Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) Representative flow cytometry gating strategy of differentiated primary human bronchial epithelial cells (BEpC; donor 2). The cells were stained for the following cell surface markers: microtubules for ciliated cells, CD271 for basal cells, and CD66c for secretory cells. Cellular doublets were excluded by size, and dead cells with a cell viability marker. B) The relative cell type compositions of the bronchial cells from donors 1-3 were determined using the flow cytometry panel and gating strategy shown in A). Unstained or double-positive cells were categorized as ‘undefined’. C) BEpC from donors 1 or 2 were infected with H1N1 A/Duck/Alberta/35/1976 or A/Hawaii/70/2019, respectively, with a multiplicity of infection (MOI) of 1, 5, or 10. At 8 hours post-infection (hpi), the cells were fixed and stained for viral nucleoprotein (anti-NP antibody), tight junctions (anti-ZO-1 antibody), and nuclei (DAPI). The infections were quantified by microscopy as the percentage of NP-positive cells relative to the total number of nuclei imaged. D) BEpC from donors 1-3 were infected for 8 hours with H1N1 A/Duck/Alberta/35/1976 or A/Hawaii/70/2019 (MOI of 5). The cells were prepared for flow cytometry with the panel and gating strategy shown in A). The infected cells were defined as surface HA-positive cells. The relative cell type compositions are shown as the percentage of each cell type in the total populations and within the infected populations. For each donor, the values were normalized to the average across the experimental repeats. Data from donors 1-3 were pooled. One repeat of each infection was previously published in . E) The normalized infection rates of the samples described in D), as well as of BEpC from donor 1 infected under identical conditions with H1N1 Netherlands/602/2009 or Pennsylvania/02/2021. The infection rates represent the percentage of infected ciliated or secretory cells normalized to the total infected cell population. Data from donors 1-3 were pooled and distinguished by symbol shape: donor 1 (square), donor 2 (triangle), donor 3 (circle). F) BEpC from donor 2 were infected at an MOI of 3 with the following influenza A viruses: H1N1 Hawaii/70/2019, H3N2 Tasmania/503/2020, H3N2 Darwin/6/2021, H11N6 Duck/England/1/1956, or H3N8 Duck/Ukraine/1/1963. At 7hpi, the cells were fixed and stained for viral nucleoproteins (anti-NP antibody), ciliated cells (microtubule dye), tight junctions (anti-ZO-1 antibody), and nuclei (DAPI). The infections were quantified as the percentage of NP-positive cells relative to all apical cells and normalized to the total infected cell population. G) BEpC from donor 2 were infected with Hawaii/70/2019 or Duck/Alberta/35/1976 (MOI 5). At 7hpi, cells were prepared for flow cytometry and stained for infected cells (NP-positive cells). The median fluorescence intensity (MFI) of the viral NP staining in infected cells was quantified. In B) - C) and F) - G), data represent means ± s.d. from n≥3 independent experiments, and in D) - E), data represent means ± s.d. from n = 3 independent experiments per donor. In E) and F), statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test. In G), statistical significance was determined using an unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Flow Cytometry, Staining, Marker, Infection, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) Uniform Manifold Approximation and Projection (UMAP) visualization of single-cell RNA sequencing (scRNAseq) data from pooled mock-infected BEpC from donors 1-3. Cell subtype allocation was based on the expression of canonical subtype-specific markers, with a minimum of three markers (two for suprabasal cells) required for cluster allocation. Basal cells (light blue): TP63, KRT5, DAPL1, KRT15, ITGA6, KRT17; Secretory cells (yellow): SCGB3A1, SCGB1A1, MUC5B, MUC5AC, SPDEF, TCN1, BPIFB1, SPRR3, AGR2; Ciliated cells (dark blue): FOXJ1, CAPS, TP73, CCDC78; Ionocytes (orange): CFTR, ASCL3, FOXI1, ATP6V1C2. Suprabasal cells (violet): KRT6A and KRT15. Intermediate cells (brown): clusters expressing markers of basal and secretory cells. Undefined cells (grey): clusters not assignable to known epithelial subtype. B) Relative cell-type composition of BEpC from donors 1-3 of the samples described in A). C) UMAP visualization of the pooled scRNAseq data of BEpC from donors 1-3 infected with H1N1 Hawaii/70/2019 or Duck/Alberta/35/1976 for 6 hours with an MOI of 1. Highlighted are the allocated cell subtypes as described in A) and the infected cell populations (black). Infected cells were defined by the presence of transcripts from at least four of the eight viral genome segments. D) Total percentage of infected cells of the samples described in C). E) The relative cell type compositions of the total and the infected cell populations from the samples described in C). F) The normalized infection rates of the samples mentioned in C). The rates represent the percentage of infected ciliated or secretory cells normalized to the total infected cell population. G) Total viral transcripts detected in the samples described in C). Unique molecular identifiers (UMI). In F) and G), data from donors 1-3 were pooled and distinguished by symbol shape: donor 1 (square), donor 2 (triangle), donor 3 (circle). In A) - G), data represent n=1 experiments per donor (donor 1-3). In E) - G), data represent means ± s.d.. In E) and F), statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test. In G), statistical significance was determined using an unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Single Cell, RNA Sequencing, Infection, Expressing

Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) BEpC from donors 1-3 were infected with Hawaii/70/2019 or Duck/Alberta/35/1976 (MOI 1). At 1- and 6-hours post-infection (hpi), the RNA was extracted and analyzed by RT-qPCR. The ΔCt was calculated using 18s-RNA for normalization. The enrichment of the viral M segment transcript level (ΔΔCt) is shown as a fold change relative to the 1hpi time point for each donor. B) and C) BEpC from donors 1-3 were infected with H1N1 Hawaii/70/2019 or Duck/Alberta/35/1976 for 6 hours (MOI 1) and analyzed by single-cell RNA sequencing (scRNAseq) as described in C). B) The scRNAseq data were pooled to generate pseudobulk expression profiles, and a differential expression analysis was performed of Hawaii/70/2019 infected cells over Duck/Alberta/35/1976 infected cells. Genes belonging to the gene ontology GO:0045087 (innate immune response) are highlighted (dark red). C) A hypergeometric over-representation analysis (ORA) of the differentially expressed genes (DEG) identified in B). The plot shows the top 10 enriched biological processes, ranked with the number of genes associated with each process overrepresented in the Duck/Alberta/35/1976 infected cell population. The threshold for DEG was set at *P ≤ 0.01. D) - G) BEpC from donor 2 were pretreated with DMSO (control) or ruxolitinib for 86 hours prior to infection. The cells were infected with mock, Hawaii/70/2019, or Duck/Alberta/35/1976 (MOI 1). At 6 hpi, the RNA was extracted and analyzed by RT-qPCR. The ΔCt was calculated using 18s-RNA for normalization. The enrichment (ΔΔCt) of the transcript levels of IFIT2, ISG15, MX1, and viral M segment are presented as a fold change relative to D) – F) mock DMSO control or G) Hawaii/70/2019 DMSO control. H) – I) BEpC from donor 2 were pretreated and infected as described for D). At the indicated time points post-infection, the apical viral release was harvested and plaqued in MDCK cells. J) – L) Mock-infected BEpC from donors 1-3 were prepared for scRNAseq, as described in A). Plots depicting single cell transform (SCTransform) -normalized expression of J) BTN3A3, K) ANP32A, and L) ANP32B within all cells from the bronchial mock-infected cells (donor 1-3 pooled). M) UMAP visualization of the BTN3A3 SCTransform-normalized expression within the bronchial mock-infected cells (donor 1-3 pooled). N) – P) Differential expression analysis was performed of Duck/Alberta/36/1976 N) infected cells over non-infected cells, O) infected secretory cells over non-infected secretory cells, and P) infected ciliated cells over non-infected ciliated cells. BTN3A3 is highlighted (dark red). Q) BEpC from donor 2 were infected with Netherlands/602/2009 PB2 S590-R591 or with Netherlands/602/2009 PB2 S590G-R591Q (MOI of 0.6). At 1 and 6 hpi, the RNA was extracted and analyzed by RT-qPCR. The ΔCt was calculated using 18s-RNA for normalization. The enrichment of the viral M segment transcript level (ΔΔCt) is shown as a fold change relative to the 1hpi time point for each donor. In A) - C) and J) – P) data represent n=1 experiments per donor (donor 1-3; means ± s.d.). In D) – G) and Q) data represent means ± s.d. from n=3 independent experiments. In H) – I), data represent medians from n=3 independent experiments. In A), G) – I), and Q), statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Infection, Quantitative RT-PCR, Single Cell, RNA Sequencing, Expressing, Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) BEpC from donor 2 were infected with control H1N1 WSN/1933 virus or recombinant 7+1 viruses containing 7 genes from the WSN/1933 strain and either the HA- or NA-encoding segment of Hawaii/70/2019, or of Duck/Alberta/35/1976. An MOI as calculated by titres determined on MDCK cells of 0.5-1 was used for the recombinant strains, and an MOI of 5 was used for the control WSN/1933 strain to achieve comparable infection rates. At 7 hours post-infection (hpi), the cells were fixed and stained for viral nucleoproteins (anti-NP antibody), ciliated cells (microtubule dye), tight junctions (anti-ZO-1 antibody), and nuclei (DAPI). The infections were quantified as the percentage of NP-positive cells relative to all apical cells and normalized to the total infected cell populations. B) – G) BEpC from donor 1-3 were trypsinized and stained for cell surface markers identifying ciliated cells (microtubules), basal cells (CD271), secretory cells (CD66c), α2,3-linked sialic acids (Maackia amurensis II lectins; MALII), and α2,6-linked sialic acids (Sambucus nigra lectins; SNA). The cells were analyzed by flow cytometry, cellular doublets were excluded by size, and dead cells with a cell viability marker. One repeat was previously published in . B) and E) show the percentage of α2,3-linked and α2,6-linked sialic acid-positive cells, respectively. C) and F) show the median fluorescent intensity (MFI) of the BV421 staining of α2,3-linked and α2,6-linked sialic acids, respectively. D) and G) show the MFI of the BV421 staining of α2,3-linked and α2,6-linked sialic acids, respectively, of pre-gated sialic acid-positive cells. The data of donors 1-3 were pooled and distinguished by symbol shape: donor 1 (square), donor 2 (triangle), donor 3 (circle). H) – I) BEpC from donor 2 were stained prior to trypsinization for apical H) α2,3-linked sialic acids (MALII) and I) α2,6-linked sialic acid (SNA), as well as ciliated cells (microtubule dye). The cells were analyzed by flow cytometry, cellular doublets were excluded by size, and dead cells with a cell viability marker. Shown is the MFI of the apical sialic acid staining on ciliated versus non-ciliated cells, pre-gated for sialic acid-positive cells. J) - L) BEpC from donor 2 were precooled on ice, and 6.4*10^9 viral copies of K) Duck/Alberta/35/1976 or L) Hawaii/70/2019 were added apically on ice. 1 hour post addition, the cells were fixed and stained for apically bound viral particles (anti-HA antibody), ciliated cells (microtubule dye), tight junctions (anti-ZO-1 antibody), and nuclei (DAPI). Quantification of apically bound viral signal. The summed viral signal volume on ciliated and non-ciliated apical cells was normalized to the viral signal present on all apical cells. J) Representative microscopy images of the mock-treated and Duck/Alberta/35/1976-treated sample are shown. The white arrows highlight the viral HA staining on a cell, shown with and without the cilia staining. In A), and H) - L), data represent means ± s.d. from n≥3 independent experiments. In B) – G) data represent means ± s.d. from n = 3 independent experiments per donor (donor 1-3). Statistical significance was determined in A) using two-way ANOVA with Šídák’s multiple comparisons test, in B) – G) using RM one-way ANOVA with Tukey’s multiple comparisons test, and in H) – I) and K) – L) using an unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Infection, Control, Virus, Recombinant, Staining, Flow Cytometry, Marker, Microscopy

Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) Synthetic glycans on the microarray N°1 with linear (A-N), bi-antennary (O-W), tri-antennary structures ( – ), and glycolipids (a-m). B) Microarray N°1 binding of H1N1 A/Duck/Alberta/35/1976. The microarray N°1 tested for glycans with no sialylation (yellow), α2,6-linked NeuAc (pink), α2,3-linked NeuAc (white), Lewis X (blue), sialyl Lewis X (dark blue), and glycolipids (cyan). Striped bars indicate glycans terminating in different epitopes on different arms. C) Representative microscopy images of apical sialyl Lewis X staining on BEpC from donor 2. The cells were stained for ciliated cells (microtubule dye) and surface sialyl Lewis X. D) BEpC from donor 2 were stained prior to trypsinization for ciliated cells (microtubule dye), as well as for apical sialyl Lewis X (sLx). The cells were analyzed by flow cytometry, cellular doublets were excluded by size, and dead cells with a cell viability marker. Shown is the MFI of the apical sialyl Lewis X staining on ciliated versus non-ciliated cells, pre-gated for sLx-positive cells. E) Microarray N°1 binding of A/Hawaii/70/2019. F) Synthetic glycans on the microarray N°2. G) Microarray N°2 binding of H1N1 A/Hawaii/70/2019. The microarray N°2 tested for glycans with no sialylation (yellow), α2,6-linked NeuAc (pink), α2,3-linked NeuAc (white). In B), E), and G), mean relative fluorescence units (RFU) ± s.d. from n = 4 technical repeats are shown. In D), data represent means ± s.d. from n=3 replicates.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Microarray, Binding Assay, Microscopy, Staining, Flow Cytometry, Marker, Fluorescence

Journal: bioRxiv

Article Title: Opposing cell type preferences for binding and replication shape influenza A virus infection in human airways

doi: 10.64898/2026.05.04.722582

Figure Lengend Snippet: A) BEpC from donors 1-3 were infected with H1N1 Hawaii/70/2019 or Duck/Alberta/35/1976 for 6 hours (MOI1) and analyzed by single-cell RNA sequencing (scRNAseq), as described in C). Total unique molecular identifiers (UMI) detected in infected secretory and ciliated cells. Data from donors 1-3 were pooled and distinguished by symbol shape: donor 1 (square), donor 2 (triangle), donor 3 (circle). B) BEpC from donor 2 were infected with Hawaii/70/2019 or Duck/Alberta/35/1976 (MOI 5; 7hpi) and prepared for flow cytometry as described in G). The cells were stained for infected cells (NP-positive cells) and ciliated cells (microtubule staining). The median fluorescence intensity (MFI) of the viral NP staining in infected ciliated and non-ciliated cells was quantified. C) The scRNAseq mock data was pooled to generate pseudobulk expression profiles, and a differential expression analysis of mock secretory cells over mock ciliated cells was performed. D) – E) A gene ontology analysis of the differentially expressed genes (DEG) identified in C). The plot shows the top 10 enriched biological processes, ranked by the adjusted p -value that were overrepresented in D) the ciliated cell and E) the secretory cell population. The threshold for DEG was set at *P ≤0.05, and an absolute log2 fold change of 0.5. F) The scRNAseq data was pooled to generate pseudobulk expression profiles, and a differential expression analysis of Hawaii/70/2019 infected samples over mock samples was performed for ciliated and secretory cell. G) – H) Single cell transform (SCTransform) -normalized expression of G) ANP32A and H) ANP32B within each subtype of the scRNAseq data set from bronchial mock-infected cells. I) BEpC from donor 2 were infected with Netherlands/602/2009 PB2 S590-R591 or with Netherlands/602/2009 PB2 S590G-R591Q (MOI of 0.45). At 8hpi, cells were prepared for flow cytometry and stained for infected cells (NP-positive cells) and ciliated cells (microtubule staining). The median fluorescence intensity (MFI) of the viral NP staining in infected ciliated and non-ciliated cells was quantified. In A) and C) – H), data represent n =1 experiments per donor (donor 1-3). The scRNAseq mock data was pooled to generate pseudobulk expression profiles, and a differential expression analysis of mock secretory cells over mock ciliated cells was performed to calculate the fold change and the statistical significance. In B) and I), data represent n ≥3 independent experiments. In A), B), and I), data represent means ± s.d. and statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Primary human bronchial epithelial cells (BEpC) were purchased from Epithelix (#EP51AB).

Techniques: Infection, Single Cell, RNA Sequencing, Flow Cytometry, Staining, Fluorescence, Expressing, Quantitative Proteomics

Journal: Nature Communications

Article Title: AI-guided multi-omics analysis identifies NPC1-modulated susceptibility to SARS-CoV-2 infection under PM 2.5 exposure

doi: 10.1038/s41467-026-71196-3

Figure Lengend Snippet: Verification of angiotensin-converting enzyme 2 (ACE2) inhibition using a series of DX600 concentrations and the corresponding viral infection efficiency in wild-type A549 (alveolar basal epithelial adenocarcinoma cells) ( a ), rs1788783 knockout (KO) A549 ( b ), primary human bronchial epithelial cells (HBECs) ( c ), and BEAS-2B (human bronchial epithelial cells) ( d ), respectively. Relative luminescence intensities are presented as mean ± standard deviation ( n = 10) from 10 biological replicates. Exact P values are shown in the figure. Reduced NPC1 expression levels using 3 distinct small interfering RNAs (siRNAs) and negative control siRNA (NC siRNA), and the corresponding viral infection efficiency in primary human bronchial epithelial cells (HBECs) ( e ) and BEAS-2B (human bronchial epithelial cells) ( f ), respectively. Viral infection was quantified by the expression levels of open reading frame 1 (ORF1) and nucleocapsid (N) genes. Data are presented as mean ± standard deviation ( n = 3) from 3 biological replicates. Statistical significance was assessed using two-sided t-tests. Source data are available in the file.

Article Snippet: Primary human bronchial epithelial cells (HBECs) were provided by Procell Life Science & Technology Co., Ltd (China, #CP-H009).

Techniques: Inhibition, Infection, Knock-Out, Standard Deviation, Expressing, Negative Control