One Health Viral Metagenomics for Pandemic Preparedness: Validated mNGS Workflows for Viral Detection and Genome Recovery from Swab and Tissue Specimens

Background Metagenomic next-generation sequencing (mNGS) is an untargeted approach that enables detection of pathogens directly from samples without prior knowledge of their genetic sequences. In the context of pandemic preparedness and One Health surveillance, there is a pressing need for validated viral mNGS workflows that perform reliably across diverse hosts sample types and pre-analytical conditions. Results The study designed and evaluated two mNGS workflows, one for swabs and one for complex tissue matrices, using a reference repository of clinical and post-mortem samples. The panel comprised swabs and tissue samples positive for 19 DNA and RNA viruses (including 12 species) from nine host species and nine anatomical sites, encompassing a range of transport media, storage temperatures and processing timelines. Quality control metrics were embedded throughout nucleic acid extraction, library preparation and sequencing to monitor performance and support interpretation. Overall, 89.5% of 19 known DNA and RNA viruses were detected, including from samples with low nucleic acid concentrations (< 1 ng/µl) and variable integrity and purity. The workflows identified viral co-infections that had not been detected by prior targeted testing, as well as Phocid herpesvirus 7 (PHV7) for which no complete reference genome was initially available. Conclusions These results demonstrate that the validated swab and tissue mNGS workflows are sufficiently robust and sensitive for deployment in investigations of suspected viral disease of unknown aetiology and for early detection of emerging viral threats at the animal–human interface.