Rapid pathotyping of Newcastle Disease Virus by pyrosequencing

Newcastle Disease Virus (NDV) is the only member of serotype 1 avian paramyxoviruses (APMV-1) that causes respiratory and neurological disease in chickens and other species of birds and can cause severe economic losses in the poultry sector. Due to the relevant variability of the genome and the pathogenicity of NDV isolates, their detection in a specimen is not sufficient to provide and confirm an exact diagnosis, and so the assessment of virus pathotype is required. To diagnose rapidly and pathotype NDV directly in clinical specimens, a method based on RT-PCR and pyrosequencing analysis has been developed and is reported in the present study. A pair of degenerated primers was designed to amplify a portion of the fusion (F) gene responsible for virulence and used to test 315 specimens collected from 2006 to 2011. The subsequent pyrosequencing reaction identified a 30-bp region encompassing the cleavage site. A total of 213 out of 315 samples were pyrosequenced and results were compared and confirmed by the Sanger sequencing procedure, which is traditionally performed for NDV pathotyping. The pyrosequencing reaction provided high quality results in real time and proved to be more rapid and cost-efficient than the classical sequencing procedure, indicating it as a possible valid alternative to the currently used diagnostic assays for NDV.