Journal: Frontiers in Immunology

Article Title: Neutrophil Extracellular Traps in Dengue Are Mainly Generated NOX-Independently

doi: 10.3389/fimmu.2021.629167

Figure Lengend Snippet: Platelets stimulate NOX-independent NET formation in a DENV NS1-dependent manner. Polymorphonuclear neutrophils (PMNs) of healthy volunteers were co-incubated with autologous platelets in the presence or absence of 10 µg/mL DENV2 NS1 or 156μM platelet agonist TRAP-6. Experiments were performed in triplicate with 2 donors for each condition. Depicted are the relative values to medium (unstimulated samples) for (A) Total NETs and (B) NOX-independent NETs. (C) Heat-inactivated DENV2 does not induce NET formation. PMNs isolated from three healthy donors were incubated with 0.5x10 7 TCID/mL heat-inactivated DENV2 or medium control (mock) in the presence or absence of autologus washed platelets (Plt). Depicted is the total NET formation (ng/mL). Data are presented as mean with standard error mean (SEM) and analyzed using repeated measure a one-way ANOVA and Bonferroni’s multiple comparisons test; * p < 0.05, **p < 0.01 . TCID, Tissue culture infectious dose.

Article Snippet: After 1h, cells were washed with pre-warmed PBS and co-cultured with or without washed platelets (ratio 1 PMN vs . 50 platelets) in the presence of the following stimuli: 10µg/mL Dengue virus-2 non-structural protein-1 (DENV2 NS1, strain Thailand/16681/84; Native Antigen, Oxford, UK) or 156 µM Thrombin Receptor Activator Peptide 6 (TRAP-6) (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 3 hours at 37°C.

Techniques: Incubation, Isolation

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Suspension multiplex immunoassay for IgM reactivity with tick-borne encephalitis whole virus vs non-structural protein 1 antigens in infected (n = 50) vs vaccinated individuals (n = 50) at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Suspension, Multiplex Assay, Virus, Infection

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Tick-borne encephalitis virus reactivity of sera from acutely infected patients (n = 50) vs vaccines (n = 50), at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Virus, Infection, Vaccines

Journal: Eurosurveillance

Article Title: Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017

doi: 10.2807/1560-7917.ES.2018.23.3.17-00838

Figure Lengend Snippet: Suspension multiplex immunoassay for IgG reactivity with tick-borne encephalitis whole virus vs non-structural protein 1 antigens in infected (n = 50) vs vaccinated individuals (n = 50) at 0, 120 and 390 days after first vaccination, Sweden, 2017

Article Snippet: TBEV whole virus (WV) antigen was purchased from Jena Bioscience, Jena, Germany (Cat. No. PR-BA112) and TBEV non-structural protein 1 (NS1) antigen from Native Antigen Company, Heyford Park, United Kingdom (Cat. No. TBEV-NS1–100).

Techniques: Suspension, Multiplex Assay, Virus, Infection

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 2. Characterization of the NS1– population during MNoV infection. (A) Depiction of the NS1 positivity and negativity in CR6-infected BV2 cells. (B) Intracellular flow cytometry plots of CR6-infected BV2 cells costained with NS6/7 and NS1 (measured with clone CM79) at 16 hpi. (C) Intracellular flow cytometry plots of CR6-infected BV2 cells costained with NS6/7 and NS1 (measured with clone 2A9) at 8 or 18 hpi. Q-VD-OPh (20 μM) was added along with the virus when indicated. (D and E) Intracel- lular flow cytometry plots of WT or Casp3 KO BV2 cells infected with CR6 (D) or BV2 cells infected with WT CR6 or CR6D121/131G mutant virus (E) and costained with NS6/7 and NS1 at 18 hpi. (F) Comparison of caspase-3 activation in NS1– and NS1+ cells, measured with an antibody targeting cleaved caspase-3. (G) Surface staining with an- nexin V compared in NS1– and NS1+ cells. (H) BV2 cells were infected with CR6, harvested at 18 hpi, and costained with NS1, NS6/7, and other viral proteins. Intracellular levels of indicated viral proteins measured by flow cytometry in NS1– and NS1+ cells are shown.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Infection, Flow Cytometry, Virus, Mutagenesis, Comparison, Activation Assay, Staining

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 3. NINJ1-mediated membrane rupture is essential for NS1 secretion. (A) Volcano plot showing sgRNAs depleted or enriched in NS1– cells compared to NS1+ cells. (B to E) WT or Ninj1 clonal KO BV2 cells were infected with MNoV CR6 at 5 multiplicity of infection (MOI). (B) NS1 protein levels in the supernatant were measured at 14 hpi by enzyme-linked immunosorbent assay (ELISA) (n = 3). (C) LDH release was measured at 16 hpi (n = 3). (D) Sytox uptake was measured at 12 ~ 20 hpi (n = 3). (E) Cells (WCL) and supernatants (Sup) were analyzed by immunoblotting at 10 ~ 16 hpi. (F and G) WT or Ninj1 KO iBMDMs (F) or IEC-M2C-CD300lf cells transduced with indicated sgRNAs (G) were infected with CR6, and NS1 secretion was measured at 14 hpi (n = 3). (H to K) WT or Ninj1 KO BV2 cells were transduced with NINJ1 WT or oligomerization- defective mutant (K45Q) and infected with CR6. (H) Cells and supernatants were analyzed by immunoblotting at 14 hpi. (I) NS1 secretion was measured by ELISA at 14 hpi (n = 6). (J) Sytox uptake was measured at 14 ~ 20 hpi (n = 3). (K) LDH release was measured at 16 hpi (n = 3). (L) WT or Ninj1 KO BV2 cells were infected with CR6 and treated with glycine (5 mM). NS1 secretion was measured at 14 hpi (n = 3). (M) BV2 cells were infected at 1 MOI with WT CR6 or mutant virus (CR6∆N) and analyzed by ELISA and immunoblotting at 10 hpi (n = 6 from two independent experiments). Data represent means ± SEM. Data were normalized to Tween-20–treated control [(C), (D), (J), and (K)]. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test [(B), (C), (F), (G), and (I)] and Tukey’s multiple comparisons test [(K) and (L)] or two-tailed unpaired Mann-Whitney test (M). ns, not significant; **P < 0.01; ****P < 0.0001. Data are representative of three independent experiments.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Membrane, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Transduction, Mutagenesis, Virus, Control, Two Tailed Test, MANN-WHITNEY

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 4. Amino acid residues within NS1 required for the specific secretion of NS1. (A) Mutant studies revealed residues required for NS1 secretion. (B) BV2 cells were transfected with a mock vector or 2xStrep-NS1/2 and caspase-3 activation was measured by flow cytometry. (C) WT or Ninj1 KO BV2 cells were transfected with 2xStrep- NS1/2. Cells and supernatants were harvested at 24 hours posttransfection and analyzed by immunoblotting. (D) BV2 cells were transfected with 2xStrep-tagged NS1/2 constructs with N-terminal truncation mutations, and cells and supernatants were analyzed by immunoblotting at 24 hours posttransfection. Red arrowheads indicate different truncation mutants. (E) BV2 cells were transfected with D121/131G cleavage mutant and triple-alanine scanning mutants and analyzed as in (D). Numbers indi- cate amino acid residues substituted by alanine. Mutants showing defect in secretion are highlighted in red. (F) BV2 cells were transfected with NS1/2 constructs with indicated point mutations and analyzed as in (D). (G) 3D structure of CR6 NS1 (58 to 114 amino acids; PDB: 2MCK) highlighting the key residues identified from mutagen- esis study. (H and I) BV2 cells with doxycycline-inducible expression of 2xStrep-tagged WT or mutant NS1/2 were infected with MNoV CR6 and incubated with doxycycline (2 μg/ml) for 12 hours. (H) Cells (WCL) and supernatants (Sup) were analyzed by immunoblotting. (I) LDH release was measured and normalized to Tween-20–treated control (n = 3). Data represent means ± SEM and were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Activation Assay, Flow Cytometry, Western Blot, Construct, Expressing, Infection, Incubation, Control

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 5. NS1 directly binds to NINJ1. (A) WT or Ninj1 KO BV2 cells were infected with CR6 at 5 MOI and harvested at 10 hpi. Cell lysates were immunoprecipitated with anti- NS1 and analyzed by immunoblotting. (B and C) Transmission electron microscopy image of uninfected (B) or CR6-infected (C) BV2 cells with immunogold labeling for FLAG-NINJ1 (12-nm gold, black arrowheads) and NS1 (18-nm gold, red arrowheads). RC, replication complex. Scale bar, 500 nm. (D) Representative immunofluorescence confocal imaging of FLAG-NINJ1 (red), NS1 (green), NS6/7 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI, blue) in Ninj1 KO BV2 cells reconstituted with FLAG-NINJ1 WT or K45Q at 14 hpi. White arrowhead highlights the colocalization of NINJ1 and NS1 proteins. Scale bars, 5 μm. All images are representative of three or more independent ex- periments. (E) Quantification of colocalization between NS1 and FLAG-NINJ1 WT or K45Q. Bars represent means ± SEM, n = 10 to 11 cells. (F) Dose-dependent binding of Fc-tagged ectodomain of human NINJ1 (hNINJ1) or mouse NINJ1 (mNINJ1) protein to recombinant NS1 protein was determined by ELISA. A monoclonal anti-NS1 (clone CM79) and isotype control were included as a positive and negative control, respectively. Data represent means ± SEM of duplicates, and the dissociation constants (Kd) are shown. (G to I) Direct binding between recombinant NS1 and mNINJ1 protein was measured by MST. ΔFnorm indicates the change in thermophoresis, and Kd is shown. n.d., not determined. (G) Dose-response curve showing bindings of WT NS1 protein to fluorescence-labeled WT or mutated mNINJ1 proteins. (H) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled mNINJ1. Maltose-binding protein (MBP) was included as a negative control. (I) Dose-response curve show- ing bindings of WT or mutated NS1 proteins to fluorescence-labeled monoclonal anti-NS1 or isotype control. Data are shown in means ± SEM (n = 3).

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Infection, Immunoprecipitation, Western Blot, Transmission Assay, Electron Microscopy, Labeling, Immunofluorescence, Imaging, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Fluorescence

Journal: Science advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion.

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: Fig. 6. HSP90 inhibitors prevent NS1 secretion by blocking caspase-3 activation. (A) BV2 cells were infected with CR6 at 5 MOI in the presence of 2321 FDA-approved drugs (dots represent individual drugs tested). NS1 levels in the supernatant were measured by ELISA at 16 hpi and normalized to vehicle control. Representative results from two independent experiments are shown. (B to D) Cells were infected with CR6 in the presence of serial dilutions of drugs. NS1 secretion was measured by ELISA at 16 hpi and normalized to no-drug control. IC50 values (nM) determined by four biological replicates are shown. BV2 cells were treated with 17-AAG (left), AT13385 (middle), or ganetespib (right) (B). IEC-M2C-CD300lf (C) or IEC-Mx2LUC-CD300lf (D) cells were treated with ganetespib. (E) Caspase-3 activation in CR6-infected BV2 cells with gane- tespib (10 μM) was measured with an antibody targeting cleaved caspase-3. For (B) to (E), data are representative of two to three independent experiments. (F to H) WT or Ifnlr1−/− B6 mice were infected with 106 PFU CR6 perorally. Ganetespib was given intravenously (50 mg/kg, iv) as experimental treatment scheme (F). MNoV genomes in stool (G), ileum, colon, and MLN (H) at indicated time points were quantified by qRT-PCR (n = 7 to 10 mice per group, combined from two independent experiments). Data represent means ± SEM and analyzed by two-tailed unpaired Mann-Whitney test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: A 2- mg volume of total protein was mixed with 10 μg of anti- NS1 monoclonal antibody (2A9) or mouse IgG2b isotype control (BioXCell), which was preincubated with Dynabeads Protein G (Thermo Fisher Scientific) for 1 hour at 4°C.

Techniques: Blocking Assay, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Reactivity of clones selected by different strategies. The supernatant of 24 Nb clones selected using the A, B, and C panning conditions—clones 1–24, 25–48, and 49–72, respectively—were tested by ELISA on plates coated with Zika virus NS1 (ZVNS1) immobilized using the A (dark gray), B (light gray), or C (gray) strategies. ill-IgG: immune llama immunoglobulin G and BtNS1: biotinylated NS1. Optical density at 450nm (OD450nm) was measured.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Virus

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Reactivity of 22 Nb clones against different concentrations of ZVNS1. The supernatant of each clone was exposed to three different concentrations of ZVNS1 (0.5 ng/mL, 5.0 ng/mL, and 100 ng/mL) passively absorbed in the ELISA wells. Measurements were done by duplicates.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Titration curves of the selected capture nanobodies. Decreasing concentrations of each nanobody were exposed to a fixed concentration of ZVNS1 (100 ng/well) directly adsorbed on the ELISA well. Measurements were done by triplicates. SC 50 : 50% signal saturation and BtNb: biotinylated nanobodies.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: Titration, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Pairwise screening of detection Nbs. The four capture Nb candidates (BtNB22, BtNb246, BtNb278, and BtNb32) were used to capture ZVNS1 (2 ng/well) and were assayed against the 72 master plate Nb supernatants. Black, gray, and white are used to denote detection Nbs selected with the A, B, and C panning strategies, respectively. The horizontal lines represent the mean OD values.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques:

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: ZVNS1-32/D6 nanobody sandwich ELISA for the detection of ZVNS1. Extended (left) and linear (right)-range calibration curves performed in phosphate-buffered saline (PBS) or serum dilutions. The results are the average values of triplicate measurements, and the error bars represent the standard deviation.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: Sandwich ELISA, Saline, Standard Deviation

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Analysis of recovery in ZVNS1 spiked serum samples.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques:

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Intraday precision of the test with ZVNS1-spiked serum.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: Serum Assay

Journal: Biomolecules

Article Title: Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

doi: 10.3390/biom10121652

Figure Lengend Snippet: Interday precision of the test with ZVNS1-spiked serum.

Article Snippet: Flaviviruses’ nonstructural protein 1 and mouse anti-ZVNS1 monoclonal antibody were purchased from The Native Antigen Company (Oxford, OX, UK).

Techniques: