The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.06.17.156471: (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
   Blots were probed with mouse anti-HA primary antibody (Miltenyi Biotech) at 1:1,000 in PBS-Tween 20 (PBS-T, 0.1%) with 5% (w/v) milk powder overnight at 4°C.	anti-HA suggested: None
   Blots were washed in PBS-T and incubated with anti-mouse secondary antibody conjugated with horseradish peroxidase (Cell Signalling) at 1:1,000 in PBS-T for 1h at room temperature.	anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   A master stock of virus …

   SciScore for 10.1101/2020.06.17.156471: (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
   Blots were probed with mouse anti-HA primary antibody (Miltenyi Biotech) at 1:1,000 in PBS-Tween 20 (PBS-T, 0.1%) with 5% (w/v) milk powder overnight at 4°C.	anti-HA suggested: None
   Blots were washed in PBS-T and incubated with anti-mouse secondary antibody conjugated with horseradish peroxidase (Cell Signalling) at 1:1,000 in PBS-T for 1h at room temperature.	anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   A master stock of virus was passaged to P2 in Vero E6 at a MOI of 0.001 in DMEM/2% FBS and used for all virus assays, following a freeze-thaw cycle at −80°C.	Vero E6 suggested: RRID:CVCL_XD71)
   BHK-21 target cells were co-transfected with 500ng of different ACE2-expressing constructs (Sup.Table.2) and 250ng of rLuc-GFP 8-11 plasmid.	BHK-21 suggested: None
   Software and Algorithms
   Sentences	Resources
   Structural analysis: Molecular images were generated with an open source build of PyMOL (Schrödinger) using the crystal structure of SARS-CoV-2 RBD in complex with human ACE2 (PDB ID 6M0J) [10] that had been further refined by Dr Tristan Croll, University of Cambridge (https://twitter.com/CrollTristan/status/1240617555510919168).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   The selected sequences were aligned using MAFFT [43] and evolutionary conservation of amino acids was mapped onto the ACE2 structure using ConSurf [44].	MAFFT suggested: (MAFFT, RRID:SCR_011811)
   Phylogenetic analysis of the final dataset was inferred using the Neighbor-Joining method [45] conducted in MEGA7 [46] with ambiguous positions removed.	MEGA7 suggested: None
   Data handling and statistical analysis: GraphPad Prism v8.2.1 (	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   (GraphPad Software) was used for graphical and statistical analysis of data sets.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Flow cytometry data was analysed using FlowJo software v10.6.2 (BD).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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   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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2. Version published to 10.1371/journal.pbio.3001016

   Dec 21, 2020
3. 

   ScreenIT

   Jun 19, 2020

   SciScore for 10.1101/2020.06.17.156471: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   In the case of SARS-CoV-2, it is likely that in late 2019 the entire global population was immunologically naïve to this virus, although there is debate as to whether preexisting immunity to the endemic human-tropic coronaviruses, e.g. OC43 and HKU1, provides any cross-protective antibodies to help mitigate disease symptoms [	HKU1 suggested: None
   Blots were probed with mouse anti-HA primary antibody ( Miltenyi Biotech ) at 1:1,000 in PBS-Tween 20 ( PBS-T , 0.1 % ) with 5 % ( w/v ) milk powder overnight at 4°C .	…

   SciScore for 10.1101/2020.06.17.156471: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   In the case of SARS-CoV-2, it is likely that in late 2019 the entire global population was immunologically naïve to this virus, although there is debate as to whether preexisting immunity to the endemic human-tropic coronaviruses, e.g. OC43 and HKU1, provides any cross-protective antibodies to help mitigate disease symptoms [	HKU1 suggested: None
   Blots were probed with mouse anti-HA primary antibody ( Miltenyi Biotech ) at 1:1,000 in PBS-Tween 20 ( PBS-T , 0.1 % ) with 5 % ( w/v ) milk powder overnight at 4°C .	anti-HA suggested: None
   Blots were washed in PBS-T and incubated with anti-mouse secondary antibody conjugated with horseradish peroxidase ( Cell Signalling ) at 1:1,000 in PBS-T for 1h at room temperature.	anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Subsequent qPCR analysis of ACE2 mRNA levels in the whole panel of cell lines , assayed using a novel panel of species-specific ACE2 primers ( Sup.Table.4) , identified only two cell lines ( Vero E6 and Marc 145 ) with Ct values less than 25 , providing a strong correlative link between ACE2 receptor expression and successful virus infection .	Vero E6 suggested: None
   Accordingly , in the BHK-21 cells transfected with carrier plasmid we saw very little evidence for virus infection and/or virus production , confirming these cells do not natively support SARS-CoV-2 infection ( Fig.3C) .	BHK-21 suggested: None
   Cells used for entry studies or fusion assays: HEK293T cells stably expressing a split Renilla luciferase-GFP plasmid ( rLuc-GFP 1-7 ) and BHK-21 cells were maintained in DMEM-10 % : Dulbecco’s Modified Eagle’s Medium , DMEM ( Sigma-Aldrich ) supplemented with 10 % FBS ( Life Science Production) , 1 % 100mM sodium pyruvate ( Sigma-Aldrich) , 1 % 200mM LGlutamine ( Sigma-Aldrich) , and 1 % Penicillin/Streptomycin , 10,000U/mL ( Life Technologies Ltd)	HEK293T suggested: None
   (D) Similarly, DF-1 cells were transfected with a chicken ACE2 expression construct or a vector control (pDISPLAY) and infected at high MOI (1).	DF-1 suggested: CVCL_XF08
   Software and Algorithms
   Sentences	Resources
   The enhanced usage may arise from a salt bridge being formed between ferret ACE2 residue E325 and SARS-CoV R426 , which is not possible in SARS-CoV-2 where the equivalent residue is asparagine.	SARS-CoV-2 suggested: (Sino Biological Cat# 40143-R019, AB_2827973)
   Table.4 . Structural analysis Molecular images were generated with an open source build of PyMOL ( Schrödinger ) using the crystal structure of SARS-CoV-2 RBD in complex with human ACE2 ( PDB ID 6M0J ) [ 10 ] that had been further refined by Dr Tristan Croll , University of Cambridge ( https://twitter.com/CrollTristan/status/1240617555510919168).	PyMOL suggested: (PyMOL, SCR_000305)
   The selected sequences were aligned using MAFFT [ 43 ] and evolutionary conservation of amino acids was mapped onto the ACE2 structure using ConSurf [ 44] .	MAFFT suggested: (MAFFT, SCR_011811)
   Data handling and statistical analysis GraphPad Prism v8.2.1 (	GraphPad Prism suggested: (GraphPad Prism, SCR_002798)
   GraphPad Software ) was used for graphical and statistical analysis of data sets .	GraphPad suggested: (GraphPad Prism, SCR_002798)
   Flow cytometry data was analysed using FlowJo software v10.6.2 ( BD) .	FlowJo suggested: (FlowJo, SCR_008520)
   (A) A phylogenetic tree of ACE2 proteins assembled using the Neighbor-Joining method [45] conducted in MEGA7 [46] with ambiguous positions removed.	MEGA7 suggested: None
   (D) WebLogo [47] plots summarising the amino acid divergence within the mammalian and bird ACE2 sequences characterised in this study.	WebLogo suggested: (WEBLOGO, SCR_010236)

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   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 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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   About SciScore

   SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

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4. 

   Version published to 10.1101/2020.06.17.156471v1 on bioRxiv

   Jun 18, 2020