A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

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1. 

   ScreenIT

   Aug 4, 2021

   SciScore for 10.1101/2021.07.30.454063: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	No statistical methods were applied to predetermine sample size.
   Cell Line Authentication	Contamination: The absence of mycoplasma contamination was confirmed with the use of a PCR Mycoplasma Test Kit (Takara, Shiga, Japan). Authentication: Expression of the mCherry-tagged S proteins was confirmed by immunofluorescence analysis.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Reagents and antibodies: Antibodies to SARS-CoV S protein (#40150-R007), to RFP (#PM005), to VSV G protein (#EB0010), and to VSV M protein (#MABF2347) were obtained from Sino Biological (Beijing, China), Medical &…

   SciScore for 10.1101/2021.07.30.454063: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	No statistical methods were applied to predetermine sample size.
   Cell Line Authentication	Contamination: The absence of mycoplasma contamination was confirmed with the use of a PCR Mycoplasma Test Kit (Takara, Shiga, Japan). Authentication: Expression of the mCherry-tagged S proteins was confirmed by immunofluorescence analysis.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Reagents and antibodies: Antibodies to SARS-CoV S protein (#40150-R007), to RFP (#PM005), to VSV G protein (#EB0010), and to VSV M protein (#MABF2347) were obtained from Sino Biological (Beijing, China), Medical & Biological Laboratory (Nagoya, Japan), Kerafast (Boston, MA), and Merck (Darmstadt, Germany), respectively.	SARS-CoV S protein ( #40150-R007) suggested: None RFP suggested: (MBL International Cat# PM005, RRID:AB_591279) VSV G protein ( #EB0010) , suggested: None VSV M protein suggested: None
   For generation of VSVΔG-S particles, cells expressing S protein of SARS-CoV or SARS-CoV-2 (either stably or transiently) were exposed to VSVΔG-G at an MOI of 3 FFU per cell for 16 h at 35°C in the presence of antibodies to VSV G in order to neutralize the parental viruses.	SARS-CoV-2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Generation of stable cell lines: pCG1-mCherry-SARS-CoV-S or pCG1-mCherry-SARS-CoV-2-S was linearized with PuvI and introduced together with pBABE-puro into HEK293T or VeroE6 cells by transfection for 24 h with the use of PEI MAX.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   BEAS-2B cells stably expressing human ACE2 were generated by lentivirus-mediated gene transfer.	BEAS-2B suggested: ATCC Cat# CRL-9609, RRID:CVCL_0168)
   HEK293T cells were thus transfected with pLVX-ACE2-IRES-BLD (Daly et al., 2020), pCAG-HIVgp, and pCMV-VSVG-RSV-Rev (Miyoshi et al., 1998) for 48 h, after which the culture supernatant was collected.	HEK293T suggested: None
   In brief, for preparation of VSVΔG-G, BHK21/G43 cells (which stably express VSV G protein) were incubated with mifepristone (10 nM) for 6 h and then infected with VSVΔG at a multiplicity of infection (MOI) of 100,000 FFU per cell for 16 h at 35°C.	BHK21/G43 suggested: None
   Recombinant DNA
   Sentences	Resources
   Expression vectors for the S protein of SARS-CoV or SARS-CoV-2 (pCG1-SARS-CoV-S and pCG1-SARS-CoV-2-S) (Hoffmann et al., 2020) were kindly provided by S. Pöhlmann.	pCG1-SARS-CoV-S suggested: None pCG1-SARS-CoV-2-S suggested: None
   The coding sequence for mCherry was cleaved out of pFX-Tom20-mCherry (Kashiwagi et al., 2019a) by digestion with BamHI and BglII, and was then subcloned into the BamHI sites of the expression vectors for the S proteins to obtain pCG1-mCherry-SARS-CoV-S and pCG1-mCherry-SARS-CoV-2-S.	pFX-Tom20-mCherry suggested: None pCG1-mCherry-SARS-CoV-S suggested: None pCG1-mCherry-SARS-CoV-2-S suggested: None
   The pBABE-puro plasmid encoding a puromycin resistance gene was obtained from Addgene.	pBABE-puro suggested: RRID:Addgene_1764)
   HEK293T cells were thus transfected with pLVX-ACE2-IRES-BLD (Daly et al., 2020), pCAG-HIVgp, and pCMV-VSVG-RSV-Rev (Miyoshi et al., 1998) for 48 h, after which the culture supernatant was collected.	pLVX-ACE2-IRES-BLD suggested: None pCAG-HIVgp suggested: None pCMV-VSVG-RSV-Rev suggested: None
   In brief, cells transfected with mCherry-CoV-S or mCherry-CoV-2-S vectors were stained with Hoechst 33342 for 15 min and placed in a stage-top incubation chamber maintained at 37°C on a Nikon Ti2 microscope (Nikon, Tokyo, Japan) equipped with a Zyla5.5 scientific complementary metal oxide semiconductor camera (Oxford Instruments, Belfast, UK).	mCherry-CoV-2-S suggested: None
   Software and Algorithms
   Sentences	Resources
   The FFU value of the pseudovirus suspension was determined by counting the number of GFP-positive cells with the use of the “Multi-wavelength cell scoring” module of MetaMorph software (Molecular Devices, CA)	MetaMorph suggested: None
   A p value of <0.05 was considered statistically significant, and all statistical analysis was performed with JMP Pro software (version 15.0.0).	JMP Pro suggested: None

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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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2. 

   Version published to 10.1101/2021.07.30.454063 on bioRxiv

   Jul 30, 2021