Isolation and Molecular Characterization of Newcastle Disease Virus in Layers

Background: Newcastle disease (ND) in spite of the availability of vaccines remains a constant threat to poultry producers worldwide. It is prevalent in Indian subcontinent and leads to economic losses. The present study was aimed with isolate and identify virulent Newcastle disease virus (NDV) in layer poultry from field outbreaks.Methods: Total 47 samples consisting of nasal (05), oropharyngeal (13) and cloacal swabs (11) and tissue samples consisting of trachea (07), lungs (06), larynx (05) were collected from layer birds. For isolation of NDV swab and tissue samples were inoculated in 9-11 days old embryonated eggs via allantoic cavity route. After preparing the viral inoculum, 47 suspected samples (29 swab and 18 tissue samples) were inoculated in 141 embryonated eggs to isolate the virus.Result: Out of 47 samples 10 (21.27%) samples were positive for HA activity. All the 10 isolates showing HA activity subjected to Reverse-Transcriptase PCR of F gene and 6 were found positive in RT-PCR for F1 gene. The PCR amplified product showed amplicon at 356 bp and 254 bp positive for F1 and F2 gene, respectively. On basis of F gene, 06 (50%) isolates were considered as virulent Newcastle Disease Virus. One isolate sequence was submitted at NCBI with accession MT890653 On phylogenetic analysis MT890653 designated as Class II/ genotype II/ virulent strain and had the motif 112R-R-R-K-R-F117 at the cleavage site of the fusion protein.