Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Stephanie DeWitte-Orr

Advisor Role

Supervisor

Abstract

Canada is one of the largest beef exporters in the world. Viruses like vesicular stomatitis virus (VSV) and bovine respiratory syncytial virus (BRSV) can negatively impact the cattle industry, and a prophylactic treatment is needed to protect against these viruses. Viruses produce long (L)dsRNA (>30-40bp) during replication that is not found in healthy cells. As such, the innate antiviral immune response, including the RNA interference (dsRNAi) pathway, are stimulated by viral LdsRNA. When low concentrations of LdsRNA are present, the RNAi pathway is triggered resulting in the degradation of viral mRNA of matching sequence, halting specific viral protein production. This project studies sequence-specific LdsRNA to block the replication of VSV and BRSV in bovine turbinate (BT) cells via RNAi. Before antiviral effects of dsRNA were examined, cytotoxicity assays were performed to determine cell viability of BT cells following dsRNA treatment. In vitro transcribed dsRNA molecules encoding the nucleotide (N) protein of VSV and BRSV, and the fusion (F) protein in BRSV, or a mismatched dsRNA sequence (mCherry), which served as a negative control, were synthesized and concentrations low enough to avoid an IFN response was determined. Viral titres were quantified 3-6 days post-infection by 50% Tissue Culture Infectious Dose (TCID50) assays to study antiviral effects of dsRNA. Sequence-specific dsRNAs matching their respective virus were able to limit replication of both viruses in BT cells, while dsRNA-mCherry did not, demonstrating a sequence-dependent antiviral response. In addition, a small molecule RISC inhibitor, called aurintricarboxylic (ATA) was used to further investigate antiviral effects of RNAi. dsRNA was found to not show cytotoxic effects with concentrations up to 5000 ng/mL, and IFN stimulation was not seen in concentrations of up to 2000 ng/mL. Antiviral results showed that there was a sequence-dependent knockdown of viral infection and dsRNA concentrations of 500 ng/mL, but addition of ATA did not provide any confirmation of dsRNAi, but ATA doses need to be further explored. This study will contribute to elucidating the dsRNAi pathway in bovine cells. The RNAi pathway is a novel therapeutic platform to treat virus infections in cattle and livestock.

Convocation Year

2025

Convocation Season

Fall

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Available for download on Wednesday, August 26, 2026

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