Genomic surveillance of genes encoding the SARS-CoV-2 spike protein to monitor for emerging variants on Jeju Island, Republic of Korea

Introduction The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been fueled by new variants emerging from circulating strains. Here, we report results from a genomic surveillance study of SARS-CoV-2 on Jeju Island, Republic of Korea, from February 2021 to September 2022. Methods A total of 3,585 SARS-CoV-2 positive samples were analyzed by Sanger sequencing of the gene encoding the spike protein before performing phylogenetic analyses. Results We found that the Alpha variant (B.1.1.7) was dominant in May 2021 before being replaced by the Delta variant (B.1.617.2) in July 2021, which was dominant until December 2021 before being replaced by the Omicron variant. Mutations in the spike protein, including N440K and G446S, have been proposed to contribute to immune evasion, accelerating the spread of Omicron variants. Discussion Our results from Juju Island, Republic of Korea, are consistent with and contribute to global surveillance efforts crucial for identifying new variants of concern of SARS-CoV-2 and for monitoring the transmission dynamics and characteristics of known strains.


Introduction
The coronavirus disease 2019  is caused by numerous genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that have emerged throughout the world (Kumar et al., 2021). The rapid genetic changes of SARS-CoV-2 have been influenced by various factors, including low levels of existing immunity to each new strain and the high mutation rates caused by error-prone polymerases (Denison et al., 2011). Screening for changes in genetic characterization could help predict vaccine effectiveness and vaccine-induced immune evasion to predict outbreaks (Mengist et al., 2021;Shaibu et al., 2021).

Epidemiological data analysis
The map of South Korea was obtained from the Statistical Geographic Information Service (SIGS) offered by Statistics Korea 1 1 https://sgis.kostat.go.kr/ (Figures 1A, B). The number of confirmed cases of COVID-19 was provided by Korea Disease Control and Prevention Agency (KDCA 2 ) ( Figure 1C).

RNA isolation and reverse transcription-polymerase chain reaction
Coronavirus disease 2019 (COVID-19) positive specimens were obtained from Seoul Clinical Laboratories (SCL) and from the Jeju Special Self-Governing Province Institute of Environment Research. Specimens were handled in a Class II biosafety cabinet (Thermo Scientific 1300 Series A2) in a biosafety level 2 (BL2) laboratory. RNA was isolated using an RNA extraction kit (Qiagen, Valencia, CA, USA), as per the manufacturer's instructions. cDNA was synthesized using SuperScript IV First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), and PCR was conducted using PrimeSTAR GXL DNA Polymerase (Takara, Shiga, Japan) from January to August 2021. From September 2021 to September 2022, reverse transcription-polymerase chain reaction (RT-PCR) was conducted using Qiagen Onestep RT-PCR kit (Qiagen, Valencia, CA, USA), DiaStar 2 × OneStep RT-PCR premix kit (SolGent, Daejeon, Republic of Korea), and SEQMAX qPCR one step mastermix (Nine Korea, Republic of Korea). Sequences of primers used in RT-PCR are shown in Table 1. To detect mutations of SARS-CoV-2 spike protein, L71/R75, L76/R79, and L80/R84 primer sets were used. RT-PCR was performed under the following conditions: an initial reverse transcription step at 50 • C for 30 min followed by a denaturation step at 95 • C for 5-15 min. This was followed by 35 cycles of 30 s at 95 • C, 30 s at 58 • C, 1 min 30 s at 68 • C, and a final extension step at 68 • C for 7 min (Ha et al., 2022). To distinguish between BA.4 and BA.5, RT-PCR was instead performed on the N (nucleocapsid) gene under the following conditions: an initial reverse transcription step at 50 • C for 30 min followed by a denaturation step at 95 • C for 5-15 min. This was followed by 35 cycles of 10 s at 98 • C, 15 s at 50 • C, 2 min 30 s at 68 • C, and a final extension step at 68 • C for 7 min.

DNA sequencing and analyses
DNA sequencing (Cosmogenetech, Seoul, Republic of Korea and SolGent, Daejeon, Republic of Korea) was performed using a standard protocol. To analyze the sequences for spike protein, L71/R75, L76/R79, and L80/R84 and inner primers including L73/R73, L78/R77, and L83/R82 were used until December 2021 ( Table 1). From 2022, inner primers were used only L73, L78, and L83. To detect the mutation in the N gene, L94/R95 were used from July and September 2021. The sequence data from COVID-19 positive specimens on Jeju Island were submitted to GenBank through the National Center for Biotechnology Information (NCBI) ( Table 2). Full-length sequences of COVID-19 spike proteins of various geographical origins were downloaded in FASTA format from GISAID. The sequence of SARS-CoV-2 reference genome Wuhan Hu-1 (accession number: NC 045512.2) was retrieved from the NCBI databases. The non-coding 3 and 5 regions were trimmed using CLC Genomic Workbench 5.0.1 software (CLC bio, Denmark). Multiple sequence alignments were done using a multiple sequence alignment (MAFFT) programs. Sequences of SARS-CoV-2 with specific mutations were searched in the GISAID database. 3 Phylogenetic analysis was done using iTOL. 4 Branch support was calculated by bootstrap, consisting of 1,000 alignments. SARS-CoV-2 positive specimens from confirmed cases of Jeju Island were classified into clades and lineages using Nextclade Beta. 5

Epidemiological data analysis
Jeju Island is Korean Republic's biggest Island, located 80 km away from the mainland (Jang et al., 2019) and includes Jeju-Si and Seogwipo-Si (Figures 1A, B). The number of confirmed cases of COVID-19 was shown from January 2020 to September 2022 ( Figure 1A). The cumulative number of COVID-19 cases in Jeju was 333,722, accounting for 1.35% of the total number of cases in the Republic of Korea ( Figure 1A). From January 2020 to September 2022, the monthly number of confirmed cases of SARS-CoV-2 on Jeju Island was shown in Figure 1C. The Delta (B.1.627.2) variant emerged and began to spread on the island in May 2021, with a sharp increase in confirmed cases appearing later (e.g., from July to August 2021) ( Figure 1C). The Omicron (B.1.1.529) variant was first detected in December 2021 and confirmed cases rose beginning shortly after (e.g., January 2022) ( Figure 1C).

Phylogenetic analyses of currently and previously circulating VOCs
A phylogenetic tree of Alpha (B.1.1.7) variants isolated from patients on Jeju Island between May and early June 2021 (OP763726∼OP763643) was generated. The Alpha lineage from samples isolated on Jeju Island was more closely related to each other than the rest of Alpha lineage (Figure 4A). Similarly, we generated phylogenetic trees of samples isolated from patients on Jeju Island for Beta (B.1.531, March to May 2021, OP763744 and OP763645). Notably, the sequence from sample OP763744 closely resembled that of hCoV-19/France/IDF-ALPIGENE-2107210176 2021 (EPI ISL 11449404) ( Figure 4B). The Delta variant on Jeju Island was grouped by confirmation date of COVID-19 infection ( Figure 4C). The sequences from OP763699 to OP763709 emerged August and September 2021, and Nextclade analyses classified these as belonging to clade 20I. Notably, the sequences from OP763703 to OP763707 closely match that of hCoV-19/South Korea/KDCA29074/2022 (EPI ISL 10046128). The sequences from OP763711 to OP763719 emerged in November 2021, and Nextclade analyses classified these to clade 21A. We also report that in OP763708, an additional T95I mutation was found in Spike protein ( Table 2). The genomes sequences of B.1.1.529 (Omicron) on Jeju Island clustered several subvariants including BA.1.1, BA.2, and BA.5 ( Figure 4D).

Discussion
In this study, we performed genomic surveillance of SARS-CoV-2 samples isolated from patients on Jeju Island 1 year after that original infection, from February 2021 to September 2022, a period during which multiple variants were circulating globally. During the period of our spike protein-based genomic surveillance study on Jeju Island, the Alpha, Delta, and Omicron variants (i.e., currently and previously circulating VOCs) became dominant in turn, while the Beta and Gamma variants did not. In addition, the import and prevalence of mutations in Jeju showed different from the mainland of Korea.  On Jeju Island, the Alpha variant was still dominant in May 2021 before decreasing from 56.55 to 6.52% as the Delta variant increased in circulation (Figure 2). In the Republic of Korea, Alpha variant was decreased from 27.40 to 8.1% in May and July 2021 . Globally, the Alpha variant dominated transmission until the emergence of the Delta variant in the winter of 2020 (Dong et al., 2022). Our data suggests that the Delta variant began to spread on overseas imported case from Jeju on May 2021, with confirmed cases of COVID-19 increasing that month from 82 to 323. After that, confirmed cases of COVID-19 on Jeju Island increased from 468 to 870 from July to August 2021 ( Figure 1C  and Supplementary Figure 3). In the Republic of Korea, the Delta variant was first confirmed on April 2021 and exceeded half of all cases by July 2021 . Globally, the Delta variant has been replaced by the Omicron variant by the end of 2021 (Lista et al., 2022), while on Jeju Island, Delta variant has been dominant over half the year, only being replaced by Omicron in January 2022 (Figure 2).
The Omicron variant was declared as a VOC on 26 November 2021, after being identified earlier that month in South Africa (Gobeil et al., 2022). In the Republic of Korea, the Omicron variant was first confirmed in the end of November 2021 and exceeded half of all cases on December 2021 . By February 2022, the Omicron variant accounted for 99.1% of all COVID-19 cases in the Republic of Korea (Park et al., 2023). On Jeju Island, confirmed cases of COVID-19 increased significantly in a short period, from 784 in January 2022 to 134,559 in April 2022 ( Figure 1C). In addition, the Omicron variant accounted for more than 99.00% of all COVID-19 cases on February 2022 (Figure 2). From this result, the import and prevalence of mutations in Jeju are different from those in Korea.

Conclusion
In conclusion, we used genomic surveillance and phylogenetic analyses to follow the relative abundance of different SARS-CoV-2 variants, from January 2020 to February 2022, on Jeju Island, Republic of Korea. During this period, multiple variants and their subvariants emerged having enhanced human-tohuman transmissibility. The dynamics of variants differed on Jeju Island compared with the mainland. Because of the limited entry points compared to land-locked regions, new variants might be introduced more slowly or take different forms due to other Island characteristics. Being a major center for domestic and international travel, Jeju Island might be at risk of continuous influx of new variants. We conclude that monitoring efforts for COVID-19 cases and SARS-CoV-2 variants is essential for monitoring and controlling outbreaks on Jeju Island.

Data availability statement
The datasets presented in this study can be found in the GenBank. The accession number from OP763667 to OP763745 can be found in the article/Supplementary material.

Ethics statement
This study uses strains obtained from Jeju Special Self-Governing Province Research Institute of Public Health and Environment and Seoul Clinical Laboratories (SCL). Korea Disease Control and Prevention Agency (KDCA) did not require the study to be reviewed or approved by an ethics committee because this study belongs to the case including (1) insignificant impact on donors of human derivatives and the public, (2) research in which researchers do not collect or record personal information, and (3) cases where human material is not directly collected. The Institutional Review Board (IRB) of Korea Disease Control and Prevention Agency (KDCA) granted exemption for this study (IRB exemption number: KDCA-2023-06-01-PE-01).

Author contributions
Y-RH contributed to the conception, design, data acquisition, and drafting of the manuscript. H-JK conducted the experiment and helped sequencing data analysis. J-SP advised the experimental procedure and treatment of specimens. Y-SC conceived the entire study and helped draft the manuscript. All authors read and approved the final manuscript.

Funding
This research was supported by a fund (6137-300-210-13) of the Korea Centers for Disease Control and Prevention Agency (KDCA).