Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5

Article activity feed

1. 

   ScreenIT

   May 28, 2022

   SciScore for 10.1101/2022.05.26.493539: (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
   To measure the surface expression level of S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-112048, 1:100)	anti-SARS-CoV-2 S suggested: None
   Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) was used as negative controls, and APC-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 111-136-144, 1:50) was used as a secondary antibody.	anti-rabbit IgG suggested: (Jackson ImmunoResearch Labs Cat# 111-136-144, RRID:AB_2337987)
   The deparaffinized sections were exposed to EnVision FLEX target …

   SciScore for 10.1101/2022.05.26.493539: (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
   To measure the surface expression level of S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-112048, 1:100)	anti-SARS-CoV-2 S suggested: None
   Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) was used as negative controls, and APC-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 111-136-144, 1:50) was used as a secondary antibody.	anti-rabbit IgG suggested: (Jackson ImmunoResearch Labs Cat# 111-136-144, RRID:AB_2337987)
   The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 minutes at 97°C to activate, and mouse anti-SARS-CoV-2 N monoclonal antibody (clone 1035111, R&D systems, Cat# MAB10474-SP, 1:400) was used as a primary antibody.	anti-SARS-CoV-2 N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: HEK293T cells (a human embryonic kidney cell line; ATCC, CRL-3216)	HEK293T suggested: None
   , HEK293 cells (a human embryonic kidney cell line; ATCC, CRL-1573) and HOS-ACE2/TMPRSS2 cells (HOS cells stably expressing human ACE2 and TMPRSS2) (Ferreira et al., 2021; Ozono et al., 2021) were maintained in DMEM (high glucose) (Sigma-Aldrich, Cat# 6429-500ML) containing 10% fetal bovine serum (FBS, Sigma-Aldrich Cat# 172012-500ML), and 1% penicillin-streptomycin (PS) (Sigma-Aldrich, Cat# P4333-100ML).	HOS-ACE2/TMPRSS2 suggested: None
   HEK293-ACE2/TMPRSS2 cells (HEK293 cells stably expressing human ACE2 and TMPRSS2) (Motozono et al., 2021) was maintained in DMEM (high glucose) containing 10% FBS, 1 µg/ml puromycin, 200 ng/ml hygromycin (Nacalai Tesque, Cat# 09287-84) and 1% PS.	HEK293-ACE2/TMPRSS2 suggested: None HEK293 suggested: None
   HEK293-C34 cells (IFNAR1 KO HEK293 cells expressing human ACE2 and TMPRSS2 by doxycycline treatment) (Torii et al., 2021) were maintained in DMEM (high glucose) containing 10% FBS, 10 μg/ml blasticidin (InvivoGen, Cat# ant-bl-1) and 1% PS.	HEK293-C34 suggested: None
   Vero cells [an African green monkey (Chlorocebus sabaeus) kidney cell line; JCRB Cell Bank, JCRB0111] were maintained in Eagle’s minimum essential medium (EMEM) (Sigma-Aldrich, Cat# M4655-500ML) containing 10% FBS and 1% PS.	Vero suggested: None
   VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2; JCRB Cell Bank, JCRB1819) (Matsuyama et al., 2020) were maintained in DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Calu-3 cells (a human lung epithelial cell line; ATCC, HTB-55) were maintained in EMEM (Sigma-Aldrich, Cat# M4655-500ML) containing 20% FBS and 1% PS.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Calu-3/DSP1-7 cells (Calu-3 cells stably expressing DSP1-7) (Yamamoto et al., 2020) were maintained in EMEM (Wako, Cat# 056-08385) containing 20% FBS and 1% PS. 293S GnTI(-) cells (HEK293S cells lacking N-acetylglucosaminyltransferase (Kubota et al., 2016) were maintained in DMEM (Nacalai tesque, #08458-16 containing 2% FBS without PS.	HEK293S suggested: RRID:CVCL_A784)
   Briefly, the amount of pseudoviruses prepared was quantified by the HiBiT assay using Nano Glo HiBiT lytic detection system (Promega,Cat# N3040) as previously described (Ozono et al., 2021; Ozono et al., 2020), and the same amount of pseudoviruses (normalized to the HiBiT value, which indicates the amount of p24 HIV-1 antigen) was inoculated into HOS-ACE2/TMPRSS2 cells, HEK293-ACE2 cells or HEK293-ACE2/TMPRSS2 and viral infectivity was measured as described above (see “Neutralization assay” section).	HEK293-ACE2 suggested: None
   On day 3 (24 hours posttransfection), 16,000 effector cells were detached and reseeded into 96-well black plates (PerkinElmer, Cat# 6005225), and target cells (VeroE6/TMPRSS2 or Calu-3/DSP1-7 cells) were reseeded at a density of 1,000,000 cells/2 ml/well in 6-well plates.	Calu-3/DSP1-7 suggested: None
   SARS-CoV-2 infection: One day before infection, Vero cells (10,000 cells) and VeroE6/TMPRSS2 cells (10,000 cells) were seeded into a 96-well plate.	VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Preparation of mouse sera: BALB/c mice (female, 7 weeks old) were immunized with 1 μg SARS-CoV-2 BA.2 RBD protein in 50% AddaVax (Invivogen, Cat# vac-adx-10) at day 0 and 14.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   The resulting PCR fragment was digested with KpnI and NotI and inserted into the corresponding site of the pCAGGS vector (Niwa et al., 1991).	pCAGGS suggested: RRID:Addgene_127347)
   2 S RBD (residues 322-536) was cloned into the expression vector pHLsec containing the N-terminal secretion signal sequence and the C-terminal His6-tag sequence (Aricescu et al., 2006).	pHLsec suggested: None
   kit (Roche, Cat# KK2601) and assembled in vivo by yeast [Saccharomyces cerevisiae strain EBY100 (ATCC, MYA-4941)] homologous recombination with pJYDC1 plasmid (Addgene, Cat# 162458) as previously described (Dejnirattisai et al., 2022; Kimura et al., 2022a; Kimura et al., 2022b; Motozono et al., 2021; Yamasoba et al., 2022a; Zahradnik et al., 2021a)	pJYDC1 suggested: RRID:Addgene_162458)
   To prepare effector cells, HEK293 cells were cotransfected with the S-expression plasmids (500 ng) and pDSP8-11 (500 ng) using PEI Max (Polysciences, Cat# 24765-1).	pDSP8-11 suggested: None
   To prepare target cells, HEK293 and HEK293-ACE2/TMPRSS2 cells were transfected with pDSP1-7 (500 ng) (Kondo et al., 2011).	pDSP1-7 suggested: None
   Software and Algorithms
   Sentences	Resources
   Sequencing reads were trimmed using fastp v0.21.0 (Chen et al., 2018) and subsequently mapped to the viral genome sequences of a lineage A isolate (strain WK-521; GISAID ID: EPI_ISL_408667) (Matsuyama et al., 2020) using BWA-MEM v0.7.17 (Li and Durbin, 2009).	BWA-MEM suggested: (Sniffles, RRID:SCR_017619)
   Variant calling, filtering, and annotation were performed using SAMtools v1.9 (Li et al., 2009) and snpEff v5.0e (Cingolani et al., 2012).	SAMtools suggested: (SAMTOOLS, RRID:SCR_002105)
   The viral genome sequences were mapped to the reference sequence of Wuhan-Hu-1 (GenBank accession number: NC_045512.2) using Minimap2 v2.17 (Li, 2018) and subsequently converted to a multiple sequence alignment according to the GISAID phylogenetic analysis pipeline (https://github.com/roblanf/sarscov2phylo).	Minimap2 suggested: (Minimap2, RRID:SCR_018550)
   Tree reconstruction was performed by RAxML v8.2.12 (Stamatakis, 2014) under the GTRCAT substitution model.	RAxML suggested: (RAxML, RRID:SCR_006086)
   Parameter estimation was performed via the MCMC approach implemented in CmdStan v2.28.1 (https://mc-stan.org) with CmdStanr v0.4.0 (https://mc-stan.org/cmdstanr/).	CmdStan suggested: None https://mc-stan.org suggested: (Stan, RRID:SCR_018459) CmdStanr suggested: None
   The assay of each serum was performed in triplicate, and the 50% neutralization titer (NT50) was calculated using Prism 9 software v9.1.1 (GraphPad Software).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   RBD expression and ACE2 signal were recorded by using a FACS S3e cell sorter device (Bio-Rad), background binding signals were subtracted and data were fitted to a standard noncooperative Hill equation by nonlinear least-squares regression using Python v3.7 (https://www.python.org) as previously described (Kimura et al., 2022a; Kimura et al., 2022b; Motozono et al., 2021; Yamasoba et al., 2022a; Zahradnik et al., 2021b).	Python suggested: (IPython, RRID:SCR_001658) https://www.python.org suggested: (CVXOPT - Python Software for Convex Optimization, RRID:SCR_002918)
   Surface expression level of S proteins (Figures 3C and S2B) was measured using FACS Canto II (BD Biosciences) and the data were analyzed using FlowJo software v10.7.1 (BD Biosciences).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The size of syncytium (GFP-positive area) was measured using Fiji software v2.2.0 (ImageJ) as previously described (Suzuki et al., 2022; Yamasoba et al., 2022a).	Fiji suggested: (Fiji, RRID:SCR_002285)
   The stained cells were washed with tap water and dried, and the size of plaques was measured using Fiji software v2.2.0 (ImageJ).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Images were incorporated as virtual slide by NDP.scan software v3.2.4 (Hamamatsu Photonics).	NDP.scan suggested: None
   These analyses were performed in R v4.1.2 (https://www.r-project.org/).	https://www.r-project.org/ suggested: (R Project for Statistical Computing, RRID:SCR_001905)

   ---

   Results from OddPub: Thank you for sharing your code and data.

   ---

   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 TrialIdentifier: No clinical trial numbers were referenced.

   ---

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

   ---

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

   ---

   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • No funding statement was detected.
   • No protocol registration statement was detected.

   ---

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

   ---

   Read the original source
2. 

   Version published to 10.1101/2022.05.26.493539v1 on bioRxiv

   May 26, 2022