Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization

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1. Version published to 10.1128/spectrum.01789-21

   Jun 29, 2022
2. 

   ScreenIT

   Aug 28, 2021

   SciScore for 10.1101/2021.08.25.457627: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Animal care and ethics statement: All animal experiments were conducted in animal biosafety level 3 (BSL3) facilities at the Biosecurity Research Institute at Kansas State University according to protocols approved by the Institutional Animal Care and Use Committee at Kansas State University and the guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-SARS-CoV-2 antibodies from humans, cats, and rabbits: …

   SciScore for 10.1101/2021.08.25.457627: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Animal care and ethics statement: All animal experiments were conducted in animal biosafety level 3 (BSL3) facilities at the Biosecurity Research Institute at Kansas State University according to protocols approved by the Institutional Animal Care and Use Committee at Kansas State University and the guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti-SARS-CoV-2 antibodies from humans, cats, and rabbits: Convalescent sera (Lotus 11 and 25) from COVID-19 patients were obtained from Dr. Thomas Rogers from the Scripps Research Institute, San Diego, CA, USA. Cat sera (Cat 247 and 903) were collected from cats enrolled in SARS-CoV-2 re-infection studies [89]	Anti-SARS-CoV-2 suggested: None
   Expression of each species’ ACE2 receptor in the cells was confirmed by Western Blot analysis using antibody to human ACE2 (Abcam, Waltham, MA)	human ACE2 suggested: (Abcam Cat# 3149-1, RRID:AB_2242331)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells and plasmids: Human embryonic kidney 293 (HEK293), the Crandell-Rees feline kidney (CRFK) and Calu-3 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA).	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
   Vero E6 cells expressing human TMPRSS2 (Vero-TMPRSS2) cells were obtained from Creative Biogene (Shirley, NY)[88].	Vero E6 suggested: RRID:CVCL_XD71)
   Generation of SARS-CoV-2 S pseudotyped viruses: The 2nd generation lentiviral packaging plasmid, psPAX2 (Addgene), a reporter plasmid pUCGFP-Luc (Addgene), and parental or mutant pAbVec-SARS2-S were transfected into HEK293 cells to produce pseudotyped viruses.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   The SA/KRISP-K005325/2020 stock was subsequently passaged on Calu3 cells and NGS results showed this stock to contain only 13% of the furin site mutation; this stock was used for the in vitro virus replication kinetic experiments.	Calu3 suggested: None
   Inoculum (defined as 0 hpi) and the time point collected supernatants were then titrated on Vero-TMPRSS2 cells.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Nasal wash and lung homogenates were filtered through a 0.2 μm filter prior to virus titration on Vero E6-TMPRSS2 cells.	Vero E6-TMPRSS2 suggested: None
   Recombinant DNA
   Sentences	Resources
   The codon-optimized cDNAs of the open reading frame of the human or animal ACE2 gene with FLAG tag were synthesized by Integrated DNA Technologies (Coralville, IA) and cloned into pIRES-Neo3 (Takara Bio, Mountain View, CA).	pIRES-Neo3 suggested: None
   Pseudotyped viruses expressing SARS-CoV-2 S protein were generated by synthesizing the S gene which was truncated by 26 amino acids at the C-terminus, fused with HA tag by Integrated DNA Technologies, and cloned into plasmid pAbVec1 (Addgene, Watertown, MA), which were designated as pAbVec-SARS2-S.	pAbVec1 suggested: None
   Generation of CRFK cells stably expressing human or animal ACE2: CRFK cells, plated the previous day, were transfected with pIRES-Neo-human (or cat, dog, cattle, horse, camel, hamster, rabbit, mink, white-tailed deer) ACE2-FLAG.	pIRES-Neo-human suggested: None
   Generation of SARS-CoV-2 S pseudotyped viruses: The 2nd generation lentiviral packaging plasmid, psPAX2 (Addgene), a reporter plasmid pUCGFP-Luc (Addgene), and parental or mutant pAbVec-SARS2-S were transfected into HEK293 cells to produce pseudotyped viruses.	psPAX2 suggested: RRID:Addgene_12260) pUCGFP-Luc suggested: None pAbVec-SARS2-S suggested: None
   Software and Algorithms
   Sentences	Resources
   The SA/KRISP-K005325/2020 stock was subsequently passaged on Calu3 cells and NGS results showed this stock to contain only 13% of the furin site mutation; this stock was used for the in vitro virus replication kinetic experiments.	NGS suggested: (PM4NGS, RRID:SCR_019164)
   Structures were analyzed and images created using PyMOL [92].	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Statistical analysis: Statistical analysis was performed using GraphPad Prism Software version 6 (San Diego, CA).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.08.25.457627v1 on bioRxiv

   Aug 25, 2021