Detection of R.1 lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with spike protein W152L/E484K/G769V mutations in Japan

Abstract

We aimed to investigate novel emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages in Japan that harbor variants in the spike protein receptor-binding domain (RBD). The total nucleic acid contents of samples from 159 patients with coronavirus disease 2019 (COVID-19) were subjected to whole genome sequencing. The SARS-CoV-2 genome sequences from these patients were examined for variants in spike protein RBD. In January 2021, three family members (one aged in their 40s and two aged under 10 years old) were found to be infected with SARS-CoV-2 harboring W152L/E484K/G769V mutations. These three patients were living in Japan and had no history of traveling abroad. After identifying these cases, we developed a TaqMan assay to screen for the above hallmark mutations and identified an additional 14 patients with the same mutations. The associated virus strain was classified into the GR clade (Global Initiative on Sharing Avian Influenza Data [GISAID]), 20B clade (Nextstrain), and R.1 lineage (Phylogenetic Assignment of Named Global Outbreak [PANGO] Lineages). As of April 22, 2021, R.1 lineage SARS-CoV-2 has been identified in 2,388 SARS-CoV-2 entries in the GISAID database, many of which were from Japan (38.2%; 913/2,388) and the United States (47.1%; 1,125/2,388). Compared with that in the United States, the percentage of SARS-CoV-2 isolates belonging to the R.1 lineage in Japan increased more rapidly over the period from October 24, 2020 to April 18, 2021. R.1 lineage SARS-CoV-2 has potential escape mutations in the spike protein RBD (E484K) and N-terminal domain (W152L); therefore, it will be necessary to continue to monitor the R.1 lineage as it spreads around the world.

Fig 1. TaqMan assay for discriminating R.1 lineage SARS-CoV-2 by its spike variant allele.

We analyzed samples of R.1 lineage SARS-CoV-2 (n = 3), which harbors a spike variant with the W152L/E484K/G769V mutations and samples of SARS-CoV-2 without these three mutations (n = 5) using a TaqMan assay. An allelic discrimination plot of the results, showing the spike variants with W152L (upper), E484K (middle), and G769V (lower). The dots indicate the mutant alleles (purple), reference allele (pink), or no amplification (orange). The dotted circles indicate the mutant alleles of each variant.

Fig 2. Timeline of SARS-CoV-2 R.1 lineage emergence and country-specific percentages of global cases.

(A) Timeline of the number of confirmed cases of infection with R.1 lineage SARS-CoV-2. The plot, created based on the data in Table 1, shows the case load for Japan (pink), the United States (light blue), and other countries (gray). (B) The percentage of R.1 lineage SARS-CoV-2 strains relative to the total number of registered samples during the period is shown for Japan (upper panel) and the United States (lower panel). The arrow indicates the date of infection for the initial three individuals infected with R.1 lineage SARS-CoV-2 who were identified in this study.

Fig 3. Phylogenetic tree of R.1 lineage SARS-CoV-2.

(A) Global data on the SARS-CoV-2 R.1 lineage prevalence over time. The R.1 lineage acquired its spike W152L and G769V mutations at the root. The sublineage harbors an ORF1b G814C mutation. (B) Geographic distribution of the SARS-CoV-2 R.1 lineage. World map showing the geographic distribution of SARS-CoV-2 R.1 lineage as of April 22, 2021. The size of the circle indicates the number of samples. Nextstrain, https://nextstrain.org, CC-BY-4.0 license.