Pathotyping and Genetic Characterization of a Highly Virulent Newcastle Disease Virus Strain Isolated from Recent Outbreak in District Naushahro Froze, Sindh, Pakistan

In Pakistan, growth of poultry industry is confronted by many viral diseases and Newcastle Disease (ND) is on top. The present study was conducted for pathotyping and genetic characterization of NDV isolated from an outbreak in District Naushahro Froze region of Sindh, Pakistan. Post mortem examination of fresh carcasses (n=10) was performed and various organs like trachea, spleen, lung tissues, and cloacal contents were collected from birds showing typical lesions of the disease aseptically. Tissue homogenate was inoculated in 9 days old embryonated chicken eggs and allantoic fluid was harvested. The presence of virulent virus was checked by hemagglutination, hemagglutination inhibition using hyperimmune serum and ICPI assays. NDV was purified using sucrose gradient ultracentrifugation and confirmation was done by F-gene amplification, SDS-PAGE and mass spectrometry of fusion protein. The HA titer was 1:256, and hemagglutination inhibition was achieved using Anti NDV serum. A band of ≈ 800 bp of F gene (Acc.no Naushahro Froze /Pakistan/MH607122) was amplified which revealed 99% similarity with Sindh Agriculture university/Pak/2015/MF437287, Sindh Agriculture university /Pak/2016/KX791187 and Tehran/Iran/MG871466 strains. SDS-PAGE yielded five bands including Fusion protein with the estimated mass of 58896.0 Da. The presence of fusion protein and ICPI value was 2 which confirmed virulence of isolated strain resulting in high mortality. Thus, it is the need of the hour to characterize the prevalent indigenous strains of NDV to develop some novel vaccine strategies for effective control.