DataSheet_1_Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.pdf

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

当前新冠病毒病2019（COVID-19）大流行引发了对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）介导的发病机制的史无前例的研究努力。对于细胞类型特异性细胞病理学及其对细胞嗜性的影响，仍需进一步了解。此外，新型SARS-CoV-2突变对细胞嗜性、替代进入途径、共感染的影响以及沿呼吸道病毒复制动力学仍需在改进的模型中进行探索。大多数应用病毒学模型并不适合解决剩余问题，因为它们未能重现人类呼吸道组织的组织结构和细胞组成。本研究的总体目标是，从单个活检标本中建立一个人成干细胞来源的类器官模型，代表上呼吸道和肺部，并探讨该模型在研究呼吸道病毒感染中的适用性。首先，我们针对生长模式、组织结构和细胞组成以及功能特性对类器官模型进行了表征。接着，在细支气管和肺泡分化类器官中，确认了病毒进入受体的原位表达，包括与流感病毒相关的唾液酸以及SARS-CoV-2进入受体ACE2和TMPRSS2。我们进一步证明了类器官能够成功感染伪型流感A H7N1和H5N1病毒，并支持流感A H7N1病毒的病毒复制。最后，通过TCID50检测和免疫染色检测细胞内SARS-CoV-2特异性核壳体和双链RNA，确认了临床分离株SARS-CoV-2在类器官中的感染和复制。SARS-CoV-2感染后类器官组织中的显著合胞体形成与感染的人类组织原位发现相似。我们得出结论，所述的人类原位模型可能特别适用于病毒学研究，以评估宿主对感染的区域差异反应。该模型包含呼吸道沿线的各种细胞类型，表达呼吸道病毒进入因子，并支持流感病毒和SARS-CoV-2的成功感染和复制。因此，该模型可能作为相关且可靠的工具应用于病毒学，并有助于大流行预防和抗病毒策略的有效评估。