Viral dark matter and cross-species connectivity: divergent host–virus sharing architectures at the giant panda–livestock interface

The expansion of anthropogenic activities into natural ecosystems has intensified contact at wildlife–livestock interfaces, creating opportunities for viral exchange among wildlife, domestic animals, and humans. Although such interfaces are increasingly recognized as hotspots for emerging infectious diseases, viral diversity and cross-host viral connectivity involving threatened species remain poorly understood. In particular, little is known about how domestic animals, sympatric wildlife, and protected species jointly shape viral exposure risk in biodiversity hotspots. To address this gap, we conducted a metagenomic investigation of the virome associated with the vulnerable giant panda (Ailuropoda melanoleuca ), sympatric wildlife, and domestic animals in Southwestern China. We identified 661,837 DNA vOTUs and 176,031 RNA vOTUs, revealing extensive viral dark matter that was particularly pronounced in the RNA virome, where 95.93% of vOTUs remained taxonomically unclassified. Comparative analyses revealed contrasting host–virus sharing patterns between DNA and RNA viruses. The DNA virome was more strongly host-associated and dominated by ubiquitous environmental viral families, such as Genomoviridae, whereas the classified RNA viral fraction showed broader cross-host sharing across domestic animals, sympatric wildlife, and giant pandas. Additional PERMANOVA analyses further showed that broad ecological status was significantly associated with both DNA and RNA virome composition. Several vertebrate-associated viral lineages, including members of Retroviridae and Picornaviridae, were detected in giant panda fecal viromes, providing metagenomic evidence of viral exposure at the wildlife–livestock interface. Network topology analyses supported a network-inferred “Source–Bridge–Sink” sharing hypothesis, in which domestic animals represented major viral-sharing hubs, sympatric wild boars and cervids occupied bridge-like positions, and giant pandas were connected to viral lineages shared with both domestic animals and sympatric wildlife. Our findings reveal extensive viral dark matter and cross-host viral connectivity at the giant panda–livestock interface. Rather than demonstrating confirmed directional transmission, the observed host–virus networks identify non-directional sharing patterns and generate testable hypotheses about potential exposure pathways. The detection of domestic-animal-associated viral signals in giant panda fecal viromes highlights the need for ecosystem-level biosecurity strategies, including livestock-free buffer zones, management of free-ranging companion animals, and longitudinal One Health surveillance, to mitigate viral exposure risks and strengthen conservation of threatened wildlife. Video Abstract