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Li C, Yu Y, Wan Z, Chiu MC, Huang J, Zhang S, Zhu X, Lan Q, Deng Y, Zhou Y, Xue W, Yue M, Cai JP, Yip CCY, Wong KKY, Liu X, Yu Y, Huang L, Chu H, Chan JFW, Clevers H, Yuen KY, Zhou J. Human respiratory organoids sustained reproducible propagation of human rhinovirus C and elucidation of virus-host interaction. Nat Commun 2024; 15:10772. [PMID: 39738014 DOI: 10.1038/s41467-024-55076-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2024] [Accepted: 11/26/2024] [Indexed: 01/01/2025] Open
Abstract
The lack of a robust system to reproducibly propagate HRV-C, a family of viruses refractory to cultivation in standard cell lines, has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C, and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression, whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly, upon HRV-C infection, we observe an innate immune response that is stronger in airway organoids than in nasal organoids, which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally, an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively, we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C, followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has opened an avenue for propagating and studying other uncultivable human and animal viruses.
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Affiliation(s)
- Cun Li
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Yifei Yu
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Zhixin Wan
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Man Chun Chiu
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Jingjing Huang
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Shuxin Zhang
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Xiaoxin Zhu
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Qiaoshuai Lan
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Yanlin Deng
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Ying Zhou
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Wei Xue
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Ming Yue
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Jian-Piao Cai
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Cyril Chik-Yan Yip
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Kenneth Kak-Yuen Wong
- Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, and Queen Mary Hospital, Hong Kong, China
| | - Xiaojuan Liu
- Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China
| | - Yang Yu
- Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China
| | | | - Hin Chu
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
- State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
| | - Jasper Fuk-Woo Chan
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
- State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
- Carol Yu Centre for Infection, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Hans Clevers
- Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), and University Medical Center (UMC) Utrecht, Utrecht, the Netherlands
- Roche Pharmaceutical Research and Early Development, Basel, Switzerland
| | - Kwok Yung Yuen
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
- State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China
- Carol Yu Centre for Infection, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Jie Zhou
- Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China.
- BiomOrgan Ltd, Hong Kong, China.
- State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong, China.
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Chen Y, Ou X, Li P, Zan F, Tan L, Qian Z. Identification of the critical residues of TMPRSS2 for entry and host range of human coronavirus HKU1. J Virol 2024; 98:e0158724. [PMID: 39526774 DOI: 10.1128/jvi.01587-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Accepted: 10/10/2024] [Indexed: 11/16/2024] Open
Abstract
Human coronavirus (CoV) HKU1 infection typically causes common cold but can lead to pneumonia in children, older people, and immunosuppressed individuals. Recently, human transmembrane serine protease 2 (hTMPRSS2) was identified as the functional receptor for HKU1, but its region and residues critical for HKU1 S binding remain elusive. In this study, we find that HKU1 could utilize human and hamster, but not rat, mouse, or bat TMPRSS2 for virus entry, displaying a narrow host range. Using human-bat TMPRSS2 chimeras, we show that the serine peptidase (SP) domain of TMPRSS2 is essential for entry of HKU1. Further extensive mutagenesis analyses of the C-terminal regions of SP domains of human and bat TMPRSS2s identify residues 417 and 469 critical for entry of HKU1. Replacement of either D417 or Y469 with asparagine in hTMPRSS2 abolishes its abilities to mediate entry of HKU1 S pseudovirions and cell-cell fusion, whereas substitution of N417 with D or N469 with Y in bat TMPRSS2 (bTMPRSS2) renders it supporting HKU1 entry. Our findings contribute to a deeper understanding of coronavirus-receptor interactions and cross-species transmission.IMPORTANCEThe interactions of coronavirus (CoV) S proteins with their cognate receptors determine the host range and cross-species transmission potential. Recently, human transmembrane serine protease 2 (hTMPRSS2) was found to be the receptor for HKU1. Here, we show that the TMPRSS2 of hamster, but not rat, mouse, or bat, can serve as a functional entry receptor for HKU1. Moreover, swapping the residues at the positions of 417 and 469 of bTMPRSS2 with the corresponding residues of hTMPRSS2 confers it supporting entry of HKU1 S pseudovirions, indicating the critical role of these residues in HKU1 entry. Our study identified the critical residues in hTMPRSS2 responsible for receptor interaction and host range of HKU1.
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Affiliation(s)
- Yahan Chen
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
| | - Xiuyuan Ou
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
| | - Pei Li
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
| | - Fuwen Zan
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
| | - Lin Tan
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
| | - Zhaohui Qian
- NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
- MOE Key Laboratory of Pathogen Infection Prevention and Control, Peking Union Medical College, Beijing, China
- State Key Laboratory of Respiratory Health and Multimorbidity, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
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3
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Tomris I, Kimpel ALM, Liang R, van der Woude R, Boons GJPH, Li Z, de Vries RP. The HCoV-HKU1 N-Terminal Domain Binds a Wide Range of 9- O-Acetylated Sialic Acids Presented on Different Glycan Cores. ACS Infect Dis 2024; 10:3880-3890. [PMID: 39394950 PMCID: PMC11555679 DOI: 10.1021/acsinfecdis.4c00488] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 09/24/2024] [Accepted: 09/26/2024] [Indexed: 10/14/2024]
Abstract
Coronaviruses (CoVs) recognize a wide array of protein and glycan receptors by using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possesses an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that the HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GnTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high mannose-containing NTD proteins revealed a much broader receptor binding profile compared to that of its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated α2-3-linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding toward 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding.
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Affiliation(s)
- Ilhan Tomris
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
| | - Anne L. M. Kimpel
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
| | - Ruonan Liang
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
| | - Roosmarijn van der Woude
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
| | - Geert-Jan P. H. Boons
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
- Complex
Carbohydrate Research Center, University
of Georgia, 315 Riverbend
Road, Athens, Georgia 30602, United States
| | - Zeshi Li
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
| | - Robert P. de Vries
- Department
of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht 3584 CG, The Netherlands
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Togami K, Wolf W, Olson LC, Card M, Shen L, Schaefer A, Okuda K, Zeitlin L, Pauly M, Whaley K, Pickles RJ, Lai SK. Impact of mAb-FcRn affinity on IgG transcytosis across human well-differentiated airway epithelium. Front Immunol 2024; 15:1371156. [PMID: 39351230 PMCID: PMC11439726 DOI: 10.3389/fimmu.2024.1371156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 08/05/2024] [Indexed: 10/04/2024] Open
Abstract
Effective treatment and immunoprophylaxis of viral respiratory infections with neutralizing monoclonal antibodies (mAbs) require maintaining inhibitory concentrations of mAbs at the airway surface. While engineered mAbs with increased affinity to the neonatal Fc receptor (FcRn) are increasingly employed, little is known how increased affinity of Fc to FcRn influences basal-to-apical transepithelial transport (transcytosis) of mAbs across the airway epithelium. To investigate this, we utilized a model of well-differentiated human airway epithelium (WD-HAE) that exhibited robust FcRn expression, and measured the transepithelial transport of a mAb against SARS-CoV-2 Spike protein (CR3022) with either wildtype IgG1-Fc or Fc modified with YTE or LS mutations known to increase affinity for FcRn. Despite the marked differences in the affinity of these CR3022 variants for FcRn, we did not find substantial differences in basal-to-apical transport reflective of systemic dosing, or apical-to-basal transport reflective of inhaled dosing, compared to the transport of wildtype IgG1-Fc. These results suggest increasing FcRn affinity may only have limited influence over transcytosis rates of systemically dosed mAbs across the human airway epithelium over short time scales. Over longer time scales, the elevated circulating levels of mAbs with greater FcRn affinity, due to more effective FcRn-mediated recycling, may better resupply mAb into the respiratory tract, leading to more effective extended immunoprophylaxis.
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Affiliation(s)
- Kohei Togami
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Whitney Wolf
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Lucas C Olson
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Madison Card
- Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Limei Shen
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Alison Schaefer
- Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Kenichi Okuda
- Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | | | | | | | - Raymond J Pickles
- Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
- Department of Microbiology & Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
| | - Samuel K Lai
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
- Department of Microbiology & Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
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Tiruthani K, Cruz‐Teran C, Chan JFW, Ma A, McSweeney M, Wolf W, Yuan S, Poon VKM, Chan CCS, Botta L, Farrer B, Stewart I, Schaefer A, Edelstein J, Kumar P, Arora H, Hutchins JT, Hickey AJ, Yuen K, Lai SK. Engineering a "muco-trapping" ACE2-immunoglobulin hybrid with picomolar affinity as an inhaled, pan-variant immunotherapy for COVID-19. Bioeng Transl Med 2024; 9:e10650. [PMID: 39036085 PMCID: PMC11256170 DOI: 10.1002/btm2.10650] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Revised: 12/29/2023] [Accepted: 01/12/2024] [Indexed: 07/23/2024] Open
Abstract
Soluble angiotensin-converting enzyme 2 (ACE2) can act as a decoy molecule that neutralizes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by blocking spike (S) proteins on virions from binding ACE2 on host cells. Based on structural insights of ACE2 and S proteins, we designed a "muco-trapping" ACE2-Fc conjugate, termed ACE2-(G4S)6-Fc, comprised of the extracellular segment of ACE2 (lacking the C-terminal collectrin domain) that is linked to mucin-binding IgG1-Fc via an extended glycine-serine flexible linker. ACE2-(G4S)6-Fc exhibits substantially greater binding affinity and neutralization potency than conventional full length ACE2-Fc decoys or similar truncated ACE2-Fc decoys without flexible linkers, possessing picomolar binding affinity and strong neutralization potency against pseudovirus and live virus. ACE2-(G4S)6-Fc effectively trapped fluorescent SARS-CoV-2 virus like particles in fresh human airway mucus and was stably nebulized using a commercial vibrating mesh nebulizer. Intranasal dosing of ACE2-(G4S)6-Fc in hamsters as late as 2 days postinfection provided a 10-fold reduction in viral load in the nasal turbinate tissues by Day 4. These results strongly support further development of ACE2-(G4S)6-Fc as an inhaled immunotherapy for COVID-19, as well as other emerging viruses that bind ACE2 for cellular entry.
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Affiliation(s)
- Karthik Tiruthani
- Division of Pharmacoengineering and Molecular PharmaceuticsUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | - Carlos Cruz‐Teran
- Division of Pharmacoengineering and Molecular PharmaceuticsUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | - Jasper F. W. Chan
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulam, Hong Kong Special Administrative RegionChina
- Centre for Virology, Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong Special Administrative RegionChina
| | - Alice Ma
- UNC/NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | | | - Whitney Wolf
- Division of Pharmacoengineering and Molecular PharmaceuticsUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | - Shoufeng Yuan
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulam, Hong Kong Special Administrative RegionChina
- Centre for Virology, Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong Special Administrative RegionChina
| | - Vincent K. M. Poon
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulam, Hong Kong Special Administrative RegionChina
- Centre for Virology, Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong Special Administrative RegionChina
| | - Chris C. S. Chan
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulam, Hong Kong Special Administrative RegionChina
- Centre for Virology, Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong Special Administrative RegionChina
| | | | - Brian Farrer
- Inhalon Biopharma, Inc.MorrisvilleNorth CarolinaUSA
| | - Ian Stewart
- RTI InternationalResearch Triangle ParkNorth CarolinaUSA
| | - Alison Schaefer
- UNC/NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | - Jasmine Edelstein
- UNC/NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
| | - Priya Kumar
- Department of Anesthesiology, School of MedicineUniversity of North CarolinaChapel HillNorth CarolinaUSA
| | - Harendra Arora
- Department of Anesthesiology, School of MedicineUniversity of North CarolinaChapel HillNorth CarolinaUSA
| | | | | | - Kwok‐Yung Yuen
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of MedicineThe University of Hong KongPokfulam, Hong Kong Special Administrative RegionChina
- Centre for Virology, Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong Special Administrative RegionChina
| | - Samuel K. Lai
- Division of Pharmacoengineering and Molecular PharmaceuticsUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
- Inhalon Biopharma, Inc.MorrisvilleNorth CarolinaUSA
- Department of Microbiology and ImmunologyUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
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6
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Hoenigsperger H, Sivarajan R, Sparrer KM. Differences and similarities between innate immune evasion strategies of human coronaviruses. Curr Opin Microbiol 2024; 79:102466. [PMID: 38555743 DOI: 10.1016/j.mib.2024.102466] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Revised: 02/20/2024] [Accepted: 03/12/2024] [Indexed: 04/02/2024]
Abstract
So far, seven coronaviruses have emerged in humans. Four recurring endemic coronaviruses cause mild respiratory symptoms. Infections with epidemic Middle East respiratory syndrome-related coronavirus or severe acute respiratory syndrome coronavirus (SARS-CoV)-1 are associated with high mortality rates. SARS-CoV-2 is the causative agent of the coronavirus disease 2019 pandemic. To establish an infection, coronaviruses evade restriction by human innate immune defenses, such as the interferon system, autophagy and the inflammasome. Here, we review similar and distinct innate immune manipulation strategies employed by the seven human coronaviruses. We further discuss the impact on pathogenesis, zoonotic emergence and adaptation. Understanding the nature of the interplay between endemic/epidemic/pandemic coronaviruses and host defenses may help to better assess the pandemic potential of emerging coronaviruses.
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Affiliation(s)
- Helene Hoenigsperger
- Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany
| | - Rinu Sivarajan
- Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany
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7
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McSweeney MD, Alnajjar S, Schaefer AM, Richardson Z, Wolf W, Stewart I, Sriboonyapirat P, McCallen J, Farmer E, Nzati B, Lord S, Farrer B, Moench TR, Kumar PA, Arora H, Pickles RJ, Hickey AJ, Ackermann M, Lai SK. Inhaled "Muco-Trapping" Monoclonal Antibody Effectively Treats Established Respiratory Syncytial Virus (RSV) Infections. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2306729. [PMID: 38225749 DOI: 10.1002/advs.202306729] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Revised: 12/12/2023] [Indexed: 01/17/2024]
Abstract
Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable "muco-trapping" variant of motavizumab (Mota-MT), a potent neutralizing mAb against RSV F is engineered. Mota-MT traps RSV in AM via polyvalent Fc-mucin bonds, reducing the fraction of fast-moving RSV particles in both fresh pediatric and adult AM by ≈20-30-fold in a Fc-glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota-MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota-MT to RSV-infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000-fold and 100 000-fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota-MT-treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco-trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.
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Affiliation(s)
| | - Sarhad Alnajjar
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7AL, UK
| | - Alison M Schaefer
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
| | | | - Whitney Wolf
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Ian Stewart
- RTI International, Research Triangle Park, NC, 27709, USA
| | | | - Justin McCallen
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Ellen Farmer
- Inhalon Biopharma, Research Triangle Park, NC, 27707, USA
| | | | - Sam Lord
- Inhalon Biopharma, Research Triangle Park, NC, 27707, USA
| | - Brian Farrer
- Inhalon Biopharma, Research Triangle Park, NC, 27707, USA
| | | | - Priya A Kumar
- Department of Anesthesiology, School of Medicine, University of North Carolina, Chapel Hill, NC, 27599, USA
- Outcomes Research Consortium, Cleveland, OH, 44195, USA
| | - Harendra Arora
- Department of Anesthesiology, University of Mississippi Medical Center, Jackson, MS, 39216, USA
| | - Raymond J Pickles
- Department of Microbiology & Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
| | | | - Mark Ackermann
- USDA/ARS-National Animal Disease Center, Ames, IA, 50010, USA
| | - Samuel K Lai
- Inhalon Biopharma, Research Triangle Park, NC, 27707, USA
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
- Department of Microbiology & Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
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8
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Saunders N, Fernandez I, Planchais C, Michel V, Rajah MM, Baquero Salazar E, Postal J, Porrot F, Guivel-Benhassine F, Blanc C, Chauveau-Le Friec G, Martin A, Grzelak L, Oktavia RM, Meola A, Ahouzi O, Hoover-Watson H, Prot M, Delaune D, Cornelissen M, Deijs M, Meriaux V, Mouquet H, Simon-Lorière E, van der Hoek L, Lafaye P, Rey F, Buchrieser J, Schwartz O. TMPRSS2 is a functional receptor for human coronavirus HKU1. Nature 2023; 624:207-214. [PMID: 37879362 PMCID: PMC11331971 DOI: 10.1038/s41586-023-06761-7] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Accepted: 10/18/2023] [Indexed: 10/27/2023]
Abstract
Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.
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Affiliation(s)
- Nell Saunders
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Ignacio Fernandez
- Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Cyril Planchais
- Humoral Immunology Unit, Institut Pasteur, Université de Paris Cité, INSERM U1222, Paris, France
| | - Vincent Michel
- Pathogenesis of Vascular Infections Unit, Institut Pasteur, INSERM, Paris, France
| | - Maaran Michael Rajah
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Eduard Baquero Salazar
- Nanoimaging core, Institut Pasteur, Université de Paris Cité, INSERM U1222, Paris, France
| | - Jeanne Postal
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Francoise Porrot
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | | | - Catherine Blanc
- Pasteur-TheraVectys Joint Lab, Institut Pasteur, Université de Paris Cité, Paris, France
| | - Gaëlle Chauveau-Le Friec
- Antibody Engineering Platform, C2RT, Institut Pasteur, Université de Paris Cité, CNRS UMR 3528, Paris, France
| | - Augustin Martin
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Ludivine Grzelak
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Rischa Maya Oktavia
- Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Annalisa Meola
- Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Olivia Ahouzi
- Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Hunter Hoover-Watson
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Matthieu Prot
- G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris, France
| | - Deborah Delaune
- G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris, France
- Institut de Recherche Biomédicale des Armées, Brétigny-sur-Orge, France
| | - Marion Cornelissen
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, Molecular Diagnostic Unit, University of Amsterdam, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands
| | - Martin Deijs
- Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, Laboratory of Experimental Virology, University of Amsterdam, Amsterdam, The Netherlands
| | - Véronique Meriaux
- Antibody Engineering Platform, C2RT, Institut Pasteur, Université de Paris Cité, CNRS UMR 3528, Paris, France
| | - Hugo Mouquet
- Humoral Immunology Unit, Institut Pasteur, Université de Paris Cité, INSERM U1222, Paris, France
| | - Etienne Simon-Lorière
- G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris, France
- National Reference Center for viruses of respiratory infections, Institut Pasteur, Paris, France
| | - Lia van der Hoek
- Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, Laboratory of Experimental Virology, University of Amsterdam, Amsterdam, The Netherlands
| | - Pierre Lafaye
- Antibody Engineering Platform, C2RT, Institut Pasteur, Université de Paris Cité, CNRS UMR 3528, Paris, France
| | - Felix Rey
- Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France
| | - Julian Buchrieser
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France.
| | - Olivier Schwartz
- Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France.
- Vaccine Research Institute, Creteil, France.
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9
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Lie LK, Synowiec A, Mazur J, Rabalski L, Pyrć K. An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E. Virology 2023; 588:109889. [PMID: 37778059 DOI: 10.1016/j.virol.2023.109889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 09/14/2023] [Accepted: 09/21/2023] [Indexed: 10/03/2023]
Abstract
The lack of suitable in vitro culture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++ cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++ cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of the Coronaviridae family.
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Affiliation(s)
- Laurensius Kevin Lie
- Virogenetics Group, Malopolska Center of Biotechnology, Jagiellonian University, Poland
| | - Aleksandra Synowiec
- Virogenetics Group, Malopolska Center of Biotechnology, Jagiellonian University, Poland; Doctoral School of Exact and Natural Sciences, Jagiellonian University, Poland
| | - Jedrzej Mazur
- Virogenetics Group, Malopolska Center of Biotechnology, Jagiellonian University, Poland
| | - Lukasz Rabalski
- Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, Gdansk, Poland; Biological Threats Identification and Countermeasure Centre, Military Institute of Hygiene and Epidemiology, Pulawy, Poland
| | - Krzysztof Pyrć
- Virogenetics Group, Malopolska Center of Biotechnology, Jagiellonian University, Poland.
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10
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Zhan Y, Ye L, Ouyang Q, Yin J, Cui J, Liu K, Guo C, Zhang H, Zhai J, Zheng C, Guo A, Sun B. The binding profile of SARS-CoV-2 with human leukocyte antigen polymorphisms reveals critical alleles involved in immune evasion. J Med Virol 2023; 95:e29113. [PMID: 37750416 DOI: 10.1002/jmv.29113] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 08/26/2023] [Accepted: 09/11/2023] [Indexed: 09/27/2023]
Abstract
The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), astonished the world and led to millions of deaths. Due to viral new mutations and immune evasion, SARS-CoV-2 ranked first in transmission and influence. The binding affinity of human leukocyte antigen (HLA) polymorphisms to SARS-CoV-2 might be related to immune escape, but the mechanisms remained unclear. In this study, we obtained the binding affinity of SARS-CoV-2 strains with different HLA proteins and identified 31 risk alleles. Subsequent structural predictions identified 10 active binding sites in these HLA proteins that may promote immune evasion. Particularly, we also found that the weak binding ability with HLA class I polymorphisms could contribute to the immune evasion of Omicron. These findings suggest important implications for preventing the immune evasion of SARS-CoV-2 and providing new insights for the vaccine design.
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Affiliation(s)
- Yan Zhan
- Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China
- Hunan Key Laboratory of Pharmacogenetics, Institute of Clinical Pharmacology, Central South University, Changsha, China
- Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Beijing, China
- National Clinical Research Center for Geriatric Disorders, Changsha, China
| | - Ling Ye
- Center of Clinical Pharmacology, The Third Xiangya Hospital, Central South University, Changsha, China
| | - Qianying Ouyang
- Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China
- Hunan Key Laboratory of Pharmacogenetics, Institute of Clinical Pharmacology, Central South University, Changsha, China
- Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Beijing, China
- National Clinical Research Center for Geriatric Disorders, Changsha, China
| | - Jiye Yin
- Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China
- Hunan Key Laboratory of Pharmacogenetics, Institute of Clinical Pharmacology, Central South University, Changsha, China
- Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Beijing, China
- National Clinical Research Center for Geriatric Disorders, Changsha, China
| | - Jiajia Cui
- Department of Geriatric Surgery, Xiangya Hospital, Central South University, Changsha, China
| | - Ke Liu
- Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, China
- Hunan Key Laboratory of Pharmacogenetics, Institute of Clinical Pharmacology, Central South University, Changsha, China
- Engineering Research Center of Applied Technology of Pharmacogenomics, Ministry of Education, Beijing, China
- National Clinical Research Center for Geriatric Disorders, Changsha, China
| | - Chengxian Guo
- Center of Clinical Pharmacology, The Third Xiangya Hospital, Central South University, Changsha, China
| | | | - Jingbo Zhai
- Key Laboratory of Zoonose Prevention and Control at Universities of Inner Mongolia Autonomous Region, Medical College, Inner Mongolia Minzu University, Tongliao, China
| | - Chunfu Zheng
- Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Aoxiang Guo
- Department of Pharmacy, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China
- Shenzhen Key Laboratory of Chinese Medicine Active substance screening and Translational Research, Shenzhen, China
| | - Bao Sun
- Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, China
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11
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Semple SL, Alkie TN, Jenik K, Warner BM, Tailor N, Kobasa D, DeWitte-Orr SJ. More tools for our toolkit: The application of HEL-299 cells and dsRNA-nanoparticles to study human coronaviruses in vitro. Virus Res 2022; 321:198925. [PMID: 36115551 PMCID: PMC9474404 DOI: 10.1016/j.virusres.2022.198925] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Revised: 08/31/2022] [Accepted: 09/08/2022] [Indexed: 12/24/2022]
Abstract
Human coronaviruses (HCoVs) are important human pathogens, as exemplified by the current SARS-CoV-2 pandemic. While the ability of type I interferons (IFNs) to limit coronavirus replication has been established, the ability of double-stranded (ds)RNA, a potent IFN inducer, to inhibit coronavirus replication when conjugated to a nanoparticle is largely unexplored. Additionally, the number of IFN competent cell lines that can be used to study coronaviruses in vitro are limited. In the present study, we show that poly inosinic: poly cytidylic acid (pIC), when conjugated to a phytoglycogen nanoparticle (pIC+NDX) is able to protect IFN-competent human lung fibroblasts (HEL-299 cells) from infection with different HCoV species. HEL-299 was found to be permissive to HCoV-229E, -OC43 and MERS-CoV-GFP but not to HCoV-NL63 or SARS-CoV-2. Further investigation revealed that HEL-299 does not contain the required ACE2 receptor to enable propagation of both HCoV-NL63 and SARS-CoV-2. Following 24h exposure, pIC+NDX was observed to stimulate a significant, prolonged increase in antiviral gene expression (IFNβ, CXCL10 and ISG15) when compared to both NDX alone and pIC alone. This antiviral response translated into complete protection against virus production, for 4 days or 7 days post treatment with HCoV-229E or -OC43 when either pre-treated for 6h or 24h respectively. Moreover, the pIC+NDX combination also provided complete protection for 2d post infection when HEL-299 cells were infected with MERS-CoV-GFP following a 24h pretreatment with pIC+NDX. The significance of this study is two-fold. Firstly, it was revealed that HEL-299 cells can effectively be used as an IFN-competent model system for in vitro analysis of MERS-CoV. Secondly, pIC+NDX acts as a powerful inducer of type I IFNs in HEL-299, to levels that provide complete protection against coronavirus replication. This suggests an exciting and novel area of investigation for antiviral therapies that utilize innate immune stimulants. The results of this study will help to expand the range of available tools scientists have to investigate, and thus further understand, human coronaviruses.
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Affiliation(s)
- Shawna L Semple
- Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada
| | - Tamiru N Alkie
- Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada
| | - Kristof Jenik
- Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada
| | - Bryce M Warner
- Public Health Agency of Canada, Winnipeg, Manitoba, Canada
| | - Nikesh Tailor
- Public Health Agency of Canada, Winnipeg, Manitoba, Canada
| | - Darwyn Kobasa
- Public Health Agency of Canada, Winnipeg, Manitoba, Canada
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12
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Dorendorf A, Bachmann I, Spiegel M, Abd El Wahed A, Dame G, Hufert F. Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification. JOURNAL OF CLINICAL VIROLOGY PLUS 2022; 2:100115. [PMID: 36248766 PMCID: PMC9546502 DOI: 10.1016/j.jcvp.2022.100115] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 10/05/2022] [Accepted: 10/07/2022] [Indexed: 11/06/2022] Open
Abstract
Background Human coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. Objectives For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study design The analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. Results Our HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. Conclusions We were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.
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Affiliation(s)
- Aline Dorendorf
- Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
| | - Iris Bachmann
- Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
| | - Martin Spiegel
- Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
- Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, Göttingen, Germany
| | - Ahmed Abd El Wahed
- Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, Germany
| | - Gregory Dame
- Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
| | - Frank Hufert
- Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany
- Brandenburg University of Technology Cottbus, Senftenberg, Germany
- Faculty of Health Sciences, Joint Faculty of Brandenburg University of Technology Cottbus - Senftenberg, Brandenburg Medical School Theodor Fontane, Neuruppin and University of Potsdam, Potsdam, Germany
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13
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McSweeney MD, Stewart I, Richardson Z, Kang H, Park Y, Kim C, Tiruthani K, Wolf W, Schaefer A, Kumar P, Aurora H, Hutchins J, Cho JM, Hickey AJ, Lee SY, Lai SK. Stable nebulization and muco-trapping properties of regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19. Bioeng Transl Med 2022; 8:e10391. [PMID: 36248234 PMCID: PMC9537933 DOI: 10.1002/btm2.10391] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2022] [Revised: 07/16/2022] [Accepted: 07/27/2022] [Indexed: 01/21/2023] Open
Abstract
The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries, can effectively trap SARS-CoV-2 virus-like particles in fresh human airway mucus. IN-006, a reformulation of regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.
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Affiliation(s)
- Morgan D. McSweeney
- Inhalon Biopharma IncResearch Triangle ParkNorth CarolinaUSA
- Mucommune LLCResearch Triangle ParkNorth CarolinaUSA
| | - Ian Stewart
- RTI InternationalResearch Triangle ParkNorth CarolinaUSA
| | - Zach Richardson
- Inhalon Biopharma IncResearch Triangle ParkNorth CarolinaUSA
- Mucommune LLCResearch Triangle ParkNorth CarolinaUSA
| | - Hyunah Kang
- Biotechnology Research InstituteCelltrion IncIncheonRepublic of Korea
| | - Yoona Park
- Biotechnology Research InstituteCelltrion IncIncheonRepublic of Korea
| | - Cheolmin Kim
- Biotechnology Research InstituteCelltrion IncIncheonRepublic of Korea
| | - Karthik Tiruthani
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of PharmacyUniversity of North Carolina‐Chapel HillChapel HillNorth CarolinaUSA
| | - Whitney Wolf
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of PharmacyUniversity of North Carolina‐Chapel HillChapel HillNorth CarolinaUSA
| | - Alison Schaefer
- UNC/NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina‐Chapel HillChapel HillNorth CarolinaUSA
| | - Priya Kumar
- Department of Anesthesiology, School of MedicineUniversity of North CarolinaChapel HillNorth CarolinaUSA
| | - Harendra Aurora
- Department of Anesthesiology, School of MedicineUniversity of North CarolinaChapel HillNorth CarolinaUSA
| | - Jeff Hutchins
- Inhalon Biopharma IncResearch Triangle ParkNorth CarolinaUSA
| | - Jong Moon Cho
- Biotechnology Research InstituteCelltrion IncIncheonRepublic of Korea
| | | | - Soo Young Lee
- Biotechnology Research InstituteCelltrion IncIncheonRepublic of Korea
| | - Samuel K. Lai
- Inhalon Biopharma IncResearch Triangle ParkNorth CarolinaUSA
- Mucommune LLCResearch Triangle ParkNorth CarolinaUSA
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of PharmacyUniversity of North Carolina‐Chapel HillChapel HillNorth CarolinaUSA
- UNC/NCSU Joint Department of Biomedical EngineeringUniversity of North Carolina‐Chapel HillChapel HillNorth CarolinaUSA
- Department of Microbiology and Immunology, School of MedicineUniversity of North Carolina at Chapel HillChapel HillNorth CarolinaUSA
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14
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Zarkoob H, Allué-Guardia A, Chen YC, Garcia-Vilanova A, Jung O, Coon S, Song MJ, Park JG, Oladunni F, Miller J, Tung YT, Kosik I, Schultz D, Iben J, Li T, Fu J, Porter FD, Yewdell J, Martinez-Sobrido L, Cherry S, Torrelles JB, Ferrer M, Lee EM. Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents. Commun Biol 2022; 5:810. [PMID: 35962146 PMCID: PMC9373898 DOI: 10.1038/s42003-022-03753-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Accepted: 07/22/2022] [Indexed: 11/09/2022] Open
Abstract
There is a critical need for physiologically relevant, robust, and ready-to-use in vitro cellular assay platforms to rapidly model the infectivity of emerging viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.
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Affiliation(s)
- Hoda Zarkoob
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Anna Allué-Guardia
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Yu-Chi Chen
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Andreu Garcia-Vilanova
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Olive Jung
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.,Biomedical Ultrasonics & Biotherapy Laboratory, Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Headington, UK
| | - Steven Coon
- Molecular Genomics Core, National Institute of Child Health and Human Development, National Institutes of Health, Rockville, MD, USA
| | - Min Jae Song
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Jun-Gyu Park
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Fatai Oladunni
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Jesse Miller
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA
| | - Yen-Ting Tung
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Ivan Kosik
- National Institute for Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - David Schultz
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.,High Throughput Screening Core, University of Pennsylvania, Philadelphia, PA, USA
| | - James Iben
- Molecular Genomics Core, National Institute of Child Health and Human Development, National Institutes of Health, Rockville, MD, USA
| | - Tianwei Li
- Molecular Genomics Core, National Institute of Child Health and Human Development, National Institutes of Health, Rockville, MD, USA
| | - Jiaqi Fu
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Forbes D Porter
- Section on Molecular Dysmorphology, Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, 20892, USA
| | - Jonathan Yewdell
- National Institute for Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
| | - Luis Martinez-Sobrido
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Sara Cherry
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.,Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.,Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA
| | - Jordi B Torrelles
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX, USA
| | - Marc Ferrer
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
| | - Emily M Lee
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
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15
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Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells. Microbiol Spectr 2022; 10:e0163922. [PMID: 35863002 PMCID: PMC9431431 DOI: 10.1128/spectrum.01639-22] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.
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16
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Weil T, Lawrenz J, Seidel A, Münch J, Müller JA. Immunodetection assays for the quantification of seasonal common cold coronaviruses OC43, NL63, or 229E infection confirm nirmatrelvir as broad coronavirus inhibitor. Antiviral Res 2022; 203:105343. [PMID: 35598779 PMCID: PMC9119192 DOI: 10.1016/j.antiviral.2022.105343] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 05/05/2022] [Accepted: 05/16/2022] [Indexed: 01/21/2023]
Abstract
Besides pandemic SARS-CoV-2, also endemic seasonal human common cold coronaviruses (hCoVs) have a significant impact on human health and economy. Studies on hCoVs and the identification of antivirals are therefore crucial to improve human well-being. However, hCoVs have long been neglected and the methodology to study virus infection, replication and inhibition warrants being updated. We here evaluated the established plaque-based assays to determine viral titers and cell-to-cell spread and developed protocols for the immunodetection of the viral nucleocapsid protein by flow cytometry and in-cell ELISA to study infection rates at early time points. The developed protocols allow detection of hCoV-229E infection after 2, and hCoV-NL63 and -OC43 infection after 3 days at a single cell level or in a 96 well microtiter format, in large sample numbers without being laborious or expensive. Both assays can be applied to assess the susceptibility of cells to hCoV infection and replication, and to determine the efficacy of antiviral compounds as well as neutralizing antibodies in a sensitive and quantitative manner. Application revealed that clinically applied SARS-CoV-2 targeting monoclonal antibodies are inactive against hCoVs, but that the viral polymerase targeting antivirals remdesivir and molnupiravir are broadly active also against all three hCoVs. Further, the in-cell ELISA provided evidence that nirmatrelvir, previously shown to broadly inhibit coronavirus proteases, also prevents replication of authentic hCoVs. Importantly, the protocols described here can be easily adapted to other coronavirus strains and species as well as viruses of other families within a short time. This will facilitate future research on known and emerging (corona)viruses, support the identification of antivirals and increase the preparedness for future virus outbreaks.
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Affiliation(s)
- Tatjana Weil
- Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany
| | - Jan Lawrenz
- Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany
| | - Alina Seidel
- Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany
| | - Jan Münch
- Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany; Core Facility Functional Peptidomics, Ulm University Medical Center, 89081, Ulm, Germany
| | - Janis A Müller
- Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany; Institute of Virology, Philipps University of Marburg, 35043, Marburg, Germany.
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17
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Chitcharoen S, Sivapornnukul P, Payungporn S. Revolutionized virome research using systems microbiology approaches. Exp Biol Med (Maywood) 2022; 247:1135-1147. [PMID: 35723062 PMCID: PMC9335507 DOI: 10.1177/15353702221102895] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Currently, both pathogenic and commensal viruses are continuously being discovered and acknowledged as ubiquitous components of microbial communities. The advancements of systems microbiological approaches have changed the face of virome research. Here, we focus on viral metagenomic approach to study virus community and their interactions with other microbial members as well as their hosts. This review also summarizes challenges, limitations, and benefits of the current virome approaches. Potentially, the studies of virome can be further applied in various biological and clinical fields.
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Affiliation(s)
- Suwalak Chitcharoen
- Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand,Research Unit of Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Pavaret Sivapornnukul
- Research Unit of Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand,Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
| | - Sunchai Payungporn
- Research Unit of Systems Microbiology, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand,Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand,Sunchai Payungporn.
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18
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Harb A, Fakhreddine M, Zaraket H, Saleh FA. Three-Dimensional Cell Culture Models to Study Respiratory Virus Infections Including COVID-19. Biomimetics (Basel) 2021; 7:3. [PMID: 35076456 PMCID: PMC8788432 DOI: 10.3390/biomimetics7010003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 12/16/2021] [Accepted: 12/20/2021] [Indexed: 12/12/2022] Open
Abstract
Respiratory viral infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are among the most common illnesses and a leading cause of morbidity and mortality worldwide. Due to the severe effects on health, the need of new tools to study the pathogenesis of respiratory viruses as well as to test for new antiviral drugs and vaccines is urgent. In vitro culture model systems, such as three-dimensional (3D) cultures, are emerging as a desirable approach to understand the virus host interactions and to identify novel therapeutic agents. In the first part of the article, we address the various scaffold-free and scaffold-based 3D culture models such as hydrogels, bioreactors, spheroids and 3D bioprinting as well as present their properties and advantages over conventional 2D methods. Then, we review the 3D models that have been used to study the most common respiratory viruses including influenza, parainfluenza, respiratory syncytial virus (RSV) and coronaviruses. Herein, we also explain how 3D models have been applied to understand the novel SARS-CoV-2 infectivity and to develop potential therapies.
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Affiliation(s)
- Aya Harb
- Department of Experimental Pathology, Immunology & Microbiology, Faculty of Medicine, American University of Beirut, Beirut 11-0236, Lebanon; (A.H.); (H.Z.)
| | | | - Hassan Zaraket
- Department of Experimental Pathology, Immunology & Microbiology, Faculty of Medicine, American University of Beirut, Beirut 11-0236, Lebanon; (A.H.); (H.Z.)
- Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Beirut 11-0236, Lebanon
| | - Fatima A. Saleh
- Department of Medical Laboratory Sciences, Faculty of Health Sciences, Beirut Arab University, Beirut 11-5020, Lebanon
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19
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Lam AHY, Cai JP, Leung KY, Zhang RR, Liu D, Fan Y, Tam AR, Cheng VCC, To KKW, Yuen KY, Hung IFN, Chan KH. In-House Immunofluorescence Assay for Detection of SARS-CoV-2 Antigens in Cells from Nasopharyngeal Swabs as a Diagnostic Method for COVID-19. Diagnostics (Basel) 2021; 11:diagnostics11122346. [PMID: 34943583 PMCID: PMC8700487 DOI: 10.3390/diagnostics11122346] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Revised: 12/02/2021] [Accepted: 12/10/2021] [Indexed: 12/18/2022] Open
Abstract
Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.
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Affiliation(s)
- Athene Hoi-Ying Lam
- Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (A.H.-Y.L.); (R.-R.Z.); (D.L.); (Y.F.)
| | - Jian-Piao Cai
- Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (J.-P.C.); (K.-Y.L.); (K.K.-W.T.); (K.-Y.Y.)
| | - Ka-Yi Leung
- Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (J.-P.C.); (K.-Y.L.); (K.K.-W.T.); (K.-Y.Y.)
| | - Ricky-Ruiqi Zhang
- Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (A.H.-Y.L.); (R.-R.Z.); (D.L.); (Y.F.)
| | - Danlei Liu
- Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (A.H.-Y.L.); (R.-R.Z.); (D.L.); (Y.F.)
| | - Yujing Fan
- Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (A.H.-Y.L.); (R.-R.Z.); (D.L.); (Y.F.)
| | | | | | - Kelvin Kai-Wang To
- Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (J.-P.C.); (K.-Y.L.); (K.K.-W.T.); (K.-Y.Y.)
- State Key Laboratory for Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
- Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
| | - Kwok-Yung Yuen
- Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (J.-P.C.); (K.-Y.L.); (K.K.-W.T.); (K.-Y.Y.)
- Department of Microbiology, Queen Mary Hospital, Hospital Authority, Hong Kong, China;
- State Key Laboratory for Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
- Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
| | - Ivan Fan-Ngai Hung
- Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (A.H.-Y.L.); (R.-R.Z.); (D.L.); (Y.F.)
- Department of Medicine, Queen Mary Hospital, Hong Kong, China;
- State Key Laboratory for Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
- Correspondence: (I.F.-N.H.); (K.-H.C.)
| | - Kwok-Hung Chan
- Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; (J.-P.C.); (K.-Y.L.); (K.K.-W.T.); (K.-Y.Y.)
- State Key Laboratory for Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
- Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
- Correspondence: (I.F.-N.H.); (K.-H.C.)
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20
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Sang Y, Miller LC, Nelli RK, Giménez-Lirola LG. Harness Organoid Models for Virological Studies in Animals: A Cross-Species Perspective. Front Microbiol 2021; 12:725074. [PMID: 34603253 PMCID: PMC8481363 DOI: 10.3389/fmicb.2021.725074] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Accepted: 08/27/2021] [Indexed: 02/02/2023] Open
Abstract
Animal models and cell culture in vitro are primarily used in virus and antiviral immune research. Whereas the limitation of these models to recapitulate the viral pathogenesis in humans has been made well aware, it is imperative to introduce more efficient systems to validate emerging viruses in both domestic and wild animals. Organoids ascribe to representative miniatures of organs (i.e., mini-organs), which are derived from three-dimensional culture of stem cells under respective differential conditions mimicking endogenous organogenetic niches. Organoids have broadened virological studies in the human context, particularly in recent uses for COVID19 research. This review examines the status and potential for cross-species applied organotypic culture in validating emerging animal, particularly zoonotic, viruses in domestic and wild animals.
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Affiliation(s)
- Yongming Sang
- Department of Agricultural and Environmental Sciences, College of Agriculture, Tennessee State University, Nashville, TN, United States
| | - Laura C Miller
- Virus and Prion Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, IA, United States
| | - Rahul K Nelli
- Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
| | - Luis Gabriel Giménez-Lirola
- Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
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21
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Rijsbergen LC, van Dijk LLA, Engel MFM, de Vries RD, de Swart RL. In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells - A Systematic Review. Front Immunol 2021; 12:683002. [PMID: 34489934 PMCID: PMC8418200 DOI: 10.3389/fimmu.2021.683002] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 07/30/2021] [Indexed: 12/12/2022] Open
Abstract
Respiratory tract infections (RTI) are a major cause of morbidity and mortality in humans. A large number of RTIs is caused by viruses, often resulting in more severe disease in infants, elderly and the immunocompromised. Upon viral infection, most individuals experience common cold-like symptoms associated with an upper RTI. However, in some cases a severe and sometimes life-threatening lower RTI may develop. Reproducible and scalable in vitro culture models that accurately reflect the human respiratory tract are needed to study interactions between respiratory viruses and the host, and to test novel therapeutic interventions. Multiple in vitro respiratory cell culture systems have been described, but the majority of these are based on immortalized cell lines. Although useful for studying certain aspects of viral infections, such monomorphic, unicellular systems fall short in creating an understanding of the processes that occur at an integrated tissue level. Novel in vitro models involving primary human airway epithelial cells and, more recently, human airway organoids, are now in use. In this review, we describe the evolution of in vitro cell culture systems and their characteristics in the context of viral RTIs, starting from advances after immortalized cell cultures to more recently developed organoid systems. Furthermore, we describe how these models are used in studying virus-host interactions, e.g. tropism and receptor studies as well as interactions with the innate immune system. Finally, we provide an outlook for future developments in this field, including co-factors that mimic the microenvironment in the respiratory tract.
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Affiliation(s)
- Laurine C. Rijsbergen
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Laura L. A. van Dijk
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Maarten F. M. Engel
- Medical Library, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Rory D. de Vries
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
| | - Rik L. de Swart
- Department of Viroscience, Postgraduate School of Molecular Medicine, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, Netherlands
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22
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Kesheh MM, Hosseini P, Soltani S, Zandi M. An overview on the seven pathogenic human coronaviruses. Rev Med Virol 2021; 32:e2282. [PMID: 34339073 DOI: 10.1002/rmv.2282] [Citation(s) in RCA: 97] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 07/20/2021] [Accepted: 07/22/2021] [Indexed: 02/06/2023]
Abstract
To date, seven human coronaviruses (HCoVs) have been detected: HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2. Four of these viruses, including HCoV-NL63, -229E, -HKU1 and -OC43, usually cause mild-to-moderate respiratory diseases with a seasonal pattern. Since 2000, three new HCoVs have emerged with a significant mortality rate. Although SARS-CoV and MERS-CoV caused an epidemic in some countries, SARS-CoV-2 escalated into a pandemic. All HCoVs can cause severe complications in the elderly and immunocompromised individuals. The bat origin of HCoVs, the presence of intermediate hosts and the nature of their viral replication suggest that other new coronaviruses may emerge in the future. Despite the fact that all HCoVs share similarities in viral replication, they differ in their accessory proteins, incubation period and pathogenicity. This study aims to review these differences between the seven HCoVs.
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Affiliation(s)
- Mina Mobini Kesheh
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Parastoo Hosseini
- Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.,Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran
| | - Saber Soltani
- Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.,Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran
| | - Milad Zandi
- Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.,Research Center for Clinical Virology, Tehran University of Medical Sciences, Tehran, Iran
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23
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Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity. J Virol 2021; 95:e0237420. [PMID: 33910954 DOI: 10.1128/jvi.02374-20] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.
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24
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Cordes AK, Rehrauer WM, Accola MA, Wölk B, Hilfrich B, Heim A. Fully automated detection and differentiation of pandemic and endemic coronaviruses (NL63, 229E, HKU1, OC43 and SARS-CoV-2) on the hologic panther fusion. J Med Virol 2021; 93:4438-4445. [PMID: 33350484 DOI: 10.1002/jmv.26749] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 12/15/2020] [Accepted: 12/19/2020] [Indexed: 12/18/2022]
Abstract
The hologic panther fusion (PF) platform provides fully automated CE marked diagnostics for respiratory viruses, including the recently discovered severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) by a transcription mediated amplification (TMA) assay, but not for the endemic human coronaviruses (hCoV). Therefore, a laboratory developed test (LDT) comprising a multiplexed reverse transcription polymerase chain reaction (RT-PCR) protocol that detects and differentiates the four hCoV NL63, 229E, HKU1, and OC43 was adapted on the PF. The novel CE marked Aptima SARS-CoV-2 TMA and the LDT for hCoV were validated with 321 diagnostic specimens from the upper and lower respiratory tract in comparison to two SARS-CoV-2 RT-PCRs (PF E-gene RT-PCR and genesig RT-PCR, 157 specimens) or the R-GENE hCoV/hParaFlu RT-PCR (164 specimens), respectively. For the endemic hCoV, results were 96.3% concordant with two specimens discordantly positive in the PF and four specimens discordantly positive in the R-GENE assay. All discordantly positive samples had Ct values between 33 and 39. The PF hCoV LDT identified 23 hCoV positive specimens as NL63, 15 as 229E, 15 as HKU1, and 25 as OC43. The Aptima SARS-CoV-2 TMA gave 99.4% concordant results compared to the consensus results with a single specimen discordantly positive. Moreover, 36 samples from proficiency testing panels were detected and typed correctly by both novel methods. In conclusion, the SARS-CoV-2 TMA and the LDT for hCoV enhanced the diagnostic spectrum of the PF for all coronaviruses circulating globally for a multitude of diagnostic materials from the upper and lower respiratory tract.
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Affiliation(s)
- Anne K Cordes
- Institute of Virology, Medical School Hannover, Hannover, Germany
| | - William M Rehrauer
- Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA
| | - Molly A Accola
- Molecular Diagnostics, Clinical Labs, University of Wisconsin Hospital, Madison, Wisconsin, USA
| | - Benno Wölk
- LADR Medical Laboratory Dr Kramer and Colleagues, Geesthacht, Germany
| | | | - Albert Heim
- Institute of Virology, Medical School Hannover, Hannover, Germany
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25
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Yoo HM, Kim IH, Kim S. Nucleic Acid Testing of SARS-CoV-2. Int J Mol Sci 2021; 22:6150. [PMID: 34200331 PMCID: PMC8201071 DOI: 10.3390/ijms22116150] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Revised: 05/25/2021] [Accepted: 06/04/2021] [Indexed: 12/13/2022] Open
Abstract
The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.
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Affiliation(s)
- Hee Min Yoo
- Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea; (H.M.Y.); (I.-H.K.)
- Department of Bio-Analytical Science, University of Science & Technology (UST), Daejeon 34113, Korea
| | - Il-Hwan Kim
- Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea; (H.M.Y.); (I.-H.K.)
| | - Seil Kim
- Microbiological Analysis Team, Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea; (H.M.Y.); (I.-H.K.)
- Department of Bio-Analytical Science, University of Science & Technology (UST), Daejeon 34113, Korea
- Convergent Research Center for Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea
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26
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Buqaileh R, Saternos H, Ley S, Aranda A, Forero K, AbouAlaiwi WA. Can cilia provide an entry gateway for SARS-CoV-2 to human ciliated cells? Physiol Genomics 2021; 53:249-258. [PMID: 33855870 PMCID: PMC8213509 DOI: 10.1152/physiolgenomics.00015.2021] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 04/12/2021] [Accepted: 04/12/2021] [Indexed: 12/30/2022] Open
Abstract
A worldwide coronavirus pandemic is in full swing and, at the time of writing, there are only few treatments that have been successful in clinical trials, but no effective antiviral treatment has been approved. Because of its lethality, it is important to understand the current strain's effects and mechanisms not only in the respiratory system but also in other affected organ systems as well. Past coronavirus outbreaks caused by SARS-CoV and MERS-CoV inflicted life-threatening acute kidney injuries (AKI) on their hosts leading to significant mortality rates, which went somewhat overlooked in the face of the severe respiratory effects. Recent evidence has emphasized renal involvement in SARS-CoV-2, stressing that kidneys are damaged in patients with COVID-19. The mechanism by which this virus inflicts AKI is still unclear, but evidence from other coronavirus strains may hold some clues. Two theories exist for the proposed mechanism of AKI: 1) the AKI is a secondary effect to reduced blood and oxygen levels causing hyperinflammation and 2) the AKI is due to cytotoxic effects. Kidneys express angiotensin-converting enzyme-2 (ACE2), the confirmed SARS-CoV-2 target receptor as well as collectrin, an ACE2 homologue that localizes to the primary cilium, an organelle historically targeted by coronaviruses. Although the available literature suggests that kidney damage is leading to higher mortality rates in patients with COVID-19, especially in those with preexisting kidney and cardiovascular diseases, the pathogenesis of COVID-19 is still being investigated. Here, we present brief literature review supporting our proposed hypothesis of a possible link between SARS-CoV-2 cellular infection and cilia.
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Affiliation(s)
- Raghad Buqaileh
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
| | - Hannah Saternos
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
| | - Sidney Ley
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
| | - Arianna Aranda
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
| | - Kathleen Forero
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
| | - Wissam A AbouAlaiwi
- Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio
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de Dios-Figueroa GT, Aguilera-Marquez JDR, Camacho-Villegas TA, Lugo-Fabres PH. 3D Cell Culture Models in COVID-19 Times: A Review of 3D Technologies to Understand and Accelerate Therapeutic Drug Discovery. Biomedicines 2021; 9:602. [PMID: 34073231 PMCID: PMC8226796 DOI: 10.3390/biomedicines9060602] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 05/15/2021] [Accepted: 05/18/2021] [Indexed: 12/12/2022] Open
Abstract
In the last decades, emerging viruses have become a worldwide concern. The fast and extensive spread of the disease caused by SARS-CoV-2 (COVID-19) has impacted the economy and human activity worldwide, highlighting the human vulnerability to infectious diseases and the need to develop and optimize technologies to tackle them. The three-dimensional (3D) cell culture models emulate major tissue characteristics such as the in vivo virus-host interactions. These systems may help to generate a quick response to confront new viruses, establish a reliable evaluation of the pathophysiology, and contribute to therapeutic drug evaluation in pandemic situations such as the one that humanity is living through today. This review describes different types of 3D cell culture models, such as spheroids, scaffolds, organoids, and organs-on-a-chip, that are used in virus research, including those used to understand the new severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2).
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Affiliation(s)
- Guadalupe Tonantzin de Dios-Figueroa
- Department of Medical and Pharmaceutical Biotechnology, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ), Av. Normalistas 800, Colinas de las Normal, Guadalajara, Jalisco 44270, Mexico; (G.T.d.D.-F.); (J.d.R.A.-M.)
| | - Janette del Rocío Aguilera-Marquez
- Department of Medical and Pharmaceutical Biotechnology, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ), Av. Normalistas 800, Colinas de las Normal, Guadalajara, Jalisco 44270, Mexico; (G.T.d.D.-F.); (J.d.R.A.-M.)
| | - Tanya A. Camacho-Villegas
- CONACYT-Department of Medical and Pharmaceutical Biotechnology, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ), Av. Normalistas 800, Colinas de las Normal, Guadalajara, Jalisco 44270, Mexico;
| | - Pavel H. Lugo-Fabres
- CONACYT-Department of Medical and Pharmaceutical Biotechnology, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ), Av. Normalistas 800, Colinas de las Normal, Guadalajara, Jalisco 44270, Mexico;
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28
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Yan Z, Deng X, Qiu J. Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium. ACTA ACUST UNITED AC 2021; 58:e107. [PMID: 32639683 DOI: 10.1002/cpmc.107] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.
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Affiliation(s)
- Ziying Yan
- Department of Anatomy, University of Iowa, Iowa City, Iowa
| | - Xuefeng Deng
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas
| | - Jianming Qiu
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas
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29
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Zarkoob H, Allué-Guardia A, Chen YC, Jung O, Garcia-Vilanova A, Song MJ, Park JG, Oladunni F, Miller J, Tung YT, Kosik I, Schultz D, Yewdell J, Torrelles JB, Martinez-Sobrido L, Cherry S, Ferrer M, Lee EM. Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2021:2021.05.11.443693. [PMID: 34013274 PMCID: PMC8132232 DOI: 10.1101/2021.05.11.443693] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.
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Affiliation(s)
- Hoda Zarkoob
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
| | - Anna Allué-Guardia
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Yu-Chi Chen
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
| | - Olive Jung
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
- Biomedical Ultrasonics & Biotherapy Laboratory, Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Headington, UK
| | - Andreu Garcia-Vilanova
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Min Jae Song
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
| | - Jun-Gyu Park
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Fatai Oladunni
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Jesse Miller
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
- Department of Microbiology, University of Pennsylvania, Philadelphia, PA
| | - Yen-Ting Tung
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
| | - Ivan Kosik
- National Institute for Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD
| | - David Schultz
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA
- High Throughput Screening Core, University of Pennsylvania, Philadelphia, PA
| | - Jonathan Yewdell
- National Institute for Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD
| | - Jordi B. Torrelles
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Luis Martinez-Sobrido
- Host-Pathogen Interactions and Population Health Programs, Texas Biomedical Research Institute, San Antonio, TX
| | - Sara Cherry
- Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
- Department of Microbiology, University of Pennsylvania, Philadelphia, PA
| | - Marc Ferrer
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
| | - Emily M. Lee
- 3D Tissue Bioprinting Lab, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD
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30
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Comber L, O Murchu E, Drummond L, Carty PG, Walsh KA, De Gascun CF, Connolly MA, Smith SM, O'Neill M, Ryan M, Harrington P. Airborne transmission of SARS-CoV-2 via aerosols. Rev Med Virol 2021; 31:e2184. [PMID: 33105071 PMCID: PMC7645866 DOI: 10.1002/rmv.2184] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 10/08/2020] [Accepted: 10/10/2020] [Indexed: 01/01/2023]
Abstract
A key consideration in the Covid-19 pandemic is the dominant modes of transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The objective of this review was to synthesise the evidence for the potential airborne transmission of SARS-CoV-2 via aerosols. Systematic literature searches were conducted in PubMed, Embase, Europe PMC and National Health Service UK evidence up to 27 July 2020. A protocol was published and Cochrane guidance for rapid review methodology was adhered to throughout. Twenty-eight studies were identified. Seven out of eight epidemiological studies suggest aerosol transmission may occur, with enclosed environments and poor ventilation noted as possible contextual factors. Ten of the 16 air sampling studies detected SARS-CoV-2 ribonucleic acid; however, only three of these studies attempted to culture the virus with one being successful in a limited number of samples. Two of four virological studies using artificially generated aerosols indicated that SARS-CoV-2 is viable in aerosols. The results of this review indicate there is inconclusive evidence regarding the viability and infectivity of SARS-CoV-2 in aerosols. Epidemiological studies suggest possible transmission, with contextual factors noted. Viral particles have been detected in air sampling studies with some evidence of clinical infectivity, and virological studies indicate these particles may represent live virus, adding further plausibility. However, there is uncertainty as to the nature and impact of aerosol transmission of SARS-CoV-2, and its relative contribution to the Covid-19 pandemic compared with other modes of transmission.
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Affiliation(s)
- Laura Comber
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | - Eamon O Murchu
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | - Linda Drummond
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | - Paul G. Carty
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | - Kieran A. Walsh
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | | | - Máire A. Connolly
- School of MedicineNational University of Ireland GalwayGalwayIreland
| | - Susan M. Smith
- Department of General PracticeHealth Research Board Centre for Primary Care ResearchRoyal College of Surgeons in IrelandDublinIreland
| | - Michelle O'Neill
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
| | - Máirín Ryan
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
- Department of Pharmacology & TherapeuticsTrinity College DublinDublinIreland
| | - Patricia Harrington
- Health Technology Assessment DirectorateHealth Information and Quality AuthorityDublinIreland
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31
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Li Z, Lang Y, Liu L, Bunyatov MI, Sarmiento AI, de Groot RJ, Boons GJ. Synthetic O-acetylated sialosides facilitate functional receptor identification for human respiratory viruses. Nat Chem 2021; 13:496-503. [PMID: 33753916 DOI: 10.1038/s41557-021-00655-9] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Accepted: 02/08/2021] [Indexed: 01/31/2023]
Abstract
The transmission of viruses from animal reservoirs to humans poses major threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding of how viruses of animal origin have adapted to binding to a cell surface component and/or receptor of the new host. Here we report on the specificities of human and animal viruses that engage with O-acetylated sialic acid, which include betacoronaviruses, toroviruses and influenza C and D viruses. Key to these studies was the development of a chemoenzymatic methodology that can provide almost any sialate-acetylation pattern. A collection of O-acetylated sialoglycans was printed as a microarray for the determination of receptor specificity. These studies showed host-specific patterns of receptor recognition and revealed that three distinct human respiratory viruses uniquely bind 9-O-acetylated α2,8-linked disialoside. Immunofluorescence and cell entry studies support that such a glycotope as part of a ganglioside is a functional receptor for human coronaviruses.
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Affiliation(s)
- Zeshi Li
- Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Yifei Lang
- Virology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | - Lin Liu
- Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
| | - Mehman I Bunyatov
- Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Angelic Isaza Sarmiento
- Virology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
| | - Raoul J de Groot
- Virology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
| | - Geert-Jan Boons
- Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. .,Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA. .,Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands. .,Chemistry Department, University of Georgia, Athens, GA, USA.
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32
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Synowiec A, Szczepański A, Barreto-Duran E, Lie LK, Pyrc K. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): a Systemic Infection. Clin Microbiol Rev 2021; 34:e00133-20. [PMID: 33441314 PMCID: PMC7849242 DOI: 10.1128/cmr.00133-20] [Citation(s) in RCA: 139] [Impact Index Per Article: 34.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
To date, seven identified coronaviruses (CoVs) have been found to infect humans; of these, three highly pathogenic variants have emerged in the 21st century. The newest member of this group, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected at the end of 2019 in Hubei province, China. Since then, this novel coronavirus has spread worldwide, causing a pandemic; the respiratory disease caused by the virus is called coronavirus disease 2019 (COVID-19). The clinical presentation ranges from asymptomatic to mild respiratory tract infections and influenza-like illness to severe disease with accompanying lung injury, multiorgan failure, and death. Although the lungs are believed to be the site at which SARS-CoV-2 replicates, infected patients often report other symptoms, suggesting the involvement of the gastrointestinal tract, heart, cardiovascular system, kidneys, and other organs; therefore, the following question arises: is COVID-19 a respiratory or systemic disease? This review aims to summarize existing data on the replication of SARS-CoV-2 in different tissues in both patients and ex vivo models.
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Affiliation(s)
- Aleksandra Synowiec
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Artur Szczepański
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
- Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Emilia Barreto-Duran
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Laurensius Kevin Lie
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
| | - Krzysztof Pyrc
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
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33
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Gorgulla C, Padmanabha Das KM, Leigh KE, Cespugli M, Fischer PD, Wang ZF, Tesseyre G, Pandita S, Shnapir A, Calderaio A, Gechev M, Rose A, Lewis N, Hutcheson C, Yaffe E, Luxenburg R, Herce HD, Durmaz V, Halazonetis TD, Fackeldey K, Patten J, Chuprina A, Dziuba I, Plekhova A, Moroz Y, Radchenko D, Tarkhanova O, Yavnyuk I, Gruber C, Yust R, Payne D, Näär AM, Namchuk MN, Davey RA, Wagner G, Kinney J, Arthanari H. A multi-pronged approach targeting SARS-CoV-2 proteins using ultra-large virtual screening. iScience 2021; 24:102021. [PMID: 33426509 PMCID: PMC7783459 DOI: 10.1016/j.isci.2020.102021] [Citation(s) in RCA: 57] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 10/28/2020] [Accepted: 12/29/2020] [Indexed: 02/07/2023] Open
Abstract
The unparalleled global effort to combat the continuing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic over the last year has resulted in promising prophylactic measures. However, a need still exists for cheap, effective therapeutics, and targeting multiple points in the viral life cycle could help tackle the current, as well as future, coronaviruses. Here, we leverage our recently developed, ultra-large-scale in silico screening platform, VirtualFlow, to search for inhibitors that target SARS-CoV-2. In this unprecedented structure-based virtual campaign, we screened roughly 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets. In addition to targeting the active sites of viral enzymes, we also targeted critical auxiliary sites such as functionally important protein-protein interactions.
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Affiliation(s)
- Christoph Gorgulla
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
- Department of Physics, Faculty of Arts and Sciences, Harvard University, Cambridge, MA 02138, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Krishna M. Padmanabha Das
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Kendra E. Leigh
- Max Planck Institute of Biophysics, Frankfurt am Main, Hessen 60438, Germany
| | | | - Patrick D. Fischer
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Saarland 66123, Germany
| | - Zi-Fu Wang
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
| | | | | | | | - Anthony Calderaio
- VirtualFlow Organization, https://virtual-flow.org/, Boston, MA 02115, USA
| | | | - Alexander Rose
- Mol∗ Consortium, https://molstar.org, San Diego, CA 92109, USA
| | | | | | | | | | - Henry D. Herce
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | | | | | - Konstantin Fackeldey
- Zuse Institute Berlin (ZIB), Berlin 14195, Germany
- Institute of Mathematics, Technical University Berlin, Berlin 10587, Germany
| | - J.J. Patten
- Department of Microbiology, Boston University Medical School, Boston University, Boston, MA 02118, USA
| | | | | | | | - Yurii Moroz
- Chemspace, Kyiv 02094, Ukraine
- Taras Shevchenko National University of Kyiv, Kyiv 01601, Ukraine
| | - Dmytro Radchenko
- Enamine, Kyiv 02094, Ukraine
- Taras Shevchenko National University of Kyiv, Kyiv 01601, Ukraine
| | | | | | - Christian Gruber
- Innophore GmbH, Graz 8010, Austria
- Institute of Molecular Biosciences, University of Graz, Graz 8010, Austria
| | - Ryan Yust
- Google, Mountain View, CA 94043, USA
| | | | - Anders M. Näär
- Department of Nutritional Sciences & Toxicology, University of California Berkeley, Berkeley, CA 94720, USA
| | - Mark N. Namchuk
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
| | - Robert A. Davey
- Department of Microbiology, Boston University Medical School, Boston University, Boston, MA 02118, USA
| | - Gerhard Wagner
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
| | | | - Haribabu Arthanari
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, MA 02115, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
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34
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Cruz-Teran C, Tiruthani K, McSweeney M, Ma A, Pickles R, Lai SK. Challenges and opportunities for antiviral monoclonal antibodies as COVID-19 therapy. Adv Drug Deliv Rev 2021; 169:100-117. [PMID: 33309815 PMCID: PMC7833882 DOI: 10.1016/j.addr.2020.12.004] [Citation(s) in RCA: 48] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Revised: 11/30/2020] [Accepted: 12/05/2020] [Indexed: 01/08/2023]
Abstract
To address the COVID-19 pandemic, there has been an unprecedented global effort to advance potent neutralizing mAbs against SARS-CoV-2 as therapeutics. However, historical efforts to advance antiviral monoclonal antibodies (mAbs) for the treatment of other respiratory infections have been met with categorical failures in the clinic. By investigating the mechanism by which SARS-CoV-2 and similar viruses spread within the lung, along with available biodistribution data for systemically injected mAb, we highlight the challenges faced by current antiviral mAbs for COVID-19. We summarize some of the leading mAbs currently in development, and present the evidence supporting inhaled delivery of antiviral mAb as an early intervention against COVID-19 that could prevent important pulmonary morbidities associated with the infection.
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Affiliation(s)
- Carlos Cruz-Teran
- Division of Pharmacoengineering and Molecular Pharmaceutics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Karthik Tiruthani
- Division of Pharmacoengineering and Molecular Pharmaceutics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | | | - Alice Ma
- UNC/NCSU Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Raymond Pickles
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Samuel K Lai
- Division of Pharmacoengineering and Molecular Pharmaceutics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Inhalon Biopharma, Durham, NC 27709, USA; UNC/NCSU Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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35
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Iverson E, Kaler L, Agostino EL, Song D, Duncan GA, Scull MA. Leveraging 3D Model Systems to Understand Viral Interactions with the Respiratory Mucosa. Viruses 2020; 12:E1425. [PMID: 33322395 PMCID: PMC7763686 DOI: 10.3390/v12121425] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 11/27/2020] [Accepted: 12/02/2020] [Indexed: 02/07/2023] Open
Abstract
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus-host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air-liquid interface, organoids, or 'on-chip' technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium-the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.
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Affiliation(s)
- Ethan Iverson
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA; (E.I.); (E.L.A.)
| | - Logan Kaler
- Biophysics Program, University of Maryland, College Park, MD 20742, USA; (L.K.); (G.A.D.)
| | - Eva L. Agostino
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA; (E.I.); (E.L.A.)
| | - Daniel Song
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA;
| | - Gregg A. Duncan
- Biophysics Program, University of Maryland, College Park, MD 20742, USA; (L.K.); (G.A.D.)
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA;
| | - Margaret A. Scull
- Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA; (E.I.); (E.L.A.)
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Learning from past failures: Challenges with monoclonal antibody therapies for COVID-19. J Control Release 2020; 329:87-95. [PMID: 33276017 PMCID: PMC7836766 DOI: 10.1016/j.jconrel.2020.11.057] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Revised: 11/25/2020] [Accepted: 11/29/2020] [Indexed: 01/08/2023]
Abstract
COVID-19, the disease caused by infection with SARS-CoV-2, requires urgent development of therapeutic interventions. Due to their safety, specificity, and potential for rapid advancement into the clinic, monoclonal antibodies (mAbs) represent a highly promising class of antiviral or anti-inflammatory agents. Herein, by analyzing prior efforts to advance antiviral mAbs for other acute respiratory infections (ARIs), we highlight the challenges faced by mAb-based immunotherapies for COVID-19. We present evidence supporting early intervention immediately following a positive diagnosis via inhaled delivery of mAbs with vibrating mesh nebulizers as a promising approach for the treatment of COVID-19.
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Walsh KA, Spillane S, Comber L, Cardwell K, Harrington P, Connell J, Teljeur C, Broderick N, de Gascun CF, Smith SM, Ryan M, O'Neill M. The duration of infectiousness of individuals infected with SARS-CoV-2. J Infect 2020; 81:847-856. [PMID: 33049331 PMCID: PMC7547320 DOI: 10.1016/j.jinf.2020.10.009] [Citation(s) in RCA: 97] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Accepted: 10/07/2020] [Indexed: 12/20/2022]
Abstract
OBJECTIVES To summarise the evidence on the duration of infectiousness of individuals in whom SARS-CoV-2 ribonucleic acid is detected. METHODS A rapid review was undertaken in PubMed, Europe PubMed Central and EMBASE from 1 January 2020 to 26 August 2020. RESULTS We identified 15 relevant studies, including 13 virus culture and 2 contact tracing studies. For 5 virus culture studies, the last day on which SARS-CoV-2 was isolated occurred within 10 days of symptom onset. For another 5 studies, SARS-CoV-2 was isolated beyond day 10 for approximately 3% of included patients. The remaining 3 virus culture studies included patients with severe or critical disease; SARS-CoV-2 was isolated up to day 32 in one study. Two studies identified immunocompromised patients from whom SARS-CoV-2 was isolated for up to 20 days. Both contact tracing studies, when close contacts were first exposed greater than 5 days after symptom onset in the index case, found no evidence of laboratory-confirmed onward transmission of SARS-CoV-2. CONCLUSION COVID-19 patients with mild-to-moderate illness are highly unlikely to be infectious beyond 10 days of symptoms. However, evidence from a limited number of studies indicates that patients with severe-to-critical illness or who are immunocompromised, may shed infectious virus for longer.
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Affiliation(s)
- Kieran A Walsh
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland.
| | - Susan Spillane
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland
| | - Laura Comber
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland
| | - Karen Cardwell
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland; Health Research Board Centre for Primary Care Research, Department of General Practice, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland
| | | | - Jeff Connell
- National Virus Reference Laboratory, University College Dublin, Belfield, Dublin 4, Ireland
| | - Conor Teljeur
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland
| | - Natasha Broderick
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland
| | - Cillian F de Gascun
- National Virus Reference Laboratory, University College Dublin, Belfield, Dublin 4, Ireland
| | - Susan M Smith
- Health Research Board Centre for Primary Care Research, Department of General Practice, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland
| | - Máirín Ryan
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland; Department of Pharmacology & Therapeutics, Trinity College Dublin, Trinity Health Sciences, James Street, Dublin 8, Ireland
| | - Michelle O'Neill
- Health Information and Quality Authority, Smithfield, Dublin 7, Ireland
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38
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Komabayashi K, Matoba Y, Seto J, Ikeda Y, Tanaka W, Aoki Y, Ikeda T, Matsuzaki Y, Itagaki T, Shirato K, Mizuta K. Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture. Jpn J Infect Dis 2020; 74:285-292. [PMID: 33250494 DOI: 10.7883/yoken.jjid.2020.776] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.
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Affiliation(s)
- Kenichi Komabayashi
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Yohei Matoba
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Junji Seto
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Yoko Ikeda
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Waka Tanaka
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Yoko Aoki
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Tatsuya Ikeda
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
| | - Yoko Matsuzaki
- Department of Infectious Diseases, Yamagata University Faculty of Medicine, Japan
| | | | - Kazuya Shirato
- Department of Virology III, National Institute of Infectious Diseases, Japan
| | - Katsumi Mizuta
- Department of Microbiology, Yamagata Prefectural Institute of Public Health, Japan
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Milewska A, Falkowski K, Kulczycka M, Bielecka E, Naskalska A, Mak P, Lesner A, Ochman M, Urlik M, Diamandis E, Prassas I, Potempa J, Kantyka T, Pyrc K. Kallikrein 13 serves as a priming protease during infection by the human coronavirus HKU1. Sci Signal 2020; 13:13/659/eaba9902. [PMID: 33234691 PMCID: PMC7857416 DOI: 10.1126/scisignal.aba9902] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Unlike SARS-CoV-2, the human coronavirus HKU1 normally causes relatively mild respiratory tract infections; however, it shares with SARS-CoV-2 the mechanism of using its surface spike (S) protein to enter target cells. Because the host receptor for HCoV-HKU1 is unknown, efforts to study the virus in cell culture systems have proved difficult. Milewska et al. found that knockout of the protease kallikrein 13 (KLK13) in human airway epithelial cells blocked their infection by HCoV-HKU1, that overexpression of KLK13 in nonpermissive cells enabled their infection by the virus, and that KLK13 cleaved the viral S protein. Together, these findings suggest that KLK13 is a priming enzyme for viral entry and may help to establish cell lines that can facilitate further investigation of the mechanism of viral pathogenesis. Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.
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Affiliation(s)
- Aleksandra Milewska
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland.,Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
| | - Katherine Falkowski
- Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
| | - Magdalena Kulczycka
- Laboratory of Proteolysis and Post-translational Modification of Proteins, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
| | - Ewa Bielecka
- Laboratory of Proteolysis and Post-translational Modification of Proteins, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland
| | - Antonina Naskalska
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland
| | - Pawel Mak
- Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., 30-387 Krakow, Poland
| | - Adam Lesner
- Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
| | - Marek Ochman
- Department of Cardiac, Vascular and Endovascular Surgery and Transplantology, Medical University of Silesia in Katowice, Silesian Centre for Heart Diseases, Zabrze, Poland
| | - Maciej Urlik
- Department of Cardiac, Vascular and Endovascular Surgery and Transplantology, Medical University of Silesia in Katowice, Silesian Centre for Heart Diseases, Zabrze, Poland
| | - Elftherios Diamandis
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.,Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.,Department of Clinical Biochemistry, University Health Network, Toronto, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
| | - Ioannis Prassas
- Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.,Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
| | - Jan Potempa
- Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.,Centre for Oral Health and Systemic Diseases, University of Louisville School of Dentistry, Louisville, KY 40202, USA
| | - Tomasz Kantyka
- Laboratory of Proteolysis and Post-translational Modification of Proteins, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.,Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, 5020 Bergen, Norway
| | - Krzysztof Pyrc
- Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland.
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Sridhar A, Simmini S, Ribeiro CMS, Tapparel C, Evers MM, Pajkrt D, Wolthers K. A Perspective on Organoids for Virology Research. Viruses 2020; 12:E1341. [PMID: 33238561 PMCID: PMC7700289 DOI: 10.3390/v12111341] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Revised: 11/12/2020] [Accepted: 11/22/2020] [Indexed: 12/27/2022] Open
Abstract
Animal models and cell lines are invaluable for virology research and host-pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape.
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Affiliation(s)
- Adithya Sridhar
- OrganoVIR Labs, Department of Medical Microbiology, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands; (A.S.); (D.P.)
- Department of Pediatric Infectious Diseases, Emma Children’s Hospital, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands
| | - Salvatore Simmini
- Gastrointestinal Biology Group, STEMCELL Technologies UK Ltd., Cambridge CB28 9TL, UK;
| | - Carla M. S. Ribeiro
- Department of Experimental Immunology, Amsterdam Institute for Infection and Immunity, Amsterdam UMC, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands;
| | - Caroline Tapparel
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland;
- Division of Infectious Diseases, Geneva University Hospital, 1205 Geneva, Switzerland
| | - Melvin M. Evers
- Department of Research and Development, uniQure Biopharma B.V., 1105 BE Amsterdam, The Netherlands;
| | - Dasja Pajkrt
- OrganoVIR Labs, Department of Medical Microbiology, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands; (A.S.); (D.P.)
- Department of Pediatric Infectious Diseases, Emma Children’s Hospital, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands
| | - Katja Wolthers
- OrganoVIR Labs, Department of Medical Microbiology, Amsterdam UMC, Location Academic Medical Center, University of Amsterdam, 1100 AZ Amsterdam, The Netherlands; (A.S.); (D.P.)
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Cao X, Coyle JP, Xiong R, Wang Y, Heflich RH, Ren B, Gwinn WM, Hayden P, Rojanasakul L. Invited review: human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives. In Vitro Cell Dev Biol Anim 2020; 57:104-132. [PMID: 33175307 PMCID: PMC7657088 DOI: 10.1007/s11626-020-00517-7] [Citation(s) in RCA: 74] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Accepted: 09/29/2020] [Indexed: 02/07/2023]
Abstract
The lung is an organ that is directly exposed to the external environment. Given the large surface area and extensive ventilation of the lung, it is prone to exposure to airborne substances, such as pathogens, allergens, chemicals, and particulate matter. Highly elaborate and effective mechanisms have evolved to protect and maintain homeostasis in the lung. Despite these sophisticated defense mechanisms, the respiratory system remains highly susceptible to environmental challenges. Because of the impact of respiratory exposure on human health and disease, there has been considerable interest in developing reliable and predictive in vitro model systems for respiratory toxicology and basic research. Human air-liquid-interface (ALI) organotypic airway tissue models derived from primary tracheobronchial epithelial cells have in vivo–like structure and functions when they are fully differentiated. The presence of the air-facing surface allows conducting in vitro exposures that mimic human respiratory exposures. Exposures can be conducted using particulates, aerosols, gases, vapors generated from volatile and semi-volatile substances, and respiratory pathogens. Toxicity data have been generated using nanomaterials, cigarette smoke, e-cigarette vapors, environmental airborne chemicals, drugs given by inhalation, and respiratory viruses and bacteria. Although toxicity evaluations using human airway ALI models require further standardization and validation, this approach shows promise in supplementing or replacing in vivo animal models for conducting research on respiratory toxicants and pathogens.
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Affiliation(s)
- Xuefei Cao
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR, Jefferson, USA.
| | - Jayme P Coyle
- Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, USA
| | - Rui Xiong
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR, Jefferson, USA
| | - Yiying Wang
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR, Jefferson, USA
| | - Robert H Heflich
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR, Jefferson, USA
| | - Baiping Ren
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Rd., AR, Jefferson, USA
| | - William M Gwinn
- Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Durham, NC, USA
| | | | - Liying Rojanasakul
- Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, USA
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42
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Hao S, Ning K, Kuz CA, Vorhies K, Yan Z, Qiu J. Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium. mBio 2020; 11:e02852-20. [PMID: 33158999 PMCID: PMC7649230 DOI: 10.1128/mbio.02852-20] [Citation(s) in RCA: 70] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2020] [Accepted: 10/16/2020] [Indexed: 12/28/2022] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.
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Affiliation(s)
- Siyuan Hao
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Kang Ning
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Cagla Aksu Kuz
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Kai Vorhies
- Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa, USA
| | - Ziying Yan
- Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa, USA
| | - Jianming Qiu
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Kinsella CM, Santos PD, Postigo-Hidalgo I, Folgueiras-González A, Passchier TC, Szillat KP, Akello JO, Álvarez-Rodríguez B, Martí-Carreras J. Preparedness needs research: How fundamental science and international collaboration accelerated the response to COVID-19. PLoS Pathog 2020; 16:e1008902. [PMID: 33035262 PMCID: PMC7546461 DOI: 10.1371/journal.ppat.1008902] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The first cluster of patients suffering from coronavirus disease 2019 (COVID-19) was identified on December 21, 2019, and as of July 29, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been linked with 664,333 deaths and number at least 16,932,996 worldwide. Unprecedented in global societal impact, the COVID-19 pandemic has tested local, national, and international preparedness for viral outbreaks to the limits. Just as it will be vital to identify missed opportunities and improve contingency planning for future outbreaks, we must also highlight key successes and build on them. Concomitant to the emergence of a novel viral disease, there is a 'research and development gap' that poses a threat to the overall pace and quality of outbreak response during its most crucial early phase. Here, we outline key components of an adequate research response to novel viral outbreaks using the example of SARS-CoV-2. We highlight the exceptional recent progress made in fundamental science, resulting in the fastest scientific response to a major infectious disease outbreak or pandemic. We underline the vital role of the international research community, from the implementation of diagnostics and contact tracing procedures to the collective search for vaccines and antiviral therapies, sustained by unique information sharing efforts.
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Affiliation(s)
- Cormac M. Kinsella
- Laboratory of Experimental Virology, Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
| | - Pauline Dianne Santos
- Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
| | - Ignacio Postigo-Hidalgo
- Charité –Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin, Germany
| | | | - Tim Casper Passchier
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
| | - Kevin P. Szillat
- Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany
| | - Joyce Odeke Akello
- Institute for Infectious Diseases, University of Bern, Bern, Switzerland
- Biology Division, Spiez Laboratory, Swiss Federal Office for Civil Protection, Spiez, Switzerland
- Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
| | - Beatriz Álvarez-Rodríguez
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
| | - Joan Martí-Carreras
- Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
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44
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Hurdiss DL, Drulyte I, Lang Y, Shamorkina TM, Pronker MF, van Kuppeveld FJM, Snijder J, de Groot RJ. Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans. Nat Commun 2020; 11:4646. [PMID: 32938911 PMCID: PMC7495468 DOI: 10.1038/s41467-020-18440-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Accepted: 08/21/2020] [Indexed: 01/23/2023] Open
Abstract
The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles contain two surface projections called spike (S) and haemagglutinin-esterase (HE), with S mediating receptor binding and membrane fusion, and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors, specifically 9-O-acetylated sialic acid. Here we present the cryo-EM structure of the ~80 kDa, heavily glycosylated HKU1 HE at 3.4 Å resolution. Comparison with existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the esterase domain left intact. Cryo-EM and mass spectrometry analysis reveals a putative glycan shield on the now redundant lectin domain. The findings further our insight into the evolution and host adaptation of human embecoviruses, and demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins.
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Affiliation(s)
- Daniel L Hurdiss
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CH, Utrecht, The Netherlands. .,Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
| | - Ieva Drulyte
- Materials and Structural Analysis, Thermo Fisher Scientific, Achtseweg Noord 5, Eindhoven, 5651 GG, The Netherlands
| | - Yifei Lang
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CH, Utrecht, The Netherlands
| | - Tatiana M Shamorkina
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Matti F Pronker
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Frank J M van Kuppeveld
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CH, Utrecht, The Netherlands
| | - Joost Snijder
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Raoul J de Groot
- Virology Section, Infectious Diseases and Immunology Division, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CH, Utrecht, The Netherlands.
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45
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Tekes G, Ehmann R, Boulant S, Stanifer ML. Development of Feline Ileum- and Colon-Derived Organoids and Their Potential Use to Support Feline Coronavirus Infection. Cells 2020; 9:E2085. [PMID: 32932592 PMCID: PMC7563363 DOI: 10.3390/cells9092085] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 09/10/2020] [Accepted: 09/10/2020] [Indexed: 12/29/2022] Open
Abstract
Feline coronaviruses (FCoVs) infect both wild and domestic cat populations world-wide. FCoVs present as two main biotypes: the mild feline enteric coronavirus (FECV) and the fatal feline infectious peritonitis virus (FIPV). FIPV develops through mutations from FECV during a persistence infection. So far, the molecular mechanism of FECV-persistence and contributing factors for FIPV development may not be studied, since field FECV isolates do not grow in available cell culture models. In this work, we aimed at establishing feline ileum and colon organoids that allow the propagation of field FECVs. We have determined the best methods to isolate, culture and passage feline ileum and colon organoids. Importantly, we have demonstrated using GFP-expressing recombinant field FECV that colon organoids are able to support infection of FECV, which were unable to infect traditional feline cell culture models. These organoids in combination with recombinant FECVs can now open the door to unravel the molecular mechanisms by which FECV can persist in the gut for a longer period of time and how transition to FIPV is achieved.
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Affiliation(s)
- Gergely Tekes
- Institute of Virology, Justus Liebig University Giessen, 35390 Giessen, Germany
| | - Rosina Ehmann
- Bundeswehr Institute of Microbiology, 80937 Munich, Germany;
| | - Steeve Boulant
- Department of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany;
- Research Group “Cellular Polarity and Viral Infection”, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Megan L. Stanifer
- Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
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46
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Resnick SJ, Iketani S, Hong SJ, Zask A, Liu H, Kim S, Melore S, Nair MS, Huang Y, Tay NE, Rovis T, Yang HW, Stockwell BR, Ho DD, Chavez A. A simplified cell-based assay to identify coronavirus 3CL protease inhibitors. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2020:2020.08.29.272864. [PMID: 32869020 PMCID: PMC7457602 DOI: 10.1101/2020.08.29.272864] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.
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Affiliation(s)
- Samuel J. Resnick
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
- Medical Scientist Training Program, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Sho Iketani
- Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 10032, USA
- Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Seo Jung Hong
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Arie Zask
- Department of Biological Sciences, Columbia University, New York, NY, 10027, USA
| | - Hengrui Liu
- Department of Chemistry, Columbia University, New York, NY, 10027, USA
| | - Sungsoo Kim
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Schuyler Melore
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Manoj S. Nair
- Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Yaoxing Huang
- Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Nicholas E.S. Tay
- Department of Chemistry, Columbia University, New York, NY, 10027, USA
| | - Tomislav Rovis
- Department of Chemistry, Columbia University, New York, NY, 10027, USA
| | - Hee Won Yang
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Brent R. Stockwell
- Department of Biological Sciences, Columbia University, New York, NY, 10027, USA
- Department of Chemistry, Columbia University, New York, NY, 10027, USA
| | - David D. Ho
- Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 10032, USA
| | - Alejandro Chavez
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 10032, USA
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Hao S, Ning K, Kuz CA, Vorhies K, Yan Z, Qiu J. Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2020. [PMID: 32869024 DOI: 10.1101/2020.08.27.271130] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 10 5 virions per cm 2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.
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48
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Zhu N, Wang W, Liu Z, Liang C, Wang W, Ye F, Huang B, Zhao L, Wang H, Zhou W, Deng Y, Mao L, Su C, Qiang G, Jiang T, Zhao J, Wu G, Song J, Tan W. Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells. Nat Commun 2020; 11:3910. [PMID: 32764693 PMCID: PMC7413383 DOI: 10.1038/s41467-020-17796-z] [Citation(s) in RCA: 237] [Impact Index Per Article: 47.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 07/15/2020] [Indexed: 12/28/2022] Open
Abstract
SARS-CoV-2, a β-coronavirus, has rapidly spread across the world, highlighting its high transmissibility, but the underlying morphogenesis and pathogenesis remain poorly understood. Here, we characterize the replication dynamics, cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison, HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion, apoptosis, destruction of epithelium integrity, cilium shrinking and beaded changes are observed in the plaque regions. Taken together, our results provide important insights into SARS-CoV-2 cell tropism, replication and morphogenesis.
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Affiliation(s)
- Na Zhu
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Wenling Wang
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Zhidong Liu
- Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University (Beijing Tuberculosis and Thoracic Tumor Research Institute), 101149, Beijing, China
| | - Chaoyang Liang
- Department of Thoracic Surgery, China-Japan Friendship Hospital, Yinghua East Road No. 2, Chaoyang District, 100029, Beijing, China
| | - Wen Wang
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Fei Ye
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Baoying Huang
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Li Zhao
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Huijuan Wang
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Weimin Zhou
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Yao Deng
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Longfei Mao
- Suzhou Institute of Systems Medicine, 215123, Suzhou, Jiangsu, China
| | - Chongyu Su
- Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University (Beijing Tuberculosis and Thoracic Tumor Research Institute), 101149, Beijing, China
| | - Guangliang Qiang
- Department of Thoracic Surgery, China-Japan Friendship Hospital, Yinghua East Road No. 2, Chaoyang District, 100029, Beijing, China
| | - Taijiao Jiang
- Suzhou Institute of Systems Medicine, 215123, Suzhou, Jiangsu, China
| | - Jincun Zhao
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, 510120, Guangzhou, China
- Institute of Infectious Disease, Guangzhou Eighth People's Hospital of Guangzhou Medical University, 510120, Guangzhou, China
| | - Guizhen Wu
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China
| | - Jingdong Song
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China.
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China.
| | - Wenjie Tan
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 102206, Beijing, China.
- Center for Biosafety Mega-Science, Chinese Academy of Sciences, 430071, Wuhan, China.
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Gorgulla C, Padmanabha Das KM, Leigh KE, Cespugli M, Fischer PD, Wang ZF, Tesseyre G, Pandita S, Shnapir A, Calderaio A, Gechev M, Rose A, Lewis N, Hutcheson C, Yaffe E, Luxenburg R, Herce HD, Durmaz V, Halazonetis TD, Fackeldey K, Patten JJ, Chuprina A, Dziuba I, Plekhova A, Moroz Y, Radchenko D, Tarkhanova O, Yavnyuk I, Gruber C, Yust R, Payne D, Näär AM, Namchuk MN, Davey RA, Wagner G, Kinney J, Arthanari H. A Multi-Pronged Approach Targeting SARS-CoV-2 Proteins Using Ultra-Large Virtual Screening. CHEMRXIV : THE PREPRINT SERVER FOR CHEMISTRY 2020:12682316. [PMID: 33200116 PMCID: PMC7668741 DOI: 10.26434/chemrxiv.12682316] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Revised: 07/24/2020] [Indexed: 11/23/2022]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019 novel coronavirus (2019-nCoV), has spread rapidly across the globe, creating an unparalleled global health burden and spurring a deepening economic crisis. As of July 7th, 2020, almost seven months into the outbreak, there are no approved vaccines and few treatments available. Developing drugs that target multiple points in the viral life cycle could serve as a strategy to tackle the current as well as future coronavirus pandemics. Here we leverage the power of our recently developed in silico screening platform, VirtualFlow, to identify inhibitors that target SARS-CoV-2. VirtualFlow is able to efficiently harness the power of computing clusters and cloud-based computing platforms to carry out ultra-large scale virtual screens. In this unprecedented structure-based multi-target virtual screening campaign, we have used VirtualFlow to screen an average of approximately 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets in the cloud. In addition to targeting the active sites of viral enzymes, we also target critical auxiliary sites such as functionally important protein-protein interaction interfaces. This multi-target approach not only increases the likelihood of finding a potent inhibitor, but could also help identify a collection of anti-coronavirus drugs that would retain efficacy in the face of viral mutation. Drugs belonging to different regimen classes could be combined to develop possible combination therapies, and top hits that bind at highly conserved sites would be potential candidates for further development as coronavirus drugs. Here, we present the top 200 in silico hits for each target site. While in-house experimental validation of some of these compounds is currently underway, we want to make this array of potential inhibitor candidates available to researchers worldwide in consideration of the pressing need for fast-tracked drug development.
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Affiliation(s)
- Christoph Gorgulla
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
- Department of Physics, Faculty of Arts and Sciences, Harvard University, Cambridge, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA
| | - Krishna M. Padmanabha Das
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA
| | | | | | - Patrick D. Fischer
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA
- Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany
| | - Zi-Fu Wang
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
| | | | | | | | | | | | | | | | | | | | | | - Henry D. Herce
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA
| | | | | | - Konstantin Fackeldey
- Zuse Institute Berlin (ZIB), Berlin, Germany
- Institute of Mathematics, Technical University Berlin, Berlin, Germany
| | - Justin J. Patten
- Department of Microbiology, Boston University Medical School, Boston University, Boston, USA
| | | | | | | | - Yurii Moroz
- Chemspace, Kyiv, Ukraine
- Taras Shevchenko National University of Kyiv, Ukraine
| | - Dmytro Radchenko
- Enamine, Kyiv, Ukraine
- Taras Shevchenko National University of Kyiv, Ukraine
| | | | | | - Christian Gruber
- Innophore GmbH, Graz, Austria
- Institute of Molecular Biosciences, University of Graz, Austria
| | | | | | - Anders M. Näär
- Department of Nutritional Sciences & Toxicology, University of California Berkeley, USA
| | - Mark N. Namchuk
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
| | - Robert A. Davey
- Department of Microbiology, Boston University Medical School, Boston University, Boston, USA
| | - Gerhard Wagner
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
| | | | - Haribabu Arthanari
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, Boston, USA
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, USA
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50
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Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium. J Virol 2020; 94:JVI.00957-20. [PMID: 32434888 PMCID: PMC7375387 DOI: 10.1128/jvi.00957-20] [Citation(s) in RCA: 45] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2020] [Accepted: 05/18/2020] [Indexed: 01/08/2023] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2. Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.
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