Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient.
Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO(4) as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20-36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20-52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20-52% continuous gradient, the virus was recovered in the region of density 1.15-1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.
Gias
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The School of Clinical Medical Sciences, The Medical School, The University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom. ednagias@hotmail.co.uk
Nielsen
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Morgan
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Toms
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D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018357:Respiratory Syncytial Virus Infections / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000302:isolation & purification*; D014283:Triiodobenzoic Acids
D014283:Triiodobenzoic Acids; C044834:iodixanol