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

View ORCID ProfileJeffrey Y Lee, View ORCID ProfilePeter AC Wing, View ORCID ProfileDalia S Gala, View ORCID ProfileAino I Järvelin, View ORCID ProfileJosh Titlow, View ORCID ProfileMarko Noerenberg, Xiaodong Zhuang, Natasha Johnson, Louise Iselin, Mary Kay Thompson, Richard M Parton, Alan Wainman, Daniel Agranoff, William James, View ORCID ProfileAlfredo Castello, View ORCID ProfileJane A McKeating, View ORCID ProfileIlan Davis
doi: https://doi.org/10.1101/2021.06.29.450133
Jeffrey Y Lee
1Department of Biochemistry, The University of Oxford, UK.
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Peter AC Wing
2Nuffield Department of Medicine, University of Oxford, Oxford, UK.
3Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK.
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Dalia S Gala
1Department of Biochemistry, The University of Oxford, UK.
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Aino I Järvelin
1Department of Biochemistry, The University of Oxford, UK.
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Josh Titlow
1Department of Biochemistry, The University of Oxford, UK.
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Marko Noerenberg
4MRC-University of Glasgow Centre for Virus Research, The University of Glasgow, UK.
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Xiaodong Zhuang
2Nuffield Department of Medicine, University of Oxford, Oxford, UK.
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Natasha Johnson
4MRC-University of Glasgow Centre for Virus Research, The University of Glasgow, UK.
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Louise Iselin
1Department of Biochemistry, The University of Oxford, UK.
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Mary Kay Thompson
1Department of Biochemistry, The University of Oxford, UK.
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Richard M Parton
1Department of Biochemistry, The University of Oxford, UK.
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Alan Wainman
5Sir William Dunn School of Pathology, University of Oxford, UK.
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Daniel Agranoff
6Brighton and Sussex University Hospitals NHS Trust, UK.
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William James
5Sir William Dunn School of Pathology, University of Oxford, UK.
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Alfredo Castello
1Department of Biochemistry, The University of Oxford, UK.
4MRC-University of Glasgow Centre for Virus Research, The University of Glasgow, UK.
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  • For correspondence: Ilan.davis@bioch.ox.ac.uk Jane.mckeating@ndm.ox.ac.uk alfredo.castello@glasgow.ac.uk
Jane A McKeating
2Nuffield Department of Medicine, University of Oxford, Oxford, UK.
3Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK.
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  • For correspondence: Ilan.davis@bioch.ox.ac.uk Jane.mckeating@ndm.ox.ac.uk alfredo.castello@glasgow.ac.uk
Ilan Davis
1Department of Biochemistry, The University of Oxford, UK.
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  • For correspondence: Ilan.davis@bioch.ox.ac.uk Jane.mckeating@ndm.ox.ac.uk alfredo.castello@glasgow.ac.uk
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Summary

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 Alpha 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.

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In brief By detecting nearly all individual SARS-CoV-2 RNA molecules we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain.

Highlights

  • Single molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics

  • There is substantial heterogeneity between cells in rates of SARS-CoV-2 replication

  • Genomic RNA is stable and persistent during the initial stages of infection

  • Alpha (B.1.1.7) variant of concern replicates more slowly than the Victoria strain

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 29, 2021.
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Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences
Jeffrey Y Lee, Peter AC Wing, Dalia S Gala, Aino I Järvelin, Josh Titlow, Marko Noerenberg, Xiaodong Zhuang, Natasha Johnson, Louise Iselin, Mary Kay Thompson, Richard M Parton, Alan Wainman, Daniel Agranoff, William James, Alfredo Castello, Jane A McKeating, Ilan Davis
bioRxiv 2021.06.29.450133; doi: https://doi.org/10.1101/2021.06.29.450133
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Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences
Jeffrey Y Lee, Peter AC Wing, Dalia S Gala, Aino I Järvelin, Josh Titlow, Marko Noerenberg, Xiaodong Zhuang, Natasha Johnson, Louise Iselin, Mary Kay Thompson, Richard M Parton, Alan Wainman, Daniel Agranoff, William James, Alfredo Castello, Jane A McKeating, Ilan Davis
bioRxiv 2021.06.29.450133; doi: https://doi.org/10.1101/2021.06.29.450133

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