key: cord-0911360-cy05bf7q authors: Hirotsu, Y.; Maejima, M.; Shibusawa, M.; Natori, Y.; Nagakubo, Y.; Hosaka, K.; Sueki, H.; Mochizuki, H.; Tsutsui, T.; Kakizaki, Y.; Miyashita, Y.; Omata, M. title: SARS-CoV-2 Omicron sublineage BA.2 replaces BA.1.1: genomic surveillance in Japan from September 2021 to March 2022 date: 2022-04-06 journal: nan DOI: 10.1101/2022.04.05.22273483 sha: 53b370978c6fb44b08e59f73febca6d57e01a6da doc_id: 911360 cord_uid: cy05bf7q Objective The new emerging Omicron strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently spreading worldwide. We aimed to analyze the genomic evolution of the shifting Omicron virus subtypes. Methods The study included 1,297 individuals diagnosed as SARS-CoV-2 positive by PCR test or antigen quantification test from September 2021 to March 2022. Samples were analyzed by whole genome sequencing analysis (n=489) or TaqMan assay (n=808). Results After the outbreak of the SARS-CoV-2 Delta strain, the Omicron strain spread rapidly in Yamanashi, Japan. BA.1.1 was the predominant sublineage of the Omicron strain from January to mid-February 2022, but the number of cases of sublineage BA.2 began to increase after mid-February, and this sublineage was shown to have replaced BA.1.1 by the end of March 2022. We observed higher viral and antigen levels of sublineage BA.2 than of sublineage BA.1.1 in nasopharyngeal swab samples. However, no difference in viral load by patient age was apparent between sublineages BA.1.1 and BA.2. Conclusions A transition from sublineage BA.1.1 to sublineage BA.2 was clearly observed over approximately one month. Omicron sublineage BA.2 was found to be more transmissible owing to its higher viral load regardless of patient age. A transition from sublineage BA.1.1 to sublineage BA.2 was clearly observed over 49 approximately one month. Omicron sublineage BA.2 was found to be more transmissible 50 owing to its higher viral load regardless of patient age. 51 52 . 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 April 6, 2022. Since the discovery of severe acute respiratory syndrome coronavirus 2 54 (SARS-CoV-2) at the end of 2019, large numbers of infections and deaths have been 55 reported. The Omicron (B.1.1.529) strain of SARS-CoV-2 was first identified in South Africa, 56 and infection with Omicron has been confirmed in 169 countries to date [1, 2] . World Health 57 Organization designated the Omicron strain as a variant of concern at the end of November 58 2021 [1] . Now several Omicron strain sublineages, such as BA.1, BA.1.1, BA.2, and BA.3, 59 have been described. 60 The Omicron strain has multiple spike protein mutations compared with other 61 variants of concern, such as the Alpha and Delta strains [2] . Consequently, there is concern 62 that serum antibody activity against the Omicron strain in vaccinated or convalescent 63 persons will be weaker than that against previous SARS-CoV-2 strains [3, 4] . In addition, for 64 some antibody therapies, the level of neutralizing activity was shown to differ between 65 Omicron sublineages BA.1 and BA.2 [5, 6] . Omicron strains are considered to be highly 66 transmissible but have a relatively lower critical illness risk [7] [8] [9] [10] . In many countries, 67 Omicron strains are rapidly increasing in prevalence and affecting medical and social 68 activities. Because the characteristics of infectivity and treatment response differ among 69 Omicron sublineages, it is important to understand the evolutionary process in real time. 70 In this study, we conducted whole genome sequencing analyses and TaqMan . 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 April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273483 doi: medRxiv preprint 4 To detect SARS-CoV-2, we performed one-step RT-qPCR amplifying the 89 nucleocapsid (N) gene of SARS-CoV-2, as we described previously [16] . The human 90 ribonuclease P protein subunit p30 (RPP30) gene was used as the internal positive control 91 (Integrated DNA Technologies, Coralville, IA, USA) [16] . accordance with the manufacturer's instructions [17] [18] [19] . Sequencing reads were processed, 108 and their quality was assessed using Genexus Software with SARS-CoV-2 plugins. The 109 sequencing reads were then mapped and aligned using the torrent mapping alignment 110 program. After initial mapping, a variant call was performed using the Torrent Variant Caller. 111 The COVID19AnnotateSnpEff plugin was used to annotate the variants. Assembly was 112 performed using the Iterative Refinement Meta-Assembler [20] . 113 The viral clade and lineage classifications were conducted using Nextstrain [21] We used the pre-designed TaqMan SARS-CoV-2 Mutation Panel for detecting 120 SARS-CoV-2 spike Δ69-70, G339D, L452R, and/or Q493R (Thermo Fisher Scientific) using 121 808 SARS-CoV-2-positive samples (in submission). The TaqMan MGB probe for the 122 wild-type allele was labelled with VIC dye, and the probe for the variant allele was labelled 123 with FAM dye. This TaqMan probe system detected both wild-type and variant sequences of 124 . 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 April 6, 2022. To determine the viral lineage of SARS-CoV-2, we performed whole genome 131 sequencing analyses or TaqMan assays using SARS-CoV-2-positive samples (n = 1,297) 132 collected consecutively in Yamanashi, Japan from September 2021 to March 2022 ( Figure 133 1A). During this period, we identified Delta strain (n = 159) and Omicron strain (n = 1,139). 134 After the first case of Omicron was identified in January 2022, Omicron rapidly replaced 135 Delta as the prevalent strain of SARS-CoV-2 ( Figure 1A) . 136 137 The whole genome sequencing data were analyzed using PANGOLIN (version and Ct value ( Figure 2B , p = 1.6×10 −3 , Student's t-test). 160 . 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 April 6, 2022. Figure 2C ). These results 167 indicate that the viral load in nasopharyngeal swabs is higher for sublineage BA.2 than for 168 sublineage BA.1.1 and that sublineage BA.2 is more contagious. 169 We next examined whether the viral load varied with patient age. There was no 170 apparent correlation between patient age and viral load or Ct value for either sublineage 171 Therefore, there is concern that the prevalence of the BA.2 sublineage may increase in the 194 The relationship between age and SARS-CoV-2 viral load of other strains was 196 . 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 April 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 shown previously [29] [30] [31] [32] [33] ; however, no data on Omicron sublineages were reported. 197 Previous studies suggested that the SARS-CoV-2 viral load tends to be higher in young 198 children than in adults, whereas other data suggest that the viral load does not vary by age 199 group [29] [30] [31] [32] [33] . In this study, no obvious differences in viral load by age group were observed 200 for either the Omicron BA.1.1 or BA.2 sublineages. In general, viral load peaks in the early 201 phase of infection and then gradually declines; hence, the timing of sampling relative to the 202 onset of symptoms is an important factor [34] . Because the time between onset and 203 sampling was not taken into account in the present study, our data are limited by sampling 204 bias. However, our data are derived from random sampling, therefore these results are 205 expected to better reflect real-world conditions. Although a high incidence of household 206 indicate that the Omicron strain retains a fairly high viral load across age groups, which may 208 contribute to the high infectivity of the Omicron strain and its accelerated spread. These data We thank all medical and ancillary hospital staff for their support. We thank Katie Oakley, 227 PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. 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 April 6, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Epidemiological characterisation of the first 785 SARS-CoV-2 Omicron variant cases in Denmark, . 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 April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273483 doi: medRxiv preprint 294 coronavirus 2 (SARS-CoV-2) with spike protein W152L/E484K/G769V mutations in Japan. . 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 April 6, 2022. . 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 April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273483 doi: medRxiv preprint World Health Organization. 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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 April 6, 2022. . 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 April 6, 2022. indicates the 95% confidence interval. 381 . 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 April 6, 2022. ; https://doi.org/10.1101/2022.04.05.22273483 doi: medRxiv preprint