Prevalence of Recent COVID-19 Variants and Cell-based Omicron KP.3 Infectivity Analysis

Abstract

Since the outbreak of the coronavirus disease (COVID-19) pandemic in 2019, the Korea Disease Control and Prevention Agency (KDCA) has been conducting genomic surveillance using whole-genome sequencing to monitor SARS-CoV-2 variants in the Republic of Korea (ROK). Since the JN.1 lineage, derived from BA.2.86, was first detected in ROK in November 2023, the proportion of various JN.1 sub-lineages has continued to increase, with KP.3 accounting for 60.9% as of August 2024. In particular, the KP.3 sub-lineages with the highest shares were identified as KP.3.3.1 (22.3%), KP.3.3 (14.0%), and KP.3.1.1 (11.1%). We compared cell-based infectivity and viral replication among recently circulating JN.1, KP.2, and KP.3. KP.3 showed the highest viral shedding rate up to 48 hours, especially at 24 hours, when it was approximately 67 times higher than that of JN.1 and 23 times higher than that of KP.2. Although there was little difference in peak viral replication among JN.1, KP.2, and KP.3, that of KP.3 was approximately 66 and 16 times higher than that of JN.1 and KP.2, respectively, at 24 hours. We hypothesize that the higher initial infectious virus shedding and viral replication of KP.3 compared to those of JN.1 and KP.2 may have contributed to the increase in transmission and cases. The KDCA will continue close monitoring based on whole-genome sequencing to identify the prevalence of variants in ROK, characterize new variants, and provide scientific evidence to guide the COVID-19 response in the country.

Starting from the first warnings of the coronavirus disease 2019 (COVID-19) outbreak in late 2019, the World Health Organization (WHO) [1] declared a global pandemic status, and over time, many variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged. In May 2023, the head of the WHO declared an end to COVID-19 as a public health emergency of international concern. However, tracking of SARS-CoV-2 variants has continued, including the ongoing efforts of monitoring for emergence of novel variants and the risk of resurgence.

Since the emergence of a new SARS-CoV-2 variant of concern, named Omicron, in November 2021, it has spread worldwide, and succeeding sub-lineages have also appeared, becoming dominant epidemic strains in many countries [2]. During the earlier days of Omicron circulation in 2022, sub-lineages BA.1, BA.2, BA.5, and BA.2.75 were sequentially prevalent. In August 2022, XBB, a recombinant sub-variant of Omicron, was first identified in India, and in 2023, sub-lineages of XBB, such as XBB.1.5, and XBB.1.16 were identified as well as EG.5, a descendent lineage of XBB 1.9.2, circulating worldwide. In July 2023, BA.2.86, another new Omicron subvariant with the largest number of spike gene (amino acid) mutations, was identified in Denmark [3]. Subsequently, JN.1 [4], a descendent of BA.2.86, and the sub-lineages of JN.1 were identified, and these strains are still prevalent in late 2024, having an impact on the trend of SARS-CoV-2 infection in the Republic of Korea (ROK) and other countries.

The Omicron subvariant JN.1 (BA.2.86.1.1) was first detected and identified in Luxembourg in August 2023, and it has been classified into 404 sub-lineages, including KP.3, KP.2, JN.1.16, and LB.1, as of August 2024. As of late 2024 the variants of interest designated by the WHO [5] are BA.2.86 and JN.1, and the variants under monitoring are JN.1.7, KP.2, KP.3, KP.3.1.1, JN.1.18, and LB.1.

Among the JN.1 sub-lineages, KP.3, which has three additional mutations (F456L, Q493E, and V1104L) with spike protein substitutions, was first detected in the United States in February 2024, and since then, its percentage share of new cases of COVID-19 climbed worldwide, and it was reported as the most dominant SARS-CoV-2 variant, as of August 2024. As of late 2024, there have been no reports confirming that KP.3 is associated with increasing severity of COVID-19, but a previous study reported that neutralization titers were significantly reduced (1.3-fold to 2.1-fold) in serum samples after XBB.1.5 vaccination compared to those of JN.1, showing slightly superior immune evasion, which is likely to have an impact on the global circulation of the variant [6].

For monitoring of SARS-CoV-2 variants circulating in ROK, the Korea Disease Control and Prevention Agency (KDCA) implemented genomic surveillance through whole-genome sequencing and other methods in 2020, and from 2022, analysis of the variants has been conducted using samples obtained through the Korea Respiratory Virus Integrated Surveillance System, which has since been expanded and improved. Since the first detection of the JN.1 variant in ROK in November 2023, its prevalence has continued to increase, along with an increase in the percentage shares of different sub-lineages of JN.1. As of September 2024, the KDCA listed JN.1, KP.3, KP.2, LB.1, and JN.1.16 as the major variants for surveillance. In addition, virus stocks of JN.1, KP.2, and KP.3, which are Omicron sub-lineages that dominated circulation in ROK in late 2024, were obtained, and using these stocks, cellular-level analyses were performed with infectious virus shedding and viral replication, through which the correlation between infectivity and transmissibility was examined.

From April 2024, when KP.3 was first detected in ROK, to August 2024, whole-genome sequencing was performed for a total of 4,988 confirmed cases of COVID-19. During the sequencing period 168 Omicron sub-lineages were identified and 145 JN.1 sub-lineages were identified (including 18 KP.3 sub-lineages, 6 KP.2 sub-lineages, 10 LB.1 sub-lineages, and 9 JN.1.16 sub-lineages). In addition, the sequencing identified 23 other Omicron and recombinant Omicron sub-lineages.

Since the first detection of the JN.1 lineage in ROK in November 2023, the share of all JN.1 sub-lineages has continued to rise, accounting for more than 97% of all SARS-CoV-2 variants. From the JN.1 sub-lineages, KP.3 accounted for 45.5% in July 2024, an increase of 33.4% compared to the previous month, and 60.9% as of August 2024, a further increase of 15.4% from the percentage share in July. Along with the increase in the percentage share of the KP.3 variant, the percentage share of different KP.3 sub-lineages also showed an increasing trend. In particular, the KP.3 sub-lineages with the top three highest percentage shares were KP.3.3.1 (22.3%), KP.3.3 (14.0%), and KP.3.1.1 (11.1%). The spike protein sequences of KP.3 and KP.3.3 are identical, but one additional mutation was identified in KP.3.3.1 (T547K) and KP.3.1.1 (S31 deletion), respectively. Other sub-lineages, such as KP.2, LB.1, and JN.1.16, had decreasing trends in their percentage shares, in contrast to the increasing percentage shares of KP.3 sub-lineages (Figure 1, Table 1).

Figure 1. Proportion of SARS-CoV-2 variants in the Republic of Korea from April to August 2024.

As of 31 August 2024.

Table 1. Proportion of SARS-CoV-2 variants in the Republic of Korea from April to August 2024.

Variants	Variants proportion (%)
	April	May	June	July	August
KP.3 sub-lineages	0.3	2.5	12.1	45.5	60.9
KP.3.3.1	0.0	0.0	0.0	6.3	22.3
KP.3.3	0.1	0.5	6.4	22.3	14.0
KP.3.1.1	0.0	0.0	0.6	2.7	11.1
Other KP.3a)	0.2	2.0	5.1	14.2	13.5
JN.1 sub-lineagesb)	83.1	67.2	59.3	14.6	18.9
KP.2	1.3	5.9	5.7	15.8	8.5
LB.1	0.1	1.3	2.5	5.4	4.9
JN.1.16	9.0	17.4	19.1	10.4	4.4
Otherc)	6.2	5.7	1.3	8.3	2.4

As of 31 August 2024. ^a)Include all sub-lineages of KP.3 except KP.3.3.1, KP.3.3, and KP.3.1.1. ^b)Include all sub-lineages of JN.1 except KP.3, KP.2, LB.1, and JN.1.16. ^c)Include all Omicron sub-lineages except all sub-lineages of JN.1.

Respiratory specimens from individuals infected with SARS-CoV-2 that tested positive for the variants JN.1, KP.2, and KP.3, confirmed by whole-genome sequencing, were used to inoculate Vero E6 cells (derived from monkey kidneys) to isolate the variants. The Vero E6 cells were inoculated with the isolated viruses at a multiplicity of infection of 0.01, and the culture supernatants were harvested for 72 hours at 12-hours intervals. For hourly evaluation of the infectious virus shedding using the culture supernatants, the viral titer was measured. The infectious virus shedding increased gradually over time, and KP.3 showed higher initial viral shedding within 48 hours than KP.2 and JN.1. Notably, viral shedding of KP.3 at 24 hours was 5.4×10⁵ log₁₀ PFU/ml, which was approximately 67 times higher than for JN.1 (8.0×10³ log₁₀ PFU/ml) and 23 times higher than for KP.2 (2.4×10⁴ log₁₀ PFU/ml) (Figure 2A).

Figure 2. Viral replication kinetics and infectivity in Vero E6 cells infected at a multiplicity of infection of 0.01 for 72 hours.

Culture supernatants were harvested at the indicated time points. (A) Viral titers measured using plaque assays. (B) Viral RNA replication copy numbers determined using real-time PCR. PCR=polymerase chain reaction.

In addition, RNA was extracted from the samples, real-time polymerase chain reaction was performed on the extracted RNA, and viral copy numbers were determined using the cycle threshold values for analysis of viral replication. The three variants showed a gradual increase in viral replication over time, with the peak viral replication (KP.3; 1.9×10¹¹ log₁₀ copies/ml, KP.2; 1.1×10¹¹ log₁₀ copies/ml, JN.1; 1.5×10¹¹ log₁₀ copies/ml) at 72 hours. No significant difference was observed in the peak viral replication among the Omicron sub-lineages, JN.1, KP.2, and KP.3, but the viral replication of KP.3 was observed to be approximately 66-fold higher than that of JN.1 and approximately 16-fold higher than that of KP.2 at 24 hours (Figure 2B).

In this study, the percentage shares of the SARS-CoV-2 variants circulating in ROK from March 2024 to September 2024 were examined and comparative analysis was performed on the infectious virus shedding and viral replication between different Omicron sub-lineages to characterize the viral variants dominating circulation at that time. As the Omicron circulation continues, a variety of Omicron sub-lineages have emerged, and in particular, KP.3, a sub-lineage of the JN.1 lineage of the Omicron variant, whose increasing prevalence has been reported worldwide, shows an increasing prevalence in ROK, having the highest percentage share among the variants. The infectivity analysis for the viral variants demonstrated that circulating KP.3 and KP.2 had higher initial infectious virus shedding within 48 hours than their parent lineage, JN.1, indicating that SARS-CoV-2 is evolving, aiming for better optimized human-to-human transmission with increasing transmissibility as it adapts to different hosts. In particular, KP.3 had approximately 23 times and 67 times more infectious virus shedding 24 hours after the inoculation than KP.2 and JN.1, respectively, indicating that the variant is dominating global circulation including ROK through elevated infectivity. In addition, since KP.3 had higher infectious virus shedding within 48 hours than that for BA.5 reported in a previous study; in particular, the 12-hours infectivity of KP.3 was higher by approximately 74-fold. Therefore, it was speculated that transmissibility of KP.3 would also be higher than for other Omicron sub-lineages [7]. Thus, it is predicted that the circulation of KP.3 with increased transmissibility will continue in ROK, as was the case previously for circulation of BA.5. Furthermore, given that neutralization titers are significantly reduced in serum samples after XBB.1.5 vaccination compared to those of JN.1 [6], it could be inferred that the increase in transmissibility and immune evasion of KP.3 has an impact on the current dominance of KP.3.

In conclusion, this study confirmed that KP.3, a variant circulating in late 2024, has higher initial infectious virus shedding within 48 hours and viral replication compared to other Omicron sub-lineages at the cellular level. Thus, it can be inferred from the analysis that KP.3 has elevated transmissibility, which is likely to have had an impact on the increasing prevalence of KP.3 in ROK and elsewhere in late 2024. However, it is not possible to draw a definite conclusion as to the impact on the incidence and epidemiology of the infectious disease simply based on the results of infectious virus shedding and viral replication analyzed at the cellular level. Therefore, in the future, additional studies on the influencing factors will be necessary to examine the correlation with the results of cellular-level analyses, by evaluating more aspects of transmissibility, such as analysis of the structural stability of the binding between spike protein (S) and the receptor, angiotensin-converting enzyme 2, for the SARS-CoV-2 virus through molecular dynamics simulation.

The KDCA will continue close monitoring based on whole-genome sequencing to identify patterns in the prevalence of SARS-CoV-2 variants and to characterize the variants emerging in ROK and elsewhere through detection of novel variants and serve to continuously provide scientific evidence for COVID-19-related response actions.