Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway
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Abstract
SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.
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Alina Chan
Review 1: "Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway"
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Alina Chan
Review of "Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway"
Reviewer: Alina Chan (Broad Institute) | 📗📗📗📗◻️
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SciScore for 10.1101/2021.08.20.456972: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Animal research was carried out under a United Kingdom Home Office License, P48DAD9B4.
IRB: Ethical approval was approved by the South Central-Berkshire B Research Ethic Committee (REC ref: 20/SC/0206; IRAS 283805).Sex as a biological variable Outbred female ferrets (16–20 weeks old) weighing 750–1,000 g were used. Randomization The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Blinding The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Power Analysis No statistical method was used to predetermine sample size. Cell … SciScore for 10.1101/2021.08.20.456972: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Animal research was carried out under a United Kingdom Home Office License, P48DAD9B4.
IRB: Ethical approval was approved by the South Central-Berkshire B Research Ethic Committee (REC ref: 20/SC/0206; IRAS 283805).Sex as a biological variable Outbred female ferrets (16–20 weeks old) weighing 750–1,000 g were used. Randomization The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Blinding The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Power Analysis No statistical method was used to predetermine sample size. Cell Line Authentication Contamination: Cell lines were not tested for mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources Cells were permeabilised in methanol 0.6% H2O2 and stained for 1 h with an antibody against SARS-CoV-2 nucleocapsid protein (Sino Biological; 40143-R019, 1:300 dilution). SARS-CoV-2 nucleocapsid proteinsuggested: NoneCells were further stained with the secondary antibody anti-rabbit HRP conjugate (Sigma; 1:3000 dilution) for 1 h. anti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources Human embryonic kidney cells (293T; ATCC; ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco), 10% fetal calf serum (FCS), 293Tsuggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)Virus titration was performed by median tissue culture infectious dose (TCID50) on Vero cells as described previously 3. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Approximately 2.5 μg of the in vitro synthesized RNA was used to transfect ∼6 ×105 BHK-hACE2-N cells stably expressing the SARS-CoV-2 N and the human ACE2 genes 45 using the MessengerMax lipofection kit (Thermo Scientific) as per the manufacturer’s instructions. BHK-hACE2-Nsuggested: NoneCells were then incubated until signs of viral replication (syncytia formation) became visible (usually after 2-3 days), at which time the medium was collected (P0 stock) and used further as a source of rescued virus to infect VERO E6 cells to generate P1 and P2 stocks. VERO E6suggested: RRID:CVCL_XD71)For the CPE-based neutralisation assay (reverse genetics virus vs Pfizer antisera), serial dilutions of sera were incubated with 100 TCID50 of virus for 1 h at 37°C, 5% CO2 in 96 well plates before a suspension of Vero-ACE2-TMPRSS2 cells were added and incubated for 3 days at 37°C, 5% CO2. Vero-ACE2-TMPRSS2suggested: NoneBriefly, BHK-21 cells were transfected with 500 ng of ACE2 or empty vector (pDISPLAY) using TransIT-X2 ( BHK-21suggested: NoneRecombinant DNA Sentences Resources Briefly, a set of overlapping cDNA fragments representing the entire genome of SARS-CoV-2 Wuhan isolate (GenBank: MN908947.3) were chemically synthesized and cloned into pUC57-Kan (Bio Basic Canada Inc). pUC57-Kansuggested: RRID:Addgene_123653)The cDNA fragment representing the 5’ terminus of the viral genome contained the bacteriophage T7 RNA polymerase promoter preceded by a short sequence stretch homologous to the XhoI-cut end of the TAR in yeast vector pEB2 44. pEB2suggested: RRID:Addgene_104001)Plasmids and cloning: Lentiviral packaging constructs pCSLW and pCAGGs-GAGPOL were made as previously described. pCSLWsuggested: NoneAt ICL, 100 mm dishes of 293Ts were transfected using lipofectamine 3000 (Thermo) with a mixture of pCSFLW, pCAGGS-GAGPOL and spike proteins expressed in pcDNA3.1. pCSFLWsuggested: NonepCAGGS-GAGPOLsuggested: NoneCells were transfected using polyethyleneimine (PEI) with a mixture of pCSFLW, p8.91 and SARS-CoV-2 spikes expressed in pcDNA3.1. p8.91suggested: NonepcDNA3.1suggested: RRID:Addgene_79663)Briefly, BHK-21 cells were transfected with 500 ng of ACE2 or empty vector (pDISPLAY) using TransIT-X2 ( pDISPLAYsuggested: RRID:Addgene_51053)Software and Algorithms Sentences Resources Viruses were sequenced using Oxford Nanopore as previously described 46. Oxford Nanoporesuggested: (Oxford Nanopore Technologies, RRID:SCR_003756)The fastq files produced by Nanopore sequencing were filtered with lengths 400 and 700 using Artic-ncov2019 pipeline v1.2.1 (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.htμl) by “artic guppyplex” function. Artic-ncov2019suggested: NoneThese primer trimmed bam files were further analysed using DiversiTools (http://josephhughes.github.io/btctools/) with the “-orfs” function to generate the ratio of amino acid change in the reads and coverage at each site of protein in comparison to the reference SARS-CoV-2 genome (MN908947.3) as we previous description 53. DiversiToolssuggested: NoneSequences were aligned to the Wuhan-Hu-1 reference genome sequence (MN908947) 54 using MAFFT v7.475 55 and the alignment was then checked manually. MAFFTsuggested: (MAFFT, RRID:SCR_011811)Phylogenetic analysis was performed using the remaining 936 mink genomes rooted on the Wuhan-Hu-1 reference genome (MN908947) with a general time reversible model of nucleotide substitution, a proportion of invariant sites estimated from the data and a gamma distribution describing among-site rate variation (GTR + / + Γ) built using RAxML v8.0.0 58 with the phylogeny rooted on the sequence of the virus Wuhan-Hu-1. RAxMLsuggested: (RAxML, RRID:SCR_006086)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
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.
Results from scite Reference Check: We found no unreliable references.
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