Tracking respiratory, enteric and vector-borne viruses through wastewater: advances from one health genomic surveillance

Highlights: • Wastewater-based surveillance supports multi-pathogen monitoring • Genomic tools enhance early detection of emerging viral variants • Environmental genomics contributes to public health surveillance • Integrated One Health approach supports coordinated viral surveillance Wastewater-Based Epidemiology (WBE) has emerged as a powerful, non-invasive tool providing population-level information on viral circulation and genetic diversity. Within Work Package 4.1 – Innovative models of One Health genomic surveillance, Task 4 – Environmental surveillance through waste and superficial waters focused on the development and application of molecular and genomic approaches for the detection of respiratory, enteric, and vector-borne viruses in wastewater, contributing to the design of integrated One Health genomic surveillance models. Respiratory viruses Activities focused on the implementation and evaluation of molecular tools for variant tracking and multi-pathogen detection in wastewater. – SARS-CoV-2: a digital PCR assay for Omicron lineage tracking showed the rapid rise of BA.2.86 from 2 % (Oct 2023) to 62 % (Jan 2024) in Italian wastewater. – Influenza A and B: a CDC-based qPCR assay adapted to dPCR revealed a clear seasonal peak (Dec–Feb), consistent with clinical surveillance data provided in collaboration with Task 1. – Avian influenza virus: in collaboration with Task 5, a veterinary qPCR assay was validated for wastewater monitoring in the Veneto region; no Eurasian H5 subtype was detected during the study period. – Multi-pathogen approach: the QIAseq xHYB Respiratory Panel identified 22 respiratory viruses among 89 targets, confirming WBE’s potential for broad-spectrum surveillance. Enteric viruses Routine WBE activities were strengthened through advanced molecular tools. – Human adenovirus (2024–2025): detected in > 90 % of samples through digital PCR combined with Sanger and Nanopore sequencing, revealing predominant genotypes A12, B3 and F41. – Rotavirus A (2024–2025): detected in 97.5 % of samples; genotyping showed G3 as dominant, with sporadic G1, G2 and G9 strains. – Norovirus (2017–2024): long-term monitoring revealed a rich diversity of 8 GI and 17 GII genotypes, including persistent (GII.2, GII.4) and emerging genotypes (GII.8, GII.10). – Hepatitis E virus: complete genome (subtype 4d) was obtained through multiplex PCR and hybrid sequencing, representing the first environmental detection in Central Italy and contributing to the genomic characterization of circulating strains. Vector-borne viruses A digital RT-PCR protocol for Dengue virus serotype 2 was developed and applied during the 2024 Marche outbreak, in collaboration with Task 3. This pioneering study demonstrated, for the first time in Italy, the feasibility of wastewater detection of arboviruses during an active epidemic. Across all virus groups, the work established harmonized and validated molecular workflows, providing a framework for high-resolution genomic data generation and cross-sector integration. Collaboration with Tasks 1, 3 and 5 enabled bidirectional data exchange between environmental, human and animal domains, advancing the objective of building interoperable One Health genomic surveillance models. These results confirm that wastewater-based surveillance can serve as a scalable, multi-pathogen platform supporting early epidemic warning, variant tracking and preparedness for emerging infectious threats. The forthcoming implementation of the revised Urban Wastewater Treatment Directive, which mandates wastewater monitoring for pathogens and antimicrobial resistance, further underscores the importance of integrating environmental, clinical and veterinary data streams within national surveillance systems to strengthen the One Health genomic framework.