From sewage to genomes: Expanding our understanding of the urban and semi-urban wastewater RNA virome

Wastewater is a hotspot for viral diversity, harboring various microbial, plant, and animal viruses, including those that infect humans. However, the dynamics, resilience, and ecological roles of viral communities during treatment are largely unknown. In this study, we explored RNA virus ecogenomics using metagenomics from influent and effluent samples across three wastewater catchment areas in Chile, with a population of 7.05 million equivalent inhabitants. We identified 14,212 RNA-dependent RNA polymerase (RdRP)-coding sequences from the Orthornavirae kingdom, clustering into 4989 viral species. Using extensive databases of 14,150 family-level representative sequences, we classified 90 % of our sequences at the family level. Our analysis revealed that treatment reduced viral richness and evenness (Shannon index), but phylogenetic diversity remained unchanged. Effluents showed lower richness and evenness than influents with similar phylogenetic diversity. Species turnover, influenced by catchment area and treatment, accounted for 54 % of sample dissimilarities (Weighted Unifrac). Biomarker analysis indicated that families like Astroviridae and Fiersviridae were more abundant in influents, while Reoviridae and Virgaviridae dominated effluents. This suggests that viral resistance to treatment varies and cannot be solely attributed to genome type, size, or morphology. We traced viral genomes through time and space, identifying sequences like the Pepper Mild Mottle Virus (PMMoV) from the Virgaviridae family over large distances and periods, highlighting its wastewater marker potential. High concentrations of human pathogens, such as Rotavirus (Reoviridae) and Human Astrovirus (Astroviridae), were found in both influents and effluents, stressing the need for continuous monitoring, especially for treated wastewater reuse.