African green monkey kidney epithelial (Vero E6) cells and human hepatocellular carcinoma cell lines (Huh-7) were purchased from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. The SARS-CoV-2 (Genebank accession no. MT123290.1) was clinically isolated from the First Affiliated Hospital of Guangzhou Medical University, and HCoV-229E (ATCCⓇVR740™) was purchased from ATCC. The viruses were propagated and adapted as previously described (Zhu et al., 2020 (link)). The 50% tissue culture infective dose (TCID50) of SARS-CoV-2 and HCoV-229E were determined using the Reed–Muench method (TCID50 = 10−6/100 μl and TCID50 = 10−5.5/μl, respectively). Virus stocks were collected and stored at −80 °C. The infection experiments of SARS-CoV-2 were performed in a biosafety level-3 laboratory and those of HCoV-229E were performed in a biosafety level-2 laboratory.
Hcov 229e
Manufactured by American Type Culture Collection
Sourced in United States, Italy
HCoV-229E is a laboratory cell culture that can be used for the study of human coronavirus 229E. It is a well-characterized cell line derived from human lung cells. This product is intended for research use only and its core function is to provide a standardized model system for the cultivation and study of the human coronavirus 229E.
Automatically generated - may contain errors
Lab products found in correlation
Hcov oc43, by American Type Culture Collection (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Vero e6, by American Type Culture Collection (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Mrc 5, by American Type Culture Collection (2 mentions)
Mrc 5 human lung fibroblast host cells, by Korean Cell Line Bank (2 mentions)
Hct 8, by American Type Culture Collection (1 mentions)
Htb 55, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Tpck trypsin, by Merck Group (1 mentions)
Vero ccl81, by American Type Culture Collection (1 mentions)
Mrc 5, by Korean Cell Line Bank (1 mentions)
Fra 1, by Santa Cruz Biotechnology (1 mentions)
Calu 3, by Korean Cell Line Bank (1 mentions)
Glut3, by Abcam (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
Glut1, by Abcam (1 mentions)
Ez atp assay kit, by DoGenBio (1 mentions)
Vero cell, by American Type Culture Collection (1 mentions)
28 protocols using hcov 229e
1
SARS-CoV-2 and HCoV-229E Infection Protocols
2
Establishment of Cell Lines and Virus Stocks
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Human hepatocellular carcinoma cell lines Huh7 and Huh7.5 and human lung cancer cell line H460 were kindly provided by Dr. Zonggen Peng and Dr. Zhen Wang, respectively, at Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College. Human hepatoblastoma cell line C3A was purchased from ATCC (Manassas, VA, USA). 293T-derived cell line expressing human recombinant angiotensin I converting enzyme 2 (293T-hACE2) was purchased from Delivectory Biosciences Inc. (Beijing, China). All cells cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) or Minimum Essential Media (MEM, Invitrogen) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a 5% CO2 incubator.
HCoV-229E (strain VR740) was purchased from ATCC. HCoV-OC43 (strain VR1558) was a kind gift from Dr. Xuesen Zhao at Beijing Ditan Hospital, Capital Medical University (Beijing, China). SARS-CoV-2 (GenBank: MT123290) for immunofluorescence (IF) assay was isolated from a throat swab of a COVID-19 patient and stored in biosafety level-3 laboratory (Guangzhou Customs Technology Center, Guangzhou, China). The vesicular stomatitis virus (VSV) and SARS-CoV-2 pseudotyped viral particles were obtained from Delivectory Biosciences Inc. (Beijing, China).
HCoV-229E (strain VR740) was purchased from ATCC. HCoV-OC43 (strain VR1558) was a kind gift from Dr. Xuesen Zhao at Beijing Ditan Hospital, Capital Medical University (Beijing, China). SARS-CoV-2 (GenBank: MT123290) for immunofluorescence (IF) assay was isolated from a throat swab of a COVID-19 patient and stored in biosafety level-3 laboratory (Guangzhou Customs Technology Center, Guangzhou, China). The vesicular stomatitis virus (VSV) and SARS-CoV-2 pseudotyped viral particles were obtained from Delivectory Biosciences Inc. (Beijing, China).
3
Cultivation and Titration of SARS-CoV-2 and Common Coronaviruses
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Human lung fibroblasts MRC5 (ATCC CCL-171), the human colorectal carcinoma HCT-8 (ATCC CCL-244), the human lung adenocarcinoma Calu-3 (ATCC HTB-55), and the African green monkey kidney Vero E6 (ATCC CRL-1586) cell lines were purchased from the American Type Culture Collection (ATCC), and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Euroclone) supplemented with 10% fetal bovine serum (FBS, Euroclone), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate (P/S, both from Euroclone).
hCoV-229E (ATCC VR-740) and hCoV-OC43 (ATCC VR-1558) were purchased from ATCC, and propagated and titrated in MRC5 and HCT-8 cells, respectively. SARS-CoV-2 (2019-nCoV/Italy INMI1) was obtained from EVAg, and propagated and titrated in Vero E6 cells. SARS-CoV-2/01/human/2020/SWE was isolated on Vero E6 cells from a nasopharyngeal sample, cultivated and titrated as previously described [18 (link)]. All work with SARS-CoV-2 was performed in Biosafety laboratory level 3 (BSL3) facilities either at the University of Padua, Italy, or at the Public Health Agency of Sweden, Sweden.
hCoV-229E (ATCC VR-740) and hCoV-OC43 (ATCC VR-1558) were purchased from ATCC, and propagated and titrated in MRC5 and HCT-8 cells, respectively. SARS-CoV-2 (2019-nCoV/Italy INMI1) was obtained from EVAg, and propagated and titrated in Vero E6 cells. SARS-CoV-2/01/human/2020/SWE was isolated on Vero E6 cells from a nasopharyngeal sample, cultivated and titrated as previously described [18 (link)]. All work with SARS-CoV-2 was performed in Biosafety laboratory level 3 (BSL3) facilities either at the University of Padua, Italy, or at the Public Health Agency of Sweden, Sweden.
4
Propagation of Common Respiratory Viruses
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HCoV-OC43 (strain, ATCC VR-759) and HCoV-229E (strain, ATCC VR-740) were obtained from Zeptometrix Corporation, and HCoV-NL63 (strain, Amsterdam I) was obtained from the Biodefense and Emerging Infections Research Resources Repository. Viral stocks were generated by propagation on Huh7.5 cells. The IAV strains A/WSN/33 (H1N1), A/Netherlands/602/2009 (H1N1)pdm09 (H1N12009 Netherlands), and A/California/04/2009 (H1N1)pdm09 (H1N12009 California) were propagated in MDCK cells in the presence of 3μg/mL TPCK-trypsin (Sigma). RSV strain A2-line19F expressing the red fluorescent protein monomeric Katushka 2 (mKate2) (51 (link)) was propagated in Vero cells. Adenovirus 5 was purchased from ATCC (VR-1516) and propagated in A549 cells. VSVINDeGFP was propagated on 293T cells (52 (link)). The replication-competent chimeric recombinant vesicular stomatitis virus encoding SARS-CoV-2 S and enhanced green fluorescent protein (eGFP), rVSV/SARS-2/GFP2E1, has been described previously (53 (link)) and was propagated on 293T-ACE2 cells. rVSV/EBOV-GP was propagated on Vero cells as previously described (42 (link)).
5
VERO Cell Culture and Virus Propagation
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The cell line derived from the renal epithelium of the African green monkey (Cercopithecus aethiops) (VERO CCL-81) was purchased from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) with 4.5 g/L of glucose, 2 mM of L-glutamine, 100 IU/mL of penicillin-streptomycin solution, and supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere with 5% CO2 at 37 °C.
The viruses used were HSV-1 SC16 containing a lacZ gene driven by the cytomegalovirus IE-1 promoter to express beta-galactosidase, HSV-1 containing the Green Fluorescent Protein reporter inserted into the gene encoding the integument protein VP22 (HSV-1-GFP), HCoV-229E (ATCC VR-740), and PV-1 (Table 1 ). All viruses were propagated in the VERO CCL-81 monolayer.
The viruses used were HSV-1 SC16 containing a lacZ gene driven by the cytomegalovirus IE-1 promoter to express beta-galactosidase, HSV-1 containing the Green Fluorescent Protein reporter inserted into the gene encoding the integument protein VP22 (HSV-1-GFP), HCoV-229E (ATCC VR-740), and PV-1 (
6
Inhibition of SARS-CoV-2 Infection by 2-DG
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Epithelial lung fibroblast cells (MRC-5) and airway epithelial cells (Calu-3) [20 ] [21 (link)] were purchased from Korean Cell Line Bank (Seoul, Korea), and HCoV-229E was obtained from ATCC, Manassas, Virginia, USA. The EZ-ATP assay kit (DoGenBio, Korea) was used to measure the total ATP levels, and the alamarBlue reagent (Invitrogen, USA) was utilized to detect cellular viability. Experiments were accomplished as per the manufacturer's guidelines. Hydroxy chloroquine diphosphate (HCQ; positive control) was obtained from Merck (Yongin, Korea). The main stock of HCQ was reconstituted in DMSO and kept frozen (-20 °C) for further treatments. 2-DG was obtained from Merck (Yongin, Korea). 2-DG was first prepared in phosphate-buffered saline prior to each application and then incubated in the virus culture solution according to the experimental conditions. HCoV-S protein-specific antibodies were purchased from Sino-Biologicals (USA). GLUT1, GLUT3, and GLUT4-specific antibodies were purchased from Abcam, USA. Antibodies specific to ABCC9 (Aviva System Biology, USA), IRF1 (Cell Signaling Technology, USA), and FRA-1 (i.e., FOSL1) Santa Cruz Biotechnology, USA, were purchased as mentioned.
7
Cultivation of Diverse Viral Strains
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Vero cells (ATCC CCL-81, Manassas, VA, USA), Vero/hSLAM cells (ECACC 04091501, Porton Down, UK), Madin Darby canine kidney (MDCK, ATCC CCL-34), and HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose (Microtech, Naples, Italy). Media were complemented with 100 IU/mL penicillin and 100 μg/mL streptomycin (Himedia, Naples, Italy) and 10% fetal bovine serum (FBS) (Microtech). MeV (ATCC VR-24) was propagated on VERO/hSLAM monolayers; RSV (ATCC VR-1540), HPIV-3 (ATCC VR-93), HCoV-229E (ATCC VR-740), SARS-CoV-2 (strain VR PV10734, kindly donated by the Lazzaro Spallanzani Hospital of Rome, Italy), and coxsackie type B3 (CVB3) strain Nancy (ATCC VR-30) were grown on Vero cells, whereas influenza virus (strain H1N1, VR-1894) was grown on the MDKC cell line, as reported elsewhere [31 (link)].
8
Propagation of HCoV-229E in MRC-5 Cells
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Human coronavirus 229E (HcoV-229E) (ATCC, Cat. No. VR-740) was added at a multiplicity of infection (MOI) of 1.0 to an approximately 85% confluent T75 flask of MRC-5 cells (ATCC, Cat. No. CCL-171), 48 h after plating. The flask was maintained with DMEM (Fisher Scientific, Cat. No. 11–965-118) supplemented with 5% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500) and 1% L-Glutamine (Gemini Bioproducts, Cat. No. 400–106), incubated at 37 °C with 5% CO2 [15 (link)]. The cell culture supernatant was harvested at 72 h post infection when 80% cytopathic effect (CPE) was observed.
9
Culturing Cells and Propagating Coronaviruses
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The African green monkey kidney epithelial (Vero E6) cells, human lung fibroblasts (MRC-5) cells and human bronchial epithelial (16HBE) cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Hyclone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Sciencell, USA) and 1% penicillin/streptomycin at 37 ℃ with 5% CO2. HCoV-229E (Lot No. ATCC-VR-740) was presented by Hunan Center for Disease Control and Prevention. SARS-CoV-2 was provided by the biosafety level 3 (BSL-3) Laboratory of Guangzhou Customs Technical Center (State Key Lab of Respiratory Disease, Guangzhou, China). HCoV-229E was propagated in MRC-5 cells and SARS-CoV-2 was propagated in the Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID50) according to the cytopathic effect (CPE) by Reed–Muench method.
10
Propagation of Human Coronaviruses
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HCoV-229E (strain VR740) was purchased from ATCC. HCoV-OC43 (strain VR1558) was a kind gift from Dr. Xuesen Zhao at Beijing Ditan Hospital, Capital Medical University (Beijing, China). HCoV-229E and HCoV-OC43 propagated on Huh7.5 and HCT-8 cells respectively before they were used. SARS-CoV-2 strain BetaCoV/Beijing/IME-BJ01/2020 was originally isolated from a COVID-19 patient, and the full genome sequence is deposited in the Genome Warehouse in national Genomics Data Center, Beijing Institute of Genomic, CAS, with the accession Nos. GWHACAX01000000. SARS-CoV-2 propagated once on Vero cells before it was used for this study. Studies involving the SARS-CoV-2 were performed at the biosafety level-3laboratory.
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