Longitudinal wastewater virome surveillance unveils untapped circulating viruses in the community

Abstract Wastewater represents a complex, unstructured mixture of biological material contributed by humans, animals, and other organisms, making it a valuable resource for pathogen surveillance. This study presents a comprehensive capture-probe-based virome analysis conducted over 1 year across four metropolitan cities, with samples collected fortnightly from 24 different sites. We identified over 170 DNA and 135 RNA viral species, including viral strains associated with multiple hosts. Notably, we detected 107 DNA viruses and 495 RNA viral genomes/segments with over 90% genome coverage. Beyond detection, we explored temporal patterns, revealing that viral families such as Astroviridae, Orthomyxoviridae, and Picornaviridae exhibited seasonal trends, while Adenoviridae and Sedoreoviridae were consistently detected throughout the year. Shannon diversity was higher and more variable at several sites in Ahmedabad, likely due to its larger population size, landscape, and heavy footfall. Bray-Curtis dissimilarity analysis showed significant variation in viral communities across cities, sites, and sampling time points. The prevalence of SARS-CoV-2 and Rotavirus A from NGS showed a significant positive correlation with digital PCR (dPCR) quantification ( p < 0.05). Additionally, trends in Hepatitis A virus abundance aligned with monthly reported cases, further validating the reliability of sequencing-based surveillance.