Beak and feather disease virus (BFDV), a non-enveloped, icosahedral virus with a circular single stranded DNA (ssDNA) genome, is the causative agent behind psittacine beak and feather disease (PBFD), an often fatal disease affecting parrots. Symptoms include feathering abnormalities, loss of feathers, and occasionally beak and claw deformities. BFDV-induced immunosuppression results in an increased susceptibility to secondary microbial infections, which is often the cause of death in infected parrots. There is no cure, no effective treatment, and no protective vaccine for BFDV. The international trade in exotic parrots has facilitated the spread of BFDV, so that it now has a global presence. Given that over a quarter of the currently recognised 356 psittacine species are considered to be at risk of extinction in the wild, the worldwide presence of BFDV, coupled with its extreme environmental stability, poses serious concerns for the future of some of the worlds most endangered parrots. That genetic diversity exists among BFDV isolates has been established, yet in the 14 years since the genome was fully sequenced, very few full-length BFDV genome sequences have been deposited in GenBank, despite the technology to rapidly isolate and amplify entire circular ssDNA genomes being readily available. Most studies have sequenced just a portion of the genome, usually one of the open reading frames (ORFs) encoding the major viral proteins, to investigate phylogenetic relationships between isolates. However the two major BFDV ORFs, encoding the replication associated protein (Rep) and the capsid protein (CP), have been shown to evolve at different rates, with the functional Rep being generally more conserved while CP is more variable. When also considering the fact that ssDNA viruses are notoriously recombinant, it becomes clear that an analysis based on a portion of the genome is unlikely to accurately establish evolutionary relationships. Therefore the focus of the studies described in this thesis was on isolation and amplification of full-length BFDV genomes from avian blood and feather samples that first tested positive to a PCR-based BFDV screening method. Samples were collected by appropriately trained people in New Zealand, New Caledonia, and Poland, before being sent to the University of Canterbury for molecular and bioinformatic analysis. The sequences of the BFDV genomes from each region were compared to each other and to all other full BFDV genome sequences publically available in GenBank, to compare the genetic diversity among these isolates. Recombination analyses were also performed, to assess how recombination is impacting on the evolution of BFDV. New strains of BFDV and new subtypes of existing BFDV strains were discovered, indicating that the global genetic diversity may be greater than previously thought. Many strains also proved to be recombinants, in particular those from Poland. Europe has had a long history with importing and breeding exotic parrots, and the high degree of recombination among the Polish BFDV isolates coupled with the number of previously unsampled strains is an example of how maintaining populations of multiple species in captivity enables evolution through recombination, and emergence of novel viral strains. Full genome analyses can also enable tracking the source of an infection. A total of 78 full genome sequences from 487 samples tested were deposited into GenBank as a direct result of the work undertaken as part of this thesis, thereby adding to the existing knowledge base regarding BFDV. With continued global sampling and full genome analysis it may one day be possible to trace the history of BFDV to its original emergence.

To enable assessment of recombinant BFDV capsid protein (recBFDVcap) for vaccination to protect against PBFD, commercially available lovebirds (Agapornis sp.) were tested for evidence of past and current BFDV infection using PCR, HI and HA to identify suitable BFDV-free birds in which to test the vaccine. During this attempt, it was found that lovebirds from commercial aviaries were endemically infected with BFDV with evidence of up to 100% prevalence of BFDV DNA in blood samples from individual birds over time. Such an approach was abandoned as unlikely to yield suitable numbers of naïve birds to conduct a BFDV vaccination trial. As commercially available lovebirds were considered to be a poor source of BFDV-free birds, wild caught cockatoo nestlings and eggs (long-billed corella; Cacatua tenuirostris and galah; Eolophus roseicapillus) were used to assess the efficacy of BFDV vaccination using baculovirus recombinant BFDV capsid. Eggs were artificially incubated and 3 eggs successfully hatched and 1 was successfully hand-reared. All nestlings were screened for BFDV DNA in blood using PCR upon arrival then on days 11, 18 and 25 and tested for anti-BFDV antibody on the day of arrival. All hatched birds were determined to be free of BFDV DNA and BFDV HI antibody in the peripheral blood throughout the hand rearing period and the flock was considered to be suitable for a BFDV vaccination trial. Corellas (n=13) were injected with 1 mL of vaccine containing 10 μg recBFDVcap on day 0 and 0.4 mL vaccine containing 66.8 μg recBFDVcap on day 11. All vaccinated corellas and 5 non-vaccinated control corellas were given 0.4 mL BFDV suspension (titre = log2 12 HAU/50 μL) intramuscularly and 0.1 mL orally 16 days after booster vaccination. Blood was collected periodically during the vaccination period and blood and feathers collected before and after BFDV administration. Testing included BFDV DNA detection by PCR and qRT PCR (on blood) as well as serum antibody detection by haemagglutination inhibition (HI) and BFDV DNA and antigen was detected by qRT PCR and haemagglutination (HA) (on feathers), respectively. Four of 97 blood samples collected from vaccinated birds post BFDV challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry (IHC). Non-vaccinated control corellas developed transient feather lesions and PCR, HI and HA test results consistent with PBFD. They were BFDV PCR positive for up to 41 days post-challenge and qRT PCR demonstrated reduced virus replication in vaccinated birds compared to non-vaccinated control birds. Thus, administration of recBFDVcap vaccine alone was found to incite an adaptive immune response in BFDV-free corellas that subsequently conferred protection against inoculation with BFDV. A commonly utilized method for excising blood dried on filter paper was proven to be of high risk of carryover contamination facilitated by a hole punch used for processing several samples. Therefore a practical method of avoiding carryover contamination was developed and used in the DNA testing procedures of the vaccination trial. Finally, the haematological characteristics of the above mentioned cockatoos were studied before and for 97 days after experimental infection with BFDV. It was found that the pre-challenge haematological values were similar between the vaccinated and non-vaccinated corellas. Most pre-challenge parameters were comparable to previously reported values of other cockatoos and psittacine birds. Significant differences were seen in both groups when comparing pre-challenge values with post challenge values for total and differential leukocyte concentrations, but PCV and TSP were not significantly affected by BFDV challenge.