ACE2-independent SARS-CoV-2 infection and mouse adaption emerge after passage in cells expressing human and mouse ACE2

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   ScreenIT

   Dec 23, 2021

   SciScore for 10.1101/2021.12.16.473063: (What is this?)

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

   Table 1: Rigor

   Ethics

   IACUC: Mouse work was approved by the QIMR Berghofer Medical Research Institute animal ethics committee (P3600, A2003-607). Euthanasia Agents: For intrapulmonary inoculations, mice were anesthetized using isoflurane. Field Sample Permit: All infectious SARS-CoV-2 work was conducted in a dedicated suite in a biosafety level-3 (PC3) facility at the QIMR Berghofer MRI (Australian Department of Agriculture, Water and the Environment certification Q2326 and Office of the Gene Technology Regulator certification 3445).

   Sex as a biological variable

   Mouse intrapulmonary SARS-CoV-2 infection: Female C57BL/6J mice (∼ 6 months old at the time of infection) were purchased from Animal Resources Centre …

   SciScore for 10.1101/2021.12.16.473063: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mouse work was approved by the QIMR Berghofer Medical Research Institute animal ethics committee (P3600, A2003-607). Euthanasia Agents: For intrapulmonary inoculations, mice were anesthetized using isoflurane. Field Sample Permit: All infectious SARS-CoV-2 work was conducted in a dedicated suite in a biosafety level-3 (PC3) facility at the QIMR Berghofer MRI (Australian Department of Agriculture, Water and the Environment certification Q2326 and Office of the Gene Technology Regulator certification 3445).
   Sex as a biological variable	Mouse intrapulmonary SARS-CoV-2 infection: Female C57BL/6J mice (∼ 6 months old at the time of infection) were purchased from Animal Resources Centre (Canning Vale, WA, Australia).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were routinely checked for mycoplasma (MycoAlert Mycoplasma Detection Kit MycoAlert, Lonza) and FCS was assayed for endotoxin contamination before purchase (55).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Immunohistochemistry for SARS-CoV-2 antigen was undertaken using mouse anti-SARS-CoV-2 spike monoclonal antibody 1E8 (Hobson-Peters et al. in preparation) as described previously (2).	anti-SARS-CoV-2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines and SARS-CoV-2 culture: Vero E6 (C1008, ECACC, Wiltshire, England; obtained via Sigma Aldrich, St. Louis, MO, USA), Lenti-X 293T (Takara Bio), AE17 (a gift from Dr Delia Nelson, Faculty of Health Sciences, Curtin Medical School), NIH-3T3 (American Type Culture Collection, ATCC, CRL-1658), LLC-PK1 (a gift from Prof. Roy Hall, UQ), A549 (ATCC CCL-185) and HeLa (ATCC-CLL 2) cells were cultured in medium comprising DMEM for Lenti-X 293T and A549 cells, M199 for LLC-PK1 cells or RPMI1640 for all others (Gibco) supplemented with 10% fetal calf serum (FCS), penicillin (100□IU/ml)/streptomycin (100□μg/ml) (Gibco/Life Technologies) and L-glutamine (2 mM) (Life Technologies).	NIH-3T3 suggested: ATCC Cat# CRL-1658, RRID:CVCL_0594) HeLa suggested: RRID:CVCL_JQ54) 293T suggested: None LLC-PK1 suggested: None
   100 µl of serially diluted samples were added to Vero E6 cells and the plates cultured for 5 days at 37°C and 5% CO2.	Vero E6 suggested: None
   After one passage in HEK293T-mACE2 cells, supernatant was used to infect new HEK293T-mACE2 cells for 2 hrs, then inoculum was removed and cells were washed 3 times with PBS and media replaced.	HEK293T-mACE2 suggested: None
   For growth kinetics experiments, HEK293T, HEK293T-hACE2 and HEK293T-mACE2, NIH-3T3, AE17, A549, HeLa or LLC-PK1 cells were infected with SARS-CoV-2 (QLD02, MA1, MA2, Alpha or Beta) at MOI 0.1 for 1 hr at 37°C, cells were washed with PBS and media replaced.	A549 suggested: None
   Analysis of RNA-Seq data from cell lines: Raw data (fastq files) from RNA-Seq of HEK293T, HeLa, 3T3, A549, A549 + influenza, Caco2 and Calu3 cells was obtained from the Sequence Read Archive (SRA).	Caco2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025) Calu3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
   Suspension cell infection and crystal violet statining: HEK293T or HEK293T-hACE2 cells were detached using trypsin (ThermoFisher scientific), TrypLE (ThermoFisher scientific), citric saline (135 mM KCl, 15 mM sodium citrate), or by mechanically detaching in culture media using a serological pipette.	HEK293T suggested: None HEK293T-hACE2 suggested: RRID:CVCL_A7UK)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Cell lines and SARS-CoV-2 culture: Vero E6 (C1008, ECACC, Wiltshire, England; obtained via Sigma Aldrich, St. Louis, MO, USA), Lenti-X 293T (Takara Bio), AE17 (a gift from Dr Delia Nelson, Faculty of Health Sciences, Curtin Medical School), NIH-3T3 (American Type Culture Collection, ATCC, CRL-1658), LLC-PK1 (a gift from Prof. Roy Hall, UQ), A549 (ATCC CCL-185) and HeLa (ATCC-CLL 2) cells were cultured in medium comprising DMEM for Lenti-X 293T and A549 cells, M199 for LLC-PK1 cells or RPMI1640 for all others (Gibco) supplemented with 10% fetal calf serum (FCS), penicillin (100□IU/ml)/streptomycin (100□μg/ml) (Gibco/Life Technologies) and L-glutamine (2 mM) (Life Technologies).	SARS-CoV-2 culture: Vero E6 suggested: None
   K18-hACE2 mice (strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, JAX Stock No: 034860) (57) were purchased from The Jackson Laboratory, USA, and bred and maintained in-house at QIMRB as heterozygotes by crossing with C57BL/6J mice.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657) B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   mACE2-hACE2 mice were created by Phenomics Australia/Monash Genome Modification Platform, and bred and maintained in-house at QIMRB as heterozygotes by crossing with C57BL/6J mice.	C57BL/6J suggested: RRID:IMSR_JAX:000664)
   Recombinant DNA
   Sentences	Resources
   SARS-CoV-2 passaging in hACE2 and mACE2 co-cultures: Lentivirus encoding hACE2, mACE2 or mACE2-N31K/H353K was produced in HEK293T cells by plasmid transfection and was used to transduce HEK293T cells, as described previously (2).	hACE2 suggested: RRID:Addgene_1786)
   Software and Algorithms
   Sentences	Resources
   The quality of raw sequencing reads was assessed using FastQC (58) (v0.11.80), and trimmed using Cutadapt (59) (v2.3) to remove adapter sequences and low-quality bases.	FastQC suggested: (FastQC, RRID:SCR_014583)
   SAMtools mpileup was used to produce a consensus sequence from mapped reads (62).	SAMtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Analysis of RNA-Seq data from cell lines: Raw data (fastq files) from RNA-Seq of HEK293T, HeLa, 3T3, A549, A549 + influenza, Caco2 and Calu3 cells was obtained from the Sequence Read Archive (SRA).	Sequence Read Archive suggested: (DDBJ Sequence Read Archive, RRID:SCR_001370)
   2-3 samples from the control experimental groups from at least two studies per cell line were analyzed as follows: Fastq files were trimmed of adapter sequences using Cutadapt, mapped to the human reference genome GRCh38 or the mouse reference genome GRCm39 using STAR aligner and TPM normalized gene counts were generated using RSEM.	Cutadapt suggested: (cutadapt, RRID:SCR_011841) STAR suggested: (STAR, RRID:SCR_004463) RSEM suggested: (RSEM, RRID:SCR_013027)
   PyMOL v4.60 (Schrodinger) was used for mutagenesis of the crystal structure of SARS-CoV-2 spike bound with ACE2 from the protein data bank (7DF4) (64).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Slides were scanned using Aperio AT Turbo (Aperio, Vista, CA USA) and analyzed using Aperio ImageScope software (LeicaBiosystems, Mt Waverley, Australia) (v10) and the Positive Pixel Count v9 algorithm.	ImageScope suggested: (ImageScope, RRID:SCR_014311)
   Automatic quantitation of white space was undertaken using QuPath v0.2.3 (65).	QuPath suggested: (QuPath, RRID:SCR_018257)
   Statistics: Statistical analyses of experimental data were performed using IBM SPSS Statistics for Windows, Version 19.0 (IBM Corp., Armonk, NY, USA).	SPSS suggested: (SPSS, RRID:SCR_002865)

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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: We detected the following sentences addressing limitations in the study:

   A potential limitation of these mouse adapted SARS-CoV-2 viruses is deletion of the QTQTN furin cleavage site flanking sequence, which impairs S1/S2 cleavage by furin (47). Prior S1/S2 cleavage is required for S2’ cleavage by transmembrane protease serine 2 (TMPRSS2) at the cell surface, which is important for entry in TMPRSS2 positive cells. The QTQTN deletion commonly arises after virus propagation in TMPRSS2 negative cell lines (such as Vero E6 and HEK293T), although this deletion is also evident in some human clinical samples (47). This deletion improves cleavage by Cathepsin L, which substitutes for TMPRSS2 by cleaving S2’ in endosomes and releasing viral RNA into the cytoplasm (48). Other studies have shown that deletion of the furin cleavage site attenuates replication in hamsters and K18-hACE mice (49) and reduces transmission in ferrets (50). Our data suggests that this deletion didn’t dramatically affect mouse adapted virus replication or tropism in C57BL/6J mouse lungs compared to other studies (11), although side-by-side comparison with virus containing the same spike amino acid changes as MA1 and MA2 but maintaining furin cleavage would be needed to determine if there is any attenuation. The D614G change, which is present in all SARS-CoV-2 variants (except the ancestral Wuhan strain), also increases virus stability and entry via the cathepsin L route (48). Cathepsin L is widely expressed in human nasal and lung epithelial cells (48), thus our mouse adapted viruse...

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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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   Version published to 10.1101/2021.12.16.473063 on bioRxiv

   Dec 21, 2021