From sewage to genomes: expanding our understanding of the urban and semi- urban municipal wastewater RNA virosphere.

The study determines the RNA viral communities present in municipal wastewater to define ecological and genomic properties in the built environment. MW is composed of household and industrial sewage; it acts as a hot spot for viral biodiversity, carrying microbial, plant, and animal viruses that have been vastly unexploited. The most interesting feature includes RNA viruses, which impose risks in public health and industry, are mostly unknown for their role in ecology in wastewater.Using influent and effluent RNA viral metagenomes from three MW catchment areas, serving 7.05 million equivalent inhabitants, we identified 14,212 RNA virus sequences from the Orthornavirae kingdom encoding RdRp. These sequences clustered into 4,989 viral species. Leveraging extensive viral databases, we were able to classify 90% of the dataset to the family level, dominated by the families Virgaviridae, Picornaviridae, Dicistroviridae, and Reoviridae.We observed that the wastewater treatment process reduced viral species richness and evenness (Shannon diversity) but maintained phylogenetic diversity. Variation in viral community composition between influents and effluents was predominantly explained by catchment area and treatment process, which explained 54% of dissimilarities (Weighted UniFrac). Biomarker analysis identified families Astroviridae, Picobirnaviridae, and Fiersviridae as enriched in influents, while effluents had higher relative abundances of families such as Virgaviridae, Reoviridae, and Marnaviridae.Our results emphasize that wastewater treatment reduces overall viral diversity but maintains phylogenetic diversity, including taxa with potential risks to human health (e.g., Reoviridae) and crops (e.g., Virgaviridae). Besides, the high abundance of bacteriophage (Leviviricetes) and eukaryotic algal viruses (Picornaviridae) points out their ecological role in MW.