Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Replication kinetics of MERS-CoV in cell lines of human, nonhuman primate, hamster, mouse, and ferret origin. (A) Huh-7 (red circles), Vero (red squares), BHK (blue circles), 3T3 (blue squares), MEF C57Bl6 (blue triangles), and primary ferret (blue inverted triangles) cell lines were inoculated with MERS-CoV using an MOI of 0.01 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 h postinoculation (hpi), and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Red lines indicate cell lines originating from species known to be susceptible to MERS-CoV infection; blue lines indicate cell lines originating from species nonsusceptible to MERS-CoV infection. (B) Western blots of cellular lysates of Huh-7, Vero, BHK, primary ferret, 3T3, and MEF C57Bl6 cells probed with anti-DPP4 or anti-actin antibodies. (C) Cells were stained using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo and GraphPad software. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations.

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Titration, Infection, Western Blot, Staining, Flow Cytometry, Software

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: DPP4 in rhesus macaque, hamster, mouse, and ferret lung and kidney tissues. IHC was performed on lung and kidney tissues from rhesus macaque, hamster, mouse, and ferret tissues using an anti-DPP4 antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. IHC images, lung: closed arrow, bronchiolar epithelium; open arrow, smooth muscle; asterisk, alveolar macrophage; closed arrowhead, alveolar interstitium. IHC images, kidney: closed arrow, renal tubular epithelium; open arrow, glomerular endothelium (magnification, ×200).

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques:

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: DPP4 expression in lung and kidney tissues of different mammalian species

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Replication kinetics of MERS-CoV on hamster and ferret cell lines expressing human, hamster, or ferret DPP4. (A) Human DPP4 (red), hamster DPP4 (blue), ferret DPP4 (blue), and GFP (green) were expressed in BHK (circles) or primary ferret (squares) cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK or primary ferret cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. (C) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT.

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Expressing, Titration, Transfection, Staining, Flow Cytometry, Software, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Replication kinetics of MERS-CoV on BHK cells expressing DPP4 of livestock species. Camel (green circles), cow (green squares), goat (green triangles), or sheep (green inverted triangles) DPP4 and rhesus macaque (red squares), ferret (blue squares), or mouse (blue triangles) DPP4 were expressed on BHK cells. As a control, human (red) or hamster (blue) DPP4 was expressed on BHK cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. (C) Expression of DPP4 mRNA was measured via qRT-PCR. Relative fold increase was calculated by the comparative CT method (35), where DPP4 expression is normalized to HPRT.

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Expressing, Titration, Transfection, Staining, Flow Cytometry, Software, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Alignment of DPP4 amino acid residues of different mammalian species interacting with the MERS-CoV spike protein a

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques:

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Interaction between MERS-CoV spike protein and DPP4s of different mammalian species. (A) Cartoon representing the binding between human DPP4 or hamster DPP4 and the spike protein of MERS-CoV. DPP4 is depicted in white; the receptor binding domain (RBD) of the spike protein of MERS-CoV is depicted in magenta and cyan. The far right panel is obtained by clockwise rotation of the middle panel along a longitudinal axis. (B) Binding energies between spike protein of MERS-CoV and DPP4 of different species as well as humanized hamster DPP4 and hamsterized human DPP4. Red bars indicate the binding energies of known binders (human and rhesus macaque DPP4), blue bars indicate the binding energies of nonbinders (hamster, mouse, and ferret DPP4), green bars indicate the binding energies of unknown binders (dromedary camel, goat, cow, and sheep), and purple bars indicate the binding energies of the in silico mutagenized hamster and human DPP4s. The DPP4 homology models were constructed using the human DPP4 structure (PDB ID 4KR0, chain A) as a template and subjected to the binding energy calculation using an all-atom distance-dependent pairwise statistical potential, DFIRE.

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Binding Assay, In Silico, Construct

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Replication kinetics of MERS-CoV on BHK cells expressing mutagenized DPP4s. (A) Humanized hamster DPP4 (blue circles) or hamsterized human DPP4 (red squares) was expressed on BHK cells. As a control, human DPP4 (red circles) was expressed on BHK cells. Twenty-four hours posttransfection, cells were inoculated with MERS-CoV using an MOI of 1 TCID50/cell. Supernatants were harvested at 0, 24, 48, and 72 hpi, and viral titers were determined by endpoint titration in quadruplicate in VeroE6 cells. Mean titers were calculated from three independent experiments. Error bars indicate standard deviations. (B) BHK cells were left untransfected (red) or transfected with DPP4 (blue) and stained 24 h posttransfection using anti-DPP4 (R&D) and an FITC-conjugated secondary antibody (Life Technologies). Samples were collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques: Expressing, Titration, Transfection, Staining, Flow Cytometry, Software

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: DPP4 in camel, goat, cow, and sheep lung and kidney tissue. IHC was performed on lung and kidney tissues from camel, goat, cow, and sheep using an anti-DPP4 antibody. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin. IHC images, lung: closed arrow, bronchiolar epithelium; open arrow, smooth muscle; asterisk, alveolar macrophage; closed arrowhead, alveolar interstitium. IHC images, kidney: closed arrow, renal tubular epithelium; open arrow, glomerular endothelium (magnification, ×200).

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques:

Journal: Journal of Virology

Article Title: Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

doi: 10.1128/JVI.00676-14

Figure Lengend Snippet: Percent identity between DPP4 protein sequences

Article Snippet: Human and hamster DPP4s in pcDNA3.1(+) were restriction digested, purified, and ligated with the humanized hamster or hamsterized human DPP4 fragments, respectively, using T4 DNA ligase (New England Biolabs).

Techniques:

Journal: Virology

Article Title: Lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides

doi: 10.1016/j.virol.2017.07.033

Figure Lengend Snippet: Effects of cell-surface proteases on HR2 peptide antiviral activities. (A) Calu3 and Vero81 cells were incubated with MERS pps in the presence of increasing concentrations of HR2 peptides. (B) Total cellular RNA was isolated from Calu3and Vero81 cells, and evaluated for the expression of DPP4, TMPRSS2, furin, cathepsin L (Cat. L) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts by reverse transcription – quantitative PCR. Expression levels were plotted relative to HPRT expression levels. ND = Not Detected. (C) Calu3 and Vero81 cells were transduced with MERS pps in the presence of increasing concentrations of HR2 peptide or camostat (Camo). Camo was present from 1 h pre-transduction and HR2 was present at the time of MERS pp inoculation. (D) Calu3 cells were incubated with or without 10 μM camostat (Camo) for 1 h, then transduced with MERS pps in the presence of increasing concentrations of HR2 peptide. (E) Vero81 cells were transfected with either TMPRSS2 or vector control plasmids. At 2 d post-transfection, cells were transduced with MERS pps in the presence of increasing concentrations of HR2 peptide. For (A), (C), (D) and (E), unbound MERS pps and entry inhibitors were removed at 1 h post-transduction. Virus entry was quantified by measuring luciferase levels at 48 h post-transduction and data were normalized to control conditions lacking inhibitors. Error bars present SD from the mean (n = 3). Statistical significance was assessed by student's t -test. ***, P < 0.001.

Article Snippet: Plasmid encoding C-terminal flag-tagged human DPP4 (GenBank accession no. NM_001935, pCMV6-Entry-hDPP4) was purchased from OriGene (Rockville, MD). pcDNA3.1-SARS-S-C9 and pcDNA3.1-hACE2-C9 plasmids were provided by Michael Farzan, Scripps Research Institute. pcDNA3.1-229E-S-C9 and pcDNA3.1-hAPN plasmids were provided by Fang Li, University of Minnesota. pCAGGS-MHV-S (A59 strain) and pcDNA3.1-mCEACAM were previously constructed ( ). pHEF-VSV G was provided from BEI resources (Manassas, VA). pcDNA3.1-TMPRSS2-flag was previously constructed ( ). pNL4.3-Luc R- E-was obtained from the NIH AIDS Research and Reference Program, cat # 3418.

Techniques: Incubation, Isolation, Expressing, Real-time Polymerase Chain Reaction, Transduction, Transfection, Plasmid Preparation, Luciferase

Journal: Virology

Article Title: Lipidation increases antiviral activities of coronavirus fusion-inhibiting peptides

doi: 10.1016/j.virol.2017.07.033

Figure Lengend Snippet: Primers for real-time PCR.

Article Snippet: Plasmid encoding C-terminal flag-tagged human DPP4 (GenBank accession no. NM_001935, pCMV6-Entry-hDPP4) was purchased from OriGene (Rockville, MD). pcDNA3.1-SARS-S-C9 and pcDNA3.1-hACE2-C9 plasmids were provided by Michael Farzan, Scripps Research Institute. pcDNA3.1-229E-S-C9 and pcDNA3.1-hAPN plasmids were provided by Fang Li, University of Minnesota. pCAGGS-MHV-S (A59 strain) and pcDNA3.1-mCEACAM were previously constructed ( ). pHEF-VSV G was provided from BEI resources (Manassas, VA). pcDNA3.1-TMPRSS2-flag was previously constructed ( ). pNL4.3-Luc R- E-was obtained from the NIH AIDS Research and Reference Program, cat # 3418.

Techniques:

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 1. Generation and characterization of K18-hDPP4 mice. A, The hDPP4 coding sequence was cloned into a plasmid containing the 5′ and 3′ genomic regions of human cytokeratin 18 (K18). The K18 5′ genomic region consists of a 2.5-kb upstream genomic sequence, promoter, and first intron of the human K18 gene while the K18 3′ region consists of exon 6, intron 6, exon 7, and approximately 300 base pairs of 3′ untranslated region of the human K18 gene, including the K18 polyA signal. Immediately upstream of the hDPP4 start codon is a translational enhancer (TE) sequence from alfalfa mosaic virus. B, Four K18-hDPP4 transgenic founder lines were generated and intranasally in- oculated with 1 × 105 plaque-forming units (PFU) of Middle East respiratory syndrome coronavirus. Lung titers of founder mice were determined by plaque assay 3 days after infection. Data are mean ± standard deviation [SD] for 6–10 mice/line. C, Quantitative measurement of human DPP4 concentrations in tissues of nontransgenic or K18-hDPP4 founder line 3 by enzyme-linked immunosorbent assay. Data are mean ± SD for 3 mice. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Virus, Transgenic Assay, Generated, Plaque Assay, Infection, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 2. Middle East respiratory syndrome coronavirus (MERS-CoV) infection causes lethal disease in human cytokeratin 18–hDPP4 mice. A–C, Animals were inoculated intranasally with 1 × 105 plaque-forming units (PFU) of MERS-CoV and survival (A), weight (B), and temperature (C) monitored daily. Data are for 4 nontransgenic mice and 14 K18-hDPP4 mice. D, MERS-CoV titers in indicated tissues at days 2, 4, and 6 after infection. Data are for 3–4 mice. E, The copy numbers of viral RNA in indicated tissues at days 2 and 4 after infection were analyzed by quantitative real-time polymerase chain reaction targeting regions within open reading frame 1a. Data are for 4 mice. All results are expressed as mean ± SD.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 4. Brain disease in Middle East respiratory syndrome coronavirus (MERS-CoV)–infected human cytokeratin 18 (K18)–hDPP4 and uninfected mice. A, Normal brain from an uninfected mouse. B, MERS-CoV caused lymphocytic perivascular cuffing in the infected brain. C, Infected neuron in hippocampus 6 days after infection. Note the granular degeneration and basophilic cytoplasmic inclusions (arrow and inset). D, Dying cells undergoing degeneration (arrows and inset; 6 days after infection) are detected in highly infected regions such as the thalamus or brain stem. E, Meningeal and perivascular cuffing included neutrophilic infiltrates (arrows; 6 days after infection). F, Several degenerating cells had small to granular basophilic cytoplasmic inclusions (arrows; 6 days after infection) that were stained with anti–MERS-CoV antibody (inset; brown). Note the neuropil rarefaction. Section were stained with hematoxylin-eosin (original magnification ×600). G–I, Outcomes of K18-hDPP4 mice infected with different intranasal inocula of MERS-CoV. K18-hDPP4 mice received 1000, 100, or 10 plaque-forming units (PFU) of MERS-CoV and were monitored for survival (G) and weight (H). There were 5 mice/group. I, Lungs and brains of mice receiving 10 PFU were harvested 10 days after inoculation or when they lost 20% of body weight. A total of 3 of 7 MERS-CoV–infected mice showed high virus titers in the brains. J, MERS-CoV replicates in cells of the nervous system. Human central nervous system–derived cell lines (U-138 MG an SK-N-SH), primary porcine astrocytes, a murine astrocytoma cell line (DBT), and African green monkey kidney cells (Vero-81) were infected with MERS-CoV at a multiplicity of infection of 1. Titers from these cells immediately after infection (day 0) or 2 days after infection were determined by plaque assay. Data are mean ± SD for 3 replicates/condition. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Infection, Staining, Virus, Derivative Assay, Plaque Assay

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 5. Immunization with Venezuelan equine encephalitis replicon particles (VRPs) expressing Middle East respiratory syndrome coronavirus (MERS-CoV) spike glyco- protein (VRP–MERS-S) or passive immunization protects human cytokeratin 18 (K18)–hDPP4 mice against MERS-CoV infection. A and B, K18-hDPP4 mice were immunized with 1 × 105 infectious units (IU) of VRPs expressing green fluorescent protein (VRP-GFP) or VRP-MERS-S in the footpad and boosted with the same dose 4 weeks later. Mice were infected with 1 × 105 plaque-forming units of MERS-CoV 2 weeks after the boost. C and D, For passive immunization, nontransgenic mice were immunized as described above. Sera were obtained 2 weeks after boosting and transferred into K18-hDPP4 mice intraperitoneally 1 day before infection with MERS-CoV. Survival and weights were recorded for active immunization (A and B) and passive immunization (C and D). Data are mean ± standard deviation (SD) for 5 mice/group. E and F, MERS-CoV titers in lung tissue (E) and brain tissue (F) 2, 4, and 6 days after infection in mice with or without passive immunization. Data are mean ± SD for 3 mice/group. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Expressing, Infection, Standard Deviation

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: (A) Schematic diagram of the optimized hDPP4 expression vector cassette. The optimized hDPP4 was cloned into a pCAGGS plasmid in which hDPP4 expression was driven by the CAG promoter. hDPP4 expression was confirmed in vitro by transfection of Cos-7 cells with the pCAGGS-hDPP4 (B) or pCAGGS (C) plasmids and detected by direct immunofluorescence assay with FITC-labeled anti-human CD26–Fluorescein antibody. Confirmation of the binding between DPP4 and MERS-CoV RBD was achieved by transfecting Cos-7 cells with pCAGGS-hDPP4 (D) or pCAGGS (E) plasmid followed by indirect immunofluorescence assay with MERS-RBD-Fc protein and DyLight 549-conjugated goat anti-human IgG antibody. (F) Determination of the copy numbers of hDPP4 cDNA in four transgenic founder lines by qPCR. (G) Expression of hDPP4 mRNA in the indicated tissues of transgenic mice in two founder lines as determined by qRT-PCR. Results are mean±SEM ( n = 3). (H) Four lines of hDPP4 transgenic mice were infected with MERS-CoV and monitored for body weight changes. Results are mean±SEM ( n = 6). (I) Lung viral titer at day 5 postinfection was determined for four lines of hDPP4 transgenic mice. The data are expressed as mean±SEM ( n = 3). The dotted line indicates the limit of detection.

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Expressing, Plasmid Preparation, Clone Assay, In Vitro, Transfection, Immunofluorescence, Labeling, Binding Assay, Transgenic Assay, Quantitative RT-PCR, Infection

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: The hDPP4 transgenic mice were infected intranasally with MERS-CoV. Lungs, kidneys, liver, spleen and brain were collected on day 5 or day 9 after MERS-CoV infection, fixed in neutral formaldehyde solution and stained with hematoxylin and eosin. Histopathological analysis of hDPP4 transgenic mice was performed using sham-infected mice as controls (A, D, G, J). (B-C) In the lungs, mild inflammation was observed, with inflammatory cell infiltration (arrowheads) and focal hemorrhage and exudation (arrow) on day 5. Damage was more severe on day 9, with increased inflammatory cell infiltration, focal hemorrhage (inset) and exudation. (E-F) In the kidney, MERS-CoV-infected hDPP4 transgenic mice showed focal interstitial inflammation with inflammatory cell infiltration in the interstitium and exudates in renal tubules (arrow) on day 5. On day 9, degeneration and necrosis in the renal tubular epithelial cells (inset) and focal hemorrhage were observed. (H-I) In the liver, MERS-CoV-infected hDPP4 transgenic mice showed scattered hepatocyte necrosis and numerous activated kupffer cells and infiltrated macrophages in the hepatic sinusoid on day 5. Fatty change of hepatocytes (inset) was observed in the liver on day 9. (K-L) In the spleen, necrotic splenic cells and increased reticulum cells in the red pulp with significant amounts of hemosiderin deposition were observed in hDPP4 transgenic mice on days 5 and 9 (arrow and inset). (M-O) Neurological damage with perivascular cuffs (M) and neuronal cell necrosis in the cerebral cortex (N) including damaged neurons (inset) in the hippocampus (O) was observed in the brains of hDPP4 transgenic mice infected with MERS-CoV. ( n = 2, scale bars = 50 μm).

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Transgenic Assay, Infection, Staining

Journal: PLoS ONE

Article Title: Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus

doi: 10.1371/journal.pone.0145561

Figure Lengend Snippet: Transgenic mice were infected intranasally with MERS-CoV and sacrificed to access the expression of viral antigens by immunohistochemical staining ( n = 2). No viral antigens were detected in the sham-infected mice (A, D, G), but viral antigens were detected in type I and type II pneumocytes and infiltrated macrophages in the lungs (B-C), renal tubular epithelial cells in the kidneys (E-F), and neuron cell bodies as well as in dendrites and axons in the brain (H-I), including the hippocampus (I). (J) Viral load was detected by qRT-PCR and expressed as viral RNA copies/g of tissues. The detection limit shown as dotted line is 1×10 3 copies/g. Results are mean±SEM ( n = 2). (K) Viral titers in tissues of infected hDPP4 transgenic mice were determined by CPE-based assay and expressed as Log 10 TCID 50 /g of tissues. Results are mean±SEM ( n = 5). The dotted line indicates the detection limit.

Article Snippet: The hDPP4 DNA copy numbers were determined by quantitative PCR using Power SYBR ® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA).

Techniques: Transgenic Assay, Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Molecules

Article Title: A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

doi: 10.3390/molecules25225392

Figure Lengend Snippet: Figure 1. Overview of DPP4 purification workflow.

Article Snippet: Expression of DPP4 in Insect Sf9 Cells Soluble human DPP4 (residues 29–766; GenBank M80536) was cloned with a C-terminal His6 –tag into the pMelbac vector and expressed according to the Bac-N-Blue baculovirus expression system protocol (Thermo Fisher Scientific) [35].

Techniques:

Journal: Molecules

Article Title: A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

doi: 10.3390/molecules25225392

Figure Lengend Snippet: Figure 2. Elution profiles of DPP4 chromatography. (A) Chromatogram from Phenyl Sepharose that was equilibrated with 12% ammonium sulphate (AS) in 10 mM Tris-HCl pH 7.6 and eluted with 0% AS in 10 mM Tris-HCl pH 7.6 buffer. Inset: Fibroblast activation protein (FAP) activity. (B) Chromatogram from Nickel Sepharose, which was equilibrated with 20 mM imidazole in 200 mM NaCl, 10 mM Tris-HCl, pH 7.6 and eluted with an increasing concentration gradient of imidazole at 30 mM (a), 100 mM (b), 500 mM (c), 1000 mM (d) in 10 mM Tris-HCl pH 7.6 buffer. Inset: FAP activity. (C) Chromatogram from DEAE Sepharose that was equilibrated with 10 mM Tris-HCl pH 7.6 and eluted with 200 mM NaCl in 10 mM Tris-HCl pH 7.6 buffer. Inset: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE; 4–12% Bis-Tris gel) of the resulting purified soluble DPP4, stained with Sypro ruby Protein was measured by optical density at 280 nm in these chromatograms.

Article Snippet: Expression of DPP4 in Insect Sf9 Cells Soluble human DPP4 (residues 29–766; GenBank M80536) was cloned with a C-terminal His6 –tag into the pMelbac vector and expressed according to the Bac-N-Blue baculovirus expression system protocol (Thermo Fisher Scientific) [35].

Techniques: Chromatography, Activation Assay, Activity Assay, Concentration Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Staining

Journal: Molecules

Article Title: A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

doi: 10.3390/molecules25225392

Figure Lengend Snippet: Figure 3. ELISA and virus neutralisation assays. (A) Purified DPP4 protein was used to capture MERS-CoV spike clamp, SARS-CoV-2 spike clamp or a control clamped protein. Clamp-stabilised proteins were detected using a clamp-specific mAb HIV1281. Data shown represent means of duplicate values. (B) DPP4, ACE2, a MERS-specific mAb m336 or a non-specific mAb C05 were used at 20 µg/mL and incubated with MERS-CoV pseudovirus. Percent MERS-CoV inhibition is the percentage reduction in luciferase signal (RLU) compared to virus-only control. N.D. indicates no inhibition detected. Data shown are the mean of duplicate values with error bars representing SD.

Article Snippet: Expression of DPP4 in Insect Sf9 Cells Soluble human DPP4 (residues 29–766; GenBank M80536) was cloned with a C-terminal His6 –tag into the pMelbac vector and expressed according to the Bac-N-Blue baculovirus expression system protocol (Thermo Fisher Scientific) [35].

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Control, Incubation, Inhibition, Luciferase

Journal: Molecules

Article Title: A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

doi: 10.3390/molecules25225392

Figure Lengend Snippet: Figure 4. Surface plasmon resonance assays. Purified soluble human ACE2 (A) and DPP4 (B) were exposed to CM5 chips that had been coated with SARS-CoV-2 RBD or spike protein, or were not coated. Experimental data are shown in red. Calculated data fit using a 1:1 binding model are shown in black. Ligands were injected at increasing concentrations of (A) ACE2 at 0.50 nM, 2.5 nM, 12 nM, 62 nM and 310 nM and (B) DPP4 at 1.6 nM, 8.0 nM, 40 nM, 200 nM and 1000 nM.

Article Snippet: Expression of DPP4 in Insect Sf9 Cells Soluble human DPP4 (residues 29–766; GenBank M80536) was cloned with a C-terminal His6 –tag into the pMelbac vector and expressed according to the Bac-N-Blue baculovirus expression system protocol (Thermo Fisher Scientific) [35].

Techniques: SPR Assay, Binding Assay, Injection

Journal: Molecules

Article Title: A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

doi: 10.3390/molecules25225392

Figure Lengend Snippet: Figure 5. Protein structures. (A) DPP4 monomer (PDB ID 1W1I) [46]. (B) Adenosine Deaminase

Article Snippet: Expression of DPP4 in Insect Sf9 Cells Soluble human DPP4 (residues 29–766; GenBank M80536) was cloned with a C-terminal His6 –tag into the pMelbac vector and expressed according to the Bac-N-Blue baculovirus expression system protocol (Thermo Fisher Scientific) [35].

Techniques: