Field-flow fractionation of alkali-liberated nuclear polyhedrosis virus from gypsy moth Lymantria dispar Linnaeus.

Rod-shaped viral particles of the gypsy moth nuclear polyhedrosis virus (NPV) were obtained through alkaline dissolution of inclusion bodies isolated and purified from infected hosts. The liberated viral fraction contains a wide assortment of enveloped aggregated forms as well as enveloped monomers. This complex mixture was separated by means of sedimentation field-flow fractionation (sedimentation FFF) to give a mass spectrum of the separated particles. From the elution pattern effective molecular weights could be assigned to the various resolved components. Through electron microscopy it was possible to characterize the components as monomers, dimers, etc. In parallel experiments the same viral mixture was separated by the more commonly used density gradient sedimentation technique. The two methods are compared as to time and convenience of manipulation, as well as to the amount of physicochemical information that can be extracted from each separation. The mixture of aggregate viral structures was dialyzed against a nonionic detergent solution. Sedimentation FFF of the dialysate revealed a uniform population of non-enveloped particles with no effective molecular weight which was lower than that of the initial monomer fraction. Electron microscopic observations confirmed that this lower molecular weight fraction did indeed contain rod-like structures which were thinner than the initial enveloped monomers indicating that most if not all of the enveloped material had been removed from the viral rod structures.

Determination of the mass of viruses by quantitative electron microscopy.

The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.

Mass purification of nucleopolyhedrosis virus inclusion bodies in the K-series centrifuge.

Nucleopolyhedrosis virus inclusion bodies specific for Hemerocampa pseudotsugata, Neodiprion sertifer, Porthetria dispar, and Heliothis zea have been purified by using a continuous-sample-flow-with-isopycnic-banding centrifuge in quantities up to 6 x 10(13) polyhedral inclusion bodies per day. Continuous-flow methods for S-rho type purification have been evolved to deal with mass isolation of bioparticles.

Comparative properties of the inclusion body proteins of the nucleopolyhedrosis viruses of Neodiprion sertifer and Lymantria dispar.

A comparison was made on the properties of the inclusion body proteins of two insect viruses: the nucleopolyhedrosis viruses of the European pine sawfly, Neodiprion sertifer, Geoffroy, and the gypsy moth, Lymantria dispar, Linnaeus. The inclusion body proteins were characterized by the following parameters: amino acid composition, polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate--mercaptoethanol, isoelectric focusing, and alkaline protease activity. The properties of the inclusion body proteins of the two viruses were similar in many respects, but clear differences were observed. A principal difference was the absence of alkaline protease activity associated with the inclusion body proteins of N. sertifer nucleopolyhedrosis virus.