key: cord-0286950-yzieelyd authors: Hirotsu, Y.; Omata, M. title: SARS-CoV-2 B.1.1.7 lineage rapidly spreads and overwhelms R.1 lineage in Japan: serial and stationary observation in a community date: 2021-07-06 journal: nan DOI: 10.1101/2021.06.30.21259820 sha: 9394dad6df76719ca7deb6545383ce2935d9618a doc_id: 286950 cord_uid: yzieelyd Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulates in the world and acquires mutations during evolution. To identify the new emergent variants, the surveillance of the variants of concern (VOC) and variants of interest (VOI) is ongoing. This study aimed to determine how the transition of viral lineage occurred by stationary genome analysis in Yamanashi, Japan. Methods We performed the whole genome sequencing using SARS-CoV-2 positive samples (n=325) collected from February 2020 to the end of June 2021. The number of analyzed samples accounted for 15.4% of the total 2,109 samples identified in our community. Viral lineage was defined by the Phylogenetic Assignment of Named Global Outbreak (PANGO) lineages. Results We identified 13 types of viral lineages including R.1, P.1, B.1.1.7 (Alpha) and B.1.617.2 (Delta) These virus lineages had distinct periods of expansion and decline. After the emerging of the R.1 lineage harboring E484K variant (designated VOI in Japan), the prevalent B.1.1.214 lineage were no longer identified. The R.1 lineages were temporarily prevalent afterwards, but the influx of B.1.1.7 lineage (designated VOC) led to a decline in R.1. Currently, B.1.1.7 has become dominant after mid-April, 2021. Conclusion We clearly elucidated the transition and replacement of viral lineage by the community-based analysis. The virus completely replaced by more infectious lineages, therefore, it will be necessary to continue to monitor the VOC and VOI. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide and threatening human health. In countries where vaccination is widely available, the number of infected cases and deaths turned to be decreasing, giving a hope for in Japan also have their own designations for VOCs and VOIs [5] . These lineages have hallmark mutations in spike protein, which was suggested to affect the transmissibility, immunity, and disease severity [6] . To prevent the spread of SARS-CoV-2, these VOCs and VOIs continue to be under surveillance in many countries. In January 2021, we started to conduct genomic surveillance of SARS-CoV-2 [7-9]. We previously reported on the SARS-CoV-2 R.1 lineage harboring spike W152L, E484K and G769V mutations [8] . In Japan, the R.1 lineage was first registered in Global Initiative on Sharing All Influenza Data (GISAID) database in November 2020 and showed an increase around January 2021 [8, 10, 11] . Although the NIID in Japan has designated R.1 lineage as a VOC, it is not clear there are associations with increased infectivity and transmissibility [5]. The B.1.1.7 lineage was first identified in the United Kingdom at September 2020 and detected at airport quarantine in Japan at December 2020. The B.1.1.7 was reported to be highly transmissible and increase the disease severity [12, 13] . Actually, the B.1.1.7 spreads rapidly and is identified in 150 countries as of June 30, 2021 [14, 15] . Although B.1.1.7 is reported to be highly transmissible, the transition of other virus lineage in Japan has not been fully elucidated after the influx of B.1.1.7. In this study, we conducted whole genome analysis of SARS-CoV-2 in 325 samples . 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint collected from February 2020 to June 2021 in Kofu, Japan. The lineages other than VOC and VOI were observed until mid-January 2021, afterward, the R.1 lineage was dominant. However, the subsequent influx of B.1.1.7 increased rapidly, suggesting a rapid replacement of R.1 with B.1.1.7. We elucidated that the two major lineages have different infectivity based on genomic surveillance. 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 it being an observational study and the urgent need to collect COVID-19 data. Nasopharyngeal swab samples were collected by using cotton swabs and placed in 3 ml of viral transport media (VTM) purchased from Copan Diagnostics (Murrieta, CA, United States). We used 200 µl of VTM for nucleic acid extraction, performed within 2 h of sample collection. Total nucleic acid was isolated using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific; Waltham, MA, United States) as previously described [16] . To detect hallmark mutations found in VOC and VOI, we performed allelic discrimination analysis with a TaqMan assay. We also used a TaqMan SARS-CoV-2 Mutation Panel for detecting spike 69-70 deletion, N501Y and E484K (Thermo Fisher Scientific). We also designed a Custom TaqMan assay (Thermo Fisher Scientific) for detecting SARS-CoV-2 spike protein with the W152L and G769V mutations as previously described [8] . TaqPath 1- Step RT-qPCR Master Mix CG was used as the master mix. The TaqMan Minor Groove Binder probes for the wild-type and variant alleles were labelled with VIC dye and FAM dye fluorescence, respectively. . 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint We studied a total of 335 patients who were infected with SARS-CoV-2 determined by realtime quantitative PCR and/or quantitative antigen test from February 15, 2020 to June 30, 2021 [16] [17] [18] [19] . We subjected all these samples to whole genome analysis and successfully obtained 325 sequence data, except for 10 samples with very low viral load. SARS-CoV-2 genomic RNA was reverse transcribed into cDNA and amplified by using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific) on the Ion Torrent Genexus System in accordance with the manufacturer's instructions [8, 9] . Sequencing reads were processed, and their quality was assessed by using Genexus Software with SARS-CoV-2 plugins. The sequencing reads were mapped and aligned by using the torrent mapping alignment program. After initial mapping, a variant call was performed by using the Torrent Variant Caller. The COVID19AnnotateSnpEff plugin was used for the annotation of variants. Assembly was performed with the Iterative Refinement Meta-Assembler [20] . The viral clade and lineage classifications were conducted by using Nextstrain [14] , and Phylogenetic Assignment of Named Global Outbreak (PANGO) Lineages [21] . The sequences data was deposited in the Global Initiative on Sharing Avian Influenza Data (GISAID) EpiCoV database [22] . From February 2020 to the end of June 2021, we collected 335 SARS-CoV-2 positive samples determined by RT-qPCR and/or quantitative antigen tests [16] [17] [18] [19] . We subjected these samples to the whole genome analysis and TaqMan mutation screening assay. As a result, sequencing analysis could successfully determine viral sequence from 325 individuals, excluding 10 individuals due to the very low viral load. As of June 30, this represented 15.4% of the 2,109 infected individuals identified in our district. To characterize the viral lineage, the yielded sequence data were analyzed by PANGO lineage [15, 21] . The result showed the 325 samples were classified into 13 types of lineage (B, B.1, B.1.1, B.1.149, B.1.1.284, B.1.1.214, B.1.411, B.1.346, R.1, P.1, B.1.1.220 . 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 July 6, 2021. [23] . At the second (from July to September 2020) and third waves (from October 2020 to February 2021), the B. 1.1.214 (a total of 96 samples) and B.1.1.284 (20 samples) lineages were prevailed. Almost of the B. 1.1.214 and B.1.1.284 lineages were identified only in Japan and did not spread to other countries [24] . Afterward, several types of VOC or VOI were identified in our district (Figure 1 and Table 1 ). The R.1 was first detected in January 2021 and a total of 20 were identified so far (Table 1) The B.1.1.7 has N501Y mutation in RBD of spike protein, binds to the angiotensin-converting enzyme 2 with high affinity and acquires a high transmission rate [25] . This study revealed that the B.1.1.7 rather than the R.1 expanded in Japan during the April to June, 2021. These , which have been shown to be of concern for immune escape [26] , while there are no reports on its transmission potential. Of note, the fact that the B.1.1.214 lineage was replaced by the R.1 lineage suggests that R.1 possibly has higher transmissibility. This situation is observed by the data of genomic epidemiological study throughout Japanese dataset and in Tokyo [10, 11, 27] , indicating current study shows that the situation is not . 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint limited to one district. Recent study showed the relative instantaneous reproduction numbers of the R.1, B. 1.1.7, and B.1.617.2 compared to other strains were estimated at 1.256 (range: 1.198-1.335), 1.449 (range: 1.342-1.596) , and 1.776 (range: 1.557-2.00), respectively [28] . This data is consistent with the situation observed in our community, suggesting that a virus . 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 July 6, 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint . 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint A Discernable Increase in the Severe Acute Respiratory Syndrome Coronavirus 2 R.1 Lineage Carrying an E484K Spike Protein Mutation in Japan SARS-CoV-2 R.1 lineage variants prevailed in Tokyo in Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England Risk of mortality in patients infected with SARS-CoV-2 variant of concern 202012/1: matched cohort study Nextstrain: real-time tracking of pathogen evolution Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients Double-quencher probes improve detection Nasopharyngeal Swabs from 1,033 Patients using the LUMIPULSE SARS-CoV Antigen Test: Comparison with RT-qPCR Viral deep sequencing needs an adaptive approach: IRMA, the iterative refinement metaassembler A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology GISAID: Global initiative on sharing all influenza data -from vision to reality Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity. The Lancet Infectious Diseases Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization Introductions and evolutions of SARS-CoV-2 strains in Japan Predicted domination of variant Delta of SARS-2021:2021 BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals We also thank Masato Kondo, Ryota Tanaka, Kazuo Sakai, Manami Nagano, Takuhito Fukami, and Ryo Kitamura (Thermo Fisher Scientific) for technical help, all of the medical and ancillary hospital staff for their support, and the patients for their participation. We thank all researchers who share genome data on GISAID (http://www.gisaid.org). 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint CoV-2 before Tokyo Olympic games, Japan. medRxiv . 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 July 6, 2021. Nature Medicine 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 July 6, 2021. ; https://doi.org/10.1101/2021.06.30.21259820 doi: medRxiv preprint