Serum extracellular vesicle RNA profiles in long COVID: insights from exercise-induced gene modulation

The Persistence of SARS-CoV-2 in tissues has been proposed as a driver of prolonged symptoms in long COVID. Pulmonary rehabilitation with exercise training is a well-established intervention for improving symptoms, functional capacity, and inflammation in chronic cardiorespiratory diseases. To investigate whether long COVID is associated with persistent viral or immune-related signals, we analyzed the long RNA profile of circulating extracellular vesicles (EVs) to determine the presence of virus-related transcripts and assess changes in response to exercise training. Fourteen adults with long COVID participated in this single-center pilot clinical trial and completed a 10-week aerobic exercise training program (twenty 1.5 h sessions). Serum-derived EV RNA profiles were analyzed via sequencing at rest (T0) and peak cardiopulmonary exercise testing (T1), before (V2) and after (V24) exercise training. Differentially expressed genes (DEGs) were identified (q < 0.05), and pathway activation analysis was performed. Serum EVs carried diverse RNA species, including protein-coding RNAs, long non-coding RNAs, short non-coding RNAs, and pseudogenes, with no virus-related RNAs detected. No significant DEGs were identified at rest between pre- and post-training, nor in response to acute exercise at pre-training. However, following training, 53 DEGs were found at peak exercise (V24T1) compared to rest (V24T0), including three upregulated genes (ANK3, FTO, FCN1) and 50 downregulated genes (TOP 5: MYL9, NRGN, H2AC6, MAP3K7CL, B2M). These genes were primarily involved in inflammation and metabolism. Pathway analysis revealed significant regulation of 100 pathways at post-training compared to pre training, predominantly inactivated, including pathways involved in inflammation (STAT3 signaling) and metabolism (O-linked glycosylation). Acute exercise and exercise training modulated EV-associated gene expression in long COVID, primarily through transcriptional downregulation. Suppression of inflammation- and immune-related genes post-training highlights potential molecular mechanisms underlying symptom improvement and identifies candidate biomarkers of recovery biology in long COVID. Importantly, while exercise training did not substantially alter EV RNA content at rest, it enhanced the body's ability to mount a dynamic EV-mediated molecular response during exertion, reflecting improved physiological adaptability.Clinical trial registration number: NCT05398692.