Purification of Herpes Simplex Virus by Chromatography of Calcium Phosphate

Abstract

THE chemical and immunological analysis of herpes simplex virus cannot be undertaken until the virus has been purified without being disrupted or denatured. The purification of virus suspensions of strain HFEM grown in eggs¹ or in HeLa cell cultures² was therefore attempted by the method of Taverne, Marshall and Fulton³ in which virus suspensions are passed down columns of calcium phosphate at room temperature and the adsorbed material then eluted with increasing concentrations of phosphate buffer at pH 7.0. The technique used here differed in two minor details. First, before chromatography, phosphate buffer was added to the virus suspensions to a final concentration of 0.2 M ; this reduced adsorption of protein but not of virus. Secondly, the eluting buffers usually contained 0.1 per cent of bovine serum albumin (Armour fraction V) to reduce loss of virus infectivity. (Albumin did not adsorb in phosphate concentrations greater than 0.2 M.) Infective virus was titrated by pock counting, and it was shown that the infectivity titre did not drop when the virus was held in 1.0 M phosphate for 15 min. at room temperature. Complement-fixing activity was measured by the method of Fulton and Dumbell⁴ using overnight fixation at 4° C. and human immune serum. Protein was estimated colorimetrically⁵.