Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore.
Department of Otolaryngology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
PLoS Pathog. 2020 Dec 7;16(12):e1009130. doi: 10.1371/journal.ppat.1009130. eCollection 2020 Dec.
The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.
新型冠状病毒 SARS-CoV-2 是导致 2019 年冠状病毒病（COVID-19）的病原体，这是一场全球性的医疗保健和经济灾难。目前对宿主对 SARS-CoV-2 的免疫反应的了解仍处于起步阶段。2020 年 3 月，在新加坡分离到一种缺少 ORF8 的 382 个核苷酸缺失株（简称Δ382）。与感染野生型 SARS-CoV-2 相比，感染Δ382 的患者疾病症状较轻。在此，我们使用从健康鼻腔组织衍生的干细胞分化的鼻腔上皮细胞（NEC）建立了一个合适的体外模型，用于研究 SARS-CoV-2 介导的发病机制。用 SARS-CoV-2 或Δ382 感染 NEC 后，病毒颗粒仅从顶端释放，复制动力学相似。从感染的 NEC 细胞的基底外侧分泌筛选出的 49 种细胞因子的面板中，发现 CXCL10 是感染后唯一显著诱导的细胞因子，在野生型和Δ382 感染细胞中的水平相当。转录组分析显示，在感染过程中，不同的基因亚群呈现出时间依赖性上调，只有抗病毒信号通路在感染后 72 小时（hpi）才能检测到。与感染 H3N2 流感株相比，这种对 SARS-CoV-2 的免疫反应明显减弱，H3N2 感染后 8 hpi 会引发炎症反应，并在晚期时间点（hpi）引起更大幅度的抗病毒基因上调。值得注意的是，Δ382 在每个检测的感染后时间点诱导的宿主转录反应几乎与野生型 SARS-CoV-2 相同。与之前的结果一致，Δ382 感染的细胞中没有检测到 ORF8 映射的转录本，而其他 SARS-CoV-2 基因的表达则保持不变。我们的研究结果阐明了气道上皮细胞对 SARS-CoV-2 感染的反应，并证明 ORF8 在调节感染细胞的宿主基因表达和细胞因子产生方面起着非必需的作用。
