Macromolecular Component of Chick Embryo Tissue Diseased with Western Strain Equine Encephalomyelitis Virus. ∗

Abstract

A macromolecular component has been isolated 1 by ultracentrifu-gation from chick embryo tissue diseased with the Eastern strain of equine encephalomyelitis virus (E.S.). This protein, within the limits of tests thus far made, behaves as the virus and is specific 2 to the virus-diseased embryo tissue, though it is separable with difficulty from the lighter normal tissue component with s₂₀° = ca 70 × 10⁻¹³ cm sec⁻¹ dynes⁻¹. 3 In the present paper are described ultracentri-fugal studies of extracts of chick embryo tissue diseased with Western strain virus (W.S.) from which a macromolecular protein possessing the properties of this strain has been isolated.

In a typical experiment, diseased tissue was extracted 24 hours at about 5°C in 4 times its volume of 0.15 M NaCl solution made to pH 8.5 with NHiOH. Cleared of tissue debris by angle centrifugation, the extract was ultracentrifuged in 8 15-cc tubes at 67,000 g for 30 minutes. The 8 pellets were taken up in 60 cc water, pH 8.5 with NH₄OH, and the resulting suspension was spun in 4 tubes at 6000 g for 5 minutes. The supernatant fluid was then spun in 4 tubes at 17,000 g for 30 minutes. A specimen from the 4 pellets was dissolved in 0.2 M NaCl solution adjusted to pH 9.0 with NH₄OH for examination in the analytical ultracentrifuge (Fig. 1), and the remainder was taken up again in 60 cc water for repetition of the cycle of 6000 g and 17,000 g. The final pellets were dissolved in 0.2 M NaCl solution for ultracentrifugal analysis (Fig. 2).

The sedimentation diagram after the second cycle showed the Indistinct, diffuse boundary (Fig. 1, a) of persisting s₂₀° — ca 70 × 10⁻¹³ and the more prominent, somewhat diffuse boundary (Fig. 1, b) of a heavier material.