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

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 MgSO4 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.

Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2

“…To exclude the possibility that contaminating products in the Vero cell-propagated RSV activated TLR2 signaling, sucrose-purified RSV and UV-inactivated purified RSV (a generous gift from Trudy Morrison) were used to stimulate wildtype macrophages. RSV was purified using methods previously described (17,26). Purified RSV contained approximately fivefold higher viral protein concentrations than unpurified RSV based on Western blot analysis (data not shown).…”

Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

“…RSV strain A2 (ATCC VR-1540) was propagated in Hep-2 cells by infecting a freshly prepared confluent monolayer grown in MEM supplemented with 2% FCS. When the cytopathic effect involved the whole monolayer, the infected-cell suspension was collected and the clarified viral supernatant was then adjusted to 100 mM MgSO 4 and 50 mM Tris-HCl (pH 7.5) to stabilize the viruses as previously reported (17). The virus stocks were aliquoted and stored at Ϫ80°C.…”

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

“…Ultracentrifugation of viruses can be carried out in different density gradients like caesium chloride [21], sucrose [22] or iodixanol [23,24]. In general the method enables efficient purification and concentration of virus particles in one step, but requires expensive equipment, process times are long (with subsequent step of density gradient material removal by dialysis or size exclusion chromatography) and sometimes several cycles are needed [21,25] Although new generation chromatography media are very efficient, chromatography based purification of viruses intended for human use still represents only a part of the virus downstream processing (DSP) scheme which usually consists of several purification steps and needs to deliver a virus preparation of high purity and efficacy (e.g.…”

“…In addition, some density gradients are not suitable for certain viruses. For example, high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus[24] leading to loss of virus infectivity and unsatisfactory infectious virus yields[25]. Another problem is extensive virus aggregation during gradient ultracentrifugation which was observed by Peng et al[23] when trying to purify recombinant adeno vectors in CsCl density gradient.Selective precipitation uses different chemical agents like ammonium sulfate and polyethylene glycol[25] to precipitate either the virus or the impurities.…”