Partial purification of lettuce necrotic yellows virus

Abstract

Lettuce necrotic yellows virus was partially purified by clarifying leaf extracts with the fluorocarbon Freon-113, followed by differential centrifugation and rate zonal contrifugation in sucrose density gradients. Adding either chelating or reducing substances to the extraction medium of 0.1 M phosphate buffer (pH 7.4) did not increase infectivity; clarifying extracts with acid, butanol, or chloroform greatly decreased or abolished it. Emulsifying extracts with Freon gave clearer, more infective, preparations than treating them with hydrated calcium phosphate gel. Infectivity decreased with increasing number of Freon treatments, more infectivity was lost when the Freon phase solidified than when it did not, and losses increased with increasing time or vigour of emulsification. Infective material sedimented in 10 minutes at 40,000 g or 40 minutes at 10,000 g. After density-gradient centrifugation of preparations from lettuce, infectivity was associated with one light-scattering band specific to preparations from diseased plants; with preparations of one virus isolate from Nicoliana glutinosa, infectivity was associated with two light-scattering bands. The final virus preparations scattered light strongly; those from lettuce were almost colourless, and those from N. glutinosa pale green. They infected N. glutinosa when diluted to 10 −5 but not at 10 −6.