Spatial heterogeneity of viral communities across the gastrointestinal tracts of ruminants

• Obtained the Ruminant Gastrointestinal Virome Catalogue (RGVC), including 43,981 vOTUs, revealing the unique and expansive nature of viral genomes within the ruminant gastrointestinal tracts. • Gastrointestinal regions, rather than ruminant species, are the primary drivers of viral community diversity in ruminants. • A strong correlation between prokaryotic hosts and host-linked viruses among gastrointestinal regions. • The prevalence of lysogenic lifestyles can contribute to the observed linkage between viruses and prokaryotic hosts. • Lysogenic viruses can enhance host metabolism by providing diverse auxiliary metabolic genes. Viruses are abundant biological entities within the gastrointestinal tract (GIT) of ruminants. Current understanding is extensive for bacterial and archaeal communities, but limited for viral communities. The study aimed to investigate viral diversity, virus-host interactions and ecological functions of viruses across GIT regions and ruminant species. We collected 373 short-read and long-read metagenomes from 10 GIT regions of seven ruminant species, combining Illumina, PacBio HiFi, and Nanopore sequencing. Viral contigs were identified using sequence homology, viral hallmark gene and machine learning, and employed to uncover community assembly of spatial heterogeneity by analyzing virus-host linkage, lifestyle, and auxiliary metabolic genes (AMGs). We constructed a Ruminant Gastrointestinal Virome Catalog (RGVC) comprising 43,981 vOTUs, revealing that viral communities were remarkably diverse and mainly driven by the GIT regions rather than by the ruminant species. Virus-host linkage analysis identified 4603 putative prokaryotic hosts across 34 classes for 5954 host-linked viruses, along with robust correlation ( R 2 = 0.91) observed between abundances of prokaryotic hosts and host-linked viruses across GIT regions. The lysogenic lifestyle was a dominant feature, with integrases being the predominant lysogenic-specific genes. We identified 864 high-confidence AMGs in lysogenic viruses that are annotated as key genes for polysaccharide degradation, glycolysis, and the Wood-Ljungdahl pathway, indicating a putative role for the viruses in supporting these host metabolic functions. The metabolic features of host-linked viruses were further verified by genomic context of selected AMGs of GH10, GPI and FHS with target function. These findings suggest that the GIT viral communities exhibit spatial heterogeneity with distinct virus-host interactions, and offer new perspectives on maintenance of complex ecological and nutritional functions in ruminant GIT.