Env Exp Biol (2012) 10: 117–123

Comparison of ultracentrifugation methods for concentration of recombinant alphaviruses: sucrose and iodixanol cushions

Abstract

Alphavirus vectors have been successfully used as a gene delivery system in animal models. A prerequisite for the efficient expression of heterologous genes is the application of high doses of recombinant viruses. The currently available methods for viral concentration are laborious, costly and often require specialised equipment. Here, we provide a comparative study of three ultracentrifugation methods used for alphavirus concentration: 20% sucrose cushion, double (20 and 50%) sucrose cushion, and double (20 and 50%) iodixanol cushion. The yield of concentrated recombinant Semliki forest virus from each method was evaluated by determination of the viral infectivity in cell culture and genome copy number as quantified using real-time PCR. The best results were obtained using ultracentrifugation on a double iodixanol cushion, a method that generated a high recovery yield (approximately 40%) and a four-fold increase in virus concentration. The methods of concentration on sucrose appeared less satisfactory. Isosmotic iodixanol-containing virus preparations did not affect alphavirus infectivity and can be used directly for in vivo applications.