Veterinarski glasnik 2008 Volume 62, Issue 3-4, Pages: 167-177
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Identification of isolated viral strains of atypical avian influenza using molecular methods of virological diagnostics

Vidanović Dejan, Šekler Milanko, Vasković Nikola, Žarković Aleksandar, Matović Kazimir, Milić Nenad, Nišavić Jakov

In addition to the implementation of standard methods of virological diagnostics used for the isolation of the Newcastle disease virus from suspect material, as well as for its identification, nowadays there is increasing use of molecular diagnostic methods, primarily reverse transcription polymerase chain reaction (RT-PCR) and the sequencing method. The objective of this work was to examine possibilities for the implementation of the above methods in the diagnosis of poultry infection caused by the Newcastle disease virus. The presence of hem agglutination antigens for the Newcastle disease virus was established in samples of allantoises liquid from 62 poultry embargoed eggs 72 h after inoculation, whose titers ranged from 1:16 to 1:2048, while the hem agglutination inhibition test (HI test) with the implementation of a referent immuno serum against the given cause provided the identification of isolated viruses in serum dilutions of 1:128 to 1:1024. The RT-PCR method and the PCR established that in eight examined samples one fragment each of viral RNA is formed in agars gel of a size of 254bp, which is characteristic for the Newcastle disease virus genome according to its nucleotide sequence. On the grounds of a comparative analysis of RNA sequences obtained from eight isolated NDV strains and the genome sequences of referent atypical poultry influenza viral strains using Mega 40 and BLAST programmers, it was established that the isolated strains of the Newcastle disease virus were highly virulent.

Keywords: Newcastle disease virus, embargoed eggs, hemagglutination test, hemagglutination inhibition test, RT-PCR, sequencing