Development of novel diagnostic and vaccine options for beak and feather disease virus (BFDV)

摘要: Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss immunosuppression leading to secondary infections eventually death in psittacine birds. The development of standardised reagents for the detection characterisation BFDV production protective vaccines has been difficult as no cell culture system yet found grow virus successfully vitro. However, consistent effective diagnostic tests now more practical through application nucleic acid-based methods recombinant technology. A quantitative real-time PCR assay DNA was developed, using primers designed amplify conserved 81 bp fragment ORFV1 SYTO9, fluorescent intercalating dye, with assays run on Corbett RotorGene 3000. A synthetic oligonucleotide used establish standard curves quantitation viral load both blood preparations. very sensitive, limit 50 copies/iL. developed BFDV-positive extracts from feathers 10 different species birds validated samples corellas vaccinated an experimental vaccine, then challenged live virus. Viral reliably detected all control (non-vaccinated) some Contamination environment dander BFDV-infected meant that HA preparations were unreliable excretion. Nonetheless, should prove be useful sensitive test range future investigations. capsid protein also produced specific monoclonal antibody against it. behaviour haemagglutination (HA) western blotting, immunohistochemistry (IHC), ELISA haemagglutination-inhibition (HI) characterised. had ability agglutinate galah erythrocytes per wild-type this agglutination inhibited by antibodies naturally immune Furthermore, self-assembled into virus-like particles determined electron microscopy. whole similar optimal titres when blotting IHC. HI activity 3 genera birds, including recently described cockatiel isolate. novel “blocking” (or “competitive”) (bELISA) anti- sera (Ab-bELISA) 166 eastern long-billed bELISA correlated strongly test, thus it have wide serodiagnosis BFDV. survey cockatiels (n=88) housed at commercial aviaries conducted investigate whether infection occurs cockatiels. All diagnosed being virus-free detectable titre assay. In addition this, genomes two isolates obtained diseased sequenced cross-reactivity performed eluted these Serological results phylogenetic analysis nucleotide sequences indicated serologically genetically other isolates. This first report antigenically distinct Since Ab-bELISA lower than assay, repeat sero-survey. No any tested questions about real prevalence possible existence serotype adapted remain unanswered. The successful PBFD pet wild depends incite strong response can efficiently large quantities. Recombinant proteins considered candidate techniques allow vaccines, vaccines. order examine potential vaccination strategy prevention BFDV, based sequence encoding mammalian expression vector pVAX1. vaccine constructs full length NLS-truncated resulted vitro vivo. Protein COS-7 cells transfected indirect immunocytochemistry Chapter 5. present nucleus full-length cytoplasm expected. Both induced levels anti-BFDV Thus, those presented here may options further are discussed.