Molecular Characterization and Genotyping of Newcastle Disease Viruses Using Whole- Genome Sequencing from Poultry Farms in Parts of Amhara Regions, and Central Ethiopia

Background Newcastle disease (ND) is a major viral infection of poultry caused by virulent strains of avian paramyxovirus type 1, resulting in considerable global economic losses. In Ethiopia, ND outbreaks persist despite vaccination efforts, and the genetic diversity of circulating Newcastle disease virus (NDV) strains remains poorly characterized. Most previous studies have relied on partial genomic data, leaving a gap in whole-genome analysis. This study aimed to provide a comprehensive genomic investigation of NDV in Ethiopia. Methodology A cross-sectional study was conducted on 14 commercial chicken farms across six districts in central Ethiopia and parts of the Amhara region. A total of 63 pooled specimens were screened using real-time qRT-PCR. Virus isolation was performed in specific-pathogen-free embryonated chicken eggs and confirmed by qRT-PCR. For molecular characterization, the sequence-independent, single-primer amplification (SISPA) technique was applied to obtain whole-genome sequences from five NDV isolates via Oxford Nanopore sequencing Technology. Result Of the 63 pooled specimens, 11 (13.8%) tested positive for NDV. Whole-genome sequencing revealed two genome sizes (15,192 and 15,186 nucleotides) and confirmed virulent NDV strains with the characteristic F-gene cleavage site motif ( ¹¹² RRQKRF ¹¹⁷ ), distinct from local vaccine strains ( ¹¹² GRQGR↓L ¹¹⁷ ). Phylogenetic analysis showed that all isolates clustered within genotype VII, with four strains (GenBank: PV189285–PV189288) exhibiting 97.24% similarity to Iranian NDV strains (accession number: ON184061) of sub-genotype VII.1.1. Conclusion This study provides the first whole-genome insight into NDV strains circulating in Ethiopia’s poultry industry. The detection of virulent NDV in vaccinated flocks highlights the need for vaccines that are genetically and antigenically matched to circulating strains. Broader genomic surveillance across production systems is essential to guide effective ND control strategies and reduce economic losses.