Effect of Different Gradient Solutions on the Buoyant Density of Scrapie Infectivity

Abstract

Summary

Scrapie-infected mouse brain was passed through a fine mesh sieve, subjected to 900 psi, and centrifuged at 3500g. The supernate was passed through 450 nm and 220 nm Millipore filters, and centrifuged at 152,000g. The pellet was resuspended to 1/5th the original volume, sonicated, and applied to cesium chloride, sucrose, and me-trizamide gradients which were simultaneously centrifuged at 152,000g for 16 hr.

Analysis of gradient fractions showed peak infectivity at a density of 1.22 g/ml in cesium chloride, 1.21 g/ml in sucrose, and 1.17 g/ml in metrizamide, with 90-95% total infectivity recovered in a well-defined band bracketing each peak titer fraction. No infectivity was observed at densities higher than 1.33 g/ml in cesium chloride, or 1.30 g/ml in sucrose and metrizamide. In each gradient the area of greatest infectivity coincided with the maximum concentration of cellular membrane vesicles and fragments, and contained between 2 and 6% of the total infective activity present in the original brain suspension. Peak infectivity fractions could be rapidly identified by spectrophotometric scanning at a wavelength of 320 nm.

We conclude that the variability in estimates of the density of scrapie infectivity observed in earlier experiments has resulted from differences in the preparation of starting material, and that density gradients composed of solutions of widely different viscosities and dipole moments do not effect the buoyant density of membrane-associated scrapie virus.