Evolutionary Trajectories and Multi-lineage Circulation of H5N1 Virus in Multiple Host Interfaces from India: Insights from the Hemagglutinin Gene

Abstract

Background:  H5N1 influenza virus circulates across multiple avian and mammalian hosts, creating genetically diverse lineages at wildlife-livestock interfaces. Aims: This study assessed hemagglutinin (HA) gene diversity and evolutionary relationships among H5N1 viruses detected in non-human animal hosts in India. Methodology: A total of 140 H5N1 HA gene sequences sampled between 2006 and 2025 were retrieved from the NCBI Virus database. Sequences of at least 1,700 bp were aligned, 111 haplotypes were identified, and a median-joining network was constructed to examine genetic affinities and host-associated connections. Results: The network revealed interconnected lineages with marked host-associated structure. Poultry-associated Gallus gallus haplotypes formed the largest and most complex component, including multiple star-like patterns consistent with substantial diversification. Turkey haplotypes were closely connected to poultry haplotypes, whereas corvid haplotypes formed an extended branch. Haplotypes associated with Felis catus, Panthera tigris, and Panthera pardus were positioned near the periphery of poultry-associated lineages, indicating repeated spillover into mammalian hosts rather than a broad mammalian transmission lineage. Median vectors connected several host-associated branches and represented potential unsampled or intermediate variants. Conclusion: The HA network showed considerable genetic diversity and complex host relationships across poultry, wild birds, and mammals. These findings support integrated genomic surveillance across wildlife-livestock interfaces to improve tracking of lineage emergence and cross-species transmission.