UNLABELLED: RNA modifications represent a critical layer of post-transcriptional gene regulation, shaping RNA fate and function through modulation of splicing, stability, translation, and immune signaling. Their role in viral infections remains insufficiently explored. We applied direct RNA sequencing to profile three key RNA modifications, 5-methylcytosine (m5C), N6-methyladenosine (m6A), and pseudouridine (psU), in whole blood samples from COVID-19 patients and healthy controls. Using CHEUI and NanoSPA, we identified condition-specific modification sites, their positional distribution, and associated functions. We detected 689 m5C, 738 m6A, and 1,201 psU sites, many uniquely enriched in infected individuals. These modifications localized predominantly to 3' UTRs and final exons. Codon-level and motif analysis revealed distinct enrichment patterns. Functional annotation indicated involvement in immune response, viral defense, inflammation, and stress. Several immune-regulatory genes, including,, and, exhibited co-occurring modifications. Pathway analysis revealed convergence on processes such as endocytosis, Fc receptor-mediated phagocytosis, and phagosome maturation, mechanisms central to viral entry and immune activation. Our findings show that RNA modifications are dynamically regulated during infection and may serve as biomarkers and therapeutic targets in COVID-19. CSF3R PIK3CD CAPZB
IMPORTANCE: RNA modifications are increasingly recognized as critical regulators of host-virus interactions, yet their specific roles in human viral infections remain largely unexplored. Here, we provide the first comprehensive epitranscriptomic map of 5-methylcytosine (m5C), N6-methyladenosine (m6A), and pseudouridine (psU) in the host transcriptome during SARS-CoV-2 infection. By combining direct RNA sequencing with advanced modification-calling algorithms, we identify hundreds of condition-specific sites, reveal their positional preferences within transcripts, and uncover their functional association with immune defense and viral entry pathways. These findings demonstrate that host RNA modifications are dynamically remodeled during infection and define molecular signatures with potential diagnostic and therapeutic value. Our work establishes RNA modification profiling as a powerful tool for dissecting viral pathogenesis and opens new avenues for targeted antiviral strategies.
