Amplification of haemagglutinin-neuraminidase gene of Newcastle disease virus using novel degenerate primers

Newcastle disease (ND) is a highly infectious disease of numerous bird species, caused by Newcastle disease virus (NDV), and is recognized as a major global poultry disease. In India, serial vaccinations using live lentogenic and mesogenic viruses have been long used as a strategy to prevent the spread of ND. However, ND still exists among the poultry flocks in Kerala, indicating the possibility of antigen mis-match between the surviving wild type viruses and those in the vaccine strains. To test this possibility, RT-PCR amplification of the HN gene was attempted with the available virulent NDV isolates as well as the LaSota strain of NDV, and the amplicons were sequenced. The HN gene was selected because of its importance in protection against ND. Surprisingly, both of the published primers failed to amplify the HN gene from the virulent field isolates, although LaSota strain was readily detected by these primers. Hence it became necessary to design new sets of primers for the detection of HN gene. Two newly designed primer pairs were used to amplify the coding region of the HN gene from field isolates as well as LaSota strain. Our results indicate that our primers are more suitable than the published primers for amplification of HN gene from virulent field isolates and might assist in the study of the genetic variation that has developed in NDV under immune selection pressure due to vaccination.