Profilling of viral and host transcriptome in SARS-CoV-2 infected A549-ACE2 cells with VIC or B.1.1.7 strains.

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Overall design: Ribosome-depleted total RNA-seq of A549-ACE2 samples infected with either VIC or B.1.1.7 (Alpha) strain of SARS-CoV-2 at timepoints 2, 8 and 24 hours post infection (hpi). 3 Replicates each.

尽管全球针对严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）开展了前所未有的研究，但病毒早期复制事件仍未得到充分解析。鉴于传播能力增强的新型病毒变异株具有重要临床价值，深入解析病毒复制与转录的早期阶段已成为迫切需求。我们采用单分子荧光原位杂交（single molecule fluorescence in situ hybridisation，smFISH）技术，以95%的检测效率对正链RNA基因组进行定量分析，同时可同步可视化负链RNA基因组、亚基因组RNA及病毒蛋白。通过对病毒RNA及复制工厂的绝对定量分析，我们发现SARS-CoV-2基因组RNA在侵入宿主细胞后可长期存留，这提示其能够规避细胞核酸酶的降解作用。此外，我们观察到SARS-CoV-2的复制在不同细胞间存在显著异质性，仅少量细胞群体呈现高病毒RNA载量。出乎意料的是，最早在英国被鉴定的B.1.1.7（Alpha）变异株，其复制动力学显著慢于维多利亚（VIC）毒株，这暗示了一种助力其更高传播能力的全新机制，且该机制具有重要临床意义。实验整体设计：对分别感染SARS-CoV-2维多利亚（VIC）毒株或B.1.1.7（Alpha）毒株的A549-ACE2细胞样本，在感染后2、8及24小时（hpi）进行核糖体去除总RNA测序，每组设置3次生物学重复。