Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences

eLife

Dalia S Gala¹  Ilan Davis¹  Richard M Parton¹  Jeffrey Y Lee¹  Aino I Järvelin¹  Louisa Iselin¹  Joshua Titlow¹  Mary Kay Thompson¹  Alfredo Castello²  Marko Noerenberg²  Daniel Agranoff³  Natasha Palmalux⁴  Xiaodong Zhuang⁵  Jane A McKeating⁶  Peter AC Wing⁶  Tammie Bishop⁷  Maria Prange-Barczynska⁷  Alan Wainman⁸  William James⁹  Francisco J Salguero^1,10 

[1] Department of Biochemistry, The University of Oxford, Oxford, United Kingdom;Department of Biochemistry, The University of Oxford, Oxford, United Kingdom;MRC-University of Glasgow Centre for Virus Research, The University of Glasgow, Glasgow, United Kingdom;Department of Infectious Diseases, University Hospitals Sussex NHS Foundation Trust, Brighton, United Kingdom;MRC-University of Glasgow Centre for Virus Research, The University of Glasgow, Glasgow, United Kingdom;Nuffield Department of Medicine, The University of Oxford, Oxford, United Kingdom;Nuffield Department of Medicine, The University of Oxford, Oxford, United Kingdom;Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), The University of Oxford, Oxford, United Kingdom;Nuffield Department of Medicine, The University of Oxford, Oxford, United Kingdom;Ludwig Institute for Cancer Research, The University of Oxford, Oxford, United Kingdom;Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom;Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom;James & Lillian Martin Centre, Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom;UK Health Security Agency, UKHSA-Porton Down, Salisbury, United Kingdom;

关键词: COVID-19;    SARS-CoV-2;    variant of concern;    B.1.1.7;    single-molecule fluorescence in situ hybridisation;    early replication;    smFISH;    Human;    Viruses;   

DOI  :  10.7554/eLife.74153

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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, subgenomic 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.

【 授权许可】

CC BY