Surveying secret diversity: pathogens in native rodents, symbiotic termites, hydrothermal microbes, and hypersaline archaea

Understanding microbial diversity and evolution in under- and unexplored biological systems has the capacity to shed important new light on evolutionary history and disease emergence. By sequencing viral and microbial communities within unexplored species and ecosystems, my thesis aims to uncover transformational biodiversity. I deployed metagenomic sequencing to reveal novel microbial and viral biodiversity in vertebrates, invertebrates, and bacteria. I also performed a detailed metatranscriptomic search for RNA viruses in archaea from extreme environments. I obtained full genomes of pathogenic Bartonella bacteria, cultured from rodents collected in the biodiversity hotspot of the Yunnan province, China. These genomes depict high bacterial diversity at the village scale and were the first metagenomically acquired genomes for Bartonella from China, providing data on how these bacteria emerge in new hosts. I identified 67 novel RNA virus species within termites, some infecting the termite itself and others infecting microbial symbionts. This study was the first investigation of the termite RNA virome and considered how aspects of termite social structure impact virome composition. Similarly, I discovered 23 viruses within the geothermal pools of Kuirau Park, located in the North Island of New Zealand. Of most note in this study was the discovery of divergent species of the expanding viral phylum Lenarviricota, that have important implications for the origins and early evolution of RNA viruses as a whole, as well as the discovery of multiple DNA viruses. This study was only the second to investigate RNA viruses from geothermal pools. Finally, I performed a metagenomic investigation of RNA viruses from hypersaline lakes in Australia and Antarctica. This study, the first of its kind, identified highly divergent RNA virus sequences that could not be placed within the global RNA virus phylogeny, but which could plausibly have bacterial or archaeal hosts

Unmapped RNA Virus Diversity in Termites and Their Symbionts

Despite their ecological importance, nothing is known about the diversity and abundance of RNA viruses in termites (Termitoidae). We used a metatranscriptomics approach to determine the RNA virome structure of 50 diverse species of termite that differ in both phylogenetic position and colony composition. From these samples, we identified 67 novel RNA viruses, characterized their genomes, quantified their abundance and inferred their evolutionary history. These viruses were found within or similar to those from the Togaviridae, Iflaviridae, Polycipiviridae, Flaviviridae, Leviviridae, Narnaviridae, Mitoviridae, Lispivirdae, Phasmaviridae, Picobirnaviridae and Partitiviridae. However, all viruses identified were novel and divergent, exhibiting only 20% to 45% amino acid identity to previously identified viruses. Our analysis suggested that 17 of the viruses identified were termite-infecting, with the remainder likely associated with the termite microbiome or diet. Unclassified sobemo-like and bunya-like viruses dominated termite viromes, while most of the phylogenetic diversity was provided by the picobirna- and mitovirus-like viruses. Of note was the identification of a novel flavi-like virus most closely related to those found in marine vertebrates and invertebrates. Notably, the sampling procedure had the strongest association with virome composition, with greater RNA virome diversity in libraries prepared from whole termite bodies than those that only sampled heads

Unmapped RNA Virus Diversity in Termites and Their Symbionts

Despite their ecological importance, nothing is known about the diversity and abundance of RNA viruses in termites (Termitoidae). We used a metatranscriptomics approach to determine the RNA virome structure of 50 diverse species of termite that differ in both phylogenetic position and colony composition. From these samples, we identified 67 novel RNA viruses, characterized their genomes, quantified their abundance and inferred their evolutionary history. These viruses were found within or similar to those from the Togaviridae, Iflaviridae, Polycipiviridae, Flaviviridae, Leviviridae, Narnaviridae, Mitoviridae, Lispivirdae, Phasmaviridae, Picobirnaviridae and Partitiviridae. However, all viruses identified were novel and divergent, exhibiting only 20% to 45% amino acid identity to previously identified viruses. Our analysis suggested that 17 of the viruses identified were termite-infecting, with the remainder likely associated with the termite microbiome or diet. Unclassified sobemo-like and bunya-like viruses dominated termite viromes, while most of the phylogenetic diversity was provided by the picobirna- and mitovirus-like viruses. Of note was the identification of a novel flavi-like virus most closely related to those found in marine vertebrates and invertebrates. Notably, the sampling procedure had the strongest association with virome composition, with greater RNA virome diversity in libraries prepared from whole termite bodies than those that only sampled heads

Novel hepatitis D-like agents in vertebrates and invertebrates

Hepatitis delta virus (HDV) is the smallest known RNA virus, encoding a single protein. Until recently, HDV had only been identified in humans, where it is strongly associated with co-infection with hepatitis B virus (HBV). However, the recent discovery of HDV-like viruses in metagenomic samples from birds and snakes suggests that this virus has a far longer evolutionary history. Herein, using additional meta-transcriptomic data, we show that highly divergent HDV-like viruses are also present in fish, amphibians, and invertebrates, with PCR and Sanger sequencing confirming the presence of the invertebrate HDV-like viruses. Notably, the novel viruses identified here share genomic features characteristic of HDV, such as a circular genome of only approximately 1.7 kb in length, and self-complementary, unbranched rod-like structures. Coiled-coil domains, leucine zippers, conserved residues with essential biological functions, and isoelectronic points similar to those in the human hepatitis delta virus antigens (HDAgs) were also identified in the putative non-human viruses. Importantly, none of these novel HDV-like viruses were associated with hepadnavirus infection, supporting the idea that the HDV-HBV association may be specific to humans. Collectively, these data not only broaden our understanding of the diversity and host range of HDV, but also shed light on its origin and evolutionary history