Grant: Master's Award: Canada Graduate Scholarships

Long double stranded ribonucleic acid (LdsRNA) is made only by viruses and is not present in healthy cells. For this reason LdsRNA is a pathogen associated molecular pattern that is detected by pattern recognition receptors that initiate innate immune responses. This project seeks to understand how the presence of viral double stranded ribonucleic acid (v-dsRNA) found in the extracellular space and in extracellular vesicles (EVs) of in vitro cells changes throughout a viral infection. The size and quantity of EVs will also be measured. Madin-Darby canine kidney cells will be infected with a PR8 influenza strain, acting as the positive control. The negative control will be rainbow trout gonadal-2 cell infected with Frog Virus 3 to produce dsRNA. Throughout the infection media will be added, collected, and put through deferential ultracentrifugation to remove debris and virus particles. RNase A will remove single stranded RNA (ssRNA) from these supernatants. Complementary DNA and the polymerase chain reaction will amplify the viral sequence. An agarose gel can then show if dsRNA is in the extracellular space. For the controls RNase A and ultracentrifugation will not be used so ssRNA and virus particles will be present. Ultracentrifugation will be used to see if the v-dsRNA is in EVs. A Tunable Resistance Pulse Stimulator will be used to characterize the EVs by size and quantity. Immunoblot analysis with dsRNA antibodies will show the presence of EV-dsRNA. The objective of this project is to better understand how cells communicate with each other using dsRNA and EVs during an infection. This research may assist in the development of early detection of a viral infection, which can lead to faster recoveries and less money spent on treatment and hospitalization.