Pseudoknots are a special kind of RNA structures that play functional roles in a wide variety of biological processes. Pseudoknots are best known for their involvement in the −1 programed ribosomal frameshifting (−1 PRF) and stop codon readthrough translational recoding events as the stimulatory structures. In this dissertation, three large scale bioinformatics investigations were carried out on the roles of pseudoknots in the −1 PRF, as well as stop codon readthrough, recoding mechanisms in viral and human mRNAs. To meet the specific needs of the bioinformatics investigations, a new algorithm and method for the detection of RNA pseudoknots has been developed. The new approach differs from all existing pseudoknot detection programs in that it is capable of identifying all potential pseudoknots in any given RNA sequence with no length limitation, in a time efficient manner. This capability is essential for large scale applications in which large datasets of long RNA sequences are analyzed. The algorithm and method have been implemented, with different flavors, in three large scale sequence analysis investigations. The three datasets of mRNA sequences are: 1) full-length genomic mRNA sequences of all animal viruses known or expected to use the −1 PRF and stop codon readthrough recoding mechanisms for viral protein production; 2) full-length genomic mRNA sequences of 4000 plus different strains of human immunodeficiency virus type-1 (HIV-1); 3) 34,000 plus full-length human mRNA sequences. Results from systematic sequence analysis on these three datasets prove the usefulness and robustness of the newly developed pseudoknot detection approach. A large number of previously unknown potential pseudoknots were detected in the viral and human mRNA sequences under investigation. Post detection analysis leads to new mechanistic insights and hypotheses of pseudoknot dependent translational recoding. Some unifying themes of RNA pseudoknot structures in general are also uncovered. The results provide solid basis for further experimental and bioinformatics studies in the future.

Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell seals, viral metagenomics led to the discovery of 152 novel anellovirus genomes, encompassing two anellovirus species. Characterizing these viruses is important for understanding the prevalence and diversity of ssDNA viruses, which have only recently been described in marine animals. Furthermore, since emerging diseases can be caused by changing conditions affecting host susceptibility to a virus that was previously not related to disease (opportunistic pathogen), having baseline data allows for quick identification of the pathogen. In addition to determining baseline data, viral metagenomics can explore the role of viruses in disease. A novel virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in the Atlantic sea star Asterias forbesi displaying symptoms of sea star wasting disease (SSWD). AfaCV was the first circular replicase-encoding ssDNA (CRESS-DNA) virus discovered in echinoderms, but it was only present in 10% of SSWD sea stars indicating it is not involved in the development of the disease. This dissertation also focuses on elucidating the role of two previously characterized viruses, chelonid fibropapillomatosis-associated herpesvirus (CHHV5; Chelonid herpesvirus 5, ChHV5) and Zalophus californianus anellovirus (ZcAV), in animal health. PCR amplicon sequencing was used to obtain large portions of the 132 kb genome of ChHV5, the putative etiological agent of the neoplastic sea turtle disease, fibropapillomatosis. Obtaining the genome of ChHV5 from Florida green, Kemp’s ridley, and loggerhead sea turtles provides data for phylogenetic analysis across geographic locations and sea turtle species, as well as a reference for designing downstream molecular assays to examine viral latency. ZcAV was first described from the lungs of captive sea lions involved in a mortality event. PCR could not detect ZcAV in the blood of infected animals, and since sea lions are a protected species, it is not possible to obtain lung biopsies from live sea lions to determine ZcAV prevalence or its role in sea lion health. To answer these important questions, an enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to ZcAV in serum from wild sea lion populations. This newly developed ELISA showed that sea lions mount an immune response to ZcAV, and was used to determine the prevalence of ZcAV among wild sea lion populations. This dissertation makes an important contribution to marine science through discovery and characterization of viruses present in healthy and diseased marine animals. Several different methods were used for virus whole-genome sequencing including viral metagenomics, PCR amplicon sequencing, and target enrichment. These findings were expanded upon by developing and using PCR assays and a serological assay to screen for virus prevalence. These methods have implications for viral surveillance and understanding the role of novel viruses in animal health.

Thesis (Ph.D. in Microbiology & Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-212).