Novel High-throughput Approach for Purification of Infectious Virions.
Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.
Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
Kevin T
KT
James
GE Healthcare, LifeSciences, Marlborough, MA, US.
Brad
B
Cooney
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
Kate
K
Agopsowicz
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Mary Ann
MA
Trevors
Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
Adil
A
Mohamed
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
Don
D
Stoltz
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
Mary
M
Hitt
Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada.
Maya
M
Shmulevitz
D000256:Adenoviridae / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods*; D008854:Microscopy, Electron; D012087:Reoviridae / Q000302:isolation & purification; D012117:Resins, Synthetic / Q000378:metabolism*; D014771:Virion / Q000302:isolation & purification*
D012117:Resins, Synthetic