Transcriptomic and RNA Modification Landscape of Severe Fever with Thrombocytopenia Syndrome Virus Revealed by Nanopore Direct RNA Sequencing

Severe Fever with Thrombocytopenia Syndrome (SFTS) is caused by the SFTS virus (SFTSV) and is associated with a high mortality rate. Although previous studies have reported RNA modifications such as m6A on SFTSV RNA, an integrated analysis of native viral transcript architecture and multiple RNA modification types within infected cells remains lacking. Here, we used Oxford Nanopore direct RNA sequencing (DRS) to analyze native SFTSV RNA in infected cells, combining strand-specific alignment, isoform reconstruction through read endpoint clustering, isoform-level quantification, and signal-level modification identification using unmodified in vitro transcripts as a baseline. This approach allowed us to construct detailed maps of the L, M, and bidirectionally encoded S segments at single-molecule, isoform-level resolution. The results reveal a “length-layering” pattern in SFTSV transcription, anchored by recurrent 3′ termination hotspots: only a few full-length transcripts dominate expression, whereas multiple reproducible truncated isoforms were associated with discrete termination windows, a pattern less consistent with random degradation alone and suggestive of regulated transcript termination. At the single-nucleotide level, the modification landscape is predominantly Ψ (pseudouridine), followed by m5C (5-methylcytosine), with sparse m6A (N6-methyladenosine). Modification hotspots are co-located across isoforms at the same genomic coordinates, exhibiting segmental/strand asymmetry, with sharper peaks on (−) RNA. These patterns provide a testable framework and raise the possibility that transcript-boundary organization and site-constrained Ψ/m5C signals may be associated with variation in viral RNA output. More broadly, isoform proportions around termination hotspots and Ψ/m5C-enriched regions at conserved sites may serve as quantitative features for characterizing viral RNA organization and prioritizing targets for future functional investigation. Our single-molecule integrated map establishes a reproducible methodological framework for studying SFTSV RNA regulation and provides a resource for future work aimed at assessing how transcript boundaries and RNA modification patterns may relate to polymerase activity and virus–host interaction.