human dpp4 fragments  (New England Biolabs)

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: