Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium
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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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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.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 34, 35, 36 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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