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: PLOS ONE

Article Title: Evaluation of the Kaira COVID-19/Flu/RSV Detection Kit for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus: A comparative study with the PowerChek SARS-CoV-2, influenza A&B, RSV Multiplex Real-time PCR Kit

doi: 10.1371/journal.pone.0278530

Figure Lengend Snippet: Analytical specificity evaluation results of the Kaira assay.

Article Snippet: Human coronavirus 229E , ATCC (VR-740D) , Negative.

Techniques: Virus

Journal: Scientific Reports

Article Title: Rapid and accurate detection of SARS-CoV-2 using the RHAM technology

doi: 10.1038/s41598-023-49733-7

Figure Lengend Snippet: Evaluation of RHAM specificity. ( A ) The specificity of RHAM targeting Orf1ab was evaluated by testing the nucleic acids of other respiratory pathogens, ( B ) The specificity of RHAM targeting N gene was evaluated by testing the nucleic acids of other respiratory pathogens. All pathogens including coronavirus (HKU1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1, H5N1, H7N9, and H9N2, influenza B virus, respiratory syncytial virus types A and B, human parainfluenza virus type II, adenovirus types 3 and 7, enterovirus EV71, Mycoplasma pneumoniae , Epstein-Barr virus, human cytomegalovirus, and Mycobacterium tuberculosis . 10 μL of nucleic acid was added to the RHAM reaction mixture (25 μL reaction volume).

Article Snippet: MERS coronavirus (ATCC, VR-3248SD), coronavirus OC43 (ATCC, VR-1558D), HKU1 (ATCC, VR-3262SD), 229E (ATCC, VR-740DQ), NL63 (ATCC, VR-3263SD), SARS coronavirus (ATCC, VR-3280SD), influenza A H1N1 (ATCC, VR-1469), H5N1 (ATCC, VR-1647), influenza B Virus (ATCC, VR-1735D), respiratory syncytial virus type A (ATCC, VR-1804DQ) and B (ATCC, 1850DQ), human parainfluenza virus type II (ATCC, VR-92DQ), adenovirus type 3 (ATCC, VR-3DQ) and 7 (ATCC, VR-7DQ), enterovirus EV71 (ATCC, VR-1775DQ), Mycoplasma pneumoniae (ATCC, 29342), human cytomegalovirus (ATCC, VR-1590), and Mycobacterium tuberculosis (ATCC 25177) were purchased from the ATCC SARS-CoV-2 RNA (GBW(E)091098) was purchased from the National Institute of Metrology (China, CNRN).

Techniques: Virus

Journal: NPJ Genomic Medicine

Article Title: Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

doi: 10.1038/s41525-017-0033-4

Figure Lengend Snippet: Detection of a 548-nucleotide deletion in a HCoV 229E strain associated with myeloablation. a Depiction of the case history surrounding myeloablation and coronavirus infection. A woman in her 40s with AML was found to be infected with a HCoV 229E just before her HCT. Due to the patient’s advanced disease, the decision was made to continue with the transplant. Four successive HCoV-positive nasal swab specimens were available from the patient; two from before treatment with myeloablative cyclophosphamide and total body irradiation and two from after. All days are reported relative to HCT. b Coverage plots of mNGS reads mapped to the reference genome for HCoV 229E (NC_002645) of three samples from the patient revealed the generation of a 548-nucleotide deletion in the ORF4a/4b gene. In addition to deleting 83% of the coding region from ORF4a/4b, the deletion resulted in a frame shift that resulted in a premature stop codon 10 amino acids before the reference genome stop codon. Primer binding sites are denoted in blue for confirmatory junction RT-PCR. c Confirmatory junction RT-PCR of the four available HCoV-positive nasal swabs revealed the absence of the deletion before myeloablation and the presence of the deletion in specimens taken after myeloablation. NTC , no template control

Article Snippet: No CPE was visualized for any of the patient’s four clinical samples and HCoV 229E RNA levels in the culture supernatant were undetectable by qRT-PCR after inoculation, while viral growth was apparent in the positive control HCoV 229E ATCC type strain (Figure ).

Techniques: Infection, Irradiation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: NPJ Genomic Medicine

Article Title: Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

doi: 10.1038/s41525-017-0033-4

Figure Lengend Snippet: Specimens sequenced in this study

Article Snippet: No CPE was visualized for any of the patient’s four clinical samples and HCoV 229E RNA levels in the culture supernatant were undetectable by qRT-PCR after inoculation, while viral growth was apparent in the positive control HCoV 229E ATCC type strain (Figure ).

Techniques:

Journal: Scientific Reports

Article Title: Commercially available SARS-CoV-2 RT-qPCR diagnostic tests need obligatory internal validation

doi: 10.1038/s41598-023-34220-w

Figure Lengend Snippet: List of commercially available molecular standards (nucleic acid solutions) used in analysis of the specificity of the three studied RT-qPCR assays.

Article Snippet: Quantitative Genomic RNA from Human coronavirus 229E , ATCC-VR-740DQ.

Techniques: Virus

Journal: Scientific Reports

Article Title: Commercially available SARS-CoV-2 RT-qPCR diagnostic tests need obligatory internal validation

doi: 10.1038/s41598-023-34220-w

Figure Lengend Snippet: RT-qPCR amplification parameters for Novel Coronavirus (2019-nCoV) Real Time Multiplex RT-PCR Kit (Liferiver), Vitassay qPCR SARS-CoV-2 (Vitassay) and TaqPath COVID‑19 CE-IVD RT-PCR Kit (Thermo Fisher Scientific).

Article Snippet: Quantitative Genomic RNA from Human coronavirus 229E , ATCC-VR-740DQ.

Techniques: Amplification, Multiplex Assay

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