Beak and feather disease virus (BFDV) is an important emerging viral pathogen of psittacine host species causing ''psittacine beak and feather disease (PBFD)'� with a global distribution in captive and wild avian species. To better understand the kinetics of BFDV replication a longitudinal study was conducted to track viral replication and mutation events occurring in BFDV over a period of four years within a flock of the critically endangered orange-bellied parrot (Neophema chrysogaster). Molecular phylogenetics using BFDV genome sequences from these birds revealed that the outbreak was linked to two unique progenitor BFDV genotypes. A predicted BFDV genome wide mutation rate of 3.41 ÃƒÂƒ' 10-3 subs/site/year was detected, akin to that for RNA viruses. Significant evidence of homologous recombination was also detected between two distinct progenitor genotypes which may have permitted BFDV adaption to this novel host. To better understand the genetic origins of BFDV in the Australian landscape, a phylogeographic study of 38 BFDV genomes from seven different host species of Australian cockatoos was analysed to appreciate host and geographically based divergence as well as probable host-switch events. The study revealed a likely host switch event in a captive orange-bellied parrot (KF188691) with BFDV derived from wild corellas in Victoria. Further estimation of broader mutation rates for BFDV confirmed previous results with a high rate of genome wide mutation detected (1.12ÃƒÂƒ'10-3 subs/site/year) along with strong support for recombination. The results indicate active cross-species transmission in various psittacine bird subpopulations across Australia. BFDV quasispecies were also identified within individual infected cockatoos particularly in splenic tissue. To further assess ongoing threats to other vulnerable and endangered parrots such as the western ground parrot (Pezoropus flaviventris), swift parrot (Lathamus discolor) and regent parrot (Polytelis anthopeplus monarchoides) BFDV genomes in parrot species throughout Australia were similarly analysed phylogenetically which again demonstrated flexible host-switching and extensive recombination amongst BFDV genomes from disparate host species. Consistent with quasispecies theory the results indicate that all endangered Australian psittacine bird species are equally likely to be infected by BFDV genotypes from any other close or distantly related host reservoir species. The presence of rich BFDV genetic diversity points to Australia as the most likely geographical origin of this virus and supports a mosaic of geographic clustering throughout the landscape with flexible host switching. The results provide evidence of Order-wide host generalism in the Psittaciformes characterised by high mutability that is buffered by frequent recombination and a slow replication strategy.A simple, rapid and inexpensive detection and genotyping method for BFDV variants using PCR and subsequent high-resolution melt (HRM) curve analysis was developed. This was achieved using PCR amplification of the relatively well conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). Melting curve profiles of this PCR-HRM technique provided results that could consistently discriminate nucleotide diversity among a range of BFDV genotypes obtained from orange-bellied parrots, lorikeets and cockatoos. Throughout the last decade several approaches has been used in frustrated attempts to yield high quality, quantity, stable and soluble recombinant BFDV capsid protein (Cap) for use in diagnostic assays and or vaccines. Accordingly, an efficient method was developed for homogenous over-production of BFDV Cap using an E. coli expression system. The purified protein was producible in approximately 14-18 fold greater concentrations (milligram amounts) than that reported previously. The recombinant protein was characterised by SDS-PAGE and antigenically authenticated by immunoassay.
|Qualification||Doctor of Philosophy|
|Award date||18 Jun 2015|
|Place of Publication||Australia|
|Publication status||Published - 2015|