An epidemiological study of the SARS-CoV-2 genome in Japan(updated: April 16, 2020)

nActive, nationwide epidemiological surveillance of COVID-19 clusters in Japan
In late December 2019, the initial outbreak of the “coronavirus disease 2019” (COVID-19)

occurred in Wuhan, China. It was caused by a new strain of beta coronavirus, known as thesevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1-3. After the identification of
the first COVID-19 patient in Japan on January 15, 2020, multiple local COVID-19 clusters wereidentified nationwide by the end of February. The Japanese government focused on identifying
and mitigating emerging COVID-19 clusters before they could spread further. In an effort tocontain these clusters and limit the number of new cases, active, nationwide epidemiologicalsurveillance of each cluster was conducted in order to identify the close contacts of existing
COVID-19 patients.
Although some of the COVID-19 clusters were successfully contained, the number of cases
continued to increase. The Japanese government declared a nationwide state of emergency inview of the worsening spread. To support the ongoing epidemiological surveillance, wecollaborated with the local public health institutes in Japan (listed below), and conducted wholegenome sequencing of SARS-CoV-2. Our goal was to apply genomic epidemiology to predict
potential routes of infection within or between clusters 4.

nImprovement of the ARTIC Network’s multiplex polymerase chain reaction (PCR) for
SARS-CoV-2 genome sequencing
Whole genome sequences of SARS-CoV-2 were obtained by means of the PrimalSeq

protocol for enriching the cDNA of the SARS-CoV-2 genome using multiplex RT-PCR, asproposed by the Wellcome Trust ARTIC Network 5. We found two amplicons that regularly showed
low to zero coverage due to primer dimerization as described by Itokawa et al. 6. Therefore, weused a modified primer for the multiplex PCR amplification 6. The next-generation sequencing
reads were mapped to the SARS-CoV-2 Wuhan-Hu-1 reference genome sequence (29.9 kb ssRNA; GenBank ID: MN908947), followed by de novo assembly to obtain the full genomesequence.

nGenomic epidemiology based on the full genome sequence of SARS-CoV-2
The nearly full-length genome sequences (≥ 29 kb, 4,511 entries) of SARS-CoV-2 wereretrieved from the GISAID EpiCoV database (updated on April 16, 2020) 7. We determined thefull genome sequences using 562 clinical specimens in Japan, and performed a haplotype
network analysis using genome-wide single nucleotide variations (SNVs) to trace potential
infection routes based on genealogy (Fig. 1). Thus far, the mutation rate and frequency has been
estimated at 25.9 SNVs/genome/year using the molecular clock technique 8, suggesting thatthroughout the whole genome sequence at least 9 SNVs may have emerged during the fourmonths since the end of 2019.
Some of the primary clusters identified through January and February in Japan (red circle ● inFig 1) have been identified around the Wuhan-Hu-1-related isolates from China. These clusters
appeared to decrease and are the most containable clusters till date.
Passengers aboard the Diamond Princess (DP) cruise ship were quarantined shortly after its
return to Yokohama, Japan on February 3, 2020, because an 80-year-old passenger whodisembarked in Hong Kong was confirmed to have COVID-19 on February 1, 2020. We
determined the SARS-CoV-2 genome sequences from 70 individuals aboard the DP cruise ship(148 positive individuals among 895 passengers) 9 and most of the isolates showed one SNV(G11083T) (magenta circle ● in Fig. 1). These DP-related isolates have not yet been detected inJapan, and have not spread throughout the country.
The aforementioned primary wave of infections originating in China appeared to have beenmostly contained through implementation of active surveillance in Japan. In contrast, the numberof COVID-19 cases increased rapidly across the Europe and North America during early March,
indicative of a pandemic. Concurrently, many sporadic COVID-19 cases were detected in Japan
from the end of March through early April. The haplotype network analysis demonstrated that anadditional large cluster (red circle ● at the upper left corner in Fig. 1) around Europe-related
isolates (originating in China or other countries) was predominantly observed in Japan during
early April, suggesting that they could have caused additional domestic spread within Japan.

nGenomic epidemiology to support active epidemiological surveillance
Analysis of whole genome sequences provides information that is useful for tracing backinfection using genome-wide SNVs among clusters. During the primary wave, local public healthcenters were able to identify likely COVID-19 patients and their close contacts within locationspecific clusters by conducting active epidemiological surveillance using the keywords “Wuhan,Hubei, and China.” Just as the situation had begun to improve around mid-March, a large numberof new COVID-19 patients were diagnosed, many of with unclear infection routes. Tracing theinfection routes was difficult because some Japanese cases had no recent history of travel toChina or any country outside Japan. The SARS-CoV-2 haplotype network analysis suggested
that the second wave of COVID-19 cases could have been imported via the returnees fromEurope, North America, or other countries.
This report suggests that haplotype network analysis using genome-wide SNVs can support
epidemiological field investigations and highlight potential infection linkages. A project todetermine viral evolution and its spread across the community is being conducted by researchers
in the US and UK 10,11. To mitigate the next wave of COVID-19 in Japan, it is necessary toformulate a more efficient containment strategy.

Acknowledgements:
We sincerely thank the COVID-19 working group in Japan listed below for the collection andtransportation of clinical specimens. We are particularly grateful to the staff of the National
Institute of Infectious Diseases, Field Epidemiology Training Program (FETP) team, the Ministryof Health, Labor and Welfare, and local governments for their assistance with administrativematters, field investigations, data collection, and laboratory testing.
We would like to thank all the researchers who have kindly deposited and shared genomic dataon GISAID.
This study was supported by a Grant-in Aid from the Japan Agency for Medical Research andDevelopment (AMED) under Grant Numbers JP19fk0108104 and JP20fk0108103).

Correspondence:
Makoto Kuroda, PhD
Director, Pathogen Genomics Center, National Institute of Infectious Diseases.
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Phone: +[81]-3-5285-1111
makokuro@niid.go.jp

Fig. 1. Haplotype network analysis using genome-wide single-nucleotide variations (HNGSNVs) of worldwide SARS-CoV-2 isolates.
Whole-genome sequences of SARS-CoV-2 isolates in Japan (n = 562) were compared with all
GISAID-available SARS-CoV-2 genomes (n = 4,511, updated on April 16, 2020) using medianjoining SNV network analysis. SARS-CoV-2 that disseminated from Wuhan City, China, at theend of December, 2019 (one of the potential origins of Wuhan-Hu-1) is plotted at the center of the
haplotype network.

Collaboration with local public health institutes in Japan:
Tokyo Metropolitan Institute of Public HealthKobe Institute of Health
Sakai City Public Health Research InstituteGunma Prefectural Institute of Public Health and Environmental Sciences
Wakayama Prefectural Institute of Public Health and Environmental Science
Nagoya City Public Health Research Institute
Saitama Prefectural Institute of Public Health,Chiba Prefectural Institute of Public Health
Oita Prefectural Institute of Public Health and Environmental Science
Gifu Prefectural Institute of Public Health and Environmental Sciences
Shizuoka City Public Health Research InstituteIshikawa Prefectural Institute of Public Health and Environmental Science
Himeji City Public Health Research Institute
Sagamihara City Public Health Research InstituteKumamoto City Public Health Research InstituteOta-ku Public Health Center
Toyama Institute of Health
Fukui Prefectural Institute of Public Health and Environmental Science
Osaka Institute of Public HealthNagasaki Prefectural Institute of Public Health and Environmental Science
Eighteen additional institutes

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4

https://www.niid.go.jp/niid/images/epi/corona/2019nCoV-02-200420.pdf.

5

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Itokawa, K., Sekizuka, T., Hashino, M., Tanaka, R. & Kuroda, M. A proposal of an alternative primer
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https://www.gisaid.org/epiflu-applications/next-hcov-19-app/.

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https://nextstrain.org/ncov?l=unrooted.

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Sekizuka, T. et al. Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise
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10

https://www.cogconsortium.uk/.

11

https://covidgenomics.org/.

,