key: cord-1032363-ix63eaa9
authors: Hirotsu, Y.; Omata, M.
title: Discovery of SARS-CoV-2 strain of P.1 lineage harboring K417T/ E484K / N501Y by whole genome sequencing in the city, Japan
date: 2021-02-26
journal: nan
DOI: 10.1101/2021.02.24.21251892
sha: 25a845534008c7ac3f58dde9b9cdcb742f792a02
doc_id: 1032363
cord_uid: ix63eaa9

On the February 2020, the very first case was an American female from Diamond Princess cruise ship. Since, we have confirmed 136 patients infected with coronavirus disease 2019 (COVID-19) until February 2021. Here, we conducted the whole genome sequencing analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on samples from 70 of 136 patients (51.5%). These patients were infected in Diamond Princess cruise ship (n=1), Africa (n=2), Japan (n=66) and Brazil (n=1). The viral genome sequence of a patient on the Diamond Princess cruise ship in February 2020 was similar to that of original strain found in Wuhan, China (19A clade). Four patients, including two returnees from Africa and two lived in Japan, confirmed at the end of March 2020 had sequences similar to those of lineage with D614G mutation, which was endemic in Europe (20A [n=3] and 20B [n=1] clade). The 64 Japanese patients confirmed from September 2020 to January 2021 had sequences similar to those of the currently prevalent lineage (20B [n=58] and 20C clade [n=6]). Subsequent analysis revealed three mutations (K417T/ E484K / N501Y) in the receptor binding domain of the spike protein in a man in his 40s. The sequence was identical to the P.1 lineage (also known as 20J/501Y.V3) reported in Brazil. This is the first report of SARS-CoV-2 P.1 lineage identified in the city, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan province, China, at December 2019. The virus is highly transmissible and its basis reproduction number (R0) is estimated to range between 2.2 and 3.9 [1] . To date, 107 million individuals have been infected with SARS-CoV-2 and 2.4 million patients have passed away from coronavirus disease 2019 (COVID-19) [2] .

SARS-CoV-2 belong to the Coronaviridae family and subdivided into genera βcoronaviruses, subgenus Sarbecovirus. SARS-CoV-2 has a single-stranded, positive-sense RNA genome with a total of 29.9 kilo base pairs in length. SARS-CoV-2 has the largest genome size among RNA virus and encodes for a 3′-5′ exoribonuclease with proofreading function, which result in low mutation rate compared to other RNA virus [3] .

It is assumed that most of the mutations occur neutrally and may not influence on virus properties. However, pandemic spread of the virus in naïve populations may affect on the selection of mutations that alter pathogenesis, virulence, and/or transmissibility [4, 5] .

Currently, an emergent D614G mutation in the spike glycoprotein of SARS-CoV-2 is prevalent globally [6, 7] . Afterward, new emerging lineages with spike protein mutations were In this study, we performed the whole genome sequencing using 70 nasopharyngeal swab samples from COVID-19 patient collected between February 2020 and February 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

A total of 136 COVID-19 patients were confirmed in our hospital. Of these, we included 70 samples for subsequent genome analysis. For these patients, one was a passenger on a Diamond cruise ship on February 2020, four was confirmed at our district on the end of March 2020, 64 was confirmed on September 2020 to January 2021, and one was on February 2020. These included one American patient infected in Diamond Princess cruise ship, two returnees from Africa, one returnee from Brazil and 66 Japanese, one year past from first confirmed case.

Nasopharyngeal swab samples were collected using cotton swabs and placed in 3 mL of viral transport media (VTM) purchased from Copan Diagnostics (Murrieta, CA, USA).

We used 200 μL of the VTM were used for nucleic acid extraction within 2 hours after sample collection.

Total nucleic acid was isolated from the samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the KingFisher Duo Prime System (Thermo Fisher Scientific) as we previously described [15, 16] . Briefly, we added 200 µL of VTM, 5 µL of proteinase K, 265 μL of binding solution, 10 μL of total nucleic acid-binding beads, 0.5 mL of wash buffer, and 0.5-1 mL of 80% ethanol to each well of a deep-well 96-well plate. The nucleic acids were eluted with 70 μL of elution buffer. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

According to the protocol developed by the National Institute of Infectious Diseases (NIID) in Japan [15, [17] [18] [19] ], we performed one-step RT-qPCR to detect SARS-CoV-2. This PCR amplifies the nucleocapsid (N) gene of SARS-CoV-2 (NC_045512.2). For the internal positive control, the human ribonuclease P protein subunit p30 (RPP30) gene was used (Integrated DNA Technologies, Coralville, IA, USA) [3] . The RT-qPCR assays were conducted on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with the following cycling conditions: 50°C for 5 min for reverse transcription, 95°C for 20 s, and 45 cycles of 95°C for 3 s and 60°C for 30 s. The absolute copy number of viral loads was determined using serial diluted DNA control targeting the N gene of SARS-CoV-2 (Integrated DNA Technologies) as previously described [18] .

The sample antigen levels were determined quantitatively with the LUMIPULSE SARS-CoV-2 Ag test (Fujirebio, Inc., Tokyo, Japan) according to the manufacturer's instructions [20] [21] [22] . In brief, 700 μL of the VTM samples were briefly vortexed, transferred into a sterile tube, and centrifuged at 2,000 ×g for 5 min. Aliquots (100 μL) of the supernatant were used for testing on the LUMIPULSE G600II automated system (Fujirebio). For samples with an antigen level > 5,000 pg/mL, the samples were diluted with the kit diluent and retested, and the antigen level was calculated taking the dilution factor into account. Samples with an antigen level ≥ 10 pg/mL were considered positive, samples with ≥ 1.0 pg/mL and < 10.0 pg/mL antigen were labeled inconclusive, while a result of <1.0 pg/mL was considered negative as per the manufacturer's guidelines [22] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint

We amplified the genomic region of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 Research Panel consists of two primer pools covering 99.7% of the viral genome (Thermo Fisher Scientific). The amplicon size ranges from 125 to 275 base pairs in length.

Extracted nucleic acids were subjected to genome sequencing on the Ion Torrent Genexus System, which automates the specimen-to-report workflow and yields results in a single day.

The data of sequencing reads and quality control were processed on Genexus Software with SARS-CoV-2 plugins. The sequencing reads were mapped and aligned on the reference genome of SARS-CoV-2 strain Wuhan-Hu-1 (Accession no NC_045512) using the torrent mapping alignment program (TMAP). After initial mapping, a variant call was performed using the Torrent Variant Caller. The COVID19AnnotateSnpEff plugin was used for annotation of variants. Assembly was performed with the Iterative Refinement Meta-Assembler (IRMA) [23], which produced the FASTA file.

Phylogenetic tree analysis and classification were conducted using Nextstrain is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. Global sequencing data of P.1 lineage was exported from GISAID EpiCoV database by 14 th February, 2021. We searched and found the 121 available datasets. We used these metadata and FASTA files were used for subsequent analysis.

The Institutional Review Board of the Clinical Research and Genome Research Committee at Yamanashi Central Hospital approved this study and the use of an opt-out consent method (Approval No. C2019-30). The requirement for written informed consent was waived owing to the observational study and the urgent need to collect data of COVID-19. Participation in the study by patients was optional. All methods were performed in accordance with the relevant guidelines and regulations, and with the Helsinki Declaration.

To determine the genomic characterization of SARS-CoV-2 identified in the Kofu is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint city, Japan, we started to whole genome sequencing analysis since 8 th January, 2021. A total of the 136 samples obtained from COVID-19 patients who were confirmed in our hospital. Of these, 70 samples were subjected to analysis, which represented 51.5% of the total confirmed patients by February 15 th 2021.

The yielded sequence data was subjected to phylogenetic analysis using the 

A man in his 40s, who had a history of staying in Brazil, attended our hospital in early February with symptoms of fever at 38.9 °C. RT-qPCR judged positive with high viral load (7.1 log10/µL) and low Ct value (Ct: 15). The antigen level was also high at 183,426 pg/mL. The patient was confirmed to be negative for SARS-CoV-2 by airport quarantine when he returned to Japan four days earlier. His symptom was mild and he was admitted to another hospital. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint 9 These mutations were perfectly matched with the mutations in P.1 lineage previously discovered in Brazil [10] (Figure 3 ). In RBD of spike protein, three mutations (K417T, E484K and N501Y) were identified. These results suggested we identified the emerging strain related to 20J/501Y.V3 (P.1 lineage) in Japan.

To examine the global data of SARS-CoV-2 of 20J/501Y.V3 (P.1 lineage), we referred the sequence data deposited in GISAID [25, 28] . By February 14 th 2021, a total of 121 sequence data was available, and 119 were derived from patient and 2 were viral strains isolated from TMPRSS2-expressing Vero E6 cell line [29] . (Table 1) , suggesting P.1 lineage began to spread in the world.

We started to the genomic surveillance to monitor the SARS-CoV-2 variants from 8 th January, 2021. In this study, we confirmed that five clades have emerged over the time.

The consecutive analysis identified SARS-CoV-2 of 20J/501Y.V3 (P.1 lineage) in a patient and detected mutations were identical to those of original P.1 lineage discovered in Brazil is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint [10]. This is the first report on the 20J/501Y.V3 (P.1 lineage) in the city, Japan.

On January 6, 2021, the NIID detected a new emerging strain (P.1 lineage) of SARS-CoV-2 at airport quarantine in four travelers who arrived from Brazil on 2 nd January 2021 [30] [31] [32] (Table 1) In particular, mutations in the RBD of spike protein are noteworthy [33, 34] . N501Y mutation increases the overall binding affinity between RBD and human ACE2 receptor [35, 36] . Recently, escape mutations from neutralizing antibody recognition were identified by SARS-CoV-2 spike protein-expression vesicular stomatitis virus (VSV) and yeast [37, 38] .

The K417N/T, E484K and N501Y mutation reduced the neutralizing activity of convalescent and mRNA vaccine-elicited serum [39] [40] [41] [42] [43] .

The SARS-CoV-2 variant is not always related to threat to human health because the virus acquires genomic diversity during the course of its life cycle [44] . However, some of these mutations would be associated to attenuate the neutralizing activity of antibody. During the ongoing evolution of SARS-CoV-2, the new emerging lineage is likely to be circulating in the human population. Thus, genomic surveillance is important for public health to monitor the emerging lineage, evaluate vaccine efficacy and virus transmissibility. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint * Four were identified at airport quarantine and one was identified in this study.

. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint

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is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted February 26, 2021. ; https://doi.org/10.1101/2021.02.24.21251892 doi: medRxiv preprint