Virome distribution of aquatic ecosystems impacted by anthropogenic activities in urban Manitoba

Municipal wastewater effluents discharged into the Red and Assiniboine Rivers of Winnipeg may not be fully treated as traditional methods that monitor the microbial quality of wastewater focus solely on the detection of fecal indicator bacteria. There is also a lack of current wastewater system effluent regulations at the federal and provincial level. Since viral DNA and RNA sequences in current genomic databases are classified as unknown, the objective of this thesis is to characterize viral DNA and RNA community structures using metagenomics and quantitative-PCR, for the purpose of establishing the virome distribution in aquatic environment’s receiving wastewater discharge. Environmental water samples were collected at 11 locations along the Red and Assiniboine rivers during the Spring, Summer and Fall 2021. Samples were filtered and underwent skimmed milk flocculation for viral concentration. Total nucleic acids were extracted, separated into half and enriched enzymatically for viral DNA and RNA to carry out culture independent approaches. Kraken 2 taxonomic sequence classification system and MG-RAST metagenomic analysis server were used to identify the taxonomic classification and functional potential of assembled reads as DNA and RNA viruses. The taxonomic classification of DNA viruses identified from the RefSeq database (available from MG-RAST) and Kraken 2 Viral Genome database were predominately DNA bacteriophages (Myoviridae, Podoviridae and Siphoviridae) belonging to the order Caudovirales which accounted for approximately 90 % of each aquatic sample location along the Red and Assiniboine rivers. Furthermore, phage related functionalities such as phage tail fiber proteins, phage replication, and phage packaging machinery accounted for 40 % of each aquatic samples collected which possibly correspond to the DNA phages that were previously identified. RNA phages (Cystoviridae and Leviviridae) were identified to a lesser extent accounting for approximately 3 % of each aquatic sample collected while other viruses such as Virgaviridae, Retroviridae, Picobirnaviridae and Partiviridae accounted for 7%–100% of aquatic samples. The functionalities of RNA viruses were primarily related to metabolic pathways such as potassium homeostasis, respiratory complexes and sialic acid metabolism, essential for RNA viruses to survive in their host.

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