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

dsRNA interference (dsRNAi) is an antiviral innate immune response that uses long dsRNA (>40bp) to inhibit gene expression through the RNA interference (RNAi) pathway. dsRNAi is an important antiviral defense mechanism in plants, invertebrates, and fungi. However, its use as an antiviral mechanism in vertebrates is not well understood. Evidence proving the significance of dsRNAi in vertebrate cells does exist, but there is still a lack of research in this area, particularly with the use of long dsRNA. Respiratory Syncytial Virus (RSV) and human coronaviruses (HCoVs) are two single stranded RNA (ssRNA) viruses that infect airway cells. This study aimed to investigate the effectiveness of the dsRNAi pathway as an antiviral response against both RSV and HCoVs using a single dsRNA molecule in human lung fibroblast (HEL-299) cells. Before exploring the effects of dsRNAi, the growing conditions of RSV expressing GFP (RSV-GFP) were optimized in HEL-299 cells. Cell viability and replication kinetic experiments demonstrated that HEL-299 cells are permissive to RSV. To induce the dsRNAi response, in vitro transcribed dsRNA coding for RSV and HCoV gene sequences were synthesized and the optimal concentrations of dsRNA too low to induce the IFN response were determined. Using TCID50 assays to quantify viral titres, it was found that soaking cells with sequence-specific dsRNA could significantly inhibit both RSV and HCoV replication. Additionally, multi-target dsRNA molecules containing sequences from both RSV and HCoV did mediate knockdown of both viruses, with effects comparable to the single-gene target dsRNAs. The results of this study provide fundamental insights into the dsRNAi response, which will be used to improve long dsRNA design and maximize dsRNAi efficiency. Further, this could aid in the development of dsRNAi-based therapeutics to improve human health.

Convocation Year

2024

Convocation Season

Fall

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Available for download on Sunday, August 16, 2026

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