Composition and Stability of Single-Stranded DNA Viral Populations in Wastewater Treatment Plants

Regular emergence and re-emergence of viral pathogens emphasizes the importance of understanding viral biogeography and migration. Single-stranded DNA (ssDNA) viruses are among the least understood groups of microbial pathogens, yet the group contains known agricultural pathogens, which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms (Microviridae). Wastewater treatment plants (WWTPs) receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a large range of hosts. Investigations utilizing high-throughput sequencing have determined that local viral diversity is extremely high but does not scale to produce an exponentially higher global diversity. It follows that similar genotypes can be found great distances apart, although they may not be permanent constituents of any single population. Transient genotypes have been observed in temporal surveys of closed systems, where genotypes migrate between individual populations. This study focused on the geographic and temporal population stability of single-stranded DNA (ssDNA) viruses in open systems. Sampling from WWTPs in three neighboring cities in Northwest Florida, which receive constant inflow and potentially receive the same viruses from the local environment, was conducted across a nine-month time span. A combination of polyethylene glycol (PEG) precipitation and filter concentration was used to isolate whole viral particles from the complex wastewater samples. The ssDNA viruses were isolated from larger viruses using a sucrose gradient for size selection and rolling circle amplification was performed to both bias the sample towards ssDNA and prepare the samples for high-throughput sequencing. Amplified genomes were sequenced using Illumina platforms and de novo assembled. Given the increased potential for migration, we expected the populations would be mostly homogenous with relatively few viruses that are unique to individual WWTPs. Viral genotypes with genetic similarity to Circoviridae, Geminiviridae, and Microviridae were recovered from all three WWTPs, however <25% of recovered genes match genotypes (>80% amino acid identity) recovered from neighboring sample sites. We determined that <10% of the genotypes were present in all three plants and the majority of genotypes were specific to one WWTP. Unexpectedly, the WWTPs that were closest to each other geographically were the least similar, and the plants geographically distant from each other had the most observed genetic overlap. These results highlight the high level of diversity within each population, while the high observed heterogeneity indicates localized genetic success and limited migration opportunities between the WWTPs. Throughout time the communities experienced a large degree of genetic turnover. Only 30% of the genotypes were present in more than one time point, 5% were recovered in three of more samplings and <1% were present in all five time points. This thesis concludes that viral genomes are continually moving through the environment and their presence in any given area may be temporary. Therefore, viruses are a continual selective force on their host species through the sheer volume of genetic potential in an area at any given time.