Examination of some biological properties of glycoprotein subunits of PHY-LMV.42 strain of Newcastle disease virus

Abstract

The objective of our work was to investigate some biological characteristics of purified glycoprotein subunits of Newcastle disease virus strain PHY-LMV.42 isolated from pigeons for the purpose of vaccine production. PHY-LMV.42 strain of Newcastle disease virus was multiplied by successive passages in embryonated eggs and identified by the methods of Reverse transcriptase PCR and Real-Time PCR along with F gene sequencing. Proving the presence of HN and F antigene in the virus subunits samples was carried out by hemagglutination inhibition method with referent immune sera. Biochemical characterization of glycoprotein subunits was performed by SDS-PAGE method as well as liquid chromatography with mass spectrometry (LC ESI-TOF-MS/MS). Testing for the virus subunits immunogenicity was carried out in biological experiment on 75 laying hens Tetra-SSL and 25 chickens Isa Brown by inducing an artificial infection with Hertz 33 strain of the virus. Low concentrations of the virus antigens of 0.36 mg/ml along with glycoprotein fractions of 77 i 58 kDa manifested a strong hemagglutination activity of 4096 HJ/0,1ml. The subunit vaccines of 256 and 128 HJ/0.5 ml induced a protective immune response in all the vaccinated animals. Based on the obtained results it can be concluded that low concentrations of purified virus subunits of PHY-LMV.42 strain can be used for preparing of effective vaccines.

Cilj našeg istraživanja je bilo ispitivanje bioloških karakteristika prečišćenih glikoproteinskih subjedinica soja PHY-LMV.42 virusa Newcastle bolesti izolovanog iz golubova radi njihovog korišćenja za pripremanje vakcine. Soj PHY-LMV.42 virusa Newcastle bolesti je umnožavan sukcesivnim pasažama u kokošijim embrionima i identifikovan metodama Reverse transcriptase PCR i Real- Time PCR uz sekvenciranje F gena. Dokazivanje prisustva HN i F antigena u uzorcima virusnih subjedinica vršeno je metodom inhibicije hemaglutinacije sa referentnim imunim serumima. Biohemijska karakterizacija glikoproteinskih subjedinica izvršena je primenom metoda SDS-PAGE i tečne hromatografije sa masenom spektrometrijom (LC ESI-TOF-MS/MS). Ispitivanje imunogenosti virusnih subjedinica sprovedeno je u biološkom ogledu na ukupno 75 kokoši nosilja Tetra-SSL i 25 pilića Isa Brown uz izvođenje veštačke infekcije sojem Hertz 33 navedenog virusa. Niske koncentracije virusnih antigena od 0,36 mg/ml sa glikoproteinskim frakcijama od 77 i 58 kDa su ispoljavale snažnu hemaglutinacionu aktivnost od 4096 HJ/0,1ml. Subjedinične vakcine od 256 i 128 HJ/0,5 ml, indukovale su imunološki odgovor zaštitnog karaktera kod svih vakcinisanih životinja. Na osnovu dobijenih rezultata može se zaključiti da se niske koncentracije prečišćenih virusnih subjedinica soja PHY-LMV.42 mogu koristiti za pripremanje efikasne vakcine. PR Projekat Ministarstva nauke Republike Srbije, br. TR 31008.