RNA bacteriophages: diversity, abundance, and applications

We live in a world created by and dominated by microbes, yet we are only beginning to understand this complex and diverse realm. This vast collection of microorganisms (the microbiome) is composed of bacteria, viruses, fungi, archaea, protozoa, and algae, and is essential to every aspect of life and are involved in countless natural processes. While interest in the microbiome has exploded in recent years, studies regarding the viral fraction has lagged behind. This viral component (virome) is dominated by bacteriophages (phages) - viruses that target and infect prokaryotes. They are intrinsically linked to the bacterial community of every ecosystem and potentially dictate the bacterial composition, function, and dynamics through a series of complex interactions. Their roles across global environments, from human gut to marine and terrestrial settings, are only just beginning to be described. While gradual improvements in virome and phageome research have provided us with some insights into the function of these viruses, much more work is needed to gauge their full importance. Of the known phages, those that encode either a double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) genome have been more intensively studied than their RNA counterparts. The RNA phages are either positive-sense, single-stranded RNA (+ssRNA; Leviviricetes) or double-stranded RNA (dsRNA; Cystoviridae) and have been understudied and underrepresented in publicly available databases. This thesis tackled the limited knowledge of these phages, their lifecycles, and our current understandings of their taxonomy. It also explored the potential biases associated with isolating and extracting RNA phages from human faecal samples which may have contributed to their under-representation in many virome studies. Properly isolating and identifying RNA phage is crucial to better understand the diversity of the global microbiome. Given that only limited numbers of +ssRNA phages are present in databases, it was timely to explore their true abundance in different environments by exploiting advances in the science of bioinformatics. Our method, utilizing specific profile hidden Markov model (HMM) search tools, is described in detail. This work greatly expanded the numbers of +ssRNA phage genomes and resulted in the submission to and acceptance of an updated taxonomy by the International Committee on Taxonomy of Viruses (ICTV). The framework depicted in this thesis allows for the expected expansion of these phages in future work. It also offers an example for potential studies looking to combine both cultured and metagenomic-derived genomes in taxonomic updates. It is important that future studies not only optimize the bioinformatic approaches used but also target and improve the isolation and extraction methods applied to enhance the recovery of RNA phages. Since the biases associated with different extraction methodologies have been pinpointed as a crucial factor, three methods were examined and assessed for their efficacy using controls spiked with MS2 and Qbeta. This work was coupled with an in-house study that, using one of these alternative phage-extraction methods, isolated +ssRNA phages from a mammalian gut for the first time in our laboratory. Over the past year, the importance of studying RNA viruses has never been so apparent as a result of the global pandemic due to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its associated disease. However, given that this virus is classified as being extremely pathogenic and subject to high rates of mutation, the idea of using another virus as a safe surrogate has been previously suggested. One such candidate virus is phi6, a dsRNA phage of the Cystoviridae family. With its enveloped structure it offers a reliable model in the examination of different treatments and therapies in terms of their potential in combating SARS-CoV-2. A chapter of this thesis is dedicated to exploring phi6 as a surrogate in lipopeptide exposure and thermotolerance assays. Overall, this thesis investigated the realm of RNA phages. From examining the current literature on their basic biology to biases associated with their recovery from virome studies, the initial two chapters offer a foundation for the four subsequent chapters. In addition, RNA phage numbers were expanded, taxonomically restructured, tracked through different extraction methods, and assessed as a surrogate for SARS-CoV-2. In 1980, Norton Zinder wrote "as long as there are bacteria, there will be RNA phage" and it is suspected that we are just beginning to realize how accurate he was