Viromic Koch's postulates resolve iron prawn syndrome etiology.

UNLABELLED: Koch's postulates remain unachievable for most crustacean viruses due to uncultivable pathogens and chronic infection dynamics. We propose viromic Koch's postulates, a culture-independent framework, to address this limitation. The modified postulates uncover comprehensive information about viruses throughout the chain from clinical samples to viral preparations to challenge trials by combining the virome from metatranscriptomic, proteomic, or molecular analyses with diagnoses from clinical or pathological observations. Applying this approach to iron prawn syndrome (IPS), a chronic disease that causes stunted growth in Macrobrachium rosenbergii, we identified infectious precocity virus (IPV) as the exclusive viral correlate through metatranscriptomic analysis. IPV showed 100% prevalence in symptomatic prawns, but was absent in controls. Viral preparations from diseased tissue reproduced IPS pathology in healthy prawns upon inoculation, with IPV replication kinetics correlating to growth retardation. Histopathology and in situ hybridization localized IPV to neuroendocrine tissues, confirming tropism and replication sites. Our findings definitively establish IPV as the causative agent of IPS. Furthermore, this study demonstrates how systematizing existing culture-independent verification steps-referred to here as viromic Koch's postulates-can serve as a practical, standardized operational roadmap for pathogen verification in uncultivable systems, facilitating the investigation of viral diseases in aquaculture.

IMPORTANCE: Koch's postulates remain difficult to apply to most crustacean and other uncultivable viruses because pathogen isolation and cell culture are often not feasible. To address this limitation, we propose "viromic Koch's postulates," a culture-independent framework that integrates whole-virome background exclusion, comprehensive inoculum characterization, and in vivo spatiotemporal validation of viral replication. Using this approach, we establish IPV as the etiological agent of IPS in Macrobrachium rosenbergii. By linking sequence-based discovery to biological causation without relying on virus culture, this work establishes an operational roadmap for rigorous pathogen identification in crustaceans and other host systems where traditional isolation-based criteria are not feasible.