Investigation of the individual genetic evolution of SARS-CoV-2 in a small cluster during the rapid spread of the BF.5 lineage in Tokyo, Japan

Abstract

There has been a decreasing trend in new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and fatalities worldwide. The virus has been evolving, indicating the potential emergence of new variants and uncertainties. These challenges necessitate continued efforts in disease control and mitigation strategies. We investigated a small cluster of SARS-CoV-2 Omicron variant infections containing a common set of genomic mutations, which provided a valuable model for investigating the transmission mechanism of genetic alterations. We conducted a study at a medical center in Japan during the Omicron surge (sub-lineage BA.5), sequencing the entire SARS-CoV-2 genomes from infected individuals and evaluating the phylogenetic tree and haplotype network among the variants. We compared the mutations present in each strain within the BA.5 strain, TKYnat2317, which was first identified in Tokyo, Japan. From June 29^th to July 4^th 2022, nine healthcare workers (HCWs) tested positive for SARS-CoV-2 by real-time PCR. During the same period, five patients also tested positive by real-time PCR. Whole genome sequencing revealed that the infected patients belonged to either the isolated BA.2 or BA.5 sub-lineage, while the healthcare worker infections were classified as BF.5. The phylogenetic tree and haplotype network clearly showed the specificity and similarity of the HCW cluster. We identified 12 common mutations in the cluster, including I110V in nonstructural protein 4 (nsp4), A1020S in the Spike protein, and H47Y in ORF7a, compared to the BA.5 reference. Additionally, one case had the extra nucleotide-deletion mutation I27* in ORF10, and low frequencies of genetic alterations were also found in certain instances. The results of genome sequencing showed that the nine HCWs shared a set of genetic mutations, indicating transmission within the cluster. Minor mutations observed in five HCW individuals suggested the emergence of new virus variants. Five amino acid substitutions occurred in nsp3, which could potentially affect virus replication or immune escape. Intra-host evolution also generated additional mutations. The cluster exhibited a mild disease course, with individuals in this case, recovering without requiring any medical treatments. Further investigation is needed to understand the relationship between the genetic evolution of the virus and the symptoms.

Keywords: BF.5 lineage; SARS-CoV-2; cluster; gene mutation; intra-host evolution; omicron variant; whole genome sequencing; within-host diversity.

Figure 1

Timeline of the Omicron BF.5 outbreak among healthcare workers (HCWs) at the JUH. On June 27^th and 28^th, six HCWs attended a dinner gathering party. Three HCWs joined the department on June 28^th and 29^th. The first symptom onset was reported on June 29^th and confirmed positive by real-time PCR the following day. All HCWs tested positive within a week after the initial infection. The duration between the gathering and diagnosis in indicated by red arrows. CT values of the test are provided.

Figure 2

Whole-genome phylogenetic analysis of SARS-CoV-2 genomes from individuals with onset between June 16^th and July 3^rd at the JUH. The HCWs (cases 1–9) cluster, highlighted in light blue, shows a distinction from the five patients (cases 10–14) in the tree. The maximum log-likelihood of this tree is −38077.05 under the best-fit model HKY + F + I selected in IQ-TREE v2.2.0 and visualized using iTOL v6.7.3. Each sample is labeled with the virus name and the case number. The ultrafast bootstrap support values are shown in the constructed tree. Pango lineage and HCW or patient classification are shown on the right. The virus name of Wuhan-Hu-1 (WIV04, GenBank accession number MN908947.3, EPI_ISL_402124), the Omicron BA.1.17 strain (NICD-N21604-DX64219, EPI_ISL_6647959), the BA.2 strain (THYhug0375, EPI_ISL_9890267), and the BA.5 strain (TKYnat231, EPI_ISL_13843385) were indicated and used as references.

Figure 3

The haplotype network for all Omicron SARS-CoV-2 sequences from individuals with onset between June 16^th and July 4^th at JUH is shown. The size of each node is proportional to the number of samples that belong to that haplotype. The color represents the PANGO lineage of references or the case samples for each haplotype. The number of lines on the branch between each node corresponds to the number of mutations between them. Each haplotype was indicated by case number or reference. Data were analyzed DnaSP v6.12.03 and drawn by PopART v1.7. The following reference viruses were used: the Wuhan-Hu-1 (GenBank accession number MN908947.3, EPI_ISL_402124), the Omicron BA.1.17 strain (EPI_ISL_6647959), and the BA.5 strain (EPI_ISL_13843385).