Whole genome sequence analysis and characterization of virulent Newcastle disease virus isolates from chicken and pheasants during 2020/21 outbreaks in Nepal

Newcastle Disease (ND) remains a major threat to poultry production worldwide, particularly in regions where it is endemic, like Southern Asia. The disease is caused by virulent forms of avian paramyxovirus-1, commonly termed Newcastle Disease Virus (NDV), a highly contagious virus with significant genetic diversity and evolving pathogenicity. This study aimed to molecularly characterize NDV isolates obtained from chickens and pheasants during the 2020/21 ND outbreaks in Nepal, to understand their genetic makeup, phylogenetic relationships, and implications for control strategies. Necropsy samples, including trachea, liver, intestine, spleen, lungs, heart, and proventriculus were collected from ten birds. Isolates from five clinical samples were typed as NDV by hemagglutination and hemagglutination inhibition (HA/HI) assays and were subjected to whole genome sequencing (WGS). Full genomes of 15,192 nucleotides were recovered from each isolate. Fusion (F) gene sequence analysis revealed the presence of multi-basic cleavage site motif ¹¹²RRQKRF¹¹⁷ in all isolates, indicative of virulent strain and suggesting a potentially velogenic or mesogenic phenotype. Phylogenetic analyses consistently classified all isolates within genotype VII.2 of class II NDV. Further comparative analysis indicated a close genetic relationship between the Nepalese isolates and strains reported from India and Bangladesh, and BEAST analysis suggested Southern Asia as the likely source of introduction into Nepal. These viral genomes provide additional insight into contemporary NDV circulating in an area of endemicity.