The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype

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1. Version published to 10.1038/s41467-022-33632-y

   Oct 7, 2022
2. 

   ScreenIT

   Apr 30, 2022

   SciScore for 10.1101/2022.04.28.489537: (What is this?)

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

   Table 1: Rigor

   Ethics

   IRB: Ethics statements for human subjects and animal experimentation: All ferret and hamster experiments were evaluated by the responsible ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg–Western Pomerania (LALLF M-V) and gained governmental approval under registration number LVL MV TSD/7221.3-1-004/21. Field Sample Permit: Mouse studies were approved by the Commission for Animal Experimentation of the Cantonal Veterinary Office of Bern and conducted in compliance with the Swiss Animal Welfare legislation and under license BE43/20. IACUC: Mouse studies were approved by the Commission for Animal Experimentation of the Cantonal Veterinary Office of …

   SciScore for 10.1101/2022.04.28.489537: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Ethics statements for human subjects and animal experimentation: All ferret and hamster experiments were evaluated by the responsible ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg–Western Pomerania (LALLF M-V) and gained governmental approval under registration number LVL MV TSD/7221.3-1-004/21. Field Sample Permit: Mouse studies were approved by the Commission for Animal Experimentation of the Cantonal Veterinary Office of Bern and conducted in compliance with the Swiss Animal Welfare legislation and under license BE43/20. IACUC: Mouse studies were approved by the Commission for Animal Experimentation of the Cantonal Veterinary Office of Bern and conducted in compliance with the Swiss Animal Welfare legislation and under license BE43/20. Consent: Written informed consent was obtained for all the patients and the study protocols were approved by the respective local Ethics Commissions (KEK-BE_2018-01801, EKSG 11/044, and EKSG 11/103). Euthanasia Agents: Nasal washing samples were obtained under a short-term isoflurane inhalation anesthesia from individual hamsters by administering 200 µl PBS to each nostril and collecting the reflux.
   Sex as a biological variable	For single-infection experiments, 7- to 17-week-old male mice were inoculated with a dose of 2×104 TCD50/mouse of either Delta (EPI_ISL_2535433) or Omicron-BA.1 (EPI_ISL_7062525) isolates.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   A 1:3000 dilution of a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, 200-401-A50) was used for the immunohistochemical (IHC) analysis of the lung and the brain.	anti-SARS-CoV nucleocapsid antibody suggested: (Rockland Cat# 200-401-A50, RRID:AB_828403)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells and culture conditions: At IVI, IBSCVeroE6 (Vero C1008, ATCC) and VeroE6/TMPRSS2 cells (NIBSC) were cultured in Dulbecco‟s modified Eagle‟s medium (DMEM).	C1008 suggested: ECACC Cat# 940606176, RRID:CVCL_K755) VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Viruses were cultivated on VeroE6, Vero-TMPRSS2, or Calu-3 cells and sequence verified by performing whole-genome NGS sequencing (see below).	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   For the hamster and ferret infection studies SARS-CoV-2 Alpha (hCoV-19/Germany/NW-RKI-I-0026/2020, L4549, SARS-CoV-2 B.1.1.7 NW-RKI-I-0026/2020 passage 3 of EPI_ISL_751799), one silent mutation in the ORF1a (sequence position 11741), SARS-CoV-2 Delta AY.	L4549 suggested: None
   1 viruses were propagated (three passages for Alpha, two passages for Omicron-BA.1, one passage for Delta) on Vero E6 cells (Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0929) using a mixture of equal volumes of Eagle MEM (Hanks‟ balanced salts solution) and Eagle MEM (Earle‟s balanced salts solution) supplemented with 2 mM L-Glutamine, nonessential amino acids adjusted to 850 mg/L, NaHCO3, 120 mg/L sodium pyruvate, 10% fetal bovine serum (FBS), pH 7.2.	Vero E6 suggested: None
   Transcribed capped mRNA was electroporated into baby hamster kidney (BHK-21) cells expressing SARS-CoV N protein.	BHK-21 suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)
   Electroporated cells were co-cultured with susceptible Vero E6TMPRSS cells to produce passage 0 (P.0) of the recombinant viruses.	Vero E6TMPRSS suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Virus titers were assessed by standard TCID50 assays on Vero-TMPRSS2 cells.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse studies: hACE2-KI mice (B6.Cg-Ace2tm1(ACE2)Dwnt) and hACE2-K18Tg mice (Tg(K18-hACE2)2Prlmn) were described previously 9,21.	hACE2-KI suggested: None B6.Cg-Ace2tm1(ACE2)Dwnt suggested: None hACE2-K18Tg suggested: None Tg(K18-hACE2)2Prlmn) suggested: None
   K18-hACE2 mice (all female, 7 to 15 weeks old) were immunized intramuscularly with a single dose of 1 μg of mRNA-Vaccine Spikevax (Moderna).	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Software and Algorithms
   Sentences	Resources
   Nanopore sequencing workflow: Virus stocks, inoculum mixtures, and samples from competition assays in NECs, BECs, and lung explants were sequenced using the MinION sequencer (Oxford Nanopore Technologies) following the ARTIC nCoV-2019 sequencing protocol V3 (LoCost) (https://protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye) with the following modifications: the Midnight primer scheme (1200 bp amplicons) was used to perform the multiplex PCR (https://www.protocols.io/view/sars-cov2-genome-sequencing-protocol-1200bp-amplic-rm7vz8q64vx1/v6) instead of the ARTIC V3 primer scheme.	MinION suggested: (MinION, RRID:SCR_017985)
   Libraries were then loaded onto a R9.4.1 flow cell on a MinION sequencer (Oxford Nanopore Technologies) and monitored using the MinKNOW software (Version 21.11.9).	MinKNOW suggested: None
   When cells reached confluence, as assessed by measuring the trans-epithelial electrical resistance (TEER) using a Volt/Ohm Meter (EVOM2/STX2, World Precision Instruments) and microscopical evaluation, the apical medium was removed, cells were washed with pre-warmed Hank‟s balanced salt solution (HBSS, ThermoFisher), and then exposed to the air.	ThermoFisher suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
   Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.	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 10.1101/2022.04.28.489537v1 on bioRxiv

   Apr 28, 2022