Co-detection and genomic characterization of avian rotavirus A, avian orthoreovirus, and chicken megrivirus-C using nontargeted metagenomic surveillance in Indian broiler chickens.

Nontargeted metagenomic surveillance of the poultry enteric virome reveals underrecognized threats to poultry health and productivity in intensive production systems. In South Asia, avian rotavirus A (AvRV-A) and avian orthoreovirus (ARV) are frequently detected in broilers by conventional diagnostics, whereas chicken megrivirus genotype C (ChMeV-C) is often identified through metagenomic surveillance. Often present in both clinical disease and coinfections, these viruses may impair gut function, immune responses, and growth performance, yet their genomic diversity and evolutionary dynamics in poultry remain poorly characterized. Here, we report complete genomes of AvRV-A, ARV, and ChMeV-C strains co-detected via nontargeted metagenomic next-generation sequencing (ntNGS) in a pooled cloacal sample comprising 150 commercial broiler chickens (19 and 33 days old) collected from three commercial farms in Kamrup Rural District, Assam, Northeast India. Despite routine vaccination, all three flocks experienced > 10% mortality, poor weight gain, and postmortem lesions including pale kidneys and hepatomegaly. Phylogenetic analyses revealed segmental clustering in ARV and AvRV-A consistent with reassortment-driven divergence, though not supported by detectable recombination, while ChMeV-C clustered within a distinct C1 sublineage, suggesting intercontinental lineage connectivity and highlighting the need to expand regional genomic baseline data. We also identified nonsynonymous single nucleotide polymorphisms in several key viral proteins, including RNA-dependent RNA polymerases (VP1 of AvRV-A, λB of ARV, and 3D of ChMeV-C), capsid proteins (VP2 and VP7 of AvRV-A, λA and σB of ARV, and VP0 and VP1 of ChMeV-C), and replication-associated nonstructural proteins. These findings expand the genomic baseline for poultry enteric viruses in South Asia, reveal novel polymorphic signatures, and underscore the value of ntNGS-based metagenomic surveillance in virus detection, diversity monitoring, and informing vaccine and biosecurity strategies.