Purification and Characterization of the Platynota idaeusalis Baculovirus Pathogenic to the Tufted Apple Bud Moth

Abstract

The polyhedral occlusion bodies (OBs) of a nuclear polyhedrosis virus were purified from larvae of the tufted apple bud moth Platynota idaeusalis. Electron micrographs of OB thin sections revealed a number of enveloped virions containing one or two rod-shaped nucleocapsids. However, fractionation of alkaline buffer released virions on sucrose gradients (10-55%, w/w) showed four closely spaced bands indicative of discrete sets of virions containing from one to four nucleocapsids per envelope and a considerably lighter band (1.084 g/ml) probably consisting of envelope material. Purified undegraded polyhedrin, which has a molecular weight of 31.7 kDa as determined by 12% SDS-PAGE, was assayed by immunodiffusion with antisera (polyclonal and monoclonal antibodies) specific to Heliothis zea SNPV (HzSNPV) polyhedrin. Precipitin bands were evident between the polyclonal antibody and the polyhedrins of P. idaeusalis MNPV (PiMNPV), HzSNPV, and Autographa californica MNPV. The type 1 reaction observed among these bands indicated a close serological relatedness of PiMNPV to baculovirus subgroup A. However, a monoclonal antibody (1003) could be used to differentiate the polyhedrin of PiMNPV from the other polyhedrin antigens. The viral DNA at a density of 1.686-1.707 g/ml was purified from 1.695 g/ml (w/w) CsCl gradient. Genetic heterogeneity of the virus was confirmed by the appearance of submolar bands following restriction endonuclease analysis. The genome size was estimated to be 79 × 10 6 Da (121.6 kb). The LD 50, determined by a dose mortality assay of 8-day-old larvae, was 334 OBs with a 95% confidence interval from 269 to 408 OBs.