Ko Ko1, Shintaro Nagashima1, Bunthen E1,2, Serge Ouoba1,3, Tomoyuki Akita1, Aya Sugiyama1, Masayuki Ohisa1, Takemasa Sakaguchi4, Hidetoshi Tahara5, Hiroki Ohge6, Hideki Ohdan7, Tatsuhiko Kubo8, Eisaku Kishita9, Masao Kuwabara10, Kazuaki Takahashi1, Junko Tanaka1. 1. Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 2. Payment Certification Agency, Ministry of Health, Phnom Penh, Cambodia. 3. Unité de Recherche Clinique de Nanoro (URCN), Nanoro, Burkina Faso. 4. Department of Virology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 5. Department of Cellular and Molecular Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 6. Department of Infectious Diseases, Hiroshima University Hospital, Hiroshima, Japan. 7. Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 8. Department of Public Health and Health Policy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 9. Hiroshima Prefecture Health and Welfare Bureau, Hiroshima, Japan. 10. Hiroshima Prefectural Center for Disease Control and Prevention, Hiroshima, Japan.
Abstract
BACKGROUND: 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. METHOD: 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. RESULTS: Viral load ranged 7.85 × 101-1.42 × 108 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 11-15 base mutations. Comparing the first- and second-wave GR strains, mutation rate was 1.17-1.36 × 10-3 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 ORF8 that causes a defect in ORF8 protein was found in one isolate (S66) from the second wave. CONCLUSION: Our findings reveal the evolutionary hierarchy of SARS-CoV-2 in Japan. The predominant D614G variants and a new form of ORF8 deletion in Hiroshima provide the clue for role of viral factor in local outbreaks of SARS-CoV-2.
BACKGROUND: 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. METHOD: 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. RESULTS: Viral load ranged 7.85 × 101-1.42 × 108 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 11-15 base mutations. Comparing the first- and second-wave GR strains, mutation rate was 1.17-1.36 × 10-3 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 ORF8 that causes a defect in ORF8 protein was found in one isolate (S66) from the second wave. CONCLUSION: Our findings reveal the evolutionary hierarchy of SARS-CoV-2 in Japan. The predominant D614G variants and a new form of ORF8 deletion in Hiroshima provide the clue for role of viral factor in local outbreaks of SARS-CoV-2.
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