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INTERNATIONAL CONGRESS OF

CancerGenetics

• Epigenetic Regulation of EBV Reactivation in Immunocompromised Patients

• Zahra Dolati,^1,* Melika Motaheyar,² Narges Safari,³ Behrad Asghari,⁴ Soodeh Tavakoli,⁵
  1. Department of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran.
  2. Faculty of Veterinary Medicine, University of Semnan, Semnan, Iran
  3. Department of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran
  4. DVM Student, Faculty of Veterinary Medicine, Babol Branch, Islamic Azad University, Mazandaran, Babol, Iran.
  5. Independent Veterinarian, Tehran, Iran
  
  
  
• Introduction: Epstein-Barr virus (EBV), a member of the Gammaherpesvirinae subfamily, persistently infects over 95% of the human population, establishing a lifelong latency primarily within memory B-lymphocytes [1]. The virus maintains a delicate balance between its latent and lytic states, a process tightly regulated by both viral factors and host immune surveillance. In immunocompetent hosts, this equilibrium is effectively controlled; however, in individuals with compromised immune function—such as solid organ or hematopoietic stem cell transplant recipients and those with HIV/AIDS—this control is lost, leading to uncontrolled EBV reactivation [2]. This uncontrolled replication is a major etiological factor in the development of severe complications, including post-transplant Lymphoproliferative disorder (PTLD) and a spectrum of B-cell lymphomas [2]. The molecular switch from latency to the lytic cycle is not stochastic but is governed by sophisticated epigenetic mechanisms, primarily DNA methylation and post-translational histone modifications [3]. These epigenetic marks act as a regulatory interface, allowing the virus to evade immune detection by silencing its genome during latency, while poised to reactivate in response to specific environmental or cellular cues. Disruption of this epigenetic regulation by external factors such as immunosuppressive therapies, environmental stressors, or co-infections can destabilise the latent state, creating a permissive environment for viral replication and, consequently, tumorigenesis [4]. While the clinical significance of EBV reactivation is well-established, a comprehensive understanding of the interplay between various epigenetic modifications in the context of specific immunocompromised states remains an area of active investigation. This study aims to bridge this gap by systematically dissecting the epigenetic landscape that drives EBV reactivation in immunocompromised patients. We further evaluate the potential of identifying robust epigenetic biomarkers for predicting reactivation risk and explore the therapeutic promise of targeted epigenetic interventions. To enhance the translational relevance of our findings, we incorporate insights from companion animal models (dogs and cats) that harbour naturally occurring herpesvirus infections, providing a valuable comparative context for understanding conserved regulatory mechanisms [5].
• Methods: Study Population and Data Acquisition We analysed publicly available and collaborator-sourced multi-omics datasets, including epigenomic and transcriptomic profiles, from a cohort of 300 immunocompromised patients. The cohort comprised 150 solid organ transplant recipients and 150 individuals with HIV (mean age ± SD: 45 ± 12 years). Additionally, to explore translational potential, we included Data from 50 companion animals (25 dogs and 25 cats) diagnosed with naturally occurring gammaherpesvirus infections. Human datasets were primarily sourced from the Gene Expression Omnibus (GEO) repository, while animal data were obtained from collaborating veterinary institutions. Epigenomic and Transcriptomic Profiling Genome-wide DNA methylation patterns were assessed using Infinium MethylationEPIC BeadChip data. Histone modifications, specifically H3K9 acetylation (H3K9ac) and H3K27 acetylation (H3K27ac), were quantified via chromatin immunoprecipitation followed by sequencing (ChIP-seq) according to established protocols. Gene expression profiles, including both host and viral transcripts, were generated using RNA sequencing (RNA-seq). Raw sequencing data were processed through standardised bioinformatics pipelines for quality control, alignment, and quantification. Machine Learning and Statistical Analysis Supervised machine learning models, specifically Random Forest and XGBoost, were employed to identify a predictive epigenetic signature of EBV reactivation. Model interpretability was enhanced using SHAP (Shapley Additive exPlanations) values to determine the contribution of individual epigenetic features. The performance of the predictive model was evaluated using the area under the receiver operating characteristic curve (AUC-ROC). Statistical analyses were performed using R software (version 4.2.0). Differences in DNA methylation levels between groups were assessed using unpaired two-tailed t-tests. The association between immunosuppressive drug use and reactivation risk was calculated using logistic regression, reported as odds ratios (OR) with 95% confidence intervals. For comparisons of multi-omics data that did not follow a normal distribution, the Mann–Whitney U test was applied. A p-value of less than 0.05 was considered statistically significant. The statistical power of the study was calculated a priori using G*Power software, targeting a power of 80% (α = 0.05).
• Results: Our multi-omics analysis revealed that epigenetic mechanisms are central regulators of EBV reactivation in immunocompromised patients. DNA Methylation Governs Promoter Silencing:During latency, the immediate-early viral promoters *BZLF1* and *BRLF1* were consistently hypermethylated, effectively silencing lytic gene expression (mean methylation difference: 42%, p<0.01). In contrast, patients experiencing EBV reactivation exhibited significant hypomethylation at these loci, indicating that loss of DNA methylation is a key permissive event for lytic cycle entry (p<0.05). Histone Acetylation Dynamics Correlate with Lytic Status: A strong positive correlation was observed between histone hyperacetylation at H3K9 and H3K27 and the expression of lytic genes (r = 0.68, p<0.05). Conversely, hypoacetylation at these marks was a hallmark of the latent state. *In vitro* experiments demonstrated that treatment with broad-spectrum HDAC inhibitors (e.g., sodium butyrate) shifted the balance toward hyperacetylation, inducing viral reactivation and confirming their utility as research tools for probing the latency–lytic switch. Immunosuppressive Drugs Enhance Reactivation Risk: The use of calcineurin inhibitors, cyclosporine and tacrolimus, was significantly associated with an increased risk of EBV reactivation (OR = 2.3, 95% CI: 1.4-3.8, p<0.01). This effect was linked to both direct T-cell functional impairment and an indirect increase in the activity of DNA demethylases, such as TET enzymes, leading to epigenetic destabilisation. EBV miRNAs Create Regulatory Feedback Loops: EBV-encoded miRNAs, particularly those from the BART cluster, were shown to target both host immune response genes (e.g., *IL-6*, *CXCL11*) and viral transcripts, including the immediate-early gene *BRLF1*. This dual Targeting creates complex feedback loops that fine-tune the reactivation process, allowing the virus to modulate its own lifecycle and evade host immune clearance (p<0.05). Synergistic Triggers Amplify Epigenetic Instability: The presence of co-infections, notably cytomegalovirus (CMV), and elevated markers of oxidative stress (e.g., 8-OHdG levels) synergistically promoted epigenetic instability. Patients with both risk factors exhibited a significantly higher rate of EBV reactivation compared to those with either factor alone (p<0.001), highlighting the multifactorial nature of reactivation triggers. Epigenetic Signature as a Predictive Biomarker: A machine learning model integrating DNA methylation patterns and histone modification marks successfully distinguished patients at high risk of reactivation from those in stable latency. The model achieved an area under the curve (AUC) of 0.88, demonstrating high sensitivity and specificity and highlighting the potential of epigenetic signatures as non-invasive clinical biomarkers. Conserved Epigenetic Patterns in Companion Animals: Analysis of samples from dogs and cats with gammaherpesvirus infections revealed epigenetic regulatory mechanisms strikingly similar to those observed in human EBV infection, including promoter methylation and histone acetylation dynamics. This finding supports the translational relevance of veterinary models, although the small sample size remains a limitation for broader generalisations.
• Conclusion: In conclusion, this study demonstrates that EBV reactivation in immunocompromised patients is a tightly regulated process governed by a triad of key epigenetic mechanisms: DNA methylation, histone modifications, and viral miRNA activity. These mechanisms are profoundly modulated by clinical factors, including immunosuppressive pharmacotherapy and environmental stressors such as co-infections and oxidative stress. Our identification of a highly accurate epigenetic biomarker signature for reactivation risk, coupled with insights from comparative animal models, paves the way for a paradigm shift toward precision medicine in managing EBV-associated diseases. The path forward requires the careful development of context-specific epigenetic therapies and their validation through rigorous longitudinal studies. Ultimately, the integration of multi-omics profiling with advanced artificial intelligence holds the revolutionary potential to transform the clinical management of EBV from a reactive to a proactive, predictive, and personalised approach, significantly improving outcomes for high-risk immunocompromised populations.
• Keywords: Epstein-Barr Virus (EBV), Epigenetic Regulation, DNA Methylation and Histone Modifications