Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000, 1,500, or 150 copies of IAV (H1N1), and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later. Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, * p < 0.05. ( c ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV (H1N1), and intracellular vRNA levels were measured with RT-qPCR 2 days later. The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. ** p < 0.01. ( d ) PK-15 ( Stat2 k/o)/TMPRSS2 #15, PK-15 ( Stat2 k/o), PK-15/TMPRSS2 #23, or normal PK-15 cells treated with one ng/mL pig IFNβ or left untreated for 24 hr and then infected with 15,000 copies of IAV (H1N1). vRNA levels in the culture supernatant were measured with RT-qPCR 2 days after infection. The relative value was calculated according to the values in normal PK-15 cells without pig IFNβ treatment. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 80,000, 8,000, or 800 copies of SIV (H1N1). and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a Mann–Whitney U -test. ** p < 0.01.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or VeroE6/TMPRSS2 cells were infected with 75,000 copies of IAV(H1N1), and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. The relative value was calculated according to the values in normal PK-15 cells. Results are the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, *** p < 0.001.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV(H1N1) in the presence of different concentrations of nafamostat mesylate, and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of measurements (n - 6) from one assay and are representative of at least three independent experiments. ( b ) The EC 50 of nafamostat mesylate against IAV (H1N1) in PK-15/TMPRSS2 #23 cells was calculated using Prism.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 1. In vitro characterization of MHC class II-targeted heterodimeric DNA vaccines containing NA and/or HA (A) MHC class II-targeted heterodimeric acid/base (A/B) vaccine proteins consist of A and B chains, each having a targeting, dimerization, and antigenic unit. The targeting unit is either an scFv that binds MHC class II on APCs (scFvaMHCII, green) or a non-targeted control that binds the hapten NIP (scFvaNIP, white). The targeting units are genetically linked to a shortened hinge from human IgG3 (exon h1), allowing covalent disulfide bond formation between the A and B chains (black lines). The hinge is followed by a duplicated A (red) or B (blue) heterodimerization unit based on a modified Jun/Fos leucine zipper motif enriched for acidic or basic amino acids, respectively. The A and B chains are linked to antigenic units composed of NA (dark blue) or HA (orange) derived from influenza H1N1 PR8 virus. The heterodimeric vaccine proteins are named as

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: In Vitro, Vaccines, Control, Derivative Assay, Virus

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 2. MHC class II-targeted NA-bivalent heterodimers efficiently induce antibody responses after a single DNA vaccination (A) BALB/c mice were vaccinated once i.m. with 5 mg of each A and B plasmids (10 mg DNA in total) encoding the indicated heterodimeric vaccine proteins (box), followed immediately by EP. Blood was sampled at the indicated time points. (B) NA-specific (top) and HA-specific (bottom) IgG (left), IgG1 (center), and IgG2a (right) titers were measured by ELISA at the indicated time points after immunization. Statistical analysis compared targeted vs. non-targeted and antigen-bivalent vs. monovalent groups. (C) BALB/c mice were immunized once i.m./EP with different amounts of the indicated DNA vaccines. Serum was analyzed for NA-specific IgG responses 4 weeks post vaccination. (D) IgG responses specific for inactivated H1N1 PR8 virus (left), rec. NA protein (center), or rec. HA protein (right) in sera harvested 6 weeks post vaccination from

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Enzyme-linked Immunosorbent Assay, Vaccines, Virus

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 4. A single DNA immunization with MHC class II-targeted NA/NA vaccines protects against homologous influenza virus challenge (A) BALB/c mice were immunized once i.m./EP and challenged with homologous influenza A H1N1 PR8 virus at week 2 (short term) or between weeks 6 and 10 (long term) after vaccination. (B–F) 50 mg of each A and B plasmids (total 100 mg, B), 5 mg/ plasmid (10 mg total, C–E), or 1 mg/plasmid (2 mg total, F) of the indicated DNA vaccines (box) were used. Mice were challenged with a lethal dose of 5 LD50 (B–D and F) or 100 LD50 (E) at week 9 (B), week 10 (C), week 2 (D and E), or week 6 (F). Weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge. n = 8 mice/group (B, C, E, and F) or n = 6 mice/group (D). Dead mice were assigned a weight of 75%. *p % 0.05, ***p % 0.001; two-way ANOVA (weight curve) or Mantel-Cox test (survival). Statistical analysis compared MHC class II- targeted NA/NA versus HA/HA as well as targeted versus non-targeted and anti- gen-bivalent versus -monovalent NA.

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Vaccines, Virus, Plasmid Preparation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 5. MHC class II-targeted NA DNA vaccine induces superior NA-specific antibody responses compared with conventional inactivated virus after one vaccination (A, B) BALB/c mice were immunized once i.m. with 0.2, 1, or 5 mg of formalin-inactivated H1N1 PR8 virus or with 5 mg/ plasmid (10 mg total) of the indicated DNA vaccines, fol- lowed by EP (box). (A) NA-specific (left) and HA-specific (right) titers were measured by ELISA at the indicated time points after immunization. Mean ± SEM is shown. (B) Mice were challenged with 5 LD50 H1N1 PR8 virus 11 week post vaccination. Weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge. Dead mice were assigned a weight of 75%; n = 8 mice/group. Statistical analysis compared the different doses of inactivated virus with DNA vaccination with MHC class II-targeted NA and/or HA. **p % 0.01, ***p % 0.001; two-way ANOVA (IgG titers, weight curve) or Mantel-Cox test (survival).

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Virus, Plasmid Preparation, Vaccines, Enzyme-linked Immunosorbent Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 6. Vaccine-induced NA-specific antibodies mediate protection against homologous influenza infection, inhibit NA enzymatic activity, and block viral replication (A–D) BALB/c mice were immunized once i.m./EP with 50 mg each of A and B plasmids (100 mg total) as indicated. (A) Pooled sera (350 mL) from mice 5 weeks after vaccination were transferred i.p. to naive BALB/c mice. One day after transfer, mice were challenged with 2.5 LD50 H1N1 PR8 virus. (B) 2 weeks after vaccination with the indicated DNA vaccines, mice were challenged with 5 LD50 H1N1 PR8 virus. Starting on day 12 after vaccination, mice were treated with anti-CD4 and anti- CD8 mAbs or isotype-matched control mAbs every second day. n = 8 mice/group; weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge, and dead mice were assigned a weight of 75% (A and B). (C) Neutrali- zation of influenza A H1N1 PR8 virus by serum from vaccinated mice (box) obtained 4 weeks after immunization was measured by microneutralization assay and is shown as IC50. (D) The same sera were tested for ability to inhibit NA enzyme activity by ELLA, using a reassorted influenza A H6N1 virus containing NA from H1N1 PR8. IC50 is indicated. (E and F) BALB/c mice were immunized once i.m./EP with 50 mg of each A and B plasmids (100 mg total, E) or 5 mg of each A and B plasmids (10 mg total, F) as indicated. Mice were challenged with homologous H1N1 PR8 virus 6 weeks after vaccination, and lungs were harvested on day 5 post challenge. Viral loads were determined by qRT-PCR (left), showing gene copy number per micro- gram total tissue RNA, and by measuring in vitro cell culture infectivity (right), pre- sented as 50% tissue culture infectious dose (TCID50) per milliliter of lung homog- enates. Individual mice and mean ± SEM are shown; n = 4–6 mice/group (C–F). *p % 0.05, **p % 0.01, ***p % 0.001; two-way ANOVA (weight curve), Mantel-Cox test (survival), Kruskal-Wallis multiple-comparisons test with Dunn’s correction (C and D, TCID50), or multiple-comparisons one-way ANOVA with Tukey’s correction (copy number).

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Infection, Activity Assay, Blocking Assay, Virus, Vaccines, Control, Microneutralization Assay, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: Respiratory Research

Article Title: Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

doi: 10.1186/s12931-023-02477-4

Figure Lengend Snippet: Anti-infection and anti-inflammation effects of VD in H1N1 infection. A Representative Alcian Blue-Periodic Acid Schiff (AB-PAS) and IF staining images for H1N1 infection and the corresponding control in both ND group and VD group. No obvious cell damage or mucus hypersecretion was observed in any group. Scale bar: 20 μm. B , C The infected organoids (cell lysate) and their supernatants were harvested to detect viral loads by quantifying the copy number of viral nucleoprotein (NP) gene. D – G mRNA levels of LL37, IFN-β, IL6, and IL10 were shown as relative fold-changes against the control samples. VD treatment significantly upregulated LL37 while downregulated the IL6, IL10 and IFN-β elevation triggered by H1N1 infection. H Analysis of cellular junctions of airway organoids via FITC-Dextran paracellular flux quantification. H1N1 infection led to the augmentation of FITC-Dextran paracellular flux, which was apt to ameliorate under the treatment of VD. I . Analysis of epithelial integrity and cellular permeability of airway organoids via TEER. VD treatment increased TEER of airway organoids and counteracted the impairment of TEER to airway organoids by H1N1 infection. J . Analysis of pathogen clearance of airway organoids via CBF. H1N1 infection disturbed the CBF of airway organoids to interfere the pathogen clearance, while VD was inclined to enhance CBF in opposite effect. Color in green represents ND control and red for 100 nM VD treatment. *: p < 0.05, **: p < 0.01, ***: p < 0.001, NA non-available

Article Snippet: Influenza H1N1 virus A/PR/8/34 strain was also purchased from ATCC.

Techniques: Infection, Staining, Control, Permeability

Journal: Respiratory Research

Article Title: Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

doi: 10.1186/s12931-023-02477-4

Figure Lengend Snippet: Transcriptomic profiling of VD treated airway organoids.① for transcriptomic profiles of VD-treated non-infected airway organoids normalized to ND control and② for H1N1-infected airway organoids normalized to ND/infection group. A Gene overlap analysis (GOA). Each arc on the outside represents the identity of each gene list. Arc on the inside in dark orange color represents the genes that appear in multiple lists and light orange color represents genes that are unique to that gene list. Purple lines link the same gene that are shared by multiple gene lists, showing how gene lists overlap. B Heatmap of top 10 upregulated and downregulated DEGs, colored by log transformation of fold change. C Heatmap for enrichment analysis based on GO/KEGG database, Reactome pathway database and Molecular signatures database, etc. D , E A subset of representative terms from the full clusters were selected to convert into a network layout. Terms from the same cluster were colored the same and shared a common term description as label. The same network was also colored according to their p values

Article Snippet: Influenza H1N1 virus A/PR/8/34 strain was also purchased from ATCC.

Techniques: Infection, Control, Transformation Assay

Journal: Respiratory Research

Article Title: Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

doi: 10.1186/s12931-023-02477-4

Figure Lengend Snippet: Pleiotropic effects of VD in transcriptome. A , B Gene set enrichment analysis (GSEA) of transcriptional modulation of H1N1 to airway organoids (NDH_NDC) and of VD treatment in counteracting H1N1 infection (VDH_NDH). C Ingenuity Canonical Pathway Analysis (ICPA) in IPA. Both GSEA And ICPA indicated the importance of TGF-β signaling and EMT. D Examination of mRNA levels of typical EMT-related genes Vimentin, Fibronectin, α-smooth muscle actin (αSMA), MMP2 and TGF-β. E . Upstream regulator analysis in IPA predicted the decreasing TGF-β under VD treatment led to the regulation of other relative genes. Network analysis of VD-modulated DEGs under H1N1 infection clustered in the development, morphogenesis, and organization of epithelial cells. Color in green represents ND control and red for 100 nM VD treatment. F Heatmap of top DEGs colored according to their expression values

Article Snippet: Influenza H1N1 virus A/PR/8/34 strain was also purchased from ATCC.

Techniques: Infection, Control, Expressing

Journal: Respiratory Research

Article Title: Vitamin D promotes epithelial tissue repair and host defense responses against influenza H1N1 virus and Staphylococcus aureus infections

doi: 10.1186/s12931-023-02477-4

Figure Lengend Snippet: Proteomic profiles of VD.① for proteomic profiles of VD-treated non-infected airway organoids normalized to ND control and ② for H1N1-infected airway organoids normalized to ND/infection group. A . Enriched Ontology Clusters in GO database. B . Ingenuity Canonical Pathway Analysis (ICPA) in IPA C . Network analysis of biological functions of DEPs in IPA

Article Snippet: Influenza H1N1 virus A/PR/8/34 strain was also purchased from ATCC.

Techniques: Infection, Control

Journal: iScience

Article Title: Down syndrome is associated with altered frequency and functioning of tracheal multiciliated cells, and response to influenza virus infection

doi: 10.1016/j.isci.2023.107361

Figure Lengend Snippet: Characterization of Influenza Viral Infection of Airway Epithelia in Down Syndrome In Vitro (A) Fast Gene Set Enrichment Analysis (fGSEA) on differentially expressed genes (DEGs) in ‘Dp16 vs. WT’ ( Table S1 ), by running against ‘Gene Ontology Biological Process’ pathways, identified enriched gene sets ( Table S2 ) for involvement in immune pathways, inflammation, and defense response to other organisms. Representative gene sets with highest Normalized Enrichment Score (NES) are shown here ( Table S3 ). (B) Influenza A virus (IAV) challenge of in vitro -differentiated primary mTECs. Representative immunostaining images of ciliated mTECs from Dp16 Mice 48 h following challenge with IAV strain A/Puerto Rico/8/1934 H1N1 at MOI of 0.1. Individual markers for cilia (green; β-tubulin IV), nuclei (magenta; TO-PRO 3), cell-cell boundaries (blue; F-actin) and viral particles (red, hemagglutinin), as well as an orthogonal section as well as a top view of the infected cells are illustrated. Scale bar, 20 μm (applies to all images). (C) Real-time qPCR revealed comparable (statistically non-significant) influenza virus genome copies shed from infected WT and Dp16 mTECs; fold change in Dp16 vs. WT (as reference) is illustrated. Data represent mean and s.e.m. compared to WT from four independent studies (indicated by different coloring) with 3–6 biological replicates – i.e., transwell inserts, per condition (WT and Dp16) per study (n = 18–20 × transwell inserts in total). Analysis was conducted by non-parametric Mann-Whitney test. n.s., not significant. See also Figures S4–S6 and Tables S1 , , , , and .

Article Snippet: All viral challenges were performed using influenza strain A/Puerto Rico/8/1934 H1N1 (IAV PR8) (ATCC).

Techniques: Infection, In Vitro, Virus, Immunostaining, MANN-WHITNEY

Journal: iScience

Article Title: Down syndrome is associated with altered frequency and functioning of tracheal multiciliated cells, and response to influenza virus infection

doi: 10.1016/j.isci.2023.107361

Figure Lengend Snippet:

Article Snippet: All viral challenges were performed using influenza strain A/Puerto Rico/8/1934 H1N1 (IAV PR8) (ATCC).

Techniques: Virus, Recombinant, Lysis, Reverse Transcription, SYBR Green Assay, RNA Sequencing, Software