Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium
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Abstract
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 × 10 5 virions per cm 2 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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SciScore for 10.1101/2020.08.27.271130: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: Primary human bronchial epithelial cells were isolated from the lungs of healthy human donors by the Cells and Tissue Core of the Center for Gene Therapy, University of Iowa and the Department of Internal Medicine, University of Kansas Medical Center with the approvals of the Institutional Review Board (IRB) of the University of Iowa and University of Kansas Medical Center, Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Virus infection, sample collection and titration: Immunofluorescent confocal microscopy: Antibodies used: … SciScore for 10.1101/2020.08.27.271130: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: Primary human bronchial epithelial cells were isolated from the lungs of healthy human donors by the Cells and Tissue Core of the Center for Gene Therapy, University of Iowa and the Department of Internal Medicine, University of Kansas Medical Center with the approvals of the Institutional Review Board (IRB) of the University of Iowa and University of Kansas Medical Center, Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Virus infection, sample collection and titration: Immunofluorescent confocal microscopy: Antibodies used: Primary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100 anti-SARS-CoV-2 nucleocapsid ( NPsuggested: Noneanti-β-tubulin IVsuggested: NoneSoftware and Algorithms Sentences Resources Statistics: Virus released kinetics were determined with the means and standard deviations obtained from at least three independent HAE-ALIB3-20, B3-20, and B9-20 and from duplicated HAE-ALIL209 by using GraphPad Prism version 8.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Airway epithelium repair is critical for the maintenance of the barrier function and the limitation of airway hyperreactivity. In a biopsy study of fresh tracheas and lungs from five deceased COVID-19 patients, it was found that the epithelium was severely damaged in some parts of the trachea, and extensive basal cell proliferation was observed in the trachea, where ciliated cells were damaged, as well as in the intrapulmonary airways 37. These data support our conclusion that basal cells are not permissive to SARS-CoV-2. As a response to these previous findings, our study observed that a subset of proliferating basal cells in the SARS-CoV-2 infected HAE-ALI, but not in the mock infected HAE-ALI (Fig. 8B). Thus, we hypothesize that SARS-CoV-2 infection induces basal cell proliferation, which accounts for the observed long-lasting infections with recurrent peaks of viral replication, which warrants future investigation. Overall, we propose a model of SARS-CoV-2-infection of HAE (Fig. 8C): SARS-CoV-2 selectively infects ciliated and goblet cells on the surface of the airway lumen (the apical side of HAE). Upon invading these cells, SARS-CoV-2 replicates and produces infectious virions, and eventually leads to the cell death and epithelial damage. Upon the destructive lesions, airway epithelium has the capacity to progressively repair and regenerate itself. Thus, basal cells (possibly also including club cells) proliferate and differentiate to ciliated cells or goblet cells to f...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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