Interpreting highly diverse dsRNA viral sequences from Picobirnaviridae within and among species for interspecies transmission inference : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Sciences at the School of Veterinary Science, Massey University, Manawatu, New Zealand

Cross-species transmission of infectious diseases has been at the forefront of our current reality considering the COVID-19 global pandemic and its repercussions. Now more than ever, we realise that the factors involved in these transmissions between species is both simple, such as the impact of increased contact between wildlife and humans, and complicated, such as the impact of biodiversity loss. Our understanding of these influences, and the complexity, has progressed considerably in the last decade though we are still far from predicting epidemics and pandemics. We do, though, understand that many of these infectious diseases cross species barriers frequently, with some types of pathogens more likely to cross between species. Picobirnaviruses (genus Picobirnavirus, family Picobirnaviridae), a genus of double-stranded RNA (dsRNA) viruses, have been identified on almost every continent, though the pathogenic potential for these viruses is still debated. Picobirnaviruses are known to have massive genetic diversity. Picobirnaviruses were first identified in rabbits and then humans and since then have been reported in many mammalian species, birds, reptiles, along with wastewater, and potentially in protozoa; prokaryotes have been implicated as potential hosts, based on conserved ribosomal-binding motifs from prokaryotic messenger RNA. Although they are highly diverse, they have often been found to have very high similarity in different host species present at the same geographic site, raising the hypothesis that cross-species transmission has occurred. The study system from Uganda described in this thesis provides a setting for the possible transmission of infectious diseases between wildlife, humans and domestic livestock. I identified multiple potential pathogens to study cross-species transmission within this system and focused on Picobirnavirus. I identified multiple picobirnaviruses in humans, wildlife and domestic animals from Uganda and New Zealand, for comparison, by using metagenomic and targeted PCR amplicon sequencing. Initial phylogenetic analyses revealed some host clustering among the picobirnaviruses studied, but host and geographical clustering was not upheld when known picobirnaviruses from global databases were included. Furthermore, the use of near-complete and two genogroup-specific viral sequences did not reveal host or geographic clustering on phylogenetic analyses. I identified multiple picobirnaviruses within the same host, detecting up to eight distinct viral sequence types in the samples. Despite the wide-ranging within-host diversity of the viruses, nearly identical virus sequences were also identified in different hosts from the study. Given the high diversity, unclear host and geographic associations, and that picobirnaviruses may infect hosts from across the natural kingdoms, including bacteria and protozoa, I sought to identify picobirnaviruses from bacterial and protozoal samples, to try to determine the host range of the viruses. I discovered picobirnavirus RNA in purified Cryptosporidium oocysts from New Zealand, suggesting a possible alternative Protist host. However, using an alternative genetic code from invertebrate mitochondria removed within putative open reading frame stop codons from some picobirnavirus sequences, supporting other studies. Further analyses of the picobirnavirus sequences revealed alternative genetic code usage or motifs in the untranslated regions that could indicate the virus has a prokaryotic rather than a eukaryotic host—which have been commonly assumed to be the definitive hosts. The discovery that prokaryotes may also be considered hosts of picobirnaviruses, as well as the presence of homologous sequence between species of this highly diverse RNA virus genera, will influence our understanding of these vexatious and under-studied viruses