Species-specific barriers restrict virus spillover potential across the Caenorhabditis genus.

Host range expansion and virus emergence depend on whether viral life-cycle processes align with the ecological, developmental and physiological traits of new hosts. Spillover therefore succeeds only when pathogens clear a hierarchy of mechanistic barriers, from entry and replication to transmission and evolutionary persistence, but how these barriers map onto whole-organism host competence remains poorly resolved. Here we dissect spillover barriers experimentally using the Caenorhabditis-Orsay virus system, integrating within-host viral kinetics, cellular progression, transmission, virulence and experimental evolution across six closely related host species. We show that host species identity deterministically reshapes the timing and completeness of the viral life cycle, generating distinct host-competence phenotypes that range from permissive to restrictive and evolutionary dead-end hosts. Alternative hosts disrupt viral life-cycle synchrony through delayed replication, imbalanced genomic segment production, impaired egress or truncated infection windows, reducing transmission despite occasional high viral loads. These mechanistic mismatches prevent sustained viral adaptation upon serial passage, revealing how multiple partially permeable barriers compound to block emergence. By resolving spillover barriers across biological scales, our results provide a mechanistic framework linking viral life-history traits to eco-evolutionary theory of host range, and show how temporal and stoichiometric mismatches can determine whether cross-species infections become epidemiologically and evolutionarily viable.