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: NPJ Vaccines

Article Title: Long-term efficacy of an inactivated H5N1 whole-particle influenza vaccine in nonhuman primates

doi: 10.1038/s41541-025-01221-x

Figure Lengend Snippet: a Cynomolgus macaques were subcutaneously vaccinated twice with inactivated whole particles of a low pathogenic avian influenza virus A/duck/Hokkaido/Vac-3/2007 (H5N1) (Vac-3) (H5 clade classical/outlier) with a two-week interval. Plasma was collected on week 0 (before vaccination), 2, 4, 8, and 32 weeks, and 1, 2, 3, 4, and 5 years after the prime-boost vaccination to measure Vac-3-specific IgG and neutralization titers. Five years later, macaques were challenged with an H5N1 highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) (H5 clade 1). Autopsy was performed 7 days after challenge. b – d Virus titers after challenge infection. Nasal swab ( b ), tracheal swab ( c ), and bronchial brush ( d ) samples were collected on the indicated days (Supplementary Table ). Left: each symbol shows the average titer calculated from titers in vaccinated macaques (red: n = 6 for those challenged 5 years after the prime-boost vaccination and blue: n = 3 for those challenged 5 weeks after the first and 3 weeks after the second vaccinations) and unvaccinated macaques (black: n = 3). Right: area under the virus titer and time curve (AUC). e Vac-3 specific IgG levels before and after challenge indicated as 50% max dilution. f Neutralization titers against Vac-3 before and after challenge infection. Each symbol indicates each macaque, with identification numbers in the graph. Neutralization titers higher than 9 and lower than 2 were calculated as 9 and 1, respectively. Statistical analysis was performed with the Mann-Whitney U test in b – d and the Wilcoxon test in e and f: * p < 0.05, ** p < 0.01.

Article Snippet: Ninety-six-well plates were coated with 50 μL of purified Vac-3 (20 mg/mL) disrupted with 0.05 M Tris-HCl (pH 7.8) containing 0.5% Triton X-100 and 0.6 M KCl and purified NA proteins (A/Thailand/1(KAN-1)/2004 (H5N1) (Cat# 40064-V07H) and A/Texas/37/2024 (H5N1) (Cat# 41016-V08B)) (1 μg/50 μL) (Sino Biological Inc., Beijing, China).

Techniques: Virus, Clinical Proteomics, Neutralization, Infection, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: Long-term efficacy of an inactivated H5N1 whole-particle influenza vaccine in nonhuman primates

doi: 10.1038/s41541-025-01221-x

Figure Lengend Snippet: Cynomolgus macaques were subcutaneously vaccinated and infected as described in Fig. . Three unvaccinated cynomolgus macaques infected with a same H5N1 strain were used for comparison. Seven days after challenge, lung tissues were collected from inflamed lesions of each lobe determined by gross pathology (redness, exudate, and hardness of the lung). RNA was extracted from each lobe and pooled for each macaque for RNA-seq. Ingenuity Pathway Analysis was performed with differentially expressed genes (q value < 0.05). A Principal component analysis. Black dots: unvaccinated macaques; Red dots: macaques challenged 5 years after vaccination. B Volcano plot of the differentially expressed genes. Red dots represent major interferon-regulated genes. C Altered canonical signaling pathways in vaccinated macaques with z score < -2. PKR: protein kinase receptor, TREM1: triggering receptor on myeloid cells-1. D Graphical summary of differential pathways in the lung of macaques challenged 5 years after vaccination vs without vaccination. Blue: predicted low activation of canonical pathways, cytokines, transcription regulators, and kinases.

Article Snippet: Ninety-six-well plates were coated with 50 μL of purified Vac-3 (20 mg/mL) disrupted with 0.05 M Tris-HCl (pH 7.8) containing 0.5% Triton X-100 and 0.6 M KCl and purified NA proteins (A/Thailand/1(KAN-1)/2004 (H5N1) (Cat# 40064-V07H) and A/Texas/37/2024 (H5N1) (Cat# 41016-V08B)) (1 μg/50 μL) (Sino Biological Inc., Beijing, China).

Techniques: Infection, Comparison, RNA Sequencing, Protein-Protein interactions, Activation Assay