Identification of isolated viral strains of atypical avian influenza using molecular methods of virological diagnostics

Abstract

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.

Pored primene standardnih metoda virusološke dijagnostike koje se koriste za izolovanje virusa Newcastle bolesti iz suspektnog materijala kao i za njegovu identifikaciju, danas su sve više u upotrebi i molekularne metode dijagnostike i to pre svega lančana reakcija polimeraze (RT-PCR) i metoda sekvenciranja. Cilj ovog rada bio je ispitivanje mogućnosti primene navedenih metoda u dijagnostici infekcije živine izazvane virusom Newcastle bolesti. Prisustvo hemaglutinacionih antigena virusa Newcastle bolesti ustanovljeno je u uzorcima alantoisne tečnosti od 62 embrionirana jaja živine posle 72h od inokulacije čiji su se titri kretali od 1:16 do 1:2048, dok je testom heminhibicije (HI testom) uz primenu referentnog imunog seruma protiv navedenog uzročnika izvršena identifikacija izolovanih virusa u razređenjima seruma od 1:128 do 1:1024. Metodom reverzne transkripcije (RT-PCT) i lančane reakcije polimeraze (PCR) ustanovljeno da se kod osam ispitivanih uzoraka formira po jedan fragment virusne RNK u gelu agaroze veličine od 254bp koji je po sekvenci nukleotida karakterističan za genom virusa Newcastle bolesti. Na osnovu uporedne analize sekvenci RNK dobijenih od osam izolovanih sojeva virusa NDV i sekvenci genoma referentnih sojeva virusa atipične kuge živine uz korišćenje Mega 40 i BLAST programa, utvrđeno je da su izolovani sojevi virusa Newcastle bolesti visoko virulentni.