Evaluation of genotype matched recombinant DNA vaccine for protection against genotype VII velogenic Newcastle disease virus in Pakistan

Abstract Newcastle disease virus (NDV) remains a major threat to the poultry industry worldwide. Recombinant DNA vaccine against NDV offers a promising solution to current Newcastle disease (ND) challenges. Present study describes the development of a DNA vaccine (rDNA-NDV-F) using the fusion (F) gene from NDV genotype VII strain isolated from Rawalpindi, Pakistan. While conventional NDV vaccines reduce mortality in commercial poultry, they do not provide complete protection or prevent viral shedding. To address this issue, genotype-matched vaccines have been proposed. Here, we developed and evaluated the efficacy of the rDNA-NDV-F vaccine against genotype VII challenge. NDV was isolated from a field strain and propagated in embryonated chicken eggs (ECE). Virus activity was confirmed using Hemagglutination assay (HA), HA inhibition (HAI), and Mean Death Time (MDT) assay. Polymerase Chain Reaction (PCR) and sequencing confirmed the genotype VII.2 strain. The DNA vaccine was constructed using the fusion (F) protein gene cloned into the expression plasmid pcDNA3.1. Gene insertion was verified by PCR and restriction digestion, while protein expression was confirmed via immunofluorescence assay. To assess vaccine efficacy, 120 chickens (14 days old) were divided into four groups: G1 (rDNA-NDV-F), G2 (empty vector), G3 (PBS control), and G4 (non-vaccinated, non-challenged control). Serological responses were measured using ELISA on days 0, 7, 14, 21, and 28. Birds were challenged with NDV genotype VII (105 EID50). Virus shedding from tracheal and cloacal swabs was analyzed on days 3, 7, and 10 post-challenge. Clinical signs and mortality rates were also recorded. The rDNA-NDV-F vaccine induced strong immune responses, with peak ELISA (6180) titers at 28 days. Virus shedding was detected in three birds on day 3 but was absent by day 10. No virus shedding was observed in cloacal swabs, indicating restriction in the digestive system. Vaccinated birds showed mild clinical signs in only two cases, with no neurological symptoms or mortality. In contrast, negative and vector control groups exhibited severe clinical signs and 90–100% mortality. Statistical analysis confirmed significant differences (P < 0.05). This study highlights the effectiveness of genotype-matched recombinant NDV vaccines in providing effective protection for poultry.