Studies on infectious bursal disease virus (IBDV) dsRNA extracted from formalin fixed paraffin embedded tissue
Hamoud, Mohamed Mamdouh
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In first study, sequencing the hypervariable region of IBDV from formalin fixed paraffin embedded tissues (FFPET) that originated from chickens experiencing immunosuppression; located both in the USA and abroad, allowed accurate strain identification of IBDV. This allows direct correlation between viral identity and tissue lesions. Several new emerging viruses that don’t group with other known IBDV along with a unique variant virus that had 63 nucleotides missing from its hypervariable region were identified. The second project investigated why some positive acute +4 lesions of FFPET yielded no RT-PCR detectable IBDV RNA. It was hypothesized that different formalin fixation conditions have negative impact on RNA detection from FFPET. To study this, bursas with high viral loads and maximum histological IBDV lesion score of 4 were fixed in formalin under various conditions. Only tissues fixed in formalin with a pH of 7.0, 5 or 10% formaldehyde, storage temperature of 25°C or less, and kept up to 2 weeks in formalin yielded detectable IBDV RNA upon extraction. No RNA could be detected from tissues fixed under extreme temperature, pH or formalin concentrations. Optimal fixation conditions for IHC detection of IBDV were in 10% formalin, pH 7.0 and 4°C, where maximum intensity of immunostaining was observed. The third project’s goal was to produce a subunit vaccine against IBDV, without manipulation of live or killed viral particles, to protect chicken against IBDV infection. The Edgar strain of IBDV VP2 gene was obtained from frozen bursas, while the hypervariable region of VP2 was obtained from FFPET. The two genes were cloned into Pichia pastoris and protein production was confirmed by western blot analysis. Specific pathogen free 1-day old chicks vaccinated with recombinant VP2 showed best protection against 3 week-old challenge with Edgar IBDV. There was no morbidity or mortality in the VP2 vaccinated birds compared to 90% morbidity and 50% mortality in placebo vaccinated birds. Recombinant vaccines don’t totally protect against viral replication in bursa as confirmed by high histopathological lesion scores and immunohistochemical detection of IBDV in infected tissue. This was probably due to the high viral dose and virulence of the challenge virus used.