Novel High-throughput Approach for Purification of Infectious Virions.

Scientific reports (2016-11-09)
Kevin T James, Brad Cooney, Kate Agopsowicz, Mary Ann Trevors, Adil Mohamed, Don Stoltz, Mary Hitt, Maya Shmulevitz
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Antibiotic Antimycotic Solution (100×), Stabilized, with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Minimum Essential Medium Eagle, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
SAFC
MEM Non-essential Amino Acid Solution (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Minimum Essential Medium Eagle, Joklik Modification, with L-glutamine, without calcium chloride and sodium bicarbonate, suitable for cell culture