Molecular characterization of nonvirulent strains of Newcastle Disease Virus and assessing their thermostability

Abstract

Newcastle disease (ND) is a big concern throughout the world. It causes devastating losses in commercial and backyard poultry. The major problem in many countries is the loss of the vaccine's effectiveness due to inadequate use or storage conditions, particularly in hot climates. Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against ND, due to their low dependence on cold chain for transport and storage. This study was therefore designed to isolate and characterize nonvirulent NDV strains from backyard chickens and then to select candidate thermostable strain by testing the strains in a series of different temperatures for a certain time period. 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 June 2022. To observe virus growth, the samples were inoculated in 9-10 days old embryonated chicken eggs through allantoic sac route. Allantoic fluids were harvested on day 4 and tested for hemagglutination (HA) 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 were found RT-PCR positive. Analysis of partial F gene sequence of RT-PCR positive samples revealed that six isolates were nonvirulent with amino acid motif 112GRQGRL117 at the fusion protein cleavage site. In the present study, thermostability of the six nonvirulent NDV strains were tested comparatively at different incubation temperatures (25℃, 37℃ and 56℃) with the use of SPF (specific pathogen free) embryonated chicken eggs. Thermostability of HA titer level was stable up to 37°C for six isolates and was reduced to zero HA titer level at 56℃. Characterization of the complete genome of these thermostable nonvirulent NDV strains may facilitate in selection of a suitable candidate for developing a thermostable ND vaccine.