Seasonal transcriptional decoupling drives microbial biosecurity risks in a large rive

Freshwater rivers are linked to human health and agricultural production and serve as natural reservoirs for human pathogens and related pathogenetic features, yet little is known about the active temporal dynamics of their distribution. Here, we integrated year-round metagenomics, metaviromics, metatranscriptomics and amplicon sequencing to resolve the seasonal dynamics and activity of bacterial and viral pathogens, antibiotic resistance genes (ARGs), virulence factors (VFs), and mobile genetic elements (MGEs) in the upper Yellow River basin. We show that microbial genomic abundance and transcriptional activity were consistently decoupled, revealing that RNA-based surveys are essential for uncovering temporal variation in active pathogen risk. We find pronounced seasonal succession in pathogens, while ARGs, VFs, and MGEs displayed asynchronous genomic and transcriptional dynamics, with greater genomic abundance in summer but elevated transcriptional activity during autumn and winter. Temperature, precipitation, hydrological variability, and viral abundance jointly explained seasonal turnover and functional activation of microbial risks, indicating that hydroclimatic conditions regulate riverine biosecurity through coordinated yet asynchronous ecological processes. Integrating complementary risk assessment frameworks demonstrated that microbial-associated risks in large rivers are primarily temporally structured and emerge from coordinated yet functionally decoupled interactions among pathogens, resistomes, and mobilomes. These findings establish transcriptional decoupling as a fundamental feature of river microbial biosecurity and demonstrate how integrated multi-omics can improve environmental surveillance and risk assessment under the One Health framework.