The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented unprecedented challenges to global health systems and economies (Filip et al., 2022; Naseer et al., 2022; Nayak et al., 2022; Albreht, 2023; Katole, 2023; McKibbin and Fernando, 2023). Since its emergence in late 2019 (Wu et al., 2020; Zhu et al., 2020), the virus has undergone rapid evolution, leading to the appearance of several variants of concern (VOCs) characterized by increased transmissibility, altered pathogenicity, and potential immune escape (Otto et al., 2021; Scovino et al., 2022; Carabelli et al., 2023; Markov et al., 2023; Zhao et al., 2024). Genomic mutations have led to the emergence of such variants, which significantly impacted the pandemic by increasing morbidity and mortality rates worldwide (Khandia et al., 2022; Markov et al., 2023). Among these, the Alpha, Delta, and Omicron VOCs have been particularly noteworthy due to their unique genetic profiles and effects on disease transmission and severity. The Alpha VOC, also designated B.1.1.7, was initially detected in the United Kingdom and rapidly propagated globally (Bayarri-Olmos et al., 2021; Stadtmüller et al., 2022). This variant is marked by mutations in the spike protein, especially N501Y, which contributed to its heightened transmissibility and raised concerns about its ability to circumvent immunity acquired from previous infection or vaccination (Bayarri-Olmos et al., 2021). Subsequently, the Delta VOC (B.1.617.2) became predominant, driving increases in COVID-19 cases worldwide. Key mutations in Delta, such as L452R and P681R, were linked to its enhanced infectivity and potentially greater severity compared to earlier strains (Zhan et al., 2022).

Among the VOCs, the Omicron variant (B.1.1.529), first identified in South Africa in late 2021 (Karim and Karim, 2021; Tegally et al., 2022; Viana et al., 2022), has swiftly become a dominant strain worldwide due to its extensive spike protein mutations. With over 50 sub-lineages identified, Omicron has shown rapid evolution and diversification since its initial discovery, posing significant challenges for vaccine efficacy and diagnostics (Alkhatib et al., 2022; Chen et al., 2022; VanBlargan et al., 2022; Zhou et al., 2022; Piubelli et al., 2024). This variant is particularly notable for its high number of spike protein mutations (Callaway, 2021; Liu et al., 2024), which is the primary target for neutralizing antibodies elicited by both natural infection and vaccination. These mutations have raised concerns about reduced vaccine efficacy and increased potential for breakthrough infection and reinfection. Initial studies suggest that Omicron exhibits enhanced transmissibility compared to previous VOCs, likely due to its ability to evade immune responses (Jung et al., 2022; Willett et al., 2022; Reuschl et al., 2024). The rapid global spread of Omicron underscores the necessity for a thorough understanding of its genomic characteristics and evolutionary dynamics across the globe, which is critical for monitoring the virus’s evolution and implementing effective public health strategies to mitigate its spread.

Genomic surveillance has been pivotal in monitoring the evolution of SARS-CoV-2 variants (Robishaw et al., 2021; Tosta et al., 2023). Through the analysis of viral genomes from diverse geographical locations and temporal points, researchers have pinpointed critical mutations linked to alterations in viral phenotype and transmission dynamics. For example, mutations in the spike protein, such as D614G, have been associated with increased viral infectivity and transmissibility (Korber et al., 2020). Furthermore, the detection of variants with mutations in the receptor-binding domain (RBD), such as E484K found in the Beta variant, has heightened concerns about immune evasion and vaccine resistance (Wang et al., 2021). Epidemiological studies leveraging genomic data have clarified the evolutionary relationships of the virus by employing whole-genome sequencing (WGS) and phylogenetic analysis. These methods have provided crucial insights into the origins and spread of the COVID-19 pandemic across different countries (Chrysostomou et al., 2023; El Mazouri et al., 2024; Gerashchenko et al., 2024; Linosefa et al., 2024). Understanding the genomic epidemiology, evolutionary history, and population dynamics of variants of concern (VOCs), such as Omicron, is critical for effective public health strategies and future pandemic preparedness. Genomic surveillance, particularly through whole-genome sequencing (WGS) and population genomics analyses, has emerged as a powerful tool in this regard. These methods offer profound insights into the transmission patterns, genetic diversity, and evolutionary history of viral pathogens. Such comprehensive analyses are indispensable for informing targeted interventions and controlling the spread of the virus.

Pakistan, like many other countries, has experienced multiple waves of COVID-19 (Umair et al., 2022a, Umair et al., 2022b; Ahmad et al., 2023; Arif and Mahmood, 2023; Javed et al., 2024), with the Omicron variant contributing significantly to the surges in cases (Khan et al., 2022; Bukhari et al., 2023). Despite the substantial impact of Omicron on the Pakistani population, detailed studies focusing on its molecular epidemiology and evolutionary trajectory within this region are limited. This gap in knowledge necessitates a comprehensive analysis to provide deeper insights into the genetic diversity, population structure, and evolutionary dynamics of the Omicron variant in Pakistan. By leveraging WGS data, we implemented a population genomics approach to elucidate the evolutionary history, population dynamics, spread patterns, and genomic epidemiology of Omicron VOC within the Pakistani context. Our analysis reveals patterns of genetic diversity and population dynamics of Omicron, highlighting notable similarities and differences compared to other VOCs, particularly the Delta variant and the earlier Wuhan strain. By providing insights into the origins, spread, and mutation patterns of the Omicron variant, this study enhances our understanding of the evolutionary dynamics of the Omicron wave in Pakistan.

In this study we elucidated genomic epidemiology and evolutionary dynamics of the Omicron variant of SARS-CoV-2 during the fifth wave of COVID-19 in Pakistan through comprehensive analysis of genetic diversity, population differentiation, and evolutionary history. The study provides crucial insights into how Omicron has evolved and spread within the Pakistani context, contributing to our understanding of its global impact.

The genomic analysis revealed a low level of genetic diversity within the population, as evidenced by the Pi value of 0.00177 ± 0.00048. This low genetic diversity suggests a recent introduction and rapid spread of the Omicron variant in Pakistan. The Omicron variant of SARS-CoV-2 harbors several mutations in its RBD and other regions, which significantly impact interactions with therapeutic antibodies and the immune system. All Omicron specific mutations (Zhao et al., 2022) were observed in our analysis including those found in RBD and RBM. These mutations result in the extremely high affinity of the Omicron RBD for the human ACE2 receptor, with notable changes including Q493R, T478K, S373P, N501Y, Q498R, S371L, and S375F (Chatterjee et al., 2023). The presence of naturally hydrophobic amino acids, such as leucine and phenylalanine, contributes to this enhanced affinity (Goutam Mukherjee et al., 2022). Some of these mutations facilitate the formation of salt bridges or hydrogen bonds, enhancing the binding of the spike protein to hACE2. However, mutations like K417N and E484A can significantly reduce polar contacts between Omicron and ACE2, counteracting some of the improved interactions created by other mutations (Cui et al., 2022; Han et al., 2022; Yin et al., 2022). A deeper examination of the crystal structures of the RBD–ACE2 complex of Omicron reveals a larger interaction surface area with the host compared to the Delta variant (Han et al., 2022). The RBD, directly involved in binding with hACE2, harbors ten significant mutations, thereby altering the affinity of the spike protein for the host receptor (Kumar et al., 2022). Among these mutations, T478K, Q498R, N440K, and Q493R are particularly important. These mutations, by replacing uncharged amino acid residues with positively charged lysine and arginine, enhance the stability of the RBD–hACE2 complex, thus improving the binding of the RBD to the human ACE2 receptor (Jung et al., 2022). In addition to these structural and binding implications, several Omicron mutations exhibit varying impacts on antibody susceptibility (Chen et al., 2022; Iketani et al., 2022; Kumar et al., 2023). G339D, a rare core mutation in the RBD, shows slight resistance to sotrovimab in laboratory assays. K417N, found in several VOCs including Beta, Gamma, and Omicron, significantly reduces ACE2 binding and confers resistance to etesivimab and casirivimab while retaining susceptibility to other monoclonal antibodies (mAbs). N440K, prevalent in Omicron, shows resistance to imdevimab and C135 mAbs targeting the RBD core but remains susceptible to sotrovimab and convalescent plasma. Conversely, S477N and T478K, common in Omicron, do not reduce susceptibility to FDA EUA-approved mAbs, whereas E484A significantly reduces susceptibility to several mAbs, including C121 and C144 (Jung et al., 2022). Overall, the mutations in the Omicron variant underscore its adaptive evolution and the ongoing challenges they pose for therapeutic and vaccine development efforts. Computational studies suggest that the RBD–hACE2 complex of Omicron is highly stable due to these mutations, contributing to its high transmissibility and significant impact on the global response to the pandemic (Jung et al., 2022).

The evolutionary history of the Omicron variant in Pakistan was elucidated through a time-calibrated phylogenetic analysis and Bayesian skyline plot. Our results indicate that Omicron was introduced into Pakistan via two distinct lineages, clades C1 (BA.2) and C2 (BA.1), which subsequently spread across the country. Specifically, C1 shared its most recent common ancestor (MRCA) in January 2022, originating from a South African lineage isolated in late December 2021. In contrast, clade C2 shared its MRCA in December 2021 and was introduced through lineages originating from Thailand, Spain, and Belgium. These findings suggest that the Omicron variant in Pakistan has multiple origins and entered the country through various sources. The observed spread of Omicron from Pakistan to other countries and vice versa underscores the necessity for global cooperation in controlling viral dissemination and monitoring emerging variants. The evolutionary history of Omicron, analyzed alongside other SARS-CoV-2 VOCs, revealed complex phylogenetic relationships and divergence patterns. The population differentiation analysis revealed that Omicron exhibited similarities with the Delta variant, indicating a potential evolutionary linkage. However, it displayed the most diversity compared to the Wuhan variant. Phylogenetic analysis highlighted that Omicron is the most divergent among all VOCs, forming a distinct monophyletic group with an extremely long branch length, sharing its MRCA with other VOCs early in 2020. This suggests that Omicron emerged from a lineage separate from other VOCs, possibly due to its accumulation of numerous mutations. Several hypotheses have been proposed regarding the origin of Omicron (Markov et al., 2023). One hypothesis posits that Omicron may have “cryptically propagated” in a population with inadequate viral monitoring and sequencing (Du et al., 2022). Another suggests that Omicron could have developed in a COVID-19 patient with a long-term infection, such as an immunocompromised individual, providing a favorable environment for intra-host viral adaptation (Callaway, 2021). A third hypothesis is that Omicron might have accumulated mutations in a nonhuman host before zoonotic transmission to humans (Shan et al., 2021).

The population dynamics analysis of SARS-CoV-2, as revealed by the BSP, offers a comprehensive view of the virus’s evolutionary trajectory. The analysis successfully recapitulated the pandemic trajectory, showing a steady growth rate before 2021, followed by a decline in mid-2021 and a subsequent sharp population expansion at the end of 2021 coinciding with the Omicron surge. The BSP showed a steady population growth rate before 2021, which is consistent with the initial rapid spread of COVID-19 following its emergence in late 2019. This rapid spread was a result of the virus’s high transmissibility and the lack of pre-existing immunity in the global population. As mass vaccination campaigns were rolled out worldwide in early 2021, a decline in the population growth rate of SARS-CoV-2 was observed starting mid-2021 (Paetzold et al., 2022; Watson et al., 2022). This decline aligns with the historical record of the pandemic, where widespread vaccination efforts significantly reduced transmission rates and curbed the spread of the virus. However, towards the end of 2021, the BSP revealed a sudden expansion in the population size of SARS-CoV-2, which corresponds to the emergence and spread of the Omicron variant (Karim and Karim, 2021; Tegally et al., 2022; Viana et al., 2022). This period marked a significant increase in cases globally, despite high levels of vaccination, due to Omicron’s enhanced transmissibility and ability to partially evade immunity conferred by previous infections and vaccinations. These results successfully recapitulate the Omicron pandemic, demonstrating a clear temporal correlation between the observed population dynamics and the known epidemiological events, thus underscore the importance of continuous genomic surveillance and population dynamics studies. Such analyses are crucial for understanding how new variants emerge and spread, providing valuable insights that can inform public health strategies and interventions.

While our study provides comprehensive insights into the genomic epidemiology and evolutionary dynamics of the Omicron variant in Pakistan, it is essential to acknowledge certain limitations, such as the potential sampling bias due to unequal geographic distribution and temporal coverage of the retrieved samples, which may not fully capture the genetic diversity of the virus across the entire population. Moreover, the reliance on publicly available sequences from databases may introduce biases due to underreporting or variability in data quality and completeness. Recognizing these limitations helps provide a balanced view of our findings and underscores the need for continuous and comprehensive genomic surveillance efforts to inform effective public health responses.

In conclusion, this study presents a comprehensive examination of the genetic diversity, genomic epidemiology, and evolutionary dynamics of the Omicron variant in Pakistan during the fifth wave of COVID-19. Our findings underscore the complex evolutionary landscape of Omicron within the Pakistani population, highlighting the introduction of two distinct lineages—clades C1 (BA.2) and C2 (BA.1)—into the country. Specifically, clade C1 originated from a South African lineage in January 2022, while clade C2 emerged in December 2021, introduced via lineages from Thailand, Spain, and Belgium. These results illustrate the multiple origins of Omicron in Pakistan and its subsequent spread, both domestically and internationally. Notably, this study offers valuable insights into the transnational transmission patterns and adaptive evolution of Omicron, with important implications for genomic surveillance and public health strategies in Pakistan.