Molecular characterisation of novel single stranded DNA viruses recovered from animal faeces

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