Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 1. The pathogenesis of SARS-CoV-2 infection is exacerbated in a viral dose-dependent manner. (A) Western blot of human ACE2 protein using various organs in WT and CAG-hACE2 mice. The top and bottom rows show hACE2 and GAPDH, respectively. (B) Schematic diagram of experimental schedule. Male and female C57BL/6 and CAG-hACE2 mice were infected via respiratory tract with SARS-CoV-2 (2 × 102 TCID50, n = 5; 2 × 103 TCID50, n = 6; 2 × 104 TCID50, n = 5) and were administrated with an equal volume of PBS for mock infection controls (WT, n = 3; CAG-hACE2 mice, n = 3). Body weight and survival were recorded daily for up to 14 days. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. White and black circles indicate WT with mock infection and WT infected with 2 × 104 TCID50, respectively. Triangles denote CAG-hACE2 mice. White, orange, blue, and red triangles represent PBS, 2 × 102 TCID50, 2 × 103 TCID50, and 2 × 104 TCID50, respectively.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Western Blot

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 7. Characterization of CAG-promoter hACE2-transgenic mice. (A) Chromosome map of annotated hACE2-containing reads. The pink horizontal bars indicate the number of annotated ACE2-containing reads. Annotated site of integrated hACE2 is indicated as a blue box. (B) Detail of annotated site in chromosome 1. The horizontal red and pink bars indicate the ACE2-containing reads mapped to the plus and minus strand of the mouse reference genome (GRCh38/mm10), respectively. The blue box represents the duplicated region. (C) Schematic diagrams and sequences are shown as the hACE2- integrated site inside the first intron region of the Colgalt2 gene locus. The blue boxes denote duplicated regions. Each yellow arrow indicates the entire hACE2 sequence. Consensus sequences of reads at 5′- and 3′-junction sites are shown.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Transgenic Assay, Sequencing

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 8. Protection against SARS-CoV-2 by immunization of SARS-CoV-2 RBD protein. (A) Schematic showing the experimental schedule. Male and female CAG-hACE2 mice were immunized with RBD-mFc/AddaVax twice before infection and were infected via respiratory tract with SARS-CoV-2 (2 × 103 TCID50, n = 4; 1 × 104 TCID50, n = 4). PBS/AddaVax–immunized CAG-hACE2 mice were used as a control (2 × 103 TCID50, n = 6; 1 × 104 TCID50, n = 6). (B) Expression of RBD-sIgG in plasma from PBS/AddaVax or RBD-mFc/AddaVax–immunized CAG-hACE2 mice at 2 days before infection. Blue and red indicate 2 × 103 TCID50 and 1 × 104 TCID50, respectively. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. White and colored circles indicate mice injected with PBS/AddaVax or RBD-mFc/AddaVax, respectively. Blue and red denote infection doses of 2 × 103 TCID50 and 1 × 104 TCID50, respectively. Data are presented as the mean (B) and the mean ± SEM (C). Statistical analyses were performed using 2-tailed unpaired t test for body weight loss (B and C) and log-rank (mantel-cox) test for survival rate (D). P < 0.05 for comparison with RBD-mFc/AddaVax (2 × 103 TCID50) or PBS/AddaVax–immunized mice, respectively.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Control, Expressing, Clinical Proteomics, Injection, Comparison

Journal: JCI insight

Article Title: Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

doi: 10.1172/jci.insight.152529

Figure Lengend Snippet: Figure 9. Protection against SARS-CoV-2 infection by the injection of RBD-Fc–immunized mouse plasma. (A) Schematic showing the experimental schedule. Male and female CAG-hACE2 mice were intravenously injected with pooled plasma from RBD-mFc/AddaVax–immunized mice at doses of 15 μg/shot (n = 6), 50 μg/shot (n = 5), and 150 μg/shot (n = 6) 1 day before SARS-CoV-2 infection and were infected via respiratory tract with SARS-CoV-2 at a dose of 1 × 104 TCID50. CAG-hACE2 mice that were injected with PBS-treated pooled plasma were used as a control (n = 6). (B) The measurement of SARS-CoV-2–neutralizing antibodies in pooled plasma. Black circles and white, violet, and gray diamonds indicate the standard, pooled plasma from PBS/ AddaVax–treated mice, pooled plasma from RBD-mFc/AddaVax–immunized mice, and mouse neutralizing antibody, respectively. (C and D) Percentage of initial body weight (C) and survival rate (D). Numbers in C represent the number of mice measured for body weight at each time. Black circles and orange, blue, and red triangles indicate plasma from PBS/AddaVax–immunized mice and RBD-mFc/AddaVax–immunized mice at doses of 150 ng/mL (n = 6), 500 ng/mL (n = 5), and 1500 ng/mL (n = 6), respectively. Data were presented as the mean (B) and the mean ± SEM (C). Statistical analyses were performed using log-rank (mantel-cox) test for survival rate (D). P < 0.05 for the comparison with PBS-treated pool plasma.

Article Snippet: Then, the membranes were incubated with HRP-conjugated goat anti-mouse polyclonal IgG for hACE2 (1:2000, catalog 172-1011, Bio-Rad) or HRP-conjugated goat anti-rabbit polyclonal IgG for GAPDH (1:2000, catalog 172-1019, Bio-Rad) for 1 hour at room temperature, and the protein signals were detected using a chemiluminescence detection reagent (Chemi-Lumi One L; Nacalai Tesque Inc.) and visualized using the ImageQuant LAS 4000 system (Cytiva).

Techniques: Infection, Injection, Clinical Proteomics, Control, Comparison

Journal: Nature Communications

Article Title: Individual bat virome analysis reveals co-infection and spillover among bats and virus zoonotic potential

doi: 10.1038/s41467-023-39835-1

Figure Lengend Snippet: a Top, homology-modeling structure of the receptor-binding domain (RBD) of Bat SARS-like coronavirus CX1 in complex with human angiotensin-converting enzyme 2 (hACE2). Blue-colored residues on RBD indicate amino acid differences compared with SARS-CoV-2 Wuhan-Hu-1. Bottom, alignment of RBD sequences (residues T333 to G526 of spike protein) of Bat SARS-like coronavirus CX1, SARS-CoV-2 Wuhan-Hu-1 and two closely related bat coronavirus. Only polymorphic sites are shown. The five amino acid differences in the RBD of Bat SARS-like coronavirus CX1 compared to SARS-CoV-2 Wuhan-Hu-1 are marked with blue dots. b Molecular dynamics simulation results of binding energy (top) and binding stability (bottom) of Bat SARS-like coronavirus CX1 RBD-hACE2 complex. c The binding capability of SARS-CoV, SARS-CoV-2 and Bat SARS-like coronavirus CX1 RBD proteins to hACE2 protein was tested with various concentrations of the RBD proteins via ELISA. d The binding kinetics was determined by the biolayer interferometry (BLI) binding analysis. The purified hACE2 were coated on the sensor followed by the injection of various concentrations of SARS-CoV, SARS-CoV-2 and Bat SARS-like coronavirus CX1 RBD proteins. Source data are provided as a Source Data file.

Article Snippet: To detect the binding of hACE2 Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (polyclonal) diluted 1:2000 (Jackson ImmunoResearch, catalog number #109-035-098) was added and incubated for 1 h at 37 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Injection

Journal: Translational Psychiatry

Article Title: Amyloid precursor protein facilitates SARS-CoV-2 virus entry into cells and enhances amyloid-β-associated pathology in APP/PS1 mouse model of Alzheimer’s disease

doi: 10.1038/s41398-023-02692-z

Figure Lengend Snippet: A and B ACE2 protein or N-terminal APP (18-612 amino acids, APP18-612), captured on the COOH chip, can bind to S protein with an affinity constant of 34.9 or 46.0 nM, respectively, as determined by localized surface plasmon resonance (LSPR) assay. C Human embryonic kidney 293T cells (HEK293T) stably overexpressing either hACE2 (HEK293T/ACE2) or hAPP (HEK293T/APP) were plated in confocal chambers at a density of 1 × 10 5 /well for 24 h. The cells were transfected with constructs encoding SARS-CoV-2 S protein for 48 h, then fixed in 4% paraformaldehyde solution and subjected to immunofluorescence staining with anti-hACE2 antibody, anti-hAPP antibody (6E10), and anti-S protein antibody. Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG) was used to detect hACE2 (green) or hAPP (green), while Alexa Fluor 555 donkey anti-rabbit IgG was used to detect S protein (red). DAPI (4’,6-diamidino-2-phenylindole) is used as a nuclear counterstain (blue) and visualized by confocal microscopy. Scale bar 20 μm. D Validation of the interaction between SARS-CoV-2 S protein (136.9 kDa) and N-terminal APP protein. SH-SY5Y cells were co-transfected with SARS-CoV-2 S protein and APP constructs for 48 h. S protein antibody or APP antibody were primarily incubated with MAg25K protein A/G agarose beads (1 h at 37 °C) to prepare the agarose beads antibody complex. The agarose beads antibody complex was then incubated with whole-cell lysates overnight at 4 °C, followed by western blotting (WB) with anti-APP antibody (6E10); An immunoprecipitation (IP) experiment in the reverse direction followed by WB with anti-S protein antibody was performed to further confirm the association of N-terminal APP protein with S protein. IP with mouse serum was used as a blank control and the lysate as the input control.

Article Snippet: Alexa Fluor 488 (1:500; Invitrogen, A-11001) goat anti-mouse IgG detected hACE2 or hAPP (green), while Alexa Fluor 555 (1:500; Invitrogen, A-31572) donkey anti-rabbit IgG detected the S protein (red).

Techniques: SPR Assay, Stable Transfection, Transfection, Construct, Immunofluorescence, Staining, Confocal Microscopy, Incubation, Western Blot, Immunoprecipitation

Journal: Translational Psychiatry

Article Title: Amyloid precursor protein facilitates SARS-CoV-2 virus entry into cells and enhances amyloid-β-associated pathology in APP/PS1 mouse model of Alzheimer’s disease

doi: 10.1038/s41398-023-02692-z

Figure Lengend Snippet: A and B APP facilitates pseudovirus (Pseudo-SARS-CoV-2 virus) infection. A HEK293T, HEK293T/hACE2, and HEK293T/hACE2 transiently transfected with constructs encoding APP (HEK293T/hACE2 transient with APP) were plated in six-well chambers at a density of 1 × 10 5 /well for 24 h. Cells were infected with the GFP-labeled pseudovirus at 10 8 /mL for 48 h at 37 °C, and visualized by fluorescence microscopy. Scale bar 100 μm. B HEK293T/hACE2 cells transfected with constructs encoding hAPP or HEK293T/ hAPP cells transfected with constructs encoding hACE2 were infected with the GFP-labeled pseudovirus at 10 8 /mL for 72 h at 37 °C. Pseudovirus mRNA from cell lysis was examined by RT-qPCR. The data are shown as the means ± SEM from three independent experiments. P values were calculated using two-way ANOVA (* P < 0.05; *** P < 0.001). C SH-SY5Y cells, SH-SY5Y cells stably overexpressing hAPP and SH-SY5Y cells with APP knockdown were infected with the GFP-labeled pseudovirus at 10 8 /mL and visualized by fluorescence microscopy at 48 h post-infection. Scale bar 100 μm. D Pseudovirus from cells lysis in C was examined by RT-qPCR. The data are shown as the means ± SEM from three independent experiments. P values were calculated using two-way ANOVA (*** P < 0.001). E The APP overexpression and knockdown efficiencies in SH-SY5Y were evaluated using WB assay with anti-APP antibody (6E10). GAPDH was selected as a reference control.

Article Snippet: Alexa Fluor 488 (1:500; Invitrogen, A-11001) goat anti-mouse IgG detected hACE2 or hAPP (green), while Alexa Fluor 555 (1:500; Invitrogen, A-31572) donkey anti-rabbit IgG detected the S protein (red).

Techniques: Virus, Infection, Transfection, Construct, Labeling, Fluorescence, Microscopy, Lysis, Quantitative RT-PCR, Stable Transfection, Over Expression

Journal: Translational Psychiatry

Article Title: Amyloid precursor protein facilitates SARS-CoV-2 virus entry into cells and enhances amyloid-β-associated pathology in APP/PS1 mouse model of Alzheimer’s disease

doi: 10.1038/s41398-023-02692-z

Figure Lengend Snippet: A N-terminal APP protein, captured on a COOH chip, can bind to AY51 with an affinity constant of 554 nM as determined by LSPR assay. B HEK293T/hACE2 cells at a density of 3 × 10 4 cells/well were plated in 96-well plates and incubated with various concentrations of AY51 (0–25 µM) at 37 °C for 2 h, followed by adding the SARS-CoV-2 pseudovirus encoding luciferase for 72 h at 37 °C. Luciferase activity was measured by using a pseudovirus neutralization assay. The results are representative of three independent experiments, with each condition triplicated and presented as means ± SD of inhibition of the pseudovirus. C Vero cells at a density of 3 × 10 4 cells/well were plated in 96-well plates for 24 h and infected with SARS-CoV-2 (at MOI of 0.05) with the presence of AY51 (2.5, 5, 10, 20, 40, and 80 μM). The virus yielded in the cell lysis was determined by qRT-PCR. Experiments were repeated twice, and the data are expressed as means ± SD. D The expression of ACE2 and APP protein in 293T/ACE2 and Vero cells was detected by WB.

Article Snippet: Alexa Fluor 488 (1:500; Invitrogen, A-11001) goat anti-mouse IgG detected hACE2 or hAPP (green), while Alexa Fluor 555 (1:500; Invitrogen, A-31572) donkey anti-rabbit IgG detected the S protein (red).

Techniques: Incubation, Luciferase, Activity Assay, Neutralization, Inhibition, Infection, Virus, Lysis, Quantitative RT-PCR, Expressing