MOLECULAR STUDY OF CIRCULATING CLASS II NEWCASTLE DISEASE VIRUS IN VACCINATED BROILER BIRDS IN RAWALPINDI DIVISION, PAKISTAN

Newcastle disease (ND), caused by Avian paramyxovirus-1 (APMV-1), continues to pose significant challenges to the poultry industry, despite widespread vaccination efforts. Outbreaks in vaccinated flocks highlight the circulation of virulent strains and genetic diversity. This study aimed to isolate and molecularly characterize local NDV isolates from field outbreaks. A total of 216 samples were collected from 72 poultry farms in the Rawalpindi region, including Rawalpindi, Attock, Chakwal, and Jhelum. The virus was cultivated in embryonated chicken eggs, and Haemagglutination Test (HA) was used for confirmation. Virulence was assessed through the mean death time (MDT) assay which use to measure the time required to kill embryo within 60hours of inoculation, it identified 54 isolates as velogenic strains. RT-PCR targeting the Fusion gene (F) confirmed 54 isolates as NDV Class II. Phylogenetic analysis revealed that the isolates were of Class II genotype NDV VII.2 (f), showing 95.4%-100% identity with virulent reference strains. Prevalence data showed Rawalpindi had the highest infection rate (80%), followed by Jhelum (71%), Attock (56%), and Chakwal (51%), with significant differences between districts (P = 0.0071). Seasonal distribution revealed the highest prevalence in summer, with Jhelum at 52.83%, followed by Chakwal (50%), Attock (42.86%), and Rawalpindi (41.54%), while winter prevalence was moderate. Sequence analysis confirmed that the NDV isolates were velogenic genotype VII.2 strains. The genetic variation between field strains and commercial vaccines likely contributes to vaccine failures, stressing the need for genotype-matched vaccines in Pakistan. This study highlights the importance of adopting locally derived or genotypically matched vaccines to improve efficacy of vaccine. The extensive genetic diversity of Newcastle Disease Virus (NDV), characterized by the emergence of distinct genotypes, poses significant challenges to effective disease control and underscores the need for continuous monitoring of circulating strains. Developing genotype-matched vaccines presents a promising strategy to enhance efficacy by aligning antigenic profiles with regionally prevalent viral genotypes, thereby reducing the risk of vaccine failure. Keywords: Newcastle disease virus, Rawalpindi, PCR, Phylogenetic analysis, F gene, genotype VII.