The COVID-19 pandemic, caused by SARS-CoV-2, led to unprecedented global health, social, and economic disruptions between 2020 and 2022, with ongoing endemic transmission into 2025. In Pakistan, approximately 1.6 million cases and 30,700 deaths were reported to the World Health Organization (WHO) from March 2020 to April 2023 ( WHO, 2024c).

Since its emergence, SARS-CoV-2 has evolved into a range of genetically distinct variants. Those posing increased risks to global public health were categorized by the WHO as Variants Under Monitoring (VUM), Variants of Interest (VOI), and Variants of Concern (VOC) ( WHO, 2023a, 2023b). VOCs are defined by their enhanced transmissibility, potential to cause more severe disease, escape immune responses, or reduce the efficacy of public health interventions, including vaccines ( WHO, 2023b). From May 2021, the WHO adopted Greek letters to label key variants, including Alpha, Beta, Delta, and Omicron—several of which have been associated with vaccine breakthrough infections ( Jamal et al., 2022; WHO, 2021).

SARS-CoV-2 genomic divergence is driven by mutation, recombination, and genetic drift ( Dvořák, Jahodářová, Stanojković, Skoupý, & Casamatta, 2023). Global sequencing efforts have enabled high-resolution tracking of variant emergence and spread, informing public health strategies. Emerging evidence suggests that SARS-CoV-2 genomes began stabilizing after widespread vaccine rollout, as shown by reduced substitution rates and viral entropy ( Ashraf et al., 2023).

Vaccination against SARS-CoV-2 has been pivotal in mitigating severe disease and mortality. During the first year of vaccine deployment (December 2020–December 2021), COVID-19 vaccines were estimated to have prevented 19.8 million deaths globally ( Watson et al., 2022). In the WHO European Region alone, 1.4 million lives were saved between December 2020 and March 2023 ( WHO, 2024b). In Pakistan, vaccines were introduced in 2021, initially prioritizing healthcare workers and the elderly. The national program deployed a mix of mRNA, inactivated virus, and viral vector vaccines. Reported vaccine effectiveness (VE) ranged from 33.8% to 67.4% depending on the platform and circulating variant ( Camacho-Moll et al., 2024; Firouzabadi, Ghasemiyeh, Moradishooli, & Mohammadi-Samani, 2023) ( Nisar et al., 2023). During the Delta wave, VE against infection was lower for inactivated vaccines ( Khan et al., 2023), though protection against severe disease remained robust across platforms ( WHO, 2024a). While vaccines clearly reduced hospitalizations and deaths ( Wang et al., 2023) ( Mwendwa et al., 2024), it remains unclear whether vaccination status altered the spectrum of infecting SARS-CoV-2 variants.

Additionally, there is limited data on SARS-CoV-2 variant dynamics in children in Pakistan. A multicenter study conducted between March 2020 and December 2021 reported an 18.6% mortality rate among hospitalized pediatric COVID-19 patients ( Abbas et al., 2023). This study noted the highest pediatric case surge and mortality early on during the Delta wave (July–September 2021), but did not explore variant-specific susceptibility in children.

In this context, we investigated SARS-CoV-2 genomic diversity in Pakistan during May 2021–October 2022, spanning the Delta and Omicron waves across the different provinces of Pakistan. We examined circulating variants in vaccinated and unvaccinated individuals, with particular attention to children under 18 years. By analyzing national sequence data alongside pediatric cases from Karachi, we aimed to assess whether age or vaccination status influenced variant distribution or viral divergence.

Vaccinations were a cornerstone of the global COVID-19 response, significantly reducing morbidity and mortality. However, the pandemic’s trajectory varied across countries due to differences in age structures, population immunity, variant circulation, and healthcare infrastructure. Pakistan reported approximately 31,000 COVID-19 deaths up to March 2023 - relatively low given its population size - prompting investigation into possible reasons. Factors including natural immunity, routine vaccination coverage (e.g., BCG, influenza), and the rollout of COVID-19 vaccines have been postulated as possible reasons across different parts of the world ( Cocco et al., 2021; Sarfraz et al., 2021; WHO, 2024a; Shrwani et al., 2021).

This study examined the phylogenetic diversity of SARS-CoV-2 variants in Pakistan between 2021 and 2022, a period marked by the dominance of Delta and Omicron variants. We analyzed genome sequences from both vaccinated and unvaccinated individuals and conducted a focused comparison on pediatric cases, who represent nearly half of Pakistan’s 240 million population ( GOP, 2024). Our results provide important insight into viral transmission dynamics in a low-resource, high seroprevalence setting.

A key finding was the lack of significant difference in variant distribution between vaccinated and unvaccinated individuals. Despite assumptions that immunological pressure might favor certain variants in vaccinated hosts, we observed similar lineage patterns and genomic divergence across groups. This suggests that during the Delta and Omicron waves, SARS-CoV-2 continued to spread through both immune and non-immune hosts without a marked shift in variant selection.

This aligns with reports of widespread subclinical transmission in Pakistan, including rural and urban areas, likely fueled by high rates of asymptomatic or mild infections ( Iqbal et al., 2025). Earlier studies found population-level SARS-CoV-2 seroprevalence reaching 81% by November 2021 ( Iqbal et al., 2025), even as testing rates remained low and many infections went undiagnosed. Such high exposure may have buffered the population against severe disease outcomes, regardless of vaccination status.

Importantly, genomic divergence values also showed no significant difference between children (<18 years) and adults, indicating that circulating variants were broadly shared across age groups. Pediatric data are often underrepresented in genomic surveillance, yet children made up 71% of our cohort due to the availability of vaccination status in this group. Our pediatric-focused analysis, both nationally and from a single-center cohort in Karachi, confirmed that Delta dominated in 2021 and Omicron in 2022 - mirroring trends seen globally ( Campbell et al., 2021; Kung et al., 2024).

Interestingly, Omicron infections in children were associated with significantly lower Ct values (i.e., higher viral loads) compared to earlier variants, consistent with increased transmissibility ( Puhach, Meyer, & Eckerle, 2023). These findings corroborate prior reports in the general population ( Nasir et al., 2023) and underscore the role of Omicron in driving large pediatric infection waves, despite lower severity.

A prior study by Abbas et al. ( Abbas et al., 2023) showed fluctuating variant dominance in hospitalized children, but did not assess genomic divergence or compare vaccinated vs. unvaccinated hosts. To our knowledge, this is the first study in Pakistan to systematically compare SARS-CoV-2 variant diversity by vaccination status and age group using phylogenetic divergence metrics.

We also observed that Islamabad and Karachi accounted for most sequence submissions during the study period - consistent with their status as international hubs and prior reports on variant introductions into Pakistan ( Aziz et al., 2023; Ali, Shah, & Siddiqui, 2020; Bukhari et al., 2023; Nasir et al., 2022). This geographic concentration reflects testing and sequencing infrastructure disparities and highlights the need for broader surveillance coverage across provinces.

This study provides novel insights into SARS-CoV-2 variant circulation in Pakistan during the Delta and Omicron waves, highlighting that viral lineages did not differ significantly between vaccinated and unvaccinated individuals, nor between children and adults. Genomic divergence analyses revealed comparable strain diversity across age groups and vaccination status, suggesting widespread community transmission of similar variants irrespective of host immune background.

Our findings underscore the importance of understanding SARS-CoV-2 dynamics in populations with high background immunity - whether from prior infection, vaccination, or both. In such settings, variant-specific susceptibility may be less pronounced, and asymptomatic or mild infections may sustain transmission. This is particularly relevant for low- and middle-income countries where young age demographics, subclinical infections, and mixed vaccine regimens influence viral evolution.

As the pandemic transitioned into an endemic phase, sustained genomic surveillance - especially in pediatric populations and underrepresented regions - is essential to track emerging variants and guide tailored public health interventions. Our results reinforce the need for integrated virologic, immunologic, and demographic approaches to inform global pandemic preparedness in the years ahead.

This study was conducted in accordance with the principles of the Declaration of Helsinki. Ethical approval was obtained from the Ethics Review Committee of Aga Khan University, which granted a waiver of written and verbal informed consent (Approval No. 2021-6316-18156 and 2022-6871-22433) on the grounds that this was a retrospective study using secondary data, for which obtaining consent was impractical. All data was de-identified before retrieval from AKUH database and therefore poses no risk of confidentiality to subjects.

Pre-print version of this article: Mwendwa, F., Ashraf, J., Kanji, A., Bukhari, A. R., Hasan, R., & Hasan, Z. (2025). Temporal Mapping of SARS-CoV-2 Variants During the Pandemic Reveals Similar Genomic Divergence in Unvaccinated and Vaccinated Cohorts, and in Both Adults and Children. Preprints. https://doi.org/10.20944/preprints202504.2203.v1

Supplementary Table 1: Data downloaded from GISAID for samples collected May 2021 to October 2022

Supplementary Table 2: Breakdown of Next clade and Pangolin lineage of the samples collected during the study period, May 2021 to October 2022

Supplementary Table 3: Genomic divergence data

Data is available under the terms of the Creative Commons Attribution 4.0 International.