RNA virome restructuring along livestock manure treatment chains: Patterns and underlying mechanisms

Livestock manure is widely treated by anaerobic digestion (AD) followed by composting or conventional thermal treatment (70 °C, 30 min) prior to land application, yet the community‑level fate of RNA viruses across treatment chains remains undefined considering their potential ecological and public health risks. Here, we systematically characterized RNA viromes in raw manure, AD digestate, compost, and thermally treated digestate (70-90 °C, 30 min) using metatranscriptomics and RT-qPCR. A total of 7472 viral operational taxonomic units (vOTUs) were identified, with hosts spanning plants, vertebrates, invertebrates, bacteria, and fungi. AD and composting acted as ecological filters, selecting for bacterial/fungal viruses while failing to eliminate animal‑ and plant‑associated viruses. Thermal treatment imposed a physicochemical barrier that progressively dismantled physical protection and RNA stability. Importantly, the widely used 70 °C was insufficient for the removal of high‑priority animal/plant viruses (about 53.7% selected virus abundance removal rate), whereas 80 °C achieved higher removal (78.9%-98.0% reduction of selected virus abundance), representing a temperature threshold beyond which no further improvement occurred at 90 °C. It was concluded that conventional AD and composting is insufficient for community‑level viral biosafety, and recommend 80 °C, 30 min as the optimized thermal treatment for AD digestate. This study revealed RNA virus transformation mechanisms in manure treatment chains and provided data-supported thermal parameters to guide pathogenic virus control for safe manure recycling.