Unveiling the Hidden RNA Virus Diversity in Organic Wastes: Shaping and Reduction Effects of Black Soldier Fly Treatment

We investigated the diversity and transmission of RNA viruses during treatment of black soldier fly larvae (BSFL) with three typical organic wastes: waste-activated sludge (WAS), food waste (FW), and pig manure (PM). Metatranscriptomic analyses identified 175 RNA viral operational taxonomic units (vOTUs), 54.3% of which were novel, mainly affiliated with the Narna-Levi and Tombus-Noda supergroups. Viral communities in BSFL and frass were shaped by both insect-associated viruses (e.g., Inseviridae and Iflaviridae ) and feedstock-derived viruses. While FW and wheat bran supported BSFL growth, the detected viruses were primarily associated with insects. PM and WAS introduced a broader range of plant-, fungus-, and vertebrate-infecting viruses, including zoonotic viruses such as Astroviridae and Picobirnaviridae . BSFL treatment eliminated Astrovirus from PM and markedly reduced human-pathogenic viruses (e.g., norovirus and enterovirus ) within 6–8 days, while picobirnavirus persisted in frass. Frass also retained plant-infecting viruses (e.g., Virgaviridae ) and novel Fiersviridae phages. Although BSFL treatment increased viral microdiversity (gene-level π ∼ 11-fold, nucleotide diversity of 7.33 × 10 –5 to 8.31 × 10 –4, decreased Tajima’s D ), most pN/pS ratios remained