Molecular Characterization and Phylogenetic Analysis of Nonvirulent Newcastle Disease Virus Strains Isolated from Backyard Chickens

Abstract

Newcastle disease (ND), popularly known as Ranikhet disease, is one of the most fatal and economically important viral diseases of backyard chickens in Bangladesh. Despite vaccination ND is endemic among indigenous chickens indicating potential failure of current vaccines. Among other causes of vaccine failure, one likely reason could be the use of thermolabile vaccine in rural areas where maintenance of cold chain is not always possible. In this context, development of a thermotolerant ND vaccine is utmost important for backyard chickens of Bangladesh to overcome losses incurred by ND. This study was therefore designed to isolate and characterize nonvirulent NDV strains from backyard chickens of Bangladesh. Cloacal and oropharyngeal swab samples (n=1282) were collected in VTM (viral transport medium) from backyard chickens of different regions during the period of July 2021 to January 2022. To observe virus growth, the samples were inoculated in 9-10 day old embryonated chicken eggs through allantoic sac route. Allantoic fluids were harvested on day 4 and tested for hemagglutination (HA) using 1% chicken RBC to see the presence of hemagglutinating virus in them. Initial ND virus identification was performed by hemagglutination inhibition (HI) test using chicken hyperimmune sera against NDV. Molecular characterization of HI positive samples was then performed by RT-PCR targeting the partial amplification of fusion protein (F) gene of NDV. Out of 55 samples, 16 (29.09%; 95% CI 17.6% - 42.9%) were found RT-PCR positive. Analysis of partial F gene sequence of RT-PCR positive samples revealed that four isolates were nonvirulent with amino acid motif 112GRQGRL117 in three isolates and motif 112GRQLRL117 in one isolate at the fusion protein cleavage site. Phylogenetic analysis disclosed the sequence homology of one isolate with those of vaccine strains reported from and/or used in Bangladesh, however other three sequences showed no resemblance with sequences used in the analysis. Isolated nonvirulent NDV strains can be used for the development of thermotolerant ND vaccine once standard criteria are fulfilled.