Lesion-anchored viromics identifies candidate viral agents of broiler stunting diseases

Background Broiler stunting diseases, including malabsorption-associated runting-stunting syndrome (RSS) and maldigestion-associated transmissible viral proventriculitis (TVP), cause significant economic losses through growth failure and increased mortality. Viral infections have been proposed as contributors, yet the aetiological roles of specific viruses remain obscure because conventional virome studies lack spatial resolution and cannot distinguish which viruses localise to lesion sites versus unaffected tissues. This limits inference regarding direct viral involvement in pathogenesis. Methods We developed a spatially informed viromic approach by coupling laser-capture microdissection of formalin-fixed paraffin-embedded tissues (duodenum, jejunum, proventriculus and gizzard) with metatranscriptomic sequencing, enabling viral communities within histological lesions to be compared with those in unaffected non-lesion regions of the same birds and flocks. Candidate viruses identified by sequencing were then verified in matched fresh-frozen tissues from the same birds by RT-PCR, RT-qPCR, amplicon sequencing, and virus isolation. Results In RSS-derived laser-microdissected samples ( n = 71), three enteric viruses were consistently enriched within gastrointestinal lesion tissues relative to non-lesion tissues: the species Gallivirus spp. (multiple strains), Avastrovirus galli , and Rotavirus deltagastroenteritidis. These viruses were detected across multiple affected farms, with co-occurrence in individual lesion samples suggesting potential co-infections. Comparative analysis with TVP-derived laser-microdissected samples ( n = 49) revealed distinct enteric virome compositions between the two syndromes, with differential enrichment of vaccine-like strains in TVP, confirming that this spatially informed approach can discriminate disease-specific viral signatures. All three RSS candidates were independently confirmed in matched fresh-frozen tissue homogenates from the same birds, and chicken astrovirus was recovered in cell culture. Conclusions By anchoring virome composition directly to lesion histopathology, we identified candidate viral agents associated with RSS pathogenesis and demonstrated that spatially informed viromics moves beyond conventional virome surveys by linking viral abundance patterns to histopathological context. This framework provides a foundation for investigating whether these candidate viruses function as primary pathogens or co-factors in stunting disease development.