Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 1. Generation of iPSC-derived alveolar epithelial type II cells (iAECIIs) as an HCoV-229E infection experimental model. (A) Outline of experimental design. (B) Bright-field images showing the morphology of iPSCs 3 and 5 days after seeding. (C) Immunofluorescent staining showing the expression of NANOG marker in iPSCs. Nuclei are stained with DAPI. (D) Schematic diagram showing a stepwise protocol of differentiation of iPSCs into iAECIIs. (E) Bright-field images showing cell morphology at the indicated time points of differentiation of iPSCs into iAECIIs.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Derivative Assay, Infection, Staining, Expressing, Marker

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 3. iAECIIs as a model of infection with HCoV-229E. (A) Immunofluorescent staining of iAECII-containing spheroids showing the co-expression of EpCAM iAECII marker and CD13 HCoV-229E receptor. (B) Bright field images of iAECIIs cultures at 24 and 48 h post-infection (hpi) with HCoV-229E. (C) Immunofluorescent staining of viral dsRNA in iAECII culture after 48 h of infection with HCoV-229E. White arrows show the dsRNA signal. In (A) and (C) – nuclei are stained with DAPI. (D) qRT-PCR analysis of the expression of HCoV-229E S protein-encoding mRNA in iAECIIs infected with HCoV-229E for the indicated time periods. Data are expressed relative to 0 h time point. Means are shown, n = 3, SD error bars, p < 0.005 (Student’s test) (E) Western blot showing the expression of HCoV-229E S and N proteins in iAECIIs infected with HCoV-229E for the indicated time periods. GAPDH – loading control.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Staining, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 4. Infected iAEClls enhance HL-60 migration and adhesion behavior. (A) Bright-field images of HL-60 neutrophils co-cultured with the medium conditioned by uninfected iAECIIs (Ctrl) and conditioned by iAECIIs infected with HCoV-229E for 48 h (48 hpi). White arrows point at granules. (B) Schematic outline of transwell migration assay. (C) Transwell migration assay showing the migration capacity of HL-60 neutrophils in response to a medium conditioned by iAECIIs infected with HCoV-229E for 24 or 48 h (24 hpi or 48 hpi). Ctrl – uninfected control. Data shown as means with SD error bars, n = 3, *p < 0.005 (ANOVA). (D) Schematic outline of adhesion assay. (E) Representative fluorescence microscopy images of adherent HL-60 neutrophils after incubation with infected iAECIIs (24 hpi and 48 hpi) for the indicated time periods. (F) Cell count of adherent HL-60 neutrophils. Mean values are shown with SD error bars, n = 3, *p < 0.005 (ANOVA). In (E) and (F), Ctrl – virus-free control.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Migration, Cell Culture, Transwell Migration Assay, Control, Cell Adhesion Assay, Fluorescence, Microscopy, Incubation, Cell Counting, Virus

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 5. Identification of cytokines secreted by infected iAECIIs and their effect on HL-60 cells. (A) Cytokine array analysis showing the levels of the indicated cy tokines in the medium conditioned by iAECIIs infected with HCoV-229E for 24 or 48 h (24 hpi and 48 hpi). Ctrl – uninfected control. Right panel: cytokine label legend. (B) Western blot analysis of the expression of IL-8 and ICAM-1 in HCoV-229E-infected iAECIIs. (C) Transwell migration assay showing the migration capacity of HL-60 neutrophils in response to a medium conditioned by iAECIIs infected with HCoV-229E for 48 h (48 hpi) in the absence or presence of an IL-8-neutralizing antibody (IL-8 nAb). Ctrl – uninfected control conditioned medium. Data shown as means with SD error bars, n = 3, *p < 0.005, (ANOVA). (D) Representative fluorescence microscopy images of adherent HL-60 neutrophils after incubation with infected iAECIIs in the absence or presence of an ICAM-1-neutralizing antibody (ICAM-1 nAb). Ctrl – uninfected control. (E) Cell count of adherent HL-60 neutrophils. Mean values are shown with SD error bars, n = 3, *p < 0.005.

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: Infection, Control, Western Blot, Expressing, Transwell Migration Assay, Migration, Fluorescence, Microscopy, Incubation, Cell Counting

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 6. RNA-seq analysis identifies upstream cytokine pathways in the immune responses by neutrophils. (A and B) Cytoscape ClueGO networks of upregulated genes by infection of iAECIIs with HCoV-229E (A) or triggered by infected HCoV-229 conditioned medium in HL-60 cells (B). (C and D) Bubble plots showing the most enriched GO-BP terms among the genes upregulated by the infection of iAECIIs with HCoV-229E (C) or the genes upregulated in HL-60 cells by cultivation in the infected HCoV-229-conditioned medium (D). (E and F) Hierarchical clustering heatmaps showing the signatures of genes differentially regulated in infected iAECIIs (E) and conditioned medium-stimulated HL-60 cells (F). (G and H) X2K network analysis showing the kinases and transcription factors predicted to regulate differentially expressed genes in infected iAECIIs (G) and conditioned medium-stimulated HL-60 cells (H).

Article Snippet: The following primary antibodies were used: SFTPB, HCoV-229E spike protein (PAB21477–100/ PAB21477–500; The Native Antigen Company, Kidlington, UK), HCoV229E nucleocapsid protein (#40,640-T62; Sino Biological, Beijing, China), GAPDH (10,494–1-AP; Rosemont, IL, USA).

Techniques: RNA Sequencing, Infection

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: The antiviral effect of Tg against HCoV-229E infection does not require IRE1, ATF6, or PERK expression. (A) Stable UPR knockdown A549 cell lines were generated via lentiviral transduction. Silencing of protein expression was confirmed via western blot. (B–F) A549 shCTRL, shIRE1, shATF6, and shPERK cells were primed with DMSO or Tg (0.05 µM) for 30 minutes, then washed prior to infection with HCoV-229E (MOI 0.05). Cell lysates (B–E) or supernatants (F) were collected at 24 hpi. (B–D) Protein expression of IRE1, ATF6, PERK and HCoV-229E N were assessed by western blot. (E) Changes in gene expression were quantified by RT-qPCR. Data are normalized to actin and set relative to shCTRL DMSO. (F) Supernatants were used to determine viral titers by plaque assay on Huh7 cells. Due to variability between replicates, titration data is expressed as a percentage relative to the shCTRL DMSO in each experiment. Graphs show means ± SD from 3 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Infection, Expressing, Knockdown, Generated, Transduction, Western Blot, Gene Expression, Quantitative RT-PCR, Plaque Assay, Titration

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological inhibition of IRE1 or ATF6 does not affect the antiviral activity of Tg against HCoV-229E infection. (A and B) A549 cells were primed with DMSO or Tg (0.05 µM) in the absence or presence of KIRA6 (10 µM) or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hours post-treatment. Xbp1s (A) or HERPUD1 (B) protein expression was assessed by western blot. (C) A549 cells were treated with DMSO, KIRA6 (10 µM), or Ceapin-A7 (6 µM) for 24 hours. Alamar blue assay was used to assess cell viability, which is expressed as a percentage relative to DMSO. (D and F) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or KIRA6 (10 µM). Cell lysates were collected at 24 hpi to assess XBP1s and N gene expression by RT-qPCR (D and F), or Xbp1s and HCoV-229E N protein expression by western blot (E). (G and I) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of Ceapin-A7 (6 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hpi to assess HERPUD1 and N expression by RT-qPCR (G and I), or HERPUD1 and HCoV-229E N protein expression by western blot (H). RT-qPCR data are normalized to actin and set relative to DMSO. (J) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM) or Ceapin-A7 (6 µM), alone or in combination, prior to infection with HCoV-229E (MOI 0.05). Inoculum was removed 2 hours later and replaced with media containing the respective inhibitor(s). Viral supernatants were collected 24 hpi and titrated by plaque assay on Huh7 cells. Graphs show mean ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate (* p < 0.05, *** p < 0.001; **** p < 0.0001). Representative western blots are shown.

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Inhibition, Activity Assay, Infection, Expressing, Western Blot, Alamar Blue Assay, Gene Expression, Quantitative RT-PCR, Plaque Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological activation of PERK inhibits HCoV-229E replication. (A–C) A549 cells were treated with DMSO or one of the selective UPR activators for 24 hours. IXA4 (10 µM) activates IRE1, AA147 (10 µM) activates ATF6, and CCT020312 (5 µM) activates PERK. RNA lysates were collected 24 hours post-treatment and the expression of genes downstream of each UPR pathway was assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (D) A549 cells were primed with Tg (0.5 µM) for 30 minutes, then washed and infected with HCoV-229E (MOI 0.5). Alternatively, cells were treated with AA147 (10 µM) or CCT020312 (5 µM) for 24 hpi. Cell lysates were collected to assess the expression of HERPUD1, PERK and 229E N protein by western blot. (F and G) A549 cells were primed with DMSO or Tg (0.05 µM) for 30 minutes prior to infection with HCoV-229E (MOI 0.05). Alternatively, cells were infected and then treated with media containing DMSO or the selective UPR activators alone or in combination. Cell lysates and supernatants were collected at 24 hpi. (F) Changes in HCoV-229E N gene expression were assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (F) Viral titer was assessed by plaque assay on Huh7 cells. (H–J) A549 cells were infected with HCoV-229E (MOI 0.05), then incubated with increasing concentrations of CCT020312 for 24 h. Cell lysates were collected to assess changes in HCoV-229E N gene (H) and CHOP (I) expression by RT-qPCR. (J) Cell viability was assessed by Alamar blue assay. Graphs show means ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way ANOVA or t -test (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Infection, Western Blot, Gene Expression, Plaque Assay, Incubation, Alamar Blue Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: UPR activation by tunicamycin does not recapitulate the antiviral effect of Tg. (A–C) A549 cells were primed with Tg (0.5 µM) for 30 minutes or with Tm (1 µg mL −1 ) for 4 hours. Cells were then washed and either mock-infected or infected with HCoV-229E (MOI 0.05). At 24 hpi, cell lysates were collected to evaluate expression of UPR target genes ( CHOP , HERPUD1 and Xbp1s ) or HCoV-229E N gene by qPCR. Graphs show means ± SD from 2 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way or two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Infection, Expressing

Journal: European Journal of Microbiology & Immunology

Article Title: Phytochemicals and micronutrients in suppressing infectivity caused by SARS-CoV-2 virions and seasonal coronavirus HCoV-229E in vivo

doi: 10.1556/1886.2023.00010

Figure Lengend Snippet: Effect of test composition on weight change, viral load and spike protein in lung tissue infected and re-infected with human coronavirus 229E (HCoV-229E) . A. Effect of four-days of oral administration of MixV in K18-hACE2 mice with no significant weight change after 3 dpi and 5 dpi and oral administration of MixV with mid-term re-infection. B. The viral load in lung tissue after 3 dpi with oral administration of MixV and 5 dpi with oral administration and mid-term re-infection, determined by RT-qPCR as described in Materials and Methods section. C. Spike protein detection by WB in representative lung tissue samples. D. Quantification of spike protein performed as described in Materials and Methods section. Data are presented as mean ± SD; infected untreated animal n = 8, infected treated animal n = 8, re-infected untreated animal n = 8, re-infected treated animal n = 8; # P ≤ 0.05, ∆ P ≤ 0.01, dpi - days post-infection

Article Snippet: HCoV-229E anti-spike antibody was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Coronavirus antigens on microarray.

Article Snippet: Coronavirus , 229E , 229E , S1+S2 , A0A1L7B942 , Insect Cells , Sino Biological , N-(AA16-1115)-His-C , 40605-V08B.

Techniques: Microarray, Expressing, Construct

Journal: Cell Reports Medicine

Article Title: SARS-CoV-2 antibodies from children exhibit broad neutralization and belong to adult public clonotypes

doi: 10.1016/j.xcrm.2023.101267

Figure Lengend Snippet:

Article Snippet: The SARS-CoV-2 S1, SARS-CoV-2 S2, SARS-CoV-2 RBD, and SARS-CoV-2 NTD subdomains as well as recombinant SARS-CoV-2 Omicron BA.4/5 S HP, and HCoV-229E S were purchased from Sino Biological.

Techniques: Virus, Recombinant, Staining, Plasmid Preparation, Conjugation Assay, Transfection, Chromatography, Next-Generation Sequencing, Software