Disparate temperature-dependent virus–host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium

Philip V’kovski
Mitra Gultom
Jenna N. Kelly
Silvio Steiner
Julie Russeil
Bastien Mangeat
Elisa Cora
Joern Pezoldt
Melle Holwerda
Annika Kratzel
Laura Laloli
Manon Wider
Jasmine Portmann
Thao Tran
Nadine Ebert
Hanspeter Stalder
Rune Hartmann
Vincent Gardeux
Daniel Alpern
Bart Deplancke
Volker Thiel
Ronald Dijkman

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Abstract

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus–host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.

Version published to 10.1371/journal.pbio.3001158
Mar 29, 2021

SciScore for 10.1101/2020.04.27.062315: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
To detect SARS-CoV and SARS-CoV-2, hAEC cultures were immunostained with a rabbit polyclonal antibody against SARS-CoV Nucleocapsid protein (Rockland, 200-401-A50), which also cross-react with SARS-CoV-2.	SARS-CoV-2 suggested: None antibody against SARS-CoV Nucleocapsid protein ( Rockland , 200-401-A50) suggested: None
Cell distribution of ACE2 were detected with a rabbit polyclonal antibody against ACE2 (ab15348, Abcam).	ACE2 suggested: None
488-labeled donkey …

SciScore for 10.1101/2020.04.27.062315: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
To detect SARS-CoV and SARS-CoV-2, hAEC cultures were immunostained with a rabbit polyclonal antibody against SARS-CoV Nucleocapsid protein (Rockland, 200-401-A50), which also cross-react with SARS-CoV-2.	SARS-CoV-2 suggested: None antibody against SARS-CoV Nucleocapsid protein ( Rockland , 200-401-A50) suggested: None
Cell distribution of ACE2 were detected with a rabbit polyclonal antibody against ACE2 (ab15348, Abcam).	ACE2 suggested: None
488-labeled donkey anti-rabbit IgG (H + L) (Jackson Immunoresearch) was used as secondary antibody.	488-labeled donkey anti-rabbit IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cells and human airway epithelial cell (hAEC) cultures: Vero-E6 cells (kindly provided by Doreen Muth, Marcel Müller, and Christian Drosten, Charité, Berlin, Germany) were propagated in Dulbecco’s Modified Eagle Medium-GlutaMAX supplemented with 1 mM sodium pyruvate, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 IU/ml penicillin, 1% (w/v) non-essential amino acids and 15 mM HEPES (Gibco).	Vero-E6 suggested: None
Viruses: SARS-CoV strain Frankfurt-1 (GenBank FJ429166) 35,54 and SARS-CoV-2 (SARS-CoV-2/München-1.1/2020/929) 55 were kindly provided by Daniela Niemeyer, Marcel Müller, and Christian Drosten, and propagated and titrated on Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
Software and Algorithms
Sentences	Resources
All images were processed using FIJI software packages 57	FIJI suggested: (Fiji, RRID:SCR_002285)
The sequencing reads were demultiplexed using the BRB-seqTools suite and were aligned against a concatenation of the human gene annotation of the human genome (hg38), SARS coronavirus Frankfurt 1 (AY291315) and SARS-CoV-2/Wuhan-Hu1/2020 (NC_045512) viral genomes using STAR and HTSeq for producing the count matrices.	STAR suggested: (STAR, RRID:SCR_015899) HTSeq suggested: (HTSeq, RRID:SCR_005514)
ComBat-seq was used with default settings to adjust for batch effects in the raw data and generate an adjusted count matrix used for downstream analyses 60 Library normalization and expression differences between uninfected and virus-infected samples were then quantified using the DESeq2 package in R (version 1.28) with a fold change (FC) cut-off of ≥ 1.5 and a False Discovery Rate (FDR) of ≤ 0.1 61.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
Venn diagrams were generated using the VennDiagram package in R for DEGs identified in approach 2 (Fig. 3a) and approach 1 (Supplementary Figure 3) 62	VennDiagram suggested: (VennDiagram, RRID:SCR_002414)
Pathway enrichment analysis was performed using the clusterProfiler and ReactomePA packages in R 63,64.	clusterProfiler suggested: (clusterProfiler, RRID:SCR_016884)
Additional data analysis and visualization was performed using a variety of packages in R, including ComplexHeatmap and ggplot2.65.	ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270)

Results from OddPub: Thank you for sharing your code and data.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Version published to 10.1101/2020.04.27.062315v2 on bioRxiv
Dec 18, 2020

SciScore for 10.1101/2020.04.27.062315: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

not detected.

Randomization

not detected.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

not detected.

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies
Sentences	Resources
To detect SARS-CoV and SARS-CoV-2 , hAEC cultures were immunostained with a rabbit polyclonal antibody against SARS-CoV nucleocapsid protein ( Rockland , 200-401-A50) , which also cross-react with SARS-CoV-2 .	SARS-CoV-2 suggested: (Sino Biological Cat# 40143-R019, AB_2827973) `<div style="margin-bottom:8px"> <div><b>SARS-CoV nucleocapsid protein ( Rockland , 200-401-A50)</b></div>`

SciScore for 10.1101/2020.04.27.062315: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

not detected.

Randomization

not detected.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

not detected.

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies

Sentences Resources

To detect SARS-CoV and SARS-CoV-2 , hAEC cultures were immunostained with a rabbit polyclonal antibody against SARS-CoV nucleocapsid protein ( Rockland , 200-401-A50) , which also cross-react with SARS-CoV-2 .

SARS-CoV-2

suggested: (Sino Biological Cat# 40143-R019, AB_2827973)

      <div style="margin-bottom:8px">
        <div><b>SARS-CoV nucleocapsid protein ( Rockland , 200-401-A50)</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell distribution of ACE2 were detected with a rabbit polyclonal antibody against ACE2 ( ab15348 , Abcam) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">488-labeled donkey anti-rabbit IgG ( H + L ) ( Jackson Immunoresearch ) was used as secondary antibody .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>488-labeled donkey anti-rabbit IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and human airway epithelial cell (hAEC) culturesVero-E6 cells (kindly provided by Doreen Muth, Marcel Müller, and Christian Drosten, Charité, Berlin, Germany) were propagated in Dulbecco’s Modified Eagle Medium-GlutaMAX supplemented with 1 mM sodium pyruvate, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 mg/ml streptomycin, 100 IU/ml penicillin, 1% (w/v) non-essential amino acids and 15 mM HEPES (Gibco).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>culturesVero-E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Titration of apical and basolateral compartmentsViruses released in the apical or basolateral compartments were titrated by plaque assay on Vero-E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero-E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All images were processed using Fiji software packages 47</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Fiji</b></div>
        <div>suggested: (Fiji, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002285">SCR_002285</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequencing reads were demultiplexed using the BRB-seqTools suite 30, and were aligned against a concatenation of the human gene annotation of the human genome (hg38), SARS coronavirus Frankfurt 1 (AY291315) and SARS-CoV-2/Wuhan-Hu1/2020 (NC_045512) viral genomes using STAR and HTSeq for producing the count matrices.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>STAR</b></div>
        <div>suggested: (STAR, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015899">SCR_015899</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HTSeq</b></div>
        <div>suggested: (HTSeq, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005514">SCR_005514</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Library normalization and expression differences between samples were quantified using the DESeq2 package, with a cut-off of fold change (FC) ≥ 1.5 and False Discovery Rate (FDR) ≤ 0.05.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>DESeq2</b></div>
        <div>suggested: (DESeq, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000154">SCR_000154</a>)</div>
      </div>
    </td></tr></table>

Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Version published to 10.1101/2020.04.27.062315v1 on bioRxiv
Apr 27, 2020

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