Detection and Characterization of Viruses in the Environment Using Established and Novel Sequencing Approaches

Abstract

Viruses play critical roles in both the environment and public health systems. This thesis integrates genomic sequencing methodologies to detect, characterize, and monitor viral diversity, advancing both fundamental ecological research and applied pathogen surveillance. Through three focused research projects, this work demonstrates how targeted and metagenomic sequencing strategies can address key knowledge gaps in environmental virology, plant pathology, and viral public health surveillance. The first objective (Chapter 2) was to develop and apply novel tiled-amplicon sequencing assays for Influenza A virus (IAV) H3N2 and respiratory syncytial virus (RSV) A in wastewater-based surveillance. These assays successfully recovered near-complete viral genomes from wastewater samples collected during the peak of the 2023/2024 respiratory virus season. Genomic coverage trends mirrored clinical case data within the associated public health region, validating the approach as a complementary tool to traditional clinical surveillance. Variant deconvolution analyses revealed distinct spatiotemporal patterns in variant distribution, demonstrating the capacity of these assays and bioinformatic tools to resolve community-level transmission dynamics and emerging viral lineages. The second objective (Chapter 3) extended this surveillance framework to the agriculturally significant Tomato brown rugose fruit virus (ToBRFV). A multiplexed tiled-amplicon assay was developed, achieving significant improvements in viral genome recovery compared to metagenomic RNA shotgun sequencing while reducing sequencing costs and resource requirements. ToBRFV clades circulating in Ontario wastewater were identified, including variants later recognized through global surveillance initiatives, underscoring the assay’s potential for early detection and the value of wastewater systems as environmental reservoirs for plant viruses. The third objective (Chapter 4) involved the assembly and characterization of the complete genome of a novel freshwater algal virus, Chrysochromulina parva virus BQ1 (CpV-BQ1). Using a hybrid Nanopore and Illumina sequencing strategy, a high-quality 165,454 bp genome was assembled and annotated, revealing hallmark nucleocytoplasmic large DNA virus (NCLDV) genes and diverse functional categories associated with viral replication, host manipulation, and capsid formation. This work offers important insights into viral infection of the ecologically important freshwater algal species C. parva. Moreover, it establishes a methodological framework for the complete sequencing, assembly, and annotation of algal virus genomes. Collectively, this thesis advances environmental virology and viral surveillance systems by developing scalable, sensitive, and cost-effective genomic workflows for virus detection and characterization in freshwater environments. By integrating virus ecology with applied public health and agricultural surveillance within a One Health framework, this work underscores the interconnectedness of environmental, human, and agricultural systems and provides practical guidance for future viral genomics research and pathogen monitoring programs.