Journal: Viruses

Article Title: The Feline calicivirus Leader of the Capsid (LC) Protein Contains a Putative Transmembrane Domain, Binds to the Cytoplasmic Membrane, and Exogenously Permeates Cells

doi: 10.3390/v16081319

Figure Lengend Snippet: The purified Histag-LC protein from FCV is associated exogenously with the cytoplasmic membrane of CrFK cells. Monolayers of CrFK cells interected or not (mock) with 1 μM of the ( A ) Histaged-LC or ( B ) ZIKV NS1 proteins were immunostained for 3 h with an anti-LC serum or the anti-flavivirus NS1 antibodies (Green). DAPI was used for nuclear (blue) staining. The cells were examined in a Zeiss LSM 700 confocal microscope. The images correspond to a z-stack of 15 slices and represent two independent experiments. Merged images are indicated.

Article Snippet: CrFK cells grown on coverslips were interacted with 1 μM of the recombinant LC protein from FCV or the recombinant NS1 protein from ZIKV (Aalto Bio Reagents, Dublin, Ireland, ROI) as a control, in DMEM for 3 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde ( v / v ) in PBS for 20 min.

Techniques: Purification, Membrane, Staining, Microscopy

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Binding of terminal complement proteins to ZIKV E. Constant amounts (5 µg/ml; A ) or serial dilutions ( B , for significance – not always depicted due to limits in space – see text) of purified complement proteins were coated onto ELISA plates and incubated with 10 µg/ml ZIKV E. To visualize binding, an E-specific Ab (4G2) followed by a HRP-goat-anti-mouse Ab and TMB as a substrate were added. To test whether already generated TCC is interacting with ZIKV E too (C) , the recombinant viral protein was coated into ELISA plates and incubated with serial dilutions of NHS. BSA and ΔC9 NHS served as controls. Binding to TCC was determined by incubation with neoepitope-specific anti-C9 (WU 13-15) followed by HRP-goat-anti-mouse Ab. Again, TMB was used as a substrate. Optical density (OD) was measured at a wavelength of 650 nm. Experiments were repeated three times and were performed in duplicates. For statistical analysis GraphPad Prism software was used ( A , 1-way ANOVA; B, C , 2-way ANOVA, respectively). *< 0.05, ** < 0.01 and *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Generated, Recombinant, Software

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of C9 polymerization after induction via C5b6 in the presence of ZIKV E. To trigger generation of the terminal pathway of complement, A549 cells were incubated with purified C5b6 (5 µg) for 2 h at 37°C. After washing, 5% NHS was added as a source of C7 to C9 in the presence of different amounts of ZIKV E. After incubation at 37°C for 50 min and additional washing steps, cells were lysed and loaded on a SDS gels under non-reducing conditions. Lysates were blotted and visualized as described in the Material & Method section. A representative Western blot out of three independent experiments is shown (A) . As a control, the effect of BSA on the C9 polymerization was used (B) .

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Incubation, Purification, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of C9 polymerization after induction of the classical pathway in the presence of ZIKV E. For the induction of complement activation, A549 cells were incubated with sublytic amounts of anti-MHC-1 (1:1,000). After washing, cells were incubated with 5% NHS, which was pre-incubated with different amounts of ZIKV E. After washing, cells were blotted and oligomerization of C9 was analyzed as described in . A representative Western blot out of three independent experiments is shown.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Activation Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Inhibition of MAC formation by ZIKV E: Viral proteins were pre-incubated with C7 to C9 and added to sheep erythrocytes pre-coated with C5b6. As a control vitronectin (VN) was used, a known inhibitor of the MAC. In the positive control (no viral protein) formation of the MAC was undisturbed and set at 100%. Hemolysis was determined by measuring the release of hemoglobin in the supernatant at an OD of 415 nm. Omission of C9 served as background control. Experiments were repeated three times performed in duplicates and analyzed by 1-way ANOVA. n.s., not significant. *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Inhibition, Incubation, Control, Positive Control

Journal: Frontiers in Immunology

Article Title: Interference of the Zika Virus E-Protein With the Membrane Attack Complex of the Complement System

doi: 10.3389/fimmu.2020.569549

Figure Lengend Snippet: Reduction of complement mediated lysis by ZIKV: Sensitized sheep erythrocytes were incubated for 30 min at 37°C with serial dilutions of NHS. As expected, lysis of the cell decreased with decreasing dilutions of NHS and was not affected by DMEM, the buffer used as mock control. Hemolysis was determined by measuring the release of hemoglobin in the supernatant at 415 nm. Experiments were performed three times in triplicates. Significance was calculated by 2-way ANOVA. *** < 0.001.

Article Snippet: ZIKV recombinant E and NS1 proteins were provided by Aviva Systems Biology (San Diego, CA); E protein PMA04848-1MG and Biozol (Eching, Germany); E protein MBS596001, and Zika NS1 MBS596002).

Techniques: Lysis, Incubation, Control

Journal: Sensors (Basel, Switzerland)

Article Title: Label-Free Electrochemical Biosensors for the Determination of Flaviviruses : Dengue, Zika, and Japanese Encephalitis

doi: 10.3390/s20164600

Figure Lengend Snippet: Survey of electrochemical label-free biosensors for Zika diagnostic.

Article Snippet: Microtiter plate precoated with ZIKV NS1/ Euroimmun anti-ZIKV IgM , Indirect ELISA , Antibody-HRP conjugates , DENV Ig M antibodies , 56% , West Nile virus , d, f , a, c , [ , ] .

Techniques: Diagnostic Assay, Virus, Polymer, Membrane, Indirect ELISA

Journal: bioRxiv

Article Title: Old Dog, New Tricks: Influenza A Virus NS1 and In Vitro Fibrillogenesis

doi: 10.1101/2020.08.17.254060

Figure Lengend Snippet: (a) – Gel-filtration results. Inset: SDS-PAGE of purified recombinant NS1-6xHis protein. Using a calibration curve, approximate molecular masses corresponding to each peak were identified. Rv – retention volume, OD 280 – optical density at 280 nm, M – molecular mass standard. (b) – SDS-PAGE of NS1-N and NS1-F after protease treatment at two protease:protein ratios (1:100 and 1:20). Ctrl – NS1-N or NS1-F incubated without trypsin, under the same conditions. Arrows and numbers indicate bands and their mass spectrometry-identified NS1-6xHis a.a. residues. The band corresponding to trypsin is marked (tr).

Article Snippet: NS1 fibrils were obtained as follows: one milliliter of NS1 recombinant protein (3 mg/ml in 2x PBS) was placed into a 1.5 ml Eppendorf tube (Eppendorf, Germany); and the tube mixed for 66 hours (55°C, 600 rpm) using a ThermoMixer Comfort orbital shaker (Eppendorf).

Techniques: Filtration, SDS Page, Purification, Recombinant, Incubation, Mass Spectrometry

Journal: bioRxiv

Article Title: Old Dog, New Tricks: Influenza A Virus NS1 and In Vitro Fibrillogenesis

doi: 10.1101/2020.08.17.254060

Figure Lengend Snippet: NS1-F with (a) ThT (fluorescence) and with (b) CR (absorbance).

Article Snippet: NS1 fibrils were obtained as follows: one milliliter of NS1 recombinant protein (3 mg/ml in 2x PBS) was placed into a 1.5 ml Eppendorf tube (Eppendorf, Germany); and the tube mixed for 66 hours (55°C, 600 rpm) using a ThermoMixer Comfort orbital shaker (Eppendorf).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Old Dog, New Tricks: Influenza A Virus NS1 and In Vitro Fibrillogenesis

doi: 10.1101/2020.08.17.254060

Figure Lengend Snippet: Monomers are indicated in dark blue and yellow. Areas homologous to NS1-N a.a.r. 1-78 (resistant to protease treatment) and to NS1-F a.a.r.149-217 are shown using surface and cartoon representation, respectively. Inset: localization of a.a.r. 149-217 (dark blue, cartoon representation) near NS1’s beta-domain (cyan and dark blue arrows, cartoon representation).

Article Snippet: NS1 fibrils were obtained as follows: one milliliter of NS1 recombinant protein (3 mg/ml in 2x PBS) was placed into a 1.5 ml Eppendorf tube (Eppendorf, Germany); and the tube mixed for 66 hours (55°C, 600 rpm) using a ThermoMixer Comfort orbital shaker (Eppendorf).

Techniques: