Introduction
Since the first case of the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in late November 2021, it has triggered another major global wave of coronavirus disease 2019 (COVID-19). The World Health Organization designated Omicron as a variant of concern following an emergency meeting, due to evidence of its rapid transmission [
1]. In the Republic of Korea, Omicron was first identified in December 2021 and has since become the dominant variant (
https://www.kdca.go.kr/board/board.es?mid=a20501010000&bid=0015&list_no=720694&cg_code=&act=view&nPage=1). Researchers studying the Omicron variant have identified at least 30 mutations in the genes that encode spike proteins, with additional mutations also reported [
2–
4]. Based on these mutations, several sublineages have been established, including BA.1, BA.2, BA.3, BA.4, and BA.5. The mutations in the Omicron spike protein have been associated with increased transmissibility, immune evasion, and enhanced binding to angiotensin-converting enzyme [
5–
9], suggesting that Omicron sublineages may be evolving to become more transmissible. In this study, we aimed to investigate the correlation between the infectivity and transmissibility of the Omicron sublineages BA.1, BA.2, BA.4, and BA.5, which have been responsible for distinct COVID-19 waves in the Republic of Korea. We assessed this relationship by examining viral replication kinetics and cellular infectivity.
Materials and Methods
Specimen Collection and Real-Time Reverse Transcription Polymerase Chain Reaction for SARS-CoV-2
Swab specimens from patients infected with the Omicron sublineages BA.1, BA.2, BA.4, and BA.5 were collected in the Republic of Korea (
Table S1). These specimens were subjected to RNA extraction and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR), as previously described [
10]. In brief, RNA was extracted from 140 μL of each sample using a Viral RNA Mini Kit (Qiagen) in accordance with the manufacturer’s instructions. Real-time RT-PCR was then performed on the extracted RNA to determine the cycle threshold values for the SARS-CoV-2 target gene. All specimens were processed in a biosafety cabinet in accordance with the biosafety guidelines of the Korea Disease Control and Prevention Agency for COVID-19.
Viral Genome Sequencing
For full-genome sequencing, complementary DNA was amplified from total RNA using the QIAseq SARS-CoV-2 Primer Panel and the QIAseq FX DNA Library UDI Kit (Qiagen). Libraries were prepared with the Nextera DNA Flex Library Prep Kit (Illumina), and sequencing was performed on a MiSeq instrument with the MiSeq reagent kit V2 (Illumina), achieving an average genome coverage exceeding 1,000× for all samples. The reads were trimmed and mapped to the reference genome Wuhan-Hu-1 (MN908947.3; GenBank) using CLC Genomics Workbench version 20.0.3 (CLC Bio) [
11]. Lineages and clades were assigned using NextClade (v1.7.1) [
12] and PANGOLIN [
4].
Virus Isolation
Samples were combined with a 1:1 mixture of nystatin (10,000 U/mL) and penicillin-streptomycin (10,000 U/mL) at a 1:4 ratio and incubated at 4 °C for 1 hour. The samples were then centrifuged at 400×g for 10 minutes, and the resulting supernatant was used as the inoculum. Vero E6 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin at 37 °C in 5% CO2. Twelve hours before infection, the cells were seeded at a density of 1×105 cells/well in 12-well plates. On the day of infection, the medium was replaced with DMEM containing 2% fetal bovine serum. A 100-μL aliquot of each sample was used to infect the cells. After 5 days of culture, the cells were harvested. The supernatant was centrifuged at 600×g for 10 minutes before collection. This procedure was repeated for secondary infections. Five days after infection, the supernatant was harvested, centrifuged at 600×g for 10 minutes, and collected. To quantify viral replication, RNA was extracted from the supernatants of the secondary infections and assessed for the presence of SARS-CoV-2 using real-time RT-PCR.
Viral Replication Kinetics and Infectivity
Vero E6 cells were seeded in 6-well plates at a density of 3×105 cells/well. After 12 to 18 hours, the cells were infected at a multiplicity of infection of 0.01 and incubated for 1 hour at 37 °C with frequent agitation. Following incubation, the inoculum was removed, and the cell monolayers were washed once before being incubated in fresh complete medium. Supernatants were harvested at 12, 24, 36, 48, 60, and 72 hours post-infection and stored in aliquots at −80 °C. Viral replication kinetics and infectivity were analyzed using 4 replicates.
Plaque Assay
Vero E6 cells were seeded as described in the preceding section and subsequently exposed to 10-fold serial dilutions of the supernatants for 1 hour at 37 °C with frequent agitation. After incubation, the inoculum was removed, and the monolayers were washed once with Minimum Essential Medium containing 5% fetal bovine serum, 1% penicillin, and 0.5% agarose. The plates were then incubated for 3 days at 37 °C in an atmosphere of 5% CO2. After incubation, the plaques were fixed with 4% formaldehyde for 4 hours and subsequently stained with 10% crystal violet for 30 minutes.
Copy Number Measurement
Plasmids carrying the SARS-CoV-2 E gene were utilized as positive controls. A standard curve was established using plasmid concentrations and the following regression equation: y=−3.5705x+39.055, where “y” represents the cycle threshold and “x” denotes the copy number. Viral copy numbers were determined using this equation and expressed as log
10 copies/mL [
13].
Statistical Analysis
Descriptive analysis was performed using SAS ver. 9.4 (SAS Institute) to compare viral RNA replication copy numbers, as determined by real-time RT-PCR, with viral titers measured by plaque assays among SARS-CoV-2 Omicron variants. Statistical significance was established at 0.05 for all copy numbers and titers.
Ethics Statement
The study received approval from the institutional review board (IRB) of the Korea Disease Control and Prevention Agency (approval number: 2020-03-01-P-A) and was designated as a public health service during the pandemic. Consequently, the IRB waived the requirement for written informed consent from the participants, as outlined in the document “Laboratory Response to COVID-19.”
Results
The viral load gradually increased over time, peaking at 72 hours. The peak values were as follows: BA.1, 2.1×10
11 log
10 copies•mL
−1; BA.2, 6.8×10
11 log
10 copies•mL
−1; BA.4, 6.2×10
11 log
10 copies•mL
−1; BA.5, 5.6×10
11 log
10 copies•mL
−1. The viral loads for BA.2, BA.4, and BA.5 were similar, whereas BA.1 exhibited a lower value (
Figure 1A).
The released infectious particles were titrated to examine viral infectivity over time. The infectious load also increased gradually, reaching its peak at 60 hours (BA.1, 1.6×10
6 log
10 plaque-forming units [PFU]•mL
−1; BA.2, 1.1×10
7 log
10 PFU•mL
−1; BA.4, 4.0×10
6 log
10 PFU•mL
−1; BA.5, 1.1×10
7 log
10 PFU•mL
−1). The peak infectious load of BA.4 was approximately 3 times lower than that of BA.2 and BA.5, while the peak load of BA.2 and BA.5 was 7 times higher than that of BA.1. Notably, BA.5 exhibited a high load at both 48 and 72 hours, while BA.1 demonstrated the lowest infectivity over the entire study period (
Figure 1B).
Discussion
In this study, we sought to understand the characteristics of variants driving waves of COVID-19 by comparing the replication kinetics and infectivity of Omicron sublineages implicated in current and past waves in the Republic of Korea. Relative to BA.1—the first Omicron sublineage—all subsequent sublineages exhibited over 10-fold higher infectivity, indicating that SARS-CoV-2 is adapting to its host and evolving to become more transmissible. Furthermore, BA.2 has been found to have higher viral infectivity than BA.1 in the Calu-3 cell line [
14]. However, in this study, BA.4 demonstrated lower infectivity than BA.2 and BA.5, suggesting it is less likely to trigger a global wave of COVID-19. In contrast, the viral infectivity of BA.5 was 3 times greater than that of BA.2 at both 48 and 72 hours after infection, a characteristic that appears key in driving the corresponding global wave. The detection of BA.5 infection was 35.1% faster than that of BA.2 infection, echoing findings from a previous study that reported higher transmissibility for BA.5 [
15]. BA.5 also displayed a growth advantage (56%) over BA.4 (17%), BA.2 (−17%), and BA.1 (−36%), which may be attributed to its intrinsic viral advantage, increased transmission, immune escape, and prolonged infectious period. Therefore, BA.5 is expected to remain the predominant variant for some time [
16]. Furthermore, BA.5 has been shown to induce neutralizing antibody titers that are 7.5-fold lower than those elicited by BA.1, contributing to immune response evasion and lower efficacy of certain antibody treatments [
17,
18].
Limitations
SARS-CoV-2 cultures can be performed using various cell lines, including Calu-3, A549, and others. However, Vero E6 cells are commonly utilized for the culture analysis of SARS-CoV-2. Consequently, we used Vero E6 cells in our study. Nevertheless, this choice may represent a limitation; further analysis utilizing various cell lines is warranted.
Conclusion
The recent global wave of BA.5 may be partly due to its higher viral replication and infectivity compared to other Omicron sublineages. However, analyzing replication and infectivity at the cellular level does not provide definitive evidence of how these properties influence the magnitude of a real-world outbreak. Therefore, it is necessary to further examine population-level immunity and epidemiological relationships to better understand the immune evasion properties of variants.
Article information
Ethics Approval
All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Declaration of Helsinki, including its later amendments or comparable ethical standards. The study received approval from the IRB of the Korea Disease Control and Prevention Agency (approval number: 2020-03-01-P-A) and was designated as a public health service during the pandemic. Consequently, the IRB waived the requirement for written informed consent from the participants, as outlined in the document “Laboratory Response to COVID-19.”
Conflicts of Interest
The authors have no conflicts of interest to declare.
Funding
This work was supported by the Korea Disease Control and Prevention Agency (grant number: 6300-6331-301).
Availability of Data
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. The whole-genome sequences of SARS-CoV-2 are accessible in the Global Initiative on Sharing All Influenza Data database (accession IDs: EPI_ISL_6959993, 13086512, 13086515, and 13086516).
Authors’ Contributions
Conceptualization: JMK, JER, EJK; Funding acquisition: CKY, EJK; Investigation: JMK, DK; Methodology: JMK, DK; Software: JMK; Validation: all authors; Writing–original draft: all authors; Writing–review & editing: all authors. All authors read and approved the final manuscript.
Additional Contributions
The authors extend their gratitude to everyone who assisted with the collection and transportation of patient specimens, as well as to the staff at the Korea Disease Control and Prevention Agency, local government officials, and private testing agencies for their contributions to controlling COVID-19.
