Since reovirus is sensitive to antiviral and stress signalling, and can loose infectivity by premature proteolysis, the purification method needs to remove cellular contaminants that could affect virus infectivity and introduce confounding effects. A limitation to high-throughput screening of virus mutants is the absence of a productive virus purification strategy that also provides high purity. Our research explores molecular aspects of reovirus replication, and makes routine use of spontaneous or reverse-engineered mutants to manipulate virus phenotypes. Our laboratory sought a straightforward and affordable method to purify many viruses at once. NA, not applicable due to absence of purification.
The choice among these three methods in laboratories is therefore dictated by trade-offs between time savings and productivity versus need for purity.ĢScalable reflects to the ability to efficiently purify virus from very small to very large sample volumes. The pitfalls of equilibrium density ultracentrifugation are that it is time consuming (typically 2–3 days) and restricted in sample number (6 samples per ultracentrifuge). This approach also allows for the separation of complete- from incompletely assembled or genome-devoid (empty) virions. Most typically, gradients of CsCl, sucrose, or iodixanol are used to separate viruses from host contaminants. To achieve ultimate virus purity, the most commonly used method in a laboratory setting is density gradient ultracentrifugation. This straightforward 60-minute step can eliminate many soluble host factors, but suffers from continued contamination with non-viral proteins. Viruses are either pelleted directly, pelleted through a low-density sucrose solution to reduce aggregation, or floated above a high-density sucrose cushion.
An intermediate-purity virus preparation is achieved by adding a high speed ultracentrifugation step. For example, cellular proteases could degrade virus, while cytokines could alter cell signalling and affect susceptibility to viruses. While this approach is quick and permits simultaneous high-throughput comparisons of a large sundry of viruses, it suffers from high contamination with non-virus factors that may affect experimental outcomes ( Table 1). To generate lysates, cells are commonly lysed by freeze-thaw, debris is removed by low speed centrifugation, and supernatants are then used for experimentation. The most rapid approach involves using crude cell lysates or cell culture media from virus-infected cells. Most laboratories use one of three methods to prepare virus for experimentation. 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. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. 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. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. 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. 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. 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. Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents.