Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

1.

2.

Viral genome sequencing analysis. The sequences of the working viruses were

verified by viral RNA-sequencing analysis. Viral RNA was extracted using QIAamp

viral RNA mini kit (Qiagen, Cat# 52906). The sequencing library for total RNA

sequencing was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina

(New England Biolabs, Cat# E7530). Paired-end, 76-bp sequencing was performed

using MiSeq (Illumina) with MiSeq reagent kit v3 (Illumina, Cat# MS-102-3001).

Sequencing reads were trimmed using fastp v0.21.037 and subsequently mapped to

the viral genome sequences of a lineage B isolate (strain Wuhan-Hu-1; GISAID ID:

EPI_ISL_402125; GenBank accession no. NC_045512.2) using BWA-MEM

v0.7.1738. Variant calling, filtering, and annotation were performed using SAMtools

v1.939 and snpEff v5.0e40. Information on the detected mutations in the working

virus stocks is summarized in Supplementary Table 2.

RNA-Seq analysis. Total RNA was extracted from tissues using the procedure

described above. The sequencing library was prepared using Illumina TruSeq

Stranded mRNA Sample Preparation Kit (Illumina). Paired-end, 150-bp sequencing was performed using Illumina NovaSeq 6000 System (Illumina).

Sequencing reads were trimmed using fastp v0.21.0. The trimmed reads were

subsequently mapped to the reference genome of Syrian hamsters (NCBI

Accession: GCF_017639785.1) with the gene annotation file, both of which were

downloaded from NCBI RefSeq (https://www.ncbi.nlm.nih.gov/refseq/), using

STAR v 2.6.1c. The read count matrix was generated using featureCounts v1.6.3.

Of the hamster genes annotated by RefSeq, genes with orthologs in humans

were analyzed in the present study. Information on the hamster-human ortholog

relationship was extracted from the NCBI RefSeq database (https://www.ncbi.nlm.

nih.gov/refseq/). Differential expression analysis was performed using DESeq2

v1.36.0. DEGs between infected and uninfected hamsters were determined using

the Wald test, and DEGs among variants were determined using the likelihood

ratio test. Genes with adjusted P-values calculated by the Benjamini–Hochberg

(BH) method <0.05 and absolute values of log2 FC >1 were regarded as DEGs in

the present study. Since the GO annotation information for hamster genes

(GCF_017639785.1) was not available, we transferred the GO annotation

information of human genes to orthologous hamster genes. GO enrichment

analysis was performed using Fisher’s exact test. GO terms with adjusted P-value

calculated by the BH method <0.1 were regarded as significant terms. The source

data is available in Supplementary Data 1, 2.

Statistics and reproducibility. Viral RNA copy, body weight, PenH, Rpef, and

SpO2, and inflammatory mRNA gene levels obtained from the in vivo experiments

were analyzed by repeated measures analysis of variance. Inflammation measures

upon infection in vivo, the mRNA of the lung hilum and periphery areas at 2 d.p.i.,

and four host genes (Cxcl10, Il-6, Isg15, and Mx-1) were compared among Omicron

subvariants using analysis of variance. Regarding PenH, Rpef, and SpO2, we

compared infected animals with each variant against uninfected animals and calculated p-values using Dunnett’s adjustment. The other measurements were tested

by Tukey’s multiplicity correction to maintain the type I error rate for comparison

among infected or uninfected animals. These analyses were conducted using SAS

Ver. 9.4 (SAS Institute, Cary, NC). The statistical significance of differences

between BA.5 and other variants or saline across timepoints from day 1 p.i. to day 7

p.i. was tested using the Holm method. The indicated analyses were performed in R

v4.1.2 (R Core Team, Vienna, Austria). The two-sided significance level was set

to 0.05.

Reporting summary. Further information on research design is available in the Nature

Portfolio Reporting Summary linked to this article.

3.

4.

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6.

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8.

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24.

25.

26.

27.

Data availability

28.

The raw data of RNA-Seq are available on Sequence Read Archive (https://www.ncbi.

nlm.nih.gov/sra; Accession PRJDB14143). The source data behind the graphs in the

figures is available in Supplementary Data 3. The raw data of immunoblotting is

deposited in Supplementary Fig. 9.

29.

30.

Code availability

Computational codes used in the present study are available on the GitHub repository

(https://github.com/TheSatoLab/Omicron_BA1_BA2_BA5_comparision).

Received: 12 October 2022; Accepted: 28 June 2023;

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Acknowledgements

We would like to thank all members belonging to The Genotype to Phenotype Japan (G2PJapan) Consortium. We also thank the National Institute for Infectious Diseases, Japan, for

providing BA.1 and BA.2 isolates, and Tokyo Metropolitan Institute of Public Health for

providing a BA.5 isolate. We are also grateful to Edanz (https://jp.edanz.com/ac) for editing

a draft of this manuscript. This study was supported in part by AMED Research Program

on Emerging and Re-emerging Infectious Diseases (JP21fk0108493, to T.F.; JP22fk0108617

to T.F.; JP22fk0108516h to T.F.; JP20fk0108146, to K.S.; JP20fk0108451, to G2P-Japan

Consortium, K.M., K.S., and T.F.; JP21fk0108494 to G2P-Japan Consortium, K.M., S.T.,

K.S., and T.F.); (JP22fk0108511, to Y.Y., G2P-Japan Consortium, K.M., K.T., S.T., K.S., and

T.F.); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to K.S.); AMED SCARDA World-leading

institutes for vaccine research and development Hokkaido Synergy Campus

(JP223fa627005, to Hirofumi Sawa); AMED Program on R&D of New Generation Vaccines Including New Modality Application (JP223fa727002, to K.S.); AMED Research

Program on HIV/AIDS (21fk0410039, to K.S.); AMED Japan Program for Infectious

Diseases Research and Infrastructure (JP22wm0125008, to Hirofumi Sawa; and

JP21wm0225003, to Hirofumi Sawa); AMED CREST (JP21gm1610005, to K.T.;

JP22gm1610008 to T.F.); JST CREST (JPMJCR20H4, to K.S.); JST PRESTO (JPMJPR22R1,

to J.I.); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to T.F.); JSPS

KAKENHI Grant-in-Aid for Early-Career Scientists (20K15767, to J.I.; 23K14526, to J.I.);

JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to

K.S.); World-leading Innovative and Smart Education (WISE) Program 1801 from the

Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to N.N.); The

Tokyo Biochemical Research Foundation (to K.S.), and Hokkaido University Support

Program for Frontier Research (to T.F.).

experiments. Y.O., L.W., M.T., and S.T. performed histopathological analysis. J.I. performed viral genome sequencing analysis. J.I. and I.Y. performed statistical and bioinformatic analyses. Y.K. and Y.S. performed RNA sequencing. T.T., J.I., Y.O., K.M., K.T.,

S.T., K.S., and T.F. designed the experiments and interpreted the results. T.T. and T.F.

wrote the original manuscript. All authors reviewed and proofread the manuscript. The

Genotype to Phenotype Japan (G2P-Japan) Consortium contributed to the project

administration.

Competing interests

Y.Y. and T.N. are founders and shareholders of HiLung, Inc. J.K. is an employee of

HiLung, Inc. Y.Y. is a co-inventor of a patent (PCT/JP2016/057254, “Method for

inducing differentiation of alveolar epithelial cells”) related to this work. I.Y. reports

speaker fees from Chugai Pharmaceutical Co, and AstraZeneca plt, outside the submitted

work. The other authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s42003-023-05081-w.

Correspondence and requests for materials should be addressed to Isao Yokota, Keita

Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato or Takasuke Fukuhara.

Peer review information This paper was previously reviewed at another Nature Portfolio

journal. Communications Biology thanks the anonymous reviewers for their contribution

to the peer review of this work. Primary Handling Editor: Gene Chong.

Reprints and permission information is available at http://www.nature.com/reprints

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© The Author(s) 2023

Author contributions

T.T., D.Y., T.K., R.H., Y.F., R.S., H.I., I.K., Y.Y., T.N., and J.K. performed cell culture

experiments. T.T., N.N., R.S., H.I., M.K., H.S., K.Y., and K.M. performed animal

The Genotype to Phenotype Japan (G2P-Japan) Consortium

Saori Suzuki1,2, Marie Kato5, Zannatul Ferdous5, Hiromi Mouri5, Kenji Shishido5, Naoko Misawa3, Keiya Uriu3,

Yusuke Kosugi3, Shigeru Fujita3, Mai Suganami3, Mika Chiba3, Ryo Yoshimura3, So Nakagawa27, Jiaqi Wu27,

Akifumi Takaori-Kondo28, Kotaro Shirakawa28, Kayoko Nagata28, Yasuhiro Kazuma28, Ryosuke Nomura28,

Yoshihito Horisawa28, Yusuke Tashiro28, Yugo Kawai28, Takao Hashiguchi28, Tateki Suzuki28, Kanako Kimura28,

Jiei Sasaki28, Yukari Nakajima28, Ayaka Sakamoto28, Naoko Yasuhara28, Takashi Irie29, Ryoko Kawabata29,

Terumasa Ikeda30, Hesham Nasser30, Ryo Shimizu30, Monira Begum30, Otowa Takahashi30, Kimiko Ichihara30,

Takamasa Ueno30, Chihiro Motozono30, Mako Toyoda30, Akatsuki Saito31, Yuri L. Tanaka31,

Erika P. Butlertanaka31, Maya Shofa31 & Kaori Tabata32

27

Tokai University School of Medicine, Isehara, Japan. 28Kyoto University, Kyoto, Japan. 29Hiroshima University, Hiroshima, Japan.

University, Kumamoto, Japan. 31University of Miayzaki, Kumamoto, Japan. 32Kyushu University, Kumamoto, Japan.

12

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Kumamoto

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