HCMV-encoded miRNAs in an EV-enriched plasma particle fraction as candidate biomarkers of HCMV DNAemia

Background Extracellular vesicle-enriched plasma particle fractions may contain viral and host RNA species that reflect infection-associated changes. However, whether HCMV-encoded miRNAs detected in circulating particle fractions are associated with HCMV DNAemia remains incompletely understood. This study analyzed selected HCMV-encoded miRNAs in an ExoQuick-derived EV-enriched plasma precipitate from patients with HCMV DNAemia and DNA-low, IgG-positive controls. Methods Plasma samples were collected from 41 patients with HCMV DNAemia, defined as HCMV DNA > 500 IU/mL, and 43 DNA-low, IgG-positive controls, defined as HCMV DNA < 500 IU/mL with HCMV IgG positivity and IgM negativity. Eight HCMV miRNAs were quantified using stem-loop RT-qPCR. HCMV DNA was measured by qPCR. A longitudinal series of 30 samples from seven treated patients was analyzed exploratorily. Results TEM and NTA showed that the ExoQuick-derived preparations contained vesicle-like nanoparticles with round or cup-shaped morphology and particle-size distributions compatible with small EV-enriched preparations. Among the eight candidate HCMV miRNAs, hcmv-miR-US4-5p and hcmv-miR-US25-2-5p remained significantly increased in the HCMV DNAemia group after false-discovery-rate correction. Hcmv-miR-US25-1-3p and hcmv-miR-UL36 showed nominal increases but did not remain significant after correction. Exploratory ROC analyses showed modest AUC values of approximately 0.64–0.73, with sensitivities below 45%. Longitudinal patterns were heterogeneous among the seven treated patients. Conclusion Selected HCMV-encoded miRNAs, particularly hcmv-miR-US4-5p and hcmv-miR-US25-2-5p were increased in an ExoQuick-derived EV-enriched plasma particle fraction from patients with HCMV DNAemia. These findings suggest potential exploratory auxiliary biomarker value, but definitive assignment to EV-encapsulated cargo and their utility for disease monitoring require further validation using EV-marker analysis, nuclease/protease protection assays, broader miRNA profiling, and independent validation cohorts are required.