Metatranscriptomics reveals urbanization-driven divergence in rodent viromes and zoonotic risks in Chinese megacities.

Metagenomic sequencing has advanced our understanding of wildlife-associated viruses and enabled identification of potential zoonotic pathogens. However, most studies remain geographically limited, with few systematic comparisons of virome compositions across urbanization gradients. To address this gap, we conducted large-scale sampling in two densely populated Chinese megacities-Wuhan and Shenzhen-with distinct climates. We collected 1,072 rodents from four species that frequently interact with humans, enabling comparative analysis of urban rectal virome dynamics and zoonotic risks in rodents. We identified 35 vertebrate-associated viruses. Among them, 9 were potentially novel species, including Norovirus and Orthopicobirnavirus species, and 3 had zoonotic potential, namely Orthohantavirus seoulense and human coronavirus OC43 (HCoV_OC43). We also discovered two viruses previously unreported in rodents, Erinaceus hedgehog Seoul orthohantavirus and Canine astrovirus, which revealed cross-order transmission risk. Additionally, 22 high-risk viruses were identified, with Wuhan and Shenzhen showing distinct prevalence patterns. Our analysis shows that inter-city rectal virome divergence is structured by meteorological variables, independently explaining 5.0% of the variation in viral community composition. Our findings highlight the importance of spatial distance in shaping the distribution and transmission of rodent-borne viruses. These insights are essential for proactive surveillance and mitigation of emerging zoonotic threats in high-density urban environments.