Host immune dysregulation associated with high plasma EBV-DNA load in children and its potential association with secondary infection.

High plasma Epstein-Barr virus (EBV)-DNA load may be associated with host immune dysregulation and secondary infection in children, although the underlying mechanisms remain uncertain. This study analyzed peripheral blood samples from 34 children with high plasma EBV-DNA load and 31 children with low plasma EBV-DNA load using transcriptomic analysis, WGCNA, CIBERSORT-based deconvolution, LASSO regression, Random Forest, qPCR, ELISA, and flow cytometry to explore immune features associated with EBV-DNA load and secondary-infection status. A total of 459 differentially expressed genes were identified. In the high-load group, antiviral and inflammatory pathways were significantly upregulated, whereas adaptive immune pathways were suppressed, revealing a pattern of innate immune hyperactivation together with impaired adaptive cellular immunity. CIBERSORT analysis estimated higher neutrophil and monocyte proportions and lower CD8⁺ T-cell and NK-cell proportions in the high-load group; these deconvolution estimates should not be interpreted as direct cell counts. Multi-strategy integrative analysis prioritized STAT1, CXCL10, and IL6 as candidate immune-related markers; their expression was positively associated with plasma EBV-DNA load and was higher in the secondary-infection group than in the non-infection group. Within this cohort, the combined three-gene model yielded an AUC of 0.85 for discriminating secondary-infection status and should be considered exploratory pending independent validation. Experimental validation was directionally consistent with the bioinformatic findings and supports an association between high plasma EBV-DNA load, circulating immune dysregulation, and secondary-infection status; it does not establish a causal mechanism.