Molecular and Phylogenetic Evidence of Endemic Genotype VII Newcastle Disease Virus Circulation and Vaccine Mismatch in Punjab, Pakistan

Abstract

Newcastle disease remains a major constraint on poultry production worldwide, with recurrent outbreaks reported even in vaccinated flocks. In Pakistan, the persistence of Newcastle disease raises concerns about the ongoing evolution of Newcastle disease virus and the effectiveness of existing vaccines. This study aimed to investigate the molecular epidemiology and genetic characteristics of Newcastle disease virus circulating in commercial poultry farms in Punjab Province, Pakistan. A cross-sectional molecular surveillance study was conducted from May to November 2025 across five major poultry-producing districts. Clinical samples consisting of pooled oropharyngeal swabs and tissue homogenates were collected from affected flocks and organized into 100 pooled samples obtained from 25 commercial farms experiencing severe disease characterized by respiratory and neurological signs with high mortality (>40%), despite routine vaccination, and clinically suspected of Newcastle disease. Reverse transcription–polymerase chain reaction targeting the fusion (F) gene detected Newcastle disease virus in 91.1% of the prioritized tissue homogenates and oropharyngeal swabs from the sampled flocks. Based on RNA quality, successful amplification of the target region, geographic distribution, and epidemiological relevance, four representative reverse transcription–polymerase chain reaction-positive clinical samples were selected for complete F-gene sequencing. All sequenced samples possessed the polybasic cleavage site motif 112RRQKR↓F117, indicating a molecular signature consistent with a velogenic pathotype. Phylogenetic analysis consistently clustered the Newcastle disease virus sequences within Class II, further divided into sub-genotypes VII.2 and VII.1.1, indicating the co-circulation of at least two endemic lineages. Comparative sequence analysis revealed substantial genetic divergence between field samples and commonly used vaccine strains, with an average nucleotide difference of 10.7% relative to the LaSota vaccine strain. Additionally, several amino acid substitutions were identified in known neutralizing epitopes, along with evidence of positive selection at antigenically significant sites. These findings demonstrate the endemic circulation of genetically diverse genotype VII Newcastle disease virus strains in Punjab and provide molecular insights into potential antigenic divergence between vaccine and field strains, highlighting the need for further functional efficacy studies. Continuous molecular surveillance and the reassessment of vaccination strategies are essential for achieving effective and sustainable control of Newcastle disease in Pakistan. Cite this article as: Naveed, M. T., Gulzar, M. W., Ahmad, M. M., Azeem, M. W., Ali, M. A., Anwar, M. N., Riaz, S., Shah, M. S., & Habib, M. (2026). Molecular and phylogenetic evidence of endemic genotype VII Newcastle disease virus circulation and vaccine mismatch in Punjab, Pakistan. Acta Veterinaria Eurasia, 52, 0072, doi: 10.5152/actavet.2026.26072.