Metagenomic Analysis of the Fecal Virome in Wild Mammals Hospitalized in Pisa, Italy

Emerging infectious diseases, particularly those of zoonotic origin, often originating from wildlife reservoirs represent a growing threat to global health. Human-driven environmental changes such as habitat fragmentation, climate change, and urban expansion have intensified interactions at the wildlife-domestic animal-human interface, facilitating cross-species viral transmission. Despite their epidemiological importance, systematic virological surveillance of wildlife remains challenging. In this study, we employed shotgun metagenomic sequencing to characterize the virome of wild animals rescued in the Pisa area and hospitalized at the "Mario Modenato" Veterinary Teaching Hospital (VTH) at the University of Pisa. Fecal samples collected from injured wildlife admitted between September 2020 and September 2021 were analyzed to detect both known and novel viruses. This approach builds upon previous PCR-based investigations of the same biological material, enabling a more comprehensive assessment of viral diversity. We adopted a shotgun approach for analyzing six sample pools-four were positive for at least one viral target-identifying diverse viral families, including Astroviridae, Circoviridae, Picornaviridae, Adenoviridae, and Retroviridae, in asymptomatic wildlife admitted to a veterinary hospital, highlighting their potential role as reservoirs. Our findings provide insights into the influence of environmental and anthropogenic factors on wildlife virome composition and highlight the value of hospital-based sampling strategies for urban viral surveillance. The results contribute to the development of integrated monitoring and prevention strategies within a One Health framework.