key: cord-0730954-scmfb0ai authors: Kodai, A.; Takako, S.; Toshiki, T.; Yuka, I.; Hirotsugu, I.; Yoshifumi, U.; Shunsuke, U.; Jun, G.; Natsuo, T.; Yuriko, T.; Junpei, K.; Hiroyuki, N.; Susumu, F.; Shikou, S.; Morio, N.; Kazuhiro, U.; Takahiko, M.; Jun, I.; Kenichiro, Y.; Toshio, K.; Shinji, M.; Kazuhiro, A.; Tamotsu, E.; Haruhiko, S.; Naoki, H.; Masayuki, A.; Yuko, K.; Makoto, S.; Kenjiro, K. title: Identification of B.1.346 lineage of SARS-CoV-2 in Japan: Genomic evidence of re-entry of Clade 20C date: 2021-02-01 journal: nan DOI: 10.1101/2021.01.29.21250798 sha: 7dfaf31a78096cb107733aecb1b607c29c983dca doc_id: 730954 cord_uid: scmfb0ai Objectives Whole SARS-CoV-2 genome sequencing from COVID-19 patients is useful for infection control and regional trends evaluation. We report a lineage data collected from hospitals in the Kanto region of Japan. Methods We performed whole genome sequencing in specimens of 198 COVID-19 patients at 13 collaborating hospitals in the Kanto region. Phylogenetic analysis and fingerprinting of the nucleotide substitutions underwent to differentiate and classify the viral lineages. Results More than 90% of the strains belonged to Clade 20B and two lineages (B.1.1.284 and B.1.1.214) have been detected predominantly in the Kanto region. However, one sample from a COVID-19 patient in November 2020, belonged to the B.1.346 lineage of Clade 20C, which has been prevalent in western United States. The patient had no history of overseas travel and no contact with anyone who had travelled abroad, suggesting that this strain appeared likely to have been imported from western United States, across the strict quarantine barrier. Conclusion B.1.1.284 and B.1.1.214 have been identified predominantly in the Kanto region and B.1.346 of clade 20C in one patient was probably imported from western United States. These results illustrate that a decentralized network of hospitals can be significantly advantageous for monitoring regional molecular epidemiologic trends. Whole SARS-CoV-2 genome sequencing of samples from COVID19 patients is useful for infection control (Takenouchi T, et al., 2020) . When multiple COVID19-positive cases occur simultaneously in a hospital, it is critical to determine whether newly diagnosed patients have nosocomial infection or community infection. Under nosocomial infection, a thorough contact tracing of healthcare workers and inpatients is essential whereas in the situation of community infection, such a persisted intrahospital surveillance may not be necessary. Datasets of whole viral genome assembled from multiple hospitals can give critical clues on regional or national trends. National surveillance system developed in the United Kingdom (UK) clarified that more than 1000 lineages spread during pre-lockdown high travel volumes and few restrictions on international travel (Du Plessis L, et al., 2021) . Recently, the genomic surveillance system successfully identified two variants of concern, 501Y.V1 (B.1.1.7 originated from the UK) and 501Y.V2 (B.1.351 originated from South Africa), both of which spread rather quickly (Davies N, et al., 2020; European Centre for Disease Prevention and Control, 2020) . In Japan, eight laboratories including our group have deposited whole viral genome data to the public sequence database GISAID (https://www.gisaid.org/): A total of 9943 sequences have been deposited and some results of national survey have been summarized (Sekizuka, et al., 2020; Sekizuka, et al., 2020) . Despite the requirement by international convention, regional origin of the samples or precise date of sampling has not been specified in most of the publicly available data derived from Japan. Here, we report the lineage summary of our data collected from 13 hospitals located in the Kanto region of Japan to document relatively recent trends in and around the Tokyo Metropolitan area. Whole genome data from 198 specimens which were accumulated and utilized for infection control showed that the lineages of the strains were limited to only two closely related strains, B.1.1.284 and B.1.1.214. The observation supports success of quarantine system. However, one strain derived from a patient with nosocomial COVID-19 infection belonged to the B.1.346 lineage which is prevalent in the western United States (USA) as of winter 2020. We suspect that the episode is attributed to undetected imported infections at the border. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 1, 2021. ; The study protocol was approved by the Institutional Review Board of Keio University School of Medicine (approval number: 20200062) with each collaborating hospitals' review board and was conducted according to the principles of the Declaration of Helsinki. Whole viral genome sequence was determined as previously reported (Takenouchi et al., 2020) . PCR-based amplification was performed using the Artic ncov-2019 primers in addition to a No.72-mutant primer, version 3 (https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-201 9/V3/nCoV-2019.tsv) in two multiplex reactions according to the globally accepted "nCoV-2019 sequencing protocol" (https://www.protocols.io/view/ncov-2019-sequencingprotocol-bbmuik6w). The sequencing library for amplicon sequencing was prepared using the Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Paired end sequencing was performed on the MiSeq platform (Illumina, CA). The bioinformatic pipeline used in this study, "Mutation calling pipeline for amplicon-based sequencing of the SARS-CoV-2 viral genome," is available at https://cmg.med.keio.ac.jp/sars-cov-2/. All single nucleotide substitutions including non-synonymous and synonymous mutations were annotated by the ANNOVAR software and were assessed using VarSifter (https://research.nhgri.nih.gov/software/VarSifter/). The analytic protocol is described previously in our publication, submitted in November 24, 2020, in preprint server medRxiv (Abe, et al., 2020) . A phylogenetic tree analysis was performed locally using the Augur program available from Nextstrain (https://nextstrain.org/) and genome sequence data obtained in the currently reported study as well as data available from the global database EpiCov hosted at GISAID (Hadfield, et al., 2020) . The Nextclade (https://clades.nextstrain.org/) was also used to generate fingerprinting patterns to visualize the SARS-CoV-2 sequence alignments and similarities/identities among samples. We used the international genetic clade nomenclature system defined by Nextstrain.org . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250798 doi: medRxiv preprint (https://virological.org/t/updated-nextstain-sars-cov-2-clade-naming-strategy/581) ( Figure 1 ). We also used the software Phylogenetic Assignment of Named Global Outbreak Lineages (Pangolin; https://cov-lineages.org/index.html) to assign viral lineages in an automatic and precise manner (Rambaut, et al., 2020) . Of the 198 strains, 189 (95.5%) belonged to Clade 20B of the Clade system. Table 1 . According to the GISAID database, 159 strains belonging to the B.1.346 lineage have been deposited in the database (acknowledgment table). Most strains derived from the western United States. Detailed examination of the phylogenetic tree revealed that 4 strains were very closely related ( Figure 2 ). The genomic data strongly suggest that this strain was presumably imported into Japan across the quarantine barrier. The GISAID data from Japan indicate that the last clade 20C strains detected in Japan were in March and May, 2020, but not thereafter, except in quarantine cases, supporting the notion that the newly identified clade 20C strain in this study was indeed imported from overseas. Comparison of the fingerprinting pattern of the B.1.346 lineage strains detected in Japan in this study and those detected in the western United States and a representative . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250798 doi: medRxiv preprint clade 20C strain detected in Japan in the spring of 2020 (Figure 3 ) further augments the notion that the strain of B.1.346 lineage belonging to clade 20C was imported into Japan across the quarantine barrier from western United States. There is no evidence that the newly detected clade 20C strain in the Kanto region shows altered transmissibility or virulence, unlike variants of concern such as 501Y .V1 (B.1.1.7) in the UK and 501Y.V2 (B.1.351) in South Africa. The fact that only two strains have been detected predominantly in the Kanto region supports a notion that Japan's quarantine system had relatively been successful after national lock down in April and May of 2020. The observation that extremely limited number of lineages (i.e., 2) makes sharp contrast to that in the UK and other countries where numerous numbers of strains are still prevailing (Popa, et al., 2020; Lemiux, et al., 2020) . In view of emergence of potentially virulent strains (501Y) in the UK and South Africa, thorough and strict quarantine system should further be warranted in Japan. States is two folds. First, no quarantine policy can be perfect. If the policy is reset to be less strict, close follow-up genomic monitoring is mandatory. Because of the internationally unprecedent uniformness of the domestic viral strains in Japan, detection of foreign strains may be relatively easy as exemplified in this single patient. Second, if entry of foreign strain does occur in the near future, it may be relatively easy to pinpoint the geographic origin of the incoming strain by the number of nucleotide substitutions in the SARS-CoV-2 genome. Overall, the viral genomic monitoring system for in-hospital infections reported herein enabled us to unravel regional or national trends with agility. Until now in Japan, a national centralized network system, composed of National Institute of Infectious Diseases in Tokyo and Public Health Centers and Public Health Institutes had been put in place for the purpose of viral genome surveillance. Our report illustrates that a decentralized network system composed of multiple hospitals mutually sharing molecular genomic data in near real-time provides robust benefits to public health with agility, under . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 the conditions of this COVID-19 pandemic. Molecular genomic data obtained through such a system could be swiftly reflected in the national decision-making process for public health practices, including the strictness of quarantine measures or implementation of lockdowns in response to the detection of SARS-CoV-2 variants of concern. KAb and TS contributed to writing of the report and data analysis. YI, HI, TT and HS Authors have no conflicts of interests. We downloaded the full nucleotide sequences of the SARS-CoV-2 genomes from the GISAID database (https://www.gisaid.org/). A table of the contributors is available . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250798 doi: medRxiv preprint (acknowledgment table) . We have uploaded the full nucleotide sequences of our cohort to the GISAID database. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted February 1, 2021. ; https://doi.org/10.1101/2021.01.29.21250798 doi: medRxiv preprint Internationally, more than 90% of Clade 20C has been detected from the USA. The whole genome sequence of a sample collected from one COVID-19 positive patient at a hospital in the Kanto area at the end of November 2020 was found to be very similar to a virus strain prevalent on the West States of the United States (a type of 20C, B.1.346) according to phylogenetic tree analysis. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 1, 2021. ; CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted February 1, 2021. ; Severity of COVID-19 is inversely correlated with increased number counts of non-synonymous mutations in Tokyo Estimated transmissibility and severity of novel SARS-CoV-2 Variant of Concern 202012/01 in England Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK European Centre for Disease Prevention and Control, Risk related to spread of new SARSCoV-2 variants of concern in the EU/EEA Nextstrain: real-time tracking of pathogen evolution Phylogenetic analysis of SARS-CoV-2 in Boston highlights the impact of superspreading events Genomic epidemiology of superspreading events in Austria reveals mutational dynamics and transmission properties of SARS-CoV-2 Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity We thank all the patients and healthcare workers who have fought against COVID-19. Shibasaburo Kitasato, the founder of Keio University School of Medicine. We also thank SRL, Inc.