Journal:

Article Title: Flock House Virus RNA Polymerase Is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion

doi: 10.1128/JVI.76.19.9856-9867.2002

Figure Lengend Snippet: Membrane association of FHV protein A deletion mutants. Flotation analysis was done as described in the Fig. ​Fig.22 legend with C-terminally HA epitope-tagged protein A deletion mutants covering all but amino acids 1 to 7 of protein A (A) or with a nested series of N-proximal deletions (B). Protein A schematic with amino acid positions and the locations of the predicted TMD (amino acids 15 to 36; see Fig. ​Fig.3),3), with viral RdRp motifs shown at the top. Deleted regions are represented by solid lines. Deletion designations are shown on the left and represent the amino acids included within the deleted region. Flotation efficiencies represent the percentage of the total protein recovered in the LD fraction after Nycodenz gradient fractionation and are averages of at least three independent experiments. Representative mouse anti-HA immunoblots are shown.

Article Snippet: Rabbit polyclonal antibodies against GFP and the influenza virus hemagglutinin (HA) epitope tag were from Santa Cruz Biotechnology (Santa Cruz, Calif.), and mouse monoclonal antibodies against HA (clone HA-7) were from Sigma (St. Louis, Mo.).

Techniques: Fractionation, Western Blot

Journal: bioRxiv

Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

doi: 10.64898/2026.01.30.702929

Figure Lengend Snippet: A) Diagram of PA-X production mechanism and mutations in the PA-X defective Perth H3N2 ΔX virus. The position of the +1 frameshift that creates PA-X is indicated, and the mutated nucleotides that reduce frameshift and generate a premature stop codon at PA-X codon 201 in the ΔX virus are marked in red. B) Schematic of ALI culture differentiation from primary basal epithelial cells. C) Human bronchial epithelial cells were cultured at ALI for 3-4 weeks and stained for tight junctions (ZO-1, top), cilia (acetylated TUB1A, middle, sagittal section), and the mucus component and goblet cell marker mucin 5AC (MUC5AC, bottom) and nuclear stain (Hoescht, all) to confirm proper differentiation of the cells and formation of the pseudostratified epithelium. D) Immunofluorescence image of infected ALI culture, with staining of influenza A virus nucleoprotein (NP), cilia (acetylated TUB1A), and nuclear stain (Hoechst) at 2 days post infection. E) Titer of virus collected by apical washes from ALI cultures. (n = 11, 5 separate donors) measured by Tissue Culture Infectious Dose 50 (TCID50) F) ALI culture barrier integrity measured using trans-epithelial electrical resistance. 1 donor shown, representative of data from data from 3 donors (Donors 2, 3, 4, more information in Methods Table 1). A single donor is shown because the time of barrier integrity loss varies between day 2 and 3 depending on the donor. G) Experimental timeline for infection and sample collection of ALI cultures.

Article Snippet: Cells were washed and then stained with MHC I HLA-ABC W6/32 pre-conjugated to Alexa Fluor 594 and Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.

Techniques: Virus, Cell Culture, Staining, Marker, Immunofluorescence, Infection

Journal: bioRxiv

Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

doi: 10.64898/2026.01.30.702929

Figure Lengend Snippet: ALI cultures were infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected and RNA was collected for scRNAseq as described in . The scRNAseq data were analyzed to identify infected cells. A) Integrated UMAP of mock, Perth H3N2 WT and Perth H3N2 ΔX infected ALI cultures colored by the percent of reads that mapped to influenza A viral genes in each cell. B-C) Bar graphs with percent influenza A viral reads per cell in each assigned cell type in WT and ΔX infected cultures at 1 (B) and 3 DPI (C). D) Bar graph showing the percentage of infected cells that belong to each epithelial cell type in influenza-positive cells in WT and ΔX infected cultures vs. the composition of the cultures derived in mock-infected samples. Influenza-positive cells were defined as cells having at least 1% reads mapping to the influenza transcriptome. The two donors were aggregated for the analysis in all panels. The two timepoints [1 and 3 days post infection [DPI]) were also aggregated for the analysis in panel A.

Article Snippet: Cells were washed and then stained with MHC I HLA-ABC W6/32 pre-conjugated to Alexa Fluor 594 and Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.

Techniques: Infection, Derivative Assay

Journal: bioRxiv

Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

doi: 10.64898/2026.01.30.702929

Figure Lengend Snippet: A-B,D) ScRNAseq data from ALI cultures infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected as shown in were analyzed for changes in type I and III IFN production and responses (n = 2, from 2 separate donors). A) Heatmap showing average expression of type I and III IFNs at 1 and 3 DPI, separated in infected (influenza A virus-positive) and bystander (influenza A virus-negative) populations. Influenza A virus-positive cells were defined as cells of any subtype with at least 1% reads mapping to the influenza transcriptome. B) Heatmap showing average expression of type I and III IFNs at 1 DPI divided by cell type (assigned with ScType). C) ALI cultures were mock-infected or infected with Perth H3N2 WT or ΔX at MOI of 0.1. The basal medium was collected at the indicated timepoints. IFN-λ in the basal media was detected using HEK-Blue IFN-λ cells reporter cells. The IFN-λ concentration and the log2 fold-change in ΔX vs. WT infection are shown. (n=5-11 independent experiments, 5 donors, Donors 1-5). ns = p > 0.05, *= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001, two-way ANOVA with Šidák correction (IFN-λ concentration) or ratio T-test (Log2 fold-change). D) Heatmap showing average expression of ISGs at 1 and 3 DPI, separated in infected and bystander populations. ISG list was based on Schoggins et al. 2011.

Article Snippet: Cells were washed and then stained with MHC I HLA-ABC W6/32 pre-conjugated to Alexa Fluor 594 and Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.

Techniques: Infection, Expressing, Virus, Concentration Assay

Journal: bioRxiv

Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

doi: 10.64898/2026.01.30.702929

Figure Lengend Snippet: A) ScRNAseq data from from ALI cultures infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected as shown in were analyzed using CytoSig for predicted cytokine responses (n = 2, from 2 separate donors). A) Heatmap showing predicted secretion of select targets at 1 and 3 DPI from bystander (influenza A virus-negative) populations. B-C) Luminex data using basal conditioned media from ALI cultures infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected as shown in (n = 5, from 5 separate donors). B) Heatmap showing mean-centered z-score of the protein concentration for each Luminex target. Black asterisks correspond to significance vs. mock (tested using two-way ANOVA) and white asterisks correspond to significance vs. WT (tested using ratio T test). C) Bar graphs showing concentrations of cytokines that were significantly altered by PA-X activity at 3 DPI. Comparisons of ΔX vs. WT are shown, (analyzed using a ratio T-test. ns = p > 0.05, *= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001.)

Article Snippet: Cells were washed and then stained with MHC I HLA-ABC W6/32 pre-conjugated to Alexa Fluor 594 and Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.

Techniques: Infection, Virus, Luminex, Protein Concentration, Activity Assay

Journal: bioRxiv

Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium

doi: 10.64898/2026.01.30.702929

Figure Lengend Snippet: A-B) Analysis of scRNAseq data from ALI cultures that were infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected as shown in (n = 2, from 2 separate donors). A) Top ten gene sets identified by gene set enrichment analysis of all differentially expressed genes (DEGs) in infected cells at 1 day post infection (DPI), with the top 10 gene sets shown. Influenza A virus-positive cells were defined as cells of any subtype with at least 1% reads mapping to the influenza transcriptome. Variable genes were found using FindAllMarkers with a cutoff of |log2FC| > 0.25 p-adjusted < 0.05, and enriched gene set cutoffs were p-adjusted < 0.05 (n = 2, from 2 separate donors, Donors 1 and 2). B) Heatmaps of DEGs in the antigen processing and presentation gene set (GO:0019882) at 1 and 3 DPI, separated by infected and bystander cells from each condition. C-D) ALI cultures were infected with Perth H3N2 WT or ΔX virus, mock-infected, or pre-treated with IFN-α at 2 ng/µL for 1 hour. Cells were collected at 3 DPI and stained for major histocompatibility complex I (MHC I) and influenza A virus nucleoprotein (IAV NP). C) Diagram of the flow cytometry staining method, where the same anti-HLA-ABC coupled to different fluorophores was used before and after permeabilization to detect surface and intracellular MHC I, respectively, gating strategy for NP+ infected and NP-bystander cells (after gating for live, single cells), and representative histogram results for MHC I HLA-ABC surface and intracellular expression. D) HLA-ABC median fluorescence intensity at 3 DPI from flow cytometry analysis plotted as fold-change relative to mock-infected ALI cultures (n = 4, from 4 separate donors, Donors 2-5). Two-way ANOVA, followed by Šídák’s multiple comparisons test, ns = p > 0.05, *= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001. E-G) Calu-3 cells were mock-infected or infected with Perth H3N2 WT or ΔX at an MOI of 0.1 for 18 hours. Surface MHC I was stripped using a citric acid buffer, then cells were collected at multiple timepoints over the course of 6 hours. Surface MHC I was measured by flow cytometry. E) Schematic of acid strip time course and MHC I surface trafficking. F-G) Log2 fold change of surface MHC I median fluorescence intensity over a 6-hour time course in all live mock-infected cells (F) and influenza A virus NP positive WT and ΔX-infected cells (G). Linear regression was performed for each condition, and the p value was calculated to compare Perth H3N2 WT and ΔX NP+ cells. Dashed lines represent the line of best fit, and the shaded regions indicate the 95% confidence interval.

Article Snippet: Cells were washed and then stained with MHC I HLA-ABC W6/32 pre-conjugated to Alexa Fluor 594 and Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.

Techniques: Infection, Virus, Staining, Immunopeptidomics, Flow Cytometry, Expressing, Fluorescence, Stripping Membranes

Journal: Analytical Chemistry

Article Title: Native Capillary Nanogel Electrophoresis Assay of Inhibitors of Neuraminidases Derived from H1N1 and H5N1 Influenza A Pandemics

doi: 10.1021/acs.analchem.4c06127

Figure Lengend Snippet: Depicts the traces obtained with H5N1 neuraminidase showing the conversion of the 6′-sialyllactose substrate to the lactose product in the presence of peramivir at concentrations listed from top to bottom as 0.010, 1.0, 3.0, 5.0, 10., 14, 75, 750 nM. The area of the product, which decreases with increasing inhibitor, is used to create the dose response curve. The K i is approximated as the IC 50 . Traces are offset to aid in visualization (see Table S13, and Figure S7 in the Supporting Information for peak areas and offsets for the x - and y -axes). The peak labeled with the asterisk is a contaminant present in the substrate preparation prior to enzyme treatment.

Article Snippet: Recombinant influenza A virus neuraminidase proteins H1N1 (4858-NM-005 lot RJJ0822031) and H5N1 (7597-NM-010 lot DCLS0121052) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Labeling

Journal: Vaccine

Article Title: Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies.

doi: 10.1016/j.vaccine.2019.09.057

Figure Lengend Snippet: Fig. 2. Screening mammalian cell types for the influenza A/California/07/2009 (H1N1) VLP production (A) HA, NA, M1 protein detection by Western Blot with specific antibodies in the supernatant of the 293 T cells expressing VLPs (1) NIBSC influenza H1N1 antigen as positive control, (2) supernatant of 293 T cell as negative control, (3) supernatant of 293 T cell expressed influenza VLPs; (B) VLPs expressed in CHO-K1 cells (1), Vero cells (2), 293 T cells (3) and A/H3N2 influenza virus as positive control (4) were observed by Cryo-TEM performed by CBMN, UMR 5248, University of Bordeaux.

Article Snippet: After geltransfer and saturation, the blots were incubated with a rabbit polyclonal antibody against H1N1 influenza A virus NA (RD System), a polyclonal sheep antibody against H1N1 M1 (ThermoFisher), or a polyclonal rabbit antibody against the HA from A/California/07/2009 (H1N1) (produced in-house) for 1 h. Blots were then incubated with a secondary sheep anti-rabbit or antisheep antibody conjugated to IRDye 800 (Rockland) for 1 h. The membranes were visualized using Odyssey Infrared Imaging System (LICOR, Bioscience).

Techniques: Western Blot, Expressing, Positive Control, Negative Control, Virus

Journal: Vaccine

Article Title: Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies.

doi: 10.1016/j.vaccine.2019.09.057

Figure Lengend Snippet: Fig. 3. Production of influenza VLP from different strains in 293 T cells in disposable 3L-Bioreactor. The results of one representative experiment for each VLP batch are presented. (A) Time course of cell viability (1) and viable cell growth (2) during VLPs production (B) HAU and NA activities measured in the clarified cell supernatant 4 days after transfection. Data displayed as the mean ± standard deviation (C) TEM on the 293 T cells producing VLPs at D4 after transfection. Ultrathin sections of the cells showed the presence of numerous VLPs (green arrow) budding from the cell membrane (1) H3N2 VLP, (2) H1N1 VLP with granules visible around the aggregated VLPs (red arrow), (3) B/Phuket VLP, (4) B/Brisbane VLP. TEM analyses performed by IBiSA, University of Tours.

Article Snippet: After geltransfer and saturation, the blots were incubated with a rabbit polyclonal antibody against H1N1 influenza A virus NA (RD System), a polyclonal sheep antibody against H1N1 M1 (ThermoFisher), or a polyclonal rabbit antibody against the HA from A/California/07/2009 (H1N1) (produced in-house) for 1 h. Blots were then incubated with a secondary sheep anti-rabbit or antisheep antibody conjugated to IRDye 800 (Rockland) for 1 h. The membranes were visualized using Odyssey Infrared Imaging System (LICOR, Bioscience).

Techniques: Transfection, Standard Deviation, Membrane