Comparative Analysis of Viral Communities in Hospital, University and Urban Wastewater by Shotgun Metagenomic Sequencing.

Wastewater-based surveillance has emerged as a powerful approach for population-level monitoring of pathogen circulation in a timely and non-invasive manner. In this study, shotgun metagenomic sequencing was applied to wastewater samples collected from a hospital (HP), a university campus (UN), and a wastewater treatment plant (WTP). Viral sequences were taxonomically classified using Kraken2. Specifically, HP samples showed the highest viral richness, followed by WTP and UN samples (HP vs. UN, p = 0.0003; WTP vs. UN, p = 0.0018). Using Jaccard distance, significant differences were observed between WTP and UN (R2 = 0.181, p < 0.001), WTP and HP (R2 = 0.159, p < 0.001), and UN and HP (R2 = 0.223, p < 0.001), and similarly, for Sørensen-Dice dissimilarity: WTP vs. UN (R2 = 0.238, p < 0.001), WTP vs. HP (R2 = 0.212, p < 0.001), and UN vs. HP (R2 = 0.307, p < 0.001). Human-associated viral families were detected across all sources, predominantly Poxviridae, Orthoherpesviridae, Polyomaviridae and Circoviridae. Furthermore, the taxonomic composition of indirectly associated viruses, mainly Anelloviridae and Crassvirales, was examined. Overall, these findings support the potential of wastewater metagenomics as a reliable tool for monitoring viral diversity within environmental and public health contexts, although further research is needed to establish its operational utility for routine surveillance applications within a One Health framework.