Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State Universitygrid.34421.30, Ames, Iowa, USA.
Microbiol Spectr. 2022 Aug 31;10(4):e0163922. doi: 10.1128/spectrum.01639-22. Epub 2022 Jul 12.
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 . 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. 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 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.
人类冠状病毒 NL63（HCoV-NL63）通常与婴儿的轻度呼吸道感染有关，呼吸道上皮细胞是该病毒感染和初始复制的主要靶标。标准永生化细胞对 HCoV-NL63 高度允许，它们通常用于从临床标本中分离和繁殖病毒。然而，这些细胞系不是病毒的天然细胞靶标，并且缺乏足够的复杂性来模拟自然感染过程。本研究比较评估了悬浮单层培养的 LLC-MK2 和原代人呼吸道上皮细胞（HRECs）以及呼吸道细胞的器官型气道培养物（ALI-HRECs）对 HCoV-NL63 感染的易感性和病毒复制效率的差异。通过细胞病变效应的形态学检查、免疫荧光、逆转录定量实时 PCR 和流式细胞术评估病毒的有效感染和生长动力学。结果显示，LLC-MK2 细胞对 HCoV-NL63 的感染和复制具有更高的易感性，其次是 ALI-HRECs，而在 HRECs 中，易感性很低，没有明显的病毒复制。这种易感性与 LLC-MK2、ALI-HRECs 和 HRECs 中血管紧张素转换酶 2（ACE2）受体蛋白的表达水平有关。值得注意的是，器官型 ALI-HREC 培养物表达的 ACE2 受体蛋白明显更多，并且比 HREC 的单层培养物更易感染 HCoV-NL63。因此，ACE2 受体是对 HCoV-NL63 感染和复制的易感性的关键因素，感染研究中使用的培养类型也是如此。HCoV-NL63 在全球广泛传播，是儿童和成人呼吸道感染的重要原因。HCoV-NL63 通过 ACE2 受体进入呼吸道上皮细胞，这是严重急性呼吸综合征冠状病毒（SARS-CoV）和 SARS-CoV-2 所使用的相同细胞受体。因此，HCoV-NL63 被认为是研究 SARS 样冠状病毒疾病机制和治疗干预的安全替代物，同时在 BSL-2 条件下进行。本研究不仅表明 ACE2 对 HCoV-NL63 感染和复制的有效性至关重要，还表明需要更精细和复杂的器官型模型来复制空气-液体呼吸道上皮细胞组成、结构和功能的代表。这些培养物拓宽了病毒学研究，以提高我们对冠状病毒如何引起疾病和传播的理解，不仅在人类中，而且在动物群体中。
