The glioblastoma virome across tumor and extracellular compartments.

Background: Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, marked by rapid progression and poor prognosis. GBM may also harbor a complex Methods: We analyzed 45 long- and small-RNA libraries from glioma stem-like cells, their extracellular fractions (exosomes, microvesicles, ribonucleoprotein complexes), and normal brain controls (BioProject PRJNA360129). Taxonomic profiling and functional annotation were performed using established pipelines (MetaPhlAn4, BLASTn, UniProtKB, InterPro), with fresh media controls to exclude background contamination. To validate discriminative power, viral RNA profiles were classified using machine learning -Random Forest models trained on nine species identified as glioblastoma-enriched by LEfSe. Results: We detected ~3.41 million viral reads, with >99% from GBM cells and extracellular compartments and <1% from normal brain. Reads spanned 36 species across 11 phyla. Nine species were consistently enriched in glioblastoma: Conclusion: Glioblastoma contains a distinct viral transcriptome; long-RNA profiles show strong diagnostic power, and compartment-specific signatures suggest biomarker potential.