Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea

The 2022 monkeypox disease (mpox) pandemic, a re-emerging infectious disease, posed a significant challenge to global health. Between May 2022 and April 2023, 87 277 confirmed cases and 128 fatalities were reported globally.1 The outbreak predominantly affected America and Europe, with 67 064 and 25 602 cases, respectively. Cases were also reported in Asia (n = 631), Oceania (n = 188), and Africa (n = 1,617).

As of late 2022, the number of global infections caused by monkeypox virus (MPXV) (order Chitovirales, family Poxviridae, genus Orthopoxvirus, species Monkeypox virus) notably decreased. However, cases of local transmission have been reported in the Far East since early 2023, consecutively in Japan (n = 183), Taiwan (n = 241), and the Republic of Korea (n = 121) from January 1, 2023, to July 16, 2023, according to the Korea Disease Control and Prevention Agency (KDCA) press report.

In the Republic of Korea, a total of 4 cases of mpox were reported in 2022; the first three cases were epidemiologically linked to foreign outbreaks, while the fourth case resulted from a needlestick injury to a physician.2, 3, 4, 5, 6, 7 MPXV isolated from the first case in 2022 was classified into Clade IIb, lineage B.1.1.8 In April 2023, the first local case without foreign travel history was reported, and subsequent local mpox cases were identified, reaching a peak of 16 new cases per week by late April 2023.

In this study, the genetic relationships between MPXV isolates in the Republic of Korea and other East Asian countries and other regions were investigated via phylogenetic analysis using full genome sequencing, with the aim to provide crucial insights into the dynamics and transmission patterns of the virus, emphasizing the unique features of isolates from the Far East.

Clinical samples collected from 8 patients diagnosed with mpox at the National Medical Center in Seoul, the Republic of Korea, between August 2022 and April 2023 were analyzed. Mpox was confirmed by the KDCA using a quantitative polymerase chain reaction assay targeting F3L, and the cycle threshold ranged from 12.8 to 24.16. DNA underwent library preparation for the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA). Viral DNA was extracted from clinical samples using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Extracted DNA was quantified, normalized, and tagmented using enrichment bead-lined transposomes for simultaneous DNA fragmentation and adapter tagging. Indexed fragments were hybridized with the Illumina Viral Surveillance Panel and enriched using capture probes and magnetic beads, following the manufacturer’s protocol. Enriched libraries were eluted, amplified, and normalized to a loading concentration of 9 pM. The prepared libraries were sequenced using an Illumina MiSeq platform in a 2 × 150 base pair format for whole-genome reconstruction. Genomic sequence analysis was conducted as follows: Human reads were filtered out using Kraken2 (version 2.1.3; https://github.com/DerrickWood/kraken2), followed by trimming with a Q30 trim threshold of 0.001. The remaining reads were mapped to the reference virus strain NC_063383.1 using the Map Reads to Reference function of QIAGEN CLC Genomics Workbench version 20 (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-genomics-workbench/). A de novo assembly method was used for genomic structure inspection, and the resulting sequences were evaluated using Quality Assessment Tool for Genome Assemblies (QUAST) (version 5.0.2; https://github.com/ablab/quast).

The completed genome was classified into clades and lineages using NextClade (version 2.14.0; NextStrain, https://clades.nextstrain.org). For more detailed phylogenetic analyses, genome data from NextStrain (https://data.nextstrain.org) and Global Initiative on Sharing All Influenza Data (GISAID) (https://www.gisaid.org) databases were analyzed via the NextStrain analysis pipeline (https://github.com/nextstrain/monkeypox). This pipeline involved the snakemake (version 7.24.1; https://github.com/snakemake/snakemake) workflow framework, along with augur (version 23.0.0; https://github.com/AugurProject/augur), mafft (version 7.475; https://mafft.cbrc.jp), iq-tree (version 2.12; http://www.iqtree.org), treetime (version 0.10.1; https://github.com/neherlab/treetime), and auspice (version 2.48.0; https://github.com/nextstrain/auspice) for visualization.

The sequencing achieved full coverage with a mean read depth of 2,223 × and 1.6 × 10⁶ reads mapped to the reference virus strain (Table 1). All genome sequences from the Republic of Korea were classified as Clade IIb. The 2022 isolates, MPXV-ROK-P002-2022 and MPXV-ROK-P003-2022, belonged to the B.1.1 and A.2.1 lineages, respectively. The 2023 isolates were all classified as B.1.3 (Table 1).

Table 1
Genomic information of monkeypox virus isolates in the Republic of Korea

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In the phylogenetic analysis, the lineage classification of the 2022 and 2023 isolates was consistent with the results obtained using NextClade. The 2022 isolates (MPXV-ROK-P002-2022 and MPXV-ROK-P003-2022) were epidemiologically imported cases (Table 2). The remaining cases were locally acquired in 2023. Specifically, 6 local 2023 isolates, belonging to the B.1.3 lineage, exhibited the closest genetic relationship with MPXV isolated in Japan in the GISAID database (Fig. 1A and C). These isolates also distinctly clustered with isolates from Taiwan. The B.1.3 lineage isolates from the Far East formed a distinct cluster compared to isolates from other continents such as Europe and North America (Fig. 1A and B). In detail, members of the B.1.3 lineage reported in Europe,9, 10 North America,11 and Far East Asia,12 a common nonsynonymous mutation G190660A; R84K in the NBT03_gp174 protein was observed. Furthermore, in the Far East Asia continent, including the Republic of Korea, Japan, Taiwan, two common mutations were observed: synonymous mutation C3987T and nonsynonymous mutation G105923A; L16F in the OPG124 protein, according to National Center for Biotechnology Information reference sequence NC_063383 coordinates.

Fig. 1
Phylogenetic tree of 8 monkeypox virus isolates in the Republic of Korea and isolates in GISAID and GenBank constructed using the NextStrain analysis pipeline. (A) The currently circulating isolates belong to the B.1 lineage. Isolates in the Republic of Korea clustered into the B.1.3 lineage indicated by the red squares within the B.1 lineage. (B) Monkeypox virus isolates from Asia (denoted by red squares) tended to cluster within the B.1.3 lineage. (C) Six monkeypox virus isolates of 2023 in the Republic of Korea within the B.1.3 lineage (in blue) were closely related to isolates from Japan (in red) and Taiwan (in yellow) with close clustering observed with isolates from Japan.

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Table 2
Clinical characteristics of the study population

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In summary, based on the characteristics of viruses reported in Far East Asian countries, we have observed that the B.1.3 lineage virus, possibly introduced from Europe and North America, has traversed borders and spread, leading to cross-border transmission within these countries, accompanied by the emergence of new common mutations. Moreover, the consistent identification of these virus strains with such attributes in the viral genomes of patients in Korea, without any travel history, serves as a crucial indicator that domestic community transmission has commenced.

The limitation of this study is that isolates were obtained from only 8 mpox-positive patients in the Republic of Korea between May 2022 and April 2023; in that, the results cannot fully represent the genetic analysis of all domestic Mpox patients, including those epidemiologically related to global travel or community transmission. Nonetheless, the study reports rapid MPXV genome sequencing and surveillance in Republic of Korea, facilitating the swift assembly of the draft genomes of local isolates. This accelerated genomic analysis enables crucial insights for public health responses to the mpox epidemic, encompassing the development of isolation strategies, vaccine production, and development of therapeutic interventions. Rapid mpox genomic surveillance not only enhances our understanding of the virus but also informs effective public health measures in response to the epidemic. Ongoing genomic surveillance remains paramount for collecting critical data to track the spread of infectious diseases, guiding the development of vaccines and the identification of novel viral variants.