized along with G1 and G4* controls mice. These mice were examined for serum IgG subtypes, saliva IgA, and cytokine release up to 14-weeks post-immunization. The specific dose and route of immunization are indicated. i.n.: intranasal. i.m.: intramuscular. The open and solid drops indicate the blood collection for animals in the batch I and II, respectively. The temporal changes in serum binding activity to MERS-S1 (B) and neutralizing activity against autologous (C) and heterologous (D) MERS-CoV variants up to 40-weeks post-immunization are shown. Live virus neutralization by immune sera from G4, G8 (E) and G6, G11 (F) at 40-weeks post-immunization. The mean ID50 for G8 and G11 immune sera are indicated. The temporal changes in IgA in saliva (G), serum IgG subtypes (H), and cytokine release (I) in G8 and G11 animals were studied for up to 14-weeks post-immunization. Red symbols represent sera from i.n. vaccination groups and blue colour indicates sera from i.m. immunized animals. The grey colour is indicative of the control groups. ED50 means dilutions of serum at which half of the binding to antigen was identified. ID50 means dilutions of serum at which half of the viruses are neutralized. G4* indicates 10 10 vp used in batch I and 10 9 in batch II.

Article Snippet: MERS-S protein in cell lysates was probed by Rabbit anti-MERS-S1 polyclonal antibody (Sino biological).

Techniques: Binding Assay, Activity Assay, Neutralization

Journal: Emerging Microbes & Infections

Article Title: Single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against MERS-CoV infection

doi: 10.1080/22221751.2019.1620083

Figure Lengend Snippet: Generation and evaluation of recombinant AdC68 expressing full-length MERS-CoV spike protein. (A) Schematic representation of the recombinant AdC68 expressing the full-length MERS-CoV spike gene (AdC68-S) (Genbank accession number: JX869059). Spike gene was inserted into the E1 region of AdC68 under the control of the CMV promoter and terminated by bovine growth hormone (BGH) polyadenylation signal sequence. (B) Western blot analysis of MERS-CoV S protein expression in 293 T cells after infection with AdC68-S (10 8 , 10 9 and 10 10 vp). MERS-S protein in cell lysates was probed by Rabbit anti-MERS-S1 polyclonal antibody (Sino biological). The solid arrow points to the band of S protein while the hollow arrow points to the band of S1 protein cleaved from S by the protease. (C) Cell surface expression of MERS-CoV S protein analysed by MERS-CoV-specific antibodies MERS-4, MERS-27, MERS-GD27. Cell lysates or cells infected by empty AdC68 (10 10 vp) were used as negative controls. 17b, an antibody against HIV-1, was used as a negative control antibody.

Article Snippet: MERS-S protein in cell lysates was probed by Rabbit anti-MERS-S1 polyclonal antibody (Sino biological).

Techniques: Recombinant, Expressing, Sequencing, Western Blot, Infection, Negative Control