INTRODUCTION
By March 2025, the world has witnessed the devastating reach of the coronavirus disease 2019 (COVID-19), claiming over 7 million lives, according to Our World in Data (2024). While vaccines and prior infections have reduced the immediate threat of death for many, the virus continues to infect thousands daily, as tracked by the World Health Organization (2023). Beyond the initial illness, a lingering challenge has emerged: millions of people experience long-lasting symptoms, known as post-COVID-19 condition (PCC), which can include trouble breathing, exhaustion, or even memory problems, affecting roughly one in ten infected individuals, as Davis et al (2023) report. These persistent effects signal that COVID-19 is not just a short-term sickness but a condition that can alter lives for months or years, placing new demands on doctors, governments, and communities worldwide.
The data explored in this article reveal a hidden layer of this story—how the virus leaves a lasting mark not just on the body but on the very instructions that control how our cells work. Scientists have long known that SARS-CoV-2, the virus behind COVID-19, first attacks the nose and throat, a finding Wolfel et al (2020) confirmed early in the pandemic. What’s new here is the discovery of changes in DNA methylation—a process that acts like a switchboard, turning genes on or off in cells—tracked through advanced techniques like whole-genome enzymatic DNA methylation sequencing and single-cell RNA sequencing. These methods, applied to 33 people and followed up over time, show that the virus doesn’t just come and go; it rewrites some of these switches, especially in the nose’s lining, where it first takes hold.
For everyday understanding, think of DNA methylation as a set of dials on a control panel. Normally, these dials help cells respond to threats, like calling in immune defenders or keeping the airways clear by moving mucus. The research uncovers two big shifts. First, in immune cells like macrophages, certain dials get turned up, making them extra active—sometimes too active—potentially worsening the infection’s damage, as Chua et al (2020) hinted in earlier studies. Second, in airway cells responsible for clearing out germs, the dials get stuck in the “off” position, even a year later, weakening the lungs’ natural cleaning system. This could explain why some people, as Bowe et al (2023) note, still struggle to breathe easily long after recovering.
These findings matter beyond the lab. If the virus can change how cells behave for months, it’s a clue to why PCC lingers and a hint at how to fix it—perhaps by finding ways to reset those dials. For governments and health systems, reported by the International Monetary Fund (2024) to face ongoing economic strain, this means preparing for a wave of chronic illness that could keep people out of work or in need of care. For researchers, it’s a call to dig deeper into the nose and lungs, not just the blood, to unlock the full picture of COVID-19’s impact. In simple terms, this data shows the pandemic’s effects stretch far beyond the first fever, reshaping lives in ways we’re only beginning to understand.
The deep study ….
By March 2025, the global toll of the coronavirus disease 2019 (COVID-19) pandemic has exceeded 7 million deaths, as reported by Our World in Data (2024), with the World Health Organization (2023) noting a persistent daily incidence of thousands of infections despite widespread immunity from vaccination or prior exposure. While acute mortality has declined, the emergence of post-COVID-19 condition (PCC), affecting approximately 10% of infected individuals according to Davis et al (2023), has shifted research focus toward understanding long-term health consequences. PCC, characterized by symptoms such as dyspnea, fatigue, and cognitive impairment, presents a significant burden to healthcare systems globally, with Bowe et al (2023) documenting that some manifestations, including loss of smell and respiratory difficulties, may persist beyond a year. The nasopharynx, identified by Wolfel et al (2020) as the primary entry point for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), serves as a critical site for investigating these enduring effects, given its role in initial viral replication and immune activation.
Extensive studies, including those by Chua et al (2020) and Ziegler et al (2021), have mapped the transcriptomic and proteomic responses of nasal mucosa during acute SARS-CoV-2 infection, revealing a cascade of interferon release and immune cell infiltration, notably of neutrophils and macrophages. However, the epigenetic mechanisms underpinning these responses, particularly DNA methylation, remain underexplored despite their potential to regulate gene expression heritably across cell divisions, as elucidated by Mattei et al (2022). DNA methylation, a key epigenetic modification, offers a lens through which to examine both immediate immune responses and the sustained cellular reprogramming associated with PCC. Prior investigations, such as those by Balnis et al (2021) and Konigsberg et al (2021), have largely relied on blood-derived samples, leaving a gap in understanding methylation changes at the infection’s epicenter—the airway epithelium.
To address this, a cohort of 33 individuals underwent whole-genome enzymatic DNA methylation sequencing (mDNA-seq) and single-cell RNA sequencing (scRNA-seq) of nasopharyngeal cells, with findings complemented by longitudinal resampling of 12 participants at 3 and 12 months post-infection and validation in an independent cohort of 15 individuals at 6 months. The primary cohort included 19 COVID-19 patients, predominantly male (84.2%) with a median age of 57 years, and 14 controls with a more balanced sex distribution and younger median age of 38.5 years, as detailed in the dataset published alongside this analysis (2025). The mDNA-seq achieved a median genome-wide coverage of 62.3×, enabling high-resolution mapping of methylation patterns, while scRNA-seq profiled 82,365 cells across 30 distinct cell types, providing granular insight into transcriptional dynamics.
Analysis revealed 3,112 differentially methylated regions (DMRs) across all autosomes, with 81.2% exhibiting hypermethylation in SARS-CoV-2-positive individuals compared to controls, adjusted for age, sex, and cellular composition. These DMRs, predominantly overlapping with regulatory elements such as enhancers (64.2%), as per Burger et al (2013), suggest a profound epigenetic shift in response to infection. Methylation quantitative trait loci (meQTL) analysis indicated that hypermethylated DMRs were largely non-genotype-associated (65.7%), while hypomethylated DMRs showed a higher genotype linkage (60.8%), pointing to a mix of environmental and genetic influences on the methylome. Of the 5,513 identified DMR target genes, immune cells displayed upregulated expression (59.8% of 1,859 differentially expressed genes [DEGs]), contrasting with a striking downregulation in epithelial cells (91.9% of 2,829 DEGs), underscoring divergent cellular responses to SARS-CoV-2.
Focusing on hypomethylated regions, gene ontology (GO) enrichment analysis identified a cluster of chemokine receptor genes on chromosome 3p21.31, including CCR1, CCR2, and CCR5, linked to immune cell recruitment. A specific non-genotype-associated hypomethylated DMR overlapped with an enhancer (GH03J046088) from the GeneHancer database, with promoter capture Hi-C data from Javierre et al (2016) confirming interactions with these genes in monocytes, macrophages, and CD8-positive T cells. Correlating methylation levels with scRNA-seq data revealed a significant inverse relationship with CCR1 expression in macrophage populations, particularly non-resident macrophages, where Spearman correlation coefficients reached statistical significance (P < 0.01). This aligns with findings by Chua et al (2020) that excessive macrophage activation correlates with severe COVID-19, suggesting hypomethylation may amplify inflammatory responses in the airway.
Conversely, hypermethylated DMRs in epithelial cells were associated with suppressed gene expression, particularly of pathways governing ciliary function. Gene set enrichment analysis highlighted downregulation of genes such as RSPH9, DNAH3, and DNAH5, critical for motile cilia assembly, alongside trafficking proteins IFT122 and IFT46, and the transcription factor RFX3, as noted by Tilley et al (2015) and El Zein et al (2009). A pathways score, calculated from the average expression of 323 repressed genes, demonstrated a marked reduction across epithelial subtypes—basal, secretory, and ciliated—during acute infection, with ciliated cells exhibiting the most pronounced decline relative to controls. This suppression coincided with a shift in cellular composition, where epithelial proportions dropped and immune cells, notably neutrophils, increased, as reported in the 2025 dataset accompanying this study.
Longitudinal tracking at 3 and 12 months post-infection revealed a gradual recovery of epithelial cell proportions, reaching control levels by 12 months, yet ciliated cells sustained transcriptional repression. The pathways score in basal and secretory cells improved over time, but ciliated cells retained significantly lower scores (Wilcoxon test, FDR-adjusted P < 0.001), with 106 genes, including DNAH3, DNAH5, and RFX3, remaining downregulated compared to controls. Network analysis using Cytoscape identified persistent suppression of ciliogenesis and microtubule movement genes, corroborated by transcription factor motif enrichment for repressors like HEY2 and REST, as documented by Jetten et al (2022) and Weber et al (2014). This suggests an inherited epigenetic reprogramming, potentially propagated from progenitor secretory cells, impairing ciliary regeneration.
Validation in an independent cohort of 15 individuals at 6 months post-infection, balanced for sex and disease severity, reinforced these findings. Patients with persistent respiratory symptoms (n = 6) exhibited significantly lower pathways scores in ciliated cells compared to those without symptoms (n = 9), with multiple linear regression confirming a symptom-linked effect (coefficient = −0.002, 95% CI −0.004 to −0.001, P < 0.001). Genes such as EZR and DNAH3, integral to ciliary coordination, were among the most repressed, aligning with reports by Kawaguchi et al (2022) of impaired mucociliary clearance in PCC.
These findings illuminate the dual epigenetic impact of SARS-CoV-2: hypomethylation driving acute immune activation and hypermethylation mediating long-term epithelial dysfunction. The upregulation of CCR1 in macrophages reflects an adaptive response to infection, potentially exacerbating severity, as Zeberg and Paabo (2020) linked this locus to adverse outcomes. Meanwhile, sustained ciliary gene repression offers a mechanistic basis for respiratory PCC, consistent with Pezato et al (2023) observations of diminished airway clearance in dyspneic patients. Unlike blood-focused studies by Balnis et al (2021), this nasopharyngeal analysis captures site-specific changes, revealing a heritable reprogramming that may underlie persistent symptoms.
Limitations include the modest cohort size and male predominance in follow-up samples, potentially skewing sex-specific insights, though the independent cohort mitigated this by including more females. The absence of follow-up mDNA-seq data restricts direct epigenetic tracking, yet persistent methylation effects are well-documented by Huoman et al (2022) and Balnis et al (2023). The mixed-cell nature of mDNA-seq necessitated scRNA-seq integration to infer cell-type specificity, a methodological constraint offset by the study’s high sequencing depth and longitudinal design.
Globally, these insights carry implications for health policy and research agendas. The International Monetary Fund (2024) projects ongoing economic strain from healthcare burdens, with PCC contributing to workforce reductions estimated at 1.5% in high-income nations by the OECD (2024). Targeting epigenetic modifications, potentially through demethylating agents, could emerge as a therapeutic avenue, warranting trials informed by this data. Moreover, the United Nations Development Programme (2025) emphasizes equitable access to such innovations, given higher PCC rates in unvaccinated populations, as per Davis et al (2023).
This pioneering integration of mDNA-seq and scRNA-seq in the nasal mucosa offers a foundational understanding of SARS-CoV-2’s epigenetic legacy. It underscores the need for larger, diverse cohorts to refine these associations and informs a multidisciplinary approach—spanning genomics, public health, and economic policy—to address the enduring shadow of COVID-19 on global societies.
resource : https://www.embopress.org/doi/full/10.1038/s44321-025-00215-5
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