Abstract
The purpose of this study is investigate Differentiation Between Vaccinal and Iranian Virulent Isolates of Newcastle Disease Virus based on F Region Genotyping by HRM Analysis. Discrimination of circulating virulent strains of Newcastle Disease Virus (NDV) from low pathogenic and vaccine stains is the basis for implementation of strategies to control and eradication of that aims at the eradication of NDV in poultry. At the present study the applicability of Real time RT-PCR followed High-Resolution Melting-Curve Analysis (HRM) was evaluated for differentiation of pathogenic ND viruses from the live vaccinal strains. Five virulent NDV isolates and 6 live vaccines samples of NDV were used to develop and evaluate the tests. Based on the nucleotide sequence of Fusion (F) gene, 3 primer pairs (B, E and H) were designed and utilized for HRM analysis. The melting temperature of vaccinal strains higher than the virulent isolates were obtained using the B and H primer pairs, but it was not the same as E. Based on the analysis of HRM curves in this study, the B and H primer pairs could better differentiate the vaccinal strains from each other and from virulent isolate than E. As it is found in this study, HRM analysis can differentiate virulent NDVs from live vaccines. In comparison to the existing methods for discrimination of different pathotypes of the ND viruses, the developed test takes a shorter time, needs less infrastructure and facilities and costs significantly less than the other traditional methods like intra cerebral pathogenicity index (ICPI).
Highlights
Newcastle Disease (ND) is one of the most contagious viral diseases affecting most species of birds with different ages in the world (Madhan et al, 2005)
Primer designing and setting up the real time Polymerase Chain Reaction (PCR): Based on the nucleotide sequence of F gene, three sets of the primers were utilized for evaluation and comparation among the patterns obtained from the vaccinal and field viruses by High-Resolution Melting-Curve Analysis (HRM) (Table 2)
By using E primer pairs, the melting temperatures of vaccines were lower than virulent virus; but with B and H primer pairs, vaccines had 0.91.5°C higher melting temperature
Summary
Newcastle Disease (ND) is one of the most contagious viral diseases affecting most species of birds with different ages in the world (Madhan et al, 2005). The NDV has numerous strains which can cause disease in birds with low to high pathogenicity and clinical signs. This disease is considered as an economically important infection in poultry industry worldwide. Newcastle disease causes death in poultry without very obvious clinical symptoms and can give 100% mortality in affected flocks. It has been reported that more than 250 species of birds are have different susceptibility to the natural or experimental infection with ND virus. It is known that the NDV strains infect all species of domestic poultry, while some species such as ducks and geese do not show severe symptoms of disease even when infected with the most acute strains of the virus (Alexander and Jones, 2008; Alexander, 2003)
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