Assay and Purification of Coxsackievirus B3
Schnurr, Michael J.
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The problem. Which of the available methodologies for the purification for Coxsackie virus B3 (CB3), a Picornavirus provide the highest degree of purity with the greatest yield. The procedure. Quantitative methods for the assay and purification of CB3 were developed. CB3 was propagated in buffalo green monkey (BGM) cells in tissue culture. The virus was released from the infected BGM cells by various methods. Purification methods included precipitations, differential, isopycnic, and rate zonal centrifugation. Virus purity was assessed by SDS-polyacrylamide gel electrophoresis. Findings: The plaque assay for CB3 was found to be reproducible and quantitative. The dose response curve of the assay was linear between 5 and 800 plaques on a 55 cm2 tissue culture dish. Maximum yields of CB3 were obtained from infected cells by treatment with sodium dodecyl sulfate (SDS). The overall yield of CB3 purified by isopycnic banding in cesium chloride was approximately 40% of the virus in the initial cell lysate. As judged by polyacrylamide gel electrophoresis in SDS, the purified virus, following rate zonal centrifugation in sucrose, was greater than 90% homogeneous. Conclusiom. The virus yields and purity were suitable for the study of virus-cell adsorption interactions. Recommendations: Further studies should be done using these techniques in an attempt to increase the amount of protein and virus applied to isopycnic and rate zonal gradients.
[vi], 74 leaves. Advisor: James C. Johnson