Monitoring viruses circulating in the human population and the environment is critical for protecting public and ecosystem health. The goal of this dissertation was to incorporate a viral metagenomic approach into virus surveillance efforts (both clinical and water quality control programs) to enhance traditional virus detection methods. Clinical surveillance programs are designed to identify and monitor etiological agents that cause disease. However, the ability to identify viruses may be compromised when novel or unsuspected viruses are causing infection since traditional virus detection methods target specific known pathogens. Here we describe the successful application of viral metagenomics in a clinical setting using samples from symptomatic patients collected through the Enterovirus Surveillance (EVS) program in the Netherlands (Appendix A). Despite extensive PCR-based testing, the viruses in a small percentage of these samples (n = 7) remained unidentified for more than 10 years after collection. Viral metagenomics allowed the identification of viruses in all seven samples within a week using minimal sequencing, thus rapidly filling the diagnostic gap. The unexplained samples contained BK polyomavirus, Herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) which dominated the unexplained samples (n = 4). This study demonstrated that metagenomic analyses can be added as a routine tool to investigate unidentified viruses in clinical samples in a public-health setting. In addition, metagenomic data gathered for SAFV was used to complete four genotype 3 SAFV (SAFV-3) genomes through primer walking, doubling the number of SAFV-3 full genomic sequences in public databases. In addition to monitoring viruses in symptomatic patients, it is also important to monitor viruses in wastewater (raw and treated) to protect the environment from biological contamination and prevent further spread of pathogens. To gain a comprehensive understanding of viruses that endure wastewater treatment, viral metagenomics was used to survey the total DNA and RNA viral community in reclaimed water (the reusable end-product of wastewater treatment) (Appendix B). Phages (viruses that infect bacteria) dominated the DNA viral community while eukaryotic viruses similar to known plant and insect viruses dominated RNA metagenomic libraries suggesting that highly stable viruses may be disseminated through this alternative water supply. A plant virus, the Pepper mild mottle virus (PMMoV), was identified as a potential indicator of wastewater contamination based on metagenomic data and quantitative PCR assays (Appendix C). The metagenomic analysis also revealed a wealth of novel single-stranded DNA (ssDNA) viruses in reclaimed water. Further investigation of sequences with low-level similarities to known ssDNA viruses led to the completion of ten novel ssDNA genomes from reclaimed water and marine environments (Appendix D). Unique genome architectures and phylogenetic analysis suggest that these ssDNA viruses belong to new viral genera and/or families. To further explore the ecology of the novel ssDNA viruses, a strategy was developed to take metagenomic analysis to the next level by combining expression analysis and immunotechnology (Appendix E). This dissertation made a significant contribution to current microbiological data regarding wastewater by uncovering viruses that endure the wastewater treatment and identifying a new viral bioindicator.

Mesozooplankton are of critical importance to marine food webs by transferring energy from the microbial food web to higher trophic levels and depositing energy to the deeper ocean layers through fecal deposition. While decades of research have shown that viruses have significant impacts in the oceans, and infect a wide range of organisms from bacteria to whales, there is still little known about the impacts of viruses on the mesozooplankton community. As copepods are the most abundant mesozooplankton group, this study sought to characterize the viruses present in natural populations of the calanoid copepods Acartia tonsa and Labidocera aestiva in Tampa Bay, Florida. Viral metagenomics revealed two virus genomes, named Acartia tonsa copepod circovirus (AcCopCV) and Labidocera aestiva copepod circovirus (LaCopCV), which were discovered in their respective copepod species. Both viruses show amino-acid similarities to known circoviruses, and phylogenetic and genomic analyses suggest they may be divergent members of the Circoviridae family. LaCopCV was found to be extremely prevalent in the L. aestiva population, with up to 100% of individuals infected. High viral loads for LaCopCV were observed by quantitative PCR, with an average viral load of 1.3x105 copies per individual. In addition, transcription of the LaCopCV replication gene was detected in L. aestiva, demonstrating active viral replication. AcCopCV could be detected sporadically in A. tonsa populations throughout the year. The circoviruses were specific to their respective hosts, and were not detected in the other copepod species or surrounding seawater. Virus-like particles were observed in A. tonsa and L. aestiva under transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue, as opposed to gut tissue, parasites, or symbionts. Preliminary results from in-situ hybridization show that the AcCopCV genome can be detected in A. tonsa tissue, linking the discovered genomes to virus propagation in copepod tissue. This is the first study describing viruses in copepods, as well as the first discovery of circoviruses infecting marine organisms. These results suggest that viruses impact marine copepod populations, necessitating further studies to determine the ecological impacts of viruses on the mesozooplankton community.