Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Production and characterization of recombinant NSDV GP38-his/FLAG. ( A ) Alignment of partial GPC sequences from different NSDV strains (Ganjam virus IG 619 = NSDV_India; NSDV 708 = NSDV_Kenya; NSDV H. longicornis China = NSDV_China) and CCHFV (IbAr10200). Numbering corresponds to NSDV_India GPC. Amino acid sequences are displayed starting from their respective N-terminus. For CCHFV, the furin protease cleavage site (RSKR) and SKI-1/S1P cleavage site (RRLL) are underlined in blue. For NSDV, arginine-containing motifs reported to be conserved across the GPCs of different orthonairoviruses are highlighted in orange. Two potential N-glycosylation sites (Asn 220 and Asn 400 ) are highlighted with an orange star. ( B ) For protein purification, the partial NSDV_India GPC sequence (aa 138–445; NSDV GP38-his/FLAG protein) was fused to a C-terminal 6×His- and FLAG-tag both highlighted with an orange underline. ( C ) SDS-PAGE of recombinant NSDV GP38-his/FLAG his-tag purified from Sf9 cells followed by Coomassie blue staining and immunoblot analysis using anti-FLAG primary and horseradish peroxidase-conjugated secondary antibodies.

Article Snippet: NSDV Gn ( NAC-REC31904-500 ) and NSDV Gc ( NAC-REC31906-500 ) from The Native Antigen Company were diluted in 0.01 M PBS and coated overnight on Maxisorp 96-well plates at a concentration of 100 ng/well.

Techniques: Recombinant, Virus, Glycoproteomics, Protein Purification, Sequencing, FLAG-tag, SDS Page, Purification, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Serological cross-reactivity between Crimean-Congo haemorrhagic fever virus and Nairobi sheep disease virus glycoprotein C

doi: 10.3389/fimmu.2024.1423474

Figure Lengend Snippet: Orthonairovirus test antigens employed during experiments.

Article Snippet: CCHFV Gc , NP_950235 (IbAr10200, Nigeria) , HEK293 (Human) , The Native Antigen Company, UK #REC31696.

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Replication-driven HBV cccDNA loss in chimeric mice with humanized livers

doi: 10.1101/2023.12.28.573542

Figure Lengend Snippet: A. Average cccDNA levels were <1 copy/cell in all 380 cccDNA samples isolated from livers of 17 mice under non-partial blocking of de novo infection (two mice: 970 and 819) or blocking of two cccDNA replenishment pathways (the remaining 15 mice). B. Average cccDNA levels were significantly lower than both untreated and treated mice with partial blocking of de novo infection . C1 and C2. Serum HBsAg and HBeAg levels were progressively reduced to undetectable levels among mice with non-partial blocking of de novo infection in mouse 819 or blocking of two pathways in the remaining 7 mice started before peak infection . C3. Kinetic serum anti-HBs antibody level . D1 and D2. Serum HBsAg and HBeAg levels were progressively reduced to undetectable levels upon boosting anti-HBs antibody levels by triweekly administrations of mouse anti-HBs antibody at a dose of 250 μg/injection starting on day 99 pi or later until termination. D3. Kinetic serum anti-HBs antibody level in the same D1/D2 group. E1 and E2. Average rcDNA levels reduced by 2-4 logs among mice through blocking of two pathways (Green) with anti-HBs antibody expressed by HBVZ10 before peak infection (E1) or with administering additional mouse anti-HBs antibody to boost the HBVZ10 expressed anti-HBs antibody level after the peak infection (E2) compared to untreated (Blue). F . Kinetic serum HBsAg levels among 3 mice (805 untreated; 833 HBVZ10 monotherapy, and 838 HBVZ10 combined with 12-week entecavir. G . Average intracellular HBsAg, rcDNA and cccDNA levels reduced by >100-fold in mouse 838 in 80 days compared to mouse 833. HBeAg, hepatitis B e antigen. The difference was considered significant at P<0.05 by Student’s t test. Error bars were plotted with standard deviations.

Article Snippet: Blood was collected tri-weekly for quantification of serum HBV DNA (qPCR see below), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA Anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA Anti-HBs 20-Calibrator kit 25219, Bio-Rad) and human albumin (Human albumin ELISA kit E-80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Isolation, Blocking Assay, Infection, Injection

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Tunable Fano-resonant Metasurfaces on a Disposable Plastic-template for Multi-modal and Multiplex Biosensing

doi: 10.1002/adma.201907160

Figure Lengend Snippet: Surface sensitivity of plastic-templated metasurface for layer-by-layer modifications. a Summary of surface chemistry steps. b Resonance peak wavelength shifts demonstrated on surfaces etched for 30, 60 and 90 sec. c Layer-by-layer binding response of bio-molecules on the 60 sec etched surface is monitored in real-time. d-f Resonance wavelength shifts as a function various concentrations of protein G, anti-gp120 antibody and rec-gp120 protein are represented via Box-Whisker plots. I-shaped box indicates 25th and 75th, red-line median, and whiskers the 95th and 5th percentiles. Dots represent data points for each concentration. Non-parametric Kruskal-Wallis analysis followed by Dunn’s multiple comparison test was performed to evaluate the collected data for each concentration at Protein G, anti-gp120 antibody and rec. gp120 protein experiments. The minimum, first quartile, median, third quartile, and maximum of each concentration were presented in Supplementary Table 1. In Protein G experiment, statistical difference was observed between 75 and 1000 μg/mL concentrations (n=3, p<0.05). In anti-gp120 antibody experiments, statistical difference was observed between 30 and 75 μg/mL concentrations (n=3, p<0.05). In rec-gp120 protein experiments, statistical difference was observed between 25 and 200 μg/mL concentrations (n=3, p<0.05).

Article Snippet: This step was followed by a 100 μL PBS wash. (2) Anti-gp120 antibody layer: 100 μL of anti-gp120 (immunoaffinity purified rabbit polyclonal IgG, 1 mg/mL, #11233-RP02, Sino Biological, Pennsylvania, US) was captured on the protein G layer and the surface was washed using 100 μL PBS. (3) HIV capture: The functionalized metasurface was used to capture recombinant gp120 proteins and HIV-1 particles.

Techniques: Binding Assay, Whisker Assay, Concentration Assay

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Production and characterization of recombinant NSDV GP38-his/FLAG. ( A ) Alignment of partial GPC sequences from different NSDV strains (Ganjam virus IG 619 = NSDV_India; NSDV 708 = NSDV_Kenya; NSDV H. longicornis China = NSDV_China) and CCHFV (IbAr10200). Numbering corresponds to NSDV_India GPC. Amino acid sequences are displayed starting from their respective N-terminus. For CCHFV, the furin protease cleavage site (RSKR) and SKI-1/S1P cleavage site (RRLL) are underlined in blue. For NSDV, arginine-containing motifs reported to be conserved across the GPCs of different orthonairoviruses are highlighted in orange. Two potential N-glycosylation sites (Asn 220 and Asn 400 ) are highlighted with an orange star. ( B ) For protein purification, the partial NSDV_India GPC sequence (aa 138–445; NSDV GP38-his/FLAG protein) was fused to a C-terminal 6×His- and FLAG-tag both highlighted with an orange underline. ( C ) SDS-PAGE of recombinant NSDV GP38-his/FLAG his-tag purified from Sf9 cells followed by Coomassie blue staining and immunoblot analysis using anti-FLAG primary and horseradish peroxidase-conjugated secondary antibodies.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Recombinant, Virus, Glycoproteomics, Protein Purification, Sequencing, FLAG-tag, SDS Page, Purification, Staining, Western Blot

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Seroconversion of NSDV- infected sheep against different NSDV antigens. ( A ) Sera from NSDV-infected sheep ( n = 6) collected at different days post infection (dpi) were tested in duplicate in an indirect in-house ELISA based on recombinant NSDV GP38-his/FLAG. Mean of corrected OD values and standard deviations are displayed. ( B ) Serum samples collected from NSDV-infected sheep ( n = 6) at 0 and 28 dpi were tested for reactivity in an indirect ELISA based on NSDV N-his/FLAG, commercially available recombinant Gn and Gc proteins, and GP38-his/FLAG.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Indirect ELISA

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Immunoblot analysis of NSDV protein expression in SW13 cells. (A) Immunoblot analysis of NSDV-infected and mock-infected SW13 cells collected at 28 h p.i. For detection, newly generated monoclonal antibody (mAb) 6F6 A3B raised against NSDV GP38-his/FLAG and goat anti-mouse IRDye 800-conjugated antibodies were used. The detection of GAPDH served as a loading control. (B) Cell lysates from multi-cycle infection kinetics were collected at the indicated time post infection (p.i.). For detection, mAb 6F6 A3B and mAb 5H11 C1 (raised against NSDV Gc) and rabbit-derived polyclonal serum (R8253) for nucleoprotein (N) detection were used as primary antibodies followed by incubation with goat anti-mouse or anti-rabbit IRDye 680 or 800CW-conjugated secondary antibodies, respectively. β-Tubulin and GAPDH served as loading controls. Representative blots from three independent experiments performed in duplicates are shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Western Blot, Expressing, Infection, Generated, Control, Derivative Assay, Incubation

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: NSDV GP38 expression in the supernatant of transfected or NSDV-infected cells. HEK 293T and SW13 cells were transfected with pcDNA NSDV MLD-GP38-Strep (−) or co-transfected with pcDNA NSDV MLD-GP38-Strep and pIR-hfurin encoding for human furin protease (+). Supernatants were harvested at 72 h post-transfection and analyzed by immunoblot using mAb 6F6 A3B. For comparison of NSDV GPC cleavage products, supernatant from NSDV-infected SW13 cells (24 h p.i.) was also analyzed by immunoblot. A representative blot from at least three independent experiments is shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Expressing, Transfection, Infection, Western Blot, Comparison

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Efficiency of cleavage of FRET substrates by furin. ( A ) FRET substrates spanning the P7-P4′ amino acid sequences of the putative cleavage site motifs containing an N-terminal o-aminobenzoyl fluorophore and a C-terminal Tyr(3 NO 2 )-NH 2 as a quenching residue were synthesized and tested in an enzyme assay with recombinant furin. Three NSDV GPC sequences were tested: GPC-1 covering the monobasic motif at position 175; GPC-2, a negative control for GPC-1 with the arginine of the motif replaced by alanine, and GPC-3 covering another putative dibasic furin motif for cleavage after residue 134. A FRET substrate with a multibasic furin cleavage site motif of hemagglutinin (HA5) of a highly pathogenic avian influenza virus (HPAIV) strain served as positive control (PC). ( B ) The cleavage of the FRET substrates by recombinant furin was measured in an enzyme kinetic assay. Mean values + SD based on three independent measurements with three independent weights of substrates are displayed.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Residue, Synthesized, Enzymatic Assay, Recombinant, Negative Control, Virus, Positive Control, Kinetic Assay

Journal: Journal of Virology

Article Title: Immunogenicity of NSDV GP38 and the role of furin in GP38 proteolytic processing

doi: 10.1128/jvi.00537-25

Figure Lengend Snippet: Impact of furin inhibition on NSDV glycoprotein processing. SW13 cells were infected with NSDV (MOI of 0.1). After removal of inoculum, fresh medium with (+) or without (−) furin inhibitor MI-1148 (30 µM in DMSO) or DMSO alone was added. At 24 h p.i., cell lysates ( A ) and supernatants ( B ) were collected and analyzed for N, Gc, and GP38 expression in immunoblot. β-Tubulin and GAPDH served as loading controls. Representative blots from three independent experiments each performed in duplicate are shown. ( C ) The signal intensities of the GP38 bands were quantified using Li-Cor software Image Studio Lite Ver 5.2. The GP38 signal intensities from inhibitor-treated samples were set in relation to the signal intensities of the untreated samples (=100%). Mean relative signal intensities and standard deviations from three independent experiments each performed in duplicate are shown.

Article Snippet: Female BALB/c mice were immunized four times in an interval of 3 weeks intraperitoneally with 25 μg of recombinant NSDV GP38-his/FLAG or NSDV Gc (NAC-REC31906-500, The Native Antigen Company), mixed with an equal amount of GERBU Adjuvant MM (GERBU Biotechnik GmbH).

Techniques: Inhibition, Infection, Expressing, Western Blot, Software

Journal: npj Viruses

Article Title: Respiratory syncytial virus glycoprotein G impedes CX 3 CR1-activation by CX 3 CL1 and monocyte function

doi: 10.1038/s44298-024-00075-9

Figure Lengend Snippet: 1 µg purified protein samples were resolved on a 10% SDS-PAGE gel and stained with PageBlue™ coomassie solution, showing total protein content (left). Western blot analyses using rabbit anti-BSA (1:1000; A11133; Thermo Fisher) verified BSA expression, rabbit anti-RSV G glycoprotein (1:1000; 40041-T62; Sino Biological) confirmed RSV G glycoprotein expression, and mouse anti-6xHis (1:1000; His.H8; Thermo Fisher) confirmed the presence of His-tagged proteins. Secondary antibodies were applied 1:20000 (goat anti-mouse IgG, Thermo Fisher; goat anti-rabbit IgG, Thermo Fisher). All membranes were treated with SuperSignal™ Western Blot Enhancer and detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher). Protein bands were visualized using a Chemidoc chemiluminescence imaging system (Bio-Rad). Black borders represent a division between separate blots.

Article Snippet: Primary antibodies were applied at a 1:1000 dilution: mouse anti-6xHis (Cat# MA1-21315; Thermo Fisher), rabbit anti-RSV G glycoprotein (Cat#40041-T62; Sino Biological), and rabbit anti-BSA (Cat#A11133; Thermo Fisher).

Techniques: Purification, SDS Page, Staining, Western Blot, Expressing, Imaging