{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T07:14:51Z","timestamp":1790493291175,"version":"4.1.0"},"reference-count":26,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2021,2,5]],"date-time":"2021-02-05T00:00:00Z","timestamp":1612483200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"A grant from the Government Academia Collaboration of Hiroshima Prefecture"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>In this study, we performed molecular characterization of SARS-CoV-2 strains in Hiroshima and its mutation pattern between the first and second waves of the outbreak.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Method<\/jats:title>\n<jats:p>A total of 55 nasal swab samples from the first wave in Hiroshima and 13 from the second wave were examined quantitatively by RT-qPCR and qualitatively by nested PCR using specific primers. Four samples from each wave underwent next-generation sequencing and phylogenetic tree analysis including controls and all sequences retrieved in Japan from GISAID and GenBank. Subsequently, mutations were examined.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>Viral load ranged 7.85 \u00d7 10<jats:sup>1<\/jats:sup>\u22121.42 \u00d7 10<jats:sup>8<\/jats:sup> copies\/ml. Of 68 samples, one was Asian type-O, 65 were European type-GR, and 2 were undetectable. Phylogenetic tree analysis indicated that Japan was infected with various Asian strains (L, S, V, O) from January through April. By second week of March, European strains (G, GH, GR) had appeared, and GR strains became predominant after mid-March. The first case in Hiroshima was classified as Asian strain O, and the rest were GR strains. Then, second wave of GR strains appeared independently with 11\u201315 base mutations. Comparing the first- and second-wave GR strains, mutation rate was 1.17\u20131.36 \u00d7 10<jats:sup>\u22123<\/jats:sup> base substitutions per site per year; in addition, amino acid changes occurred at S1361P and P3371S in ORF1a, A314V in ORF1b, and P151L in N. All seven GR strains were D614G variants with R202K and G203R mutations in N. A single-nucleotide insertion in <jats:italic>ORF8<\/jats:italic> that causes a defect in ORF8 protein was found in one isolate (S66) from the second wave.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusion<\/jats:title>\n<jats:p>Our findings reveal the evolutionary hierarchy of SARS-CoV-2 in Japan. The predominant D614G variants and a new form of <jats:italic>ORF8<\/jats:italic> deletion in Hiroshima provide the clue for role of viral factor in local outbreaks of SARS-CoV-2.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0246383","type":"journal-article","created":{"date-parts":[[2021,2,8]],"date-time":"2021-02-08T07:09:33Z","timestamp":1612768173000},"page":"e0246383","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":34,"title":["Molecular characterization and the mutation pattern of SARS-CoV-2 during first and second wave outbreaks in Hiroshima, Japan"],"prefix":"10.1371","volume":"16","author":[{"given":"Ko","family":"Ko","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shintaro","family":"Nagashima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bunthen","family":"E.","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3944-0033","authenticated-orcid":true,"given":"Serge","family":"Ouoba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoyuki","family":"Akita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aya","family":"Sugiyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masayuki","family":"Ohisa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takemasa","family":"Sakaguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hidetoshi","family":"Tahara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroki","family":"Ohge","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideki","family":"Ohdan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8822-9181","authenticated-orcid":true,"given":"Tatsuhiko","family":"Kubo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eisaku","family":"Kishita","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masao","family":"Kuwabara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Takahashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5669-4051","authenticated-orcid":true,"given":"Junko","family":"Tanaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,2,5]]},"reference":[{"issue":"4","key":"pone.0246383.ref001","first-page":"536","article-title":"The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2","volume":"5","author":"AE Gorbalenya","journal-title":"Nature Microbiology. 2020"},{"issue":"6","key":"pone.0246383.ref002","doi-asserted-by":"crossref","first-page":"1011","DOI":"10.3390\/v4061011","article-title":"Mechanisms of coronavirus cell entry mediated by the viral spike protein","volume":"4","author":"S Belouzard","year":"2012","journal-title":"Viruses"},{"issue":"6","key":"pone.0246383.ref003","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1038\/s41577-020-0311-8","article-title":"The trinity of COVID-19: immunity, inflammation and intervention","volume":"20","author":"MZ Tay","year":"2020","journal-title":"Nature Reviews Immunology"},{"issue":"4","key":"pone.0246383.ref004","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1177\/1091581815584918","article-title":"Cytokines: The Good, the Bad, and the Deadly","volume":"34","author":"T Ramani","year":"2015","journal-title":"International Journal of Toxicology"},{"key":"pone.0246383.ref005","unstructured":"(WHO) WHO. 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