Cereal Chlorotic Mottle Virus?Purification, Serology and Electron Microscopy in Plant and Insect Tissues

Abstract

Cereal chlorotic mottle virus (CCMV) accumulated in the perinuclear space and cytoplasmic vesicles of infected plant [Dinebra retroflexa] cells. The virus was present in both vascular and mesophyll tissues. In infected leafhoppers Nesoclutha pallida (Evans) the virus particles had a similar appearance and intracellular location but sometimes appeared to be in a granular matrix and were dispersed throughout some degenerated nuclei. The virions (63 by 230 nm), when viewed in cross-section, showed 17 densely staining points around the circumference and had 3 concentric ring structures but no stained axial core. Tangential sections confirmed the hexamer structure shown in negative stain. CCMV was purified by extraction in tris-HCl buffer and concentrated on a discontinuous sucrose gradient before filtering through a celite pad. The filtrate was pelleted and then given 2 sucrose gradient separations before the final pelleting. Purified virus was used to prepare an antiserum which had a titer of 128 and no detectable host reaction. In gel-diffusion tests with the homologous antiserum, CCMV produced 2 major reaction lines, with an additional line present in some tests.