Claire Fernandes, University of Toronto

Claire Fernandes1,2, Nhien Dinh1, Deepa Chaphekar1,2, Arvin Tejnarine Persaud1,2, Aiman Farheen1, Vera A Tang3, and Christina Guzzo1,2,4 1. Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada 2. Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada 3. Faculty of Medicine, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Flow Cytometry and Virometry Core Facility, Ottawa, Ontario, Canada 4. Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, ON, Canada.

Discovering & Evolving

Virus particles display a highly diverse array of surface proteins. Our lab has shown that these unique protein profiles impact viral transmission and homing in vivo. However, it is extremely challenging to use conventional lab techniques to isolate phenotypically distinct virus subpopulations that remain functional for downstream experimental analyses. Therefore, the lack of high-throughput methodology capable of sorting viable particles remains a critical barrier to understanding the impact of virus heterogeneity on transmission and disease. Herein, we demonstrate high yield sorting of virus particles built on the principles of quantitative Flow Virometry (FV) our lab has developed for investigating human proteins on the Human Immunodeficiency Virus (HIV). To evaluate the reliability of this novel strategy, we first employed a biological reference particle to standardize measurements acquired by a range of commercially available flow-cytometry analyzers and compared data to a small particle sorter. In a second set of experiments, we optimized pre- and post- sort parameters to successfully purify HIV pseudoparticles and intact infectious virions. Importantly, we demonstrated that sorted particles remained viable for downstream analyses of viral proteins, viral genomes, and infectivity assays. To our knowledge, this is the first study to achieve high-yield purification of HIV particles via standardized cytometry-based sorting methods. These novel virus sorting techniques will provide enhanced tools to study virus heterogeneity and the unique phenotypes of specific virus subpopulations. This work will open new avenues of research targeted at studying single virus subpopulations, discerning their contributions to disease progression and viral spread.