Towards a Comprehensive Wastewater Virome Atlas for Pathogen Monitoring

Background Wastewater represents a complex unstructured mixture of biological material contributed by humans, animals, and other organisms, making it a valuable resource for pathogen surveillance. Capturing the virome using a probe-based approach enables a broader understanding of viral community distribution, allows for the detection of multiple viral strains, and facilitates the identification of viral nucleic acids present in very low amounts in wastewater samples. This study presents a comprehensive virome analysis conducted over one year across four metropolitan cities, with samples collected fortnightly from 24 different sites. Results We identified over 170 DNA and 135 RNA viral species, including viral strains associated with multiple hosts humans, animals, plants, and insects. 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. Conclusions This large-scale, longitudinal study demonstrates that NGS-based wastewater virome profiling is a reliable and scalable method for detecting both circulating and sporadic viral strains. The ability to detect low-abundance viral genomes supports early variant identification and offers insights into viral prevalence in the community. The findings offer valuable insights for early public health interventions.