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.

Recent metagenomic studies have shown that there is a higher diversity of ssDNA viruses in the environment than previously thought. While some viral families are well characterised, novel ssDNA isolates discovered with sequence-independent molecular techniques are often too divergent to fit within the currently established viral taxonomy. Several factors have contributed to the gap in knowledge, including: the (previously) high cost of sequencing, the disproportionate amount of research that occurs after a threat is identified, and the use of sequence-based molecular techniques to isolate viral sequences. Recent studies have begun to explore viral diversity in the environment, however, most of these studies have occurred outside New Zealand. Several benefits would come from uncovering the true ssDNA viral diversity and global distribution including improving the resolution of the current taxonomic structure for identifying unknown isolates and inferring possible virus-host relationships, and providing baseline data for the development of disease prevention and monitoring strategies. Studies specific to the New Zealand environment are essential. With its geographical isolation and Gondwana ancestry, New Zealand will possess a unique viral sequence space. Studies on local viral diversity and the spread of ssDNA viruses are going to be most relevant if they are conducted within the established ecosystems in New Zealand. In this dissertation, a novel protocol was developed for exploring viral diversity in the New Zealand environment using basic molecular techniques and animal faecal samples. Design considerations included: identifying highly novel small circular viral sequences with DNA genomes without the use of specific primers, inflicting as little environmental impact as possible, and keeping the cost low. The faecal sampling approach does not require animal handling and therefore incorporates the use of viral reservoirs while remaining non-invasive. The molecular techniques in this protocol used non-specific rolling circle amplification (RCA) followed by restriction enzyme (RE) digests, cloning, and sequencing of the cloned genomes via sanger sequencing. This inexpensive exploratory method provided preliminary sequence information from which primers were designed for recovery of full viral genomes. The success of this protocol was demonstrated by the recovery and molecular characterisation of a novel ssDNA virus isolate from a pig faecal sample, which was tentatively named porcine stool-associated circular virus (PoSCV). This protocol was then applied to sample viruses in the faecal matter from variety of domesticated, wild, and farmed animals in New Zealand. The faecal samples were collected from the North and South Island of New Zealand as well as South East Island of the Chatham Islands (Rangatira). Several putative gemycircularviral isolates (novel viruses with similarities to geminiviruses and the recently discovered ssDNA virus infecting Sclerotinia sclerotiorum) were identified in the sequencing results based on BLASTx similarities to viral sequences available in public databases (GenBank). The full genomes of these isolates were recovered and characterised. Identification was based on genome organization, phylogenetic analysis of the replication associated protein (Rep), and full genome nucleotide pairwise identities. Fourteen novel ssDNA virus sequences relating to gemycircularviruses were discovered, of which ten were representative of new species (FaSCV-1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) and three were identified as strains of the same species (FasGCV-1). Two additional isolates were discovered to be distantly related to these viruses: Ostrich faecal associated ssDNA virus (OfaV) and Rabbit faecal associated ssDNA virus (RfaV). Additionally, this protocol was used to recover novel ssDNA viruses from the nesting material of a dead Yellow-crowned Parakeet chick found in the Poulter Valley in the South Island of New Zealand. The nesting material likely contained faecal matter and thus represented another approach strategy for exploring ssDNA viruses in the environment. Two novel ssDNA isolates were discovered and molecularly characterised: Cyanoramphus nest-associated circular X virus (CynNCXV), and Cyanoramphus nest-associated circular K virus CynNCKV.