Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Schematic diagram of dual detection of HA protein from influenza viruses H5N1 and H1N1 based on FRET combined with DNase I.

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Optimization of GO reaction conditions. (A) Fluorescence emission spectral signals of the H5N1-FAM probe. (B) H1N1-ROX probe in the presence of different concentrations of GO (0, 10, 20, 30, 40, 50, 60, 80, and 100 μg/ml). (C) Changes in trends of fluorescence signals of H5N1-FAM and H1N1-ROX probes in the presence of different concentrations of GO (0, 10, 20, 30, 40, 50, 60, 80, and 100 μg/ml). (D) Changes in trends of fluorescence signals at different time points (0, 1, 2, 3, 5, 10, 15, and 20 min) in the presence of GO (50 μg/ml).

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques: Fluorescence

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Examination of cross-reactions by monitoring fluorescence spectra of the two probes. (A) Different concentrations of HA of H5N1 in the detection system. (B) Different concentrations of HA of H1N1 in the detection system. A range of HA protein concentrations (0, 200, 500, and 1,000 ng/ml) was examined at a fixed GO concentration of 50 μg/ml.

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques: Fluorescence, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Effects of different doses of DNase I on fluorescence recovery for (A) H5N1-FAM and (B) H1N1-ROX probes in the detection system. Effects of incubation times of DNase I on fluorescence recovery for (C) H5N1-FAM and (D) H1N1-ROX probes.

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques: Fluorescence, Incubation

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Effects of DNase I on the sensitivity of detection of H1N1 HA. (A) Fluorescence spectra of a series of concentrations of H5N1 HA (0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 10, 15, and 20 ng/ml) in the presence of DNase I. (B) (F – F0)/F0 (fluorescence change) vs. concentration of H1N1 HA in the presence of DNase I. (C) Fluorescence intensities of a series of concentrations of H1N1 HA (0, 10, 20, 30, 50, 70, 100, 120, 150, and 200 ng/ml) in the absence of DNase I. (D) (F – F0)/F0 (fluorescence change) vs. concentration of H1N1 HA in the absence of DNase I. The insets depict a linear relationship between (F – F0)/F0 and the concentration of HA H1N1 in the presence of DNase I (B) and the absence of DNase I (D) .

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques: Fluorescence, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Specificity analysis of the aptamer sensor. HA proteins from H1N1 and H5N1 act as counterpoint controls for each other. Background control protein BSA and human IgG are at a concentration of 20 ng/ml.

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques: Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I

doi: 10.3389/fmicb.2022.934475

Figure Lengend Snippet: Dual detection of the HA from H1N1 and H5N1 viruses in human serum samples.

Article Snippet: Hemagglutinin proteins of influenza A H5N1 (H5N1A/Anhui/1/05) and H1N1 (H1N1A/New Caledonia/20/99), the targets for H5N1 and H1N1 AIV detection, were purchased from Sino Biology (Beijing, China).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines

doi: 10.3389/fmicb.2022.937192

Figure Lengend Snippet: The construction, expression, and cell entry ability of EΔM-tM2e or EΔM-HM2e. (A) Amino acid sequences of tM2e, including influenza virus M2e consensus from human, swine, and bird strains. (B) Amino acid sequence of HM2e, including influenza virus HA stalk and M2e from human strain. (C) EΔM plasmid containing EboGP gene with MLD deletion (aa 305–485) and EΔM-HM2e or EΔM-tM2e plasmids. (D,E) 293T cells were co-transfected Δ8.2, ΔRI/ΔE/Gluc+ with EΔM-tM2e and EΔM-HM2e or HA, NA, and M2 plasmids. WB was used to detect the expression of M2e, HA, EboGP, and HIV P24 in cells and PVPs. (F,G) Human PBMC-derived macrophages (MDMs) and dendritic cells (MDDCs) were infected with the EΔM-HM2e, EΔM-tM2e, EΔM, and HA/NA/M2-PVPs. The supernatants were collected at different time points after infection and subjected to a GLuc activity assay. Error bars represent variation between duplicate samples, and the data are representative of results obtained in three independent experiments. Statistical significance was determined using an unpaired t -test, and significant p -values were represented with asterisks *** ≤0.001. No significance (ns) was not shown.

Article Snippet: Human M2e peptide (SLLTEVETPIRNEWGCRCNDSSD) was purchased from GenScript (RP20206), while avian influenza H7N9 M2e peptide (SLLTEVETPTRTGWECNCSGSSD) and swine influenza H1N1 M2e peptide (SLLTEVETPTRSEWECRCSDSSD) were synthesized by GenScript as previously described ( ) and HA peptide was synthesized by Shanghai Royobiotech (19CL00157).

Techniques: Expressing, Virus, Sequencing, Plasmid Preparation, Transfection, Derivative Assay, Infection, Activity Assay

Journal: Frontiers in Microbiology

Article Title: Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines

doi: 10.3389/fmicb.2022.937192

Figure Lengend Snippet: The anti- M2e and anti-HA antibodies were induced by EΔM-tM2e- or EΔM-HM2e -VLPs in Balb/c mice (A) Schematic of the EΔM-tM2e, EΔM-HA M2e, or HA/NA/M2-VLP immunization protocol used in this study ( n = 4/5 mice per group). (B) Mice body weights were monitored weekly, in which 100% body weight was set at day 0. (C) The levels of anti-M2e, Left panel: OD 450 nm; Right panel: Endpoint titer. (D) Anti-HA M2e, Left panel: OD 450 nm; Right panel: Endpoint titer. (E) Anti-Ebola GP. (F) Anti-HIV P24. IgG-specific antibodies were measured by the ELISA method. Statistical significance was determined using an unpaired t -test, and significant p -values were represented with asterisks * ≤0.05, ** ≤0.01, *** ≤0.001, and **** ≤0.0001. No significance (ns) was not shown.

Article Snippet: Human M2e peptide (SLLTEVETPIRNEWGCRCNDSSD) was purchased from GenScript (RP20206), while avian influenza H7N9 M2e peptide (SLLTEVETPTRTGWECNCSGSSD) and swine influenza H1N1 M2e peptide (SLLTEVETPTRSEWECRCSDSSD) were synthesized by GenScript as previously described ( ) and HA peptide was synthesized by Shanghai Royobiotech (19CL00157).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines

doi: 10.3389/fmicb.2022.937192

Figure Lengend Snippet: The specific anti-M2e and anti-HA IgG induced by rVSV-EΔM-tM2e or rVSV-EΔM-HM2e in mice sera. (A) The Balb/c mice were injected IM with 1.0 × 10 7 TCID 50 of rVSV-EΔM -tM2e or rVSV-EΔM-HM2e on day 0 and 5 × 10 6 TCID 50 on day 21. Serum was collected on days 20 and 35 ( n = 4/5 mice per group). (B) ELISA assay was used to detect the levels of anti-Ebola GP IgG (Left panel: OD 450 nm; Right panel: Endpoint titer). (C) Anti-M2e IgG (Left panel: OD 450 nm; Middle panel: Endpoint titer; Right panel: Test against human, avian, and swine M2e peptides). (D) Anti-M2e IgG1 and IgG2a. (E) Anti-HA (HA1, HA3, and HA5) IgG (Left panel: OD 450 nm; Right panel: Endpoint titer) and anti-HA IgG1 and IgG2a (F) . Statistical significance between the two groups was determined using an unpaired t -test, and significant p -values are represented with asterisks, * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. No significance (ns) was shown.

Article Snippet: Human M2e peptide (SLLTEVETPIRNEWGCRCNDSSD) was purchased from GenScript (RP20206), while avian influenza H7N9 M2e peptide (SLLTEVETPTRTGWECNCSGSSD) and swine influenza H1N1 M2e peptide (SLLTEVETPTRSEWECRCSDSSD) were synthesized by GenScript as previously described ( ) and HA peptide was synthesized by Shanghai Royobiotech (19CL00157).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines

doi: 10.3389/fmicb.2022.937192

Figure Lengend Snippet: The rVSV EΔM-tM2e or EΔM-HM2e induced specific serological and mucosal Anti-M2e or anti-HA IgA antibodies and mediated cell immune responses. (A) The levels of Anti-M2e IgA antibody in mice sera. (B) The levels of anti-HA (H1, H3, or H5) IgA antibody in mice sera. (C) The levels of Anti-M2e and Anti-HA IgA in the nasal wash. (D) Splenocytes were isolated from the rVSV-EΔM-tM2e- or rVSV-EΔM-HM2e-immunized mice and were stimulated with Influenza M2e peptide or HA5 recombinant protein. The release of cytokines and chemokines in the supernatants of cell cultures were quantified with an MSD V-plex kit mouse cytokine kit and counted in the MAGPIX instrument. Statistical significance between the two groups was determined using an unpaired t -test, and significant p -values are represented with asterisks, * ≤0.05, *** ≤0.001. No significance (ns) was not shown.

Article Snippet: Human M2e peptide (SLLTEVETPIRNEWGCRCNDSSD) was purchased from GenScript (RP20206), while avian influenza H7N9 M2e peptide (SLLTEVETPTRTGWECNCSGSSD) and swine influenza H1N1 M2e peptide (SLLTEVETPTRSEWECRCSDSSD) were synthesized by GenScript as previously described ( ) and HA peptide was synthesized by Shanghai Royobiotech (19CL00157).

Techniques: Isolation, Recombinant