Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.
Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.
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SARS-COV-2和382-NT缺失分离株缺乏ORF8的人类鼻上皮细胞的感染揭示了类似的病毒动力学和宿主的转录谱。
DOI：
10.1371/journal.ppat.1009130
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发表时间：
2020-12
期刊：
影响因子：
6.7
通讯作者：
Wang LF
中科院分区：
文献类型：
--
作者：
Gamage AM;Tan KS;Chan WOY;Liu J;Tan CW;Ong YK;Thong M;Andiappan AK;Anderson DE;Wang Y;Wang LF
新型冠状病毒SARS-CoV-2是2019冠状病毒病（COVID-19）的病原体，这是一场全球医疗和经济灾难。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株缺乏ORF8的382-nt缺失菌株（Δ382）。与感染野生型SARS-CoV-2相比，感染Δ382与患者病情较轻相关。在这里，我们建立了从健康鼻组织来源的干细胞分化的鼻上皮细胞（NECs）作为离体研究SARS-CoV-2介导的发病机制的合适模型。感染SARS-CoV-2或Δ382的NECs导致病毒颗粒仅从顶端侧释放，复制动力学相似。对感染NECs基底外侧分泌的49种细胞因子进行筛选，发现CXCL10是唯一在感染后显著诱导的细胞因子，在野生型和Δ382感染细胞中水平相当。转录组分析显示，感染期间不同基因亚群的时间上调，抗病毒信号通路仅在较晚的时间点（感染后72小时，hpi）检测到。与感染流感毒株H3N2相比，这种对SARS-CoV-2的免疫反应明显减弱，后者在8 hpi内引起炎症反应，并且在较晚的时间点抗病毒基因上调幅度更大。值得注意的是，Δ382在检测的每个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。根据先前的结果，Δ382感染细胞显示缺乏ORF8转录本，并且其他SARS-CoV-2基因保守表达。我们的研究结果揭示了呼吸道上皮对SARS-CoV-2感染的反应，并证明ORF8在调节宿主基因表达和感染细胞的细胞因子产生方面的非必要作用。气道上皮细胞是COVID-19患者感染最早的细胞类型之一。我们发现分化的nec是体外研究SARS-CoV-2感染动力学的合适模型，并且感染的nec的免疫反应令人惊讶地有限。这种对SARS-CoV-2感染的有限早期反应可能会损害病毒清除，并延长感染的持续时间。这进一步表明浸润性免疫细胞可能是促炎细胞因子如IL-6和TNFα的来源，据报道，在感染后期患者血清中IL-6和TNFα升高。CXCL10的产生可能是限制免疫细胞浸润和随后细胞因子产生的主要治疗节点。SARS-CoV-2和Δ382体外感染在宿主反应上的相似性突出了冠状病毒基因组的可塑性，并暗示ORF8在调节宿主反应中的翻译后（而不是转录）作用。
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. Airway epithelial cells are one of the earliest cell types infected in COVID-19 patients. We show that differentiated NECs are a suitable model for studying the dynamics of SARS-CoV-2 infection in vitro, and that the immune response from infected NECs is surprisingly limited. This limited early response to SARS-CoV-2 infection could impair viral clearance, and prolong the duration of infection. This further implies that infiltrating immune cells are the likely source of pro-inflammatory cytokines such as IL-6 and TNFα reported to be elevated in patient sera later during infection. CXCL10 production could represent a major therapeutic node for limiting immune cell infiltration and subsequent cytokine production. The similarities in host-response between SARS-CoV-2 and Δ382 infection in-vitro highlight the plasticity of CoV genomes, and implicate a post-translational (and not a transcriptional) role for ORF8 in modulating the host-response.
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