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. 2008 Feb;147(2):328-32.
doi: 10.1016/j.jviromet.2007.09.013. Epub 2007 Oct 29.

Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient

E Gias  1 , S U Nielsen, L A F Morgan, G L Toms
Affiliations

Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient

E Gias et al. J Virol Methods. 2008 Feb.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Purification of PEG-concentrated hRSV strain R17532 on discontinuous iodixanol gradient. The amount of viral F glycoprotein in gradient fractions was determined by ELISA. The peak fraction 8 was diluted 1:2 in NT buffer containing MgSO4 and layered on a continuous iodixanol gradient.
Fig. 2
Fig. 2
Purification of hRSV strain R17532 on continuous iodixanol gradient. The density (g/ml, Δ), amount of hRSV genome by real-time PCR (formula image) and F glycoprotein by ELISA (●) was determined across the fractions. The infectivity titres (PFU/ml, x) were determined for fractions 5–9 only.
See this image and copyright information in PMC

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