—13 — The Nature of Nuclear-Polyhedrosis Viruses GERNOT H. BERGOLD Instituto Venezolano de Investigaciones Cientificas (I.V.I.C.), Departamento de Virologia, Apartado 1827, Caracas, Venezuela I. Introduction 413 II. Definition 414 III. Preparation and Purification of Polyhedra and Viruses .. 414 A. Preparation and Purification of Polyhedra 414 B. Preparation and Purification of Virus Particles 415 IV. Structure of Polyhedra and Morphology of Viruses 417 A. Size and Structure of Polyhedra 417 B. Morphology and Size of Viruses 418 V. Physicochemical Properties and Chemical Composition of Polyhedra 432 A. Physicochemical Properties 432 B. Chemical Composition 435 VI. Chemical Composition of Virus Particles 438 VII. Serological Properties and Relationship of Nuclear- Polyhedron Proteins, Nuclear-Polyhedrosis Viruses, and Insect Hosts 444 A. Serological Properties of Nuclear-Polyhedron Proteins 444 B. Serological Properties of Nuclear-Polyhedrosis Viruses 445 C. Serological Relationship between Nuclear-Polyhedron Proteins and Nuclear-Polyhedrosis Viruses 446 VIII. Taxonomy of Nuclear-Polyhedrosis Viruses 447 IX. List of Nuclear-Polyhedrosis Viruses 448 References 450 I. INTRODUCTION Representatives of the nuclear-polyhedrosis viruses were the first insect viruses investigated. The reason for this is that this group of insect viruses is characterized by the presence of inclusion bodies, the so-called 413 414 GERNOT Η. BERGOLD polyhedra, in infected insects. These polyhedra can be readily seen with the light microscope. Maestri (1856) and Cornalia (1856) were the first to describe them. The nature and significance of these poly­ hedra remained a mystery for a long time and is still not entirely under­ stood. Von Prowazek (1907, 1912, 1913) was probably the first to observe particles within the polyhedra. Komärek and Breindl (1924) were the first who actually demonstrated with histological methods some particles within the polyhedra of Lymantria monacha (Linnaeus). Paillot and Gratia (1939) observed, in the dark field of a light microscope, during the alkaline dissolution of polyhedra, very small highly refractive gran­ ules which they believed to be the infectious virus agent. However, it was not until the electron microscope was available that the virus parti­ cles could be isolated and identified as the infectious viral agent by Bergold (1947). II. DEFINITION According to our present-day knowledge, typical nuclear-polyhedrosis viruses occur only in Lepidoptera, in the family of Tenthredinidae of Hymenoptera, and in a species of Neuroptera. The polyhedrosis virus of Bombyx mori (Linnaeus) and Peridroma saucia (Hübner) have been successfully grown in insect tissue culture (Trager, 1935; Aizawa and Vago, 1959; Martignoni and Scallion, 1961). In the following, the word ''polyhedra'' will designate inclusion bodies found in the nucleus of cells of insects as a result of virus infection. The term "polyhedrosis virus" will be used for the infectious agent which causes nuclear polyhedroses in insects. Synonymous for the term "polyhedrosis/' which was first proposed by Fischer (1906), is Poly­ ederkrankheit suggested by Wahl (1909), which is sometimes used in German-speaking countries. Accordingly, Polyeder Virus is synonymous with polyhedrosis virus. In Italy, the terms giallume and poliedria, and in France the terms grasserie, jaunisse, and vir ose are used. III. PREPARATION AND PURIFICATION OF POLYHEDRA AND VIRUSES A. Preparation and Purification of Polyhedra Bolle (1873, 1893) found first that polyhedra are insoluble in water. Furthermore, they are not decomposed by ordinary bacterial putrefac­ tion processes. For these reasons, it is comparatively easy to obtain gram quantities—up to 10 mg per larva—in a very pure form by differential centrifugation. All that is necessary is to suspend virus-diseased insects in water, allow the polyhedra to settle to the bottom, and purify them further by centrifugation. One has to watch, however, that the pH of the medium in which the polyhedra are suspended never drops below 13. NATURE OF NUCLEAR-POLYHEDROSIS VIRUSES 415 prL 5, or rises above pH 8.5 to prevent possible dissolution of the poly­ hedra. In some cases, treatment with fluorocarbons of suspensions of insects containing smaller quantities of polyhedra (Bergold, 1959a) is helpful for the purification process. A more rapid method of obtaining quantities of polyhedra in very pure form is to use only fresh hemolymph taken from virus-diseased larvae before their death. Such a hemolymph is of a milky appearance; its purification is very simple if carried out the next day. Polyhedra can also be purified readily from infected pupae (Vago and Atger, 1961). Because of their rather unusual physicochemical properties, polyhedra can be stored in form of a dry powder or in flame- sealed glass tubes for as long as twenty years without significant changes in their solubility as well as without loss of infectivity of the enclosed virus particles (Aizawa, 1953, 1954a; Steinhaus, 1960a). B. Preparation and Purification of Virus Particles 1. Principal Considerations Virus particles characteristic of nuclear polyhedroses, as well as of those causing granuloses (see Chapter 16), are by far the easiest of all known viruses to purify. There are several reasons for this: first, the starting material consists of water-insoluble inclusion bodies which them­ selves can be obtained in a very pure form. Second, as will be shown in Section IV, B, 2, except for their own protein, polyhedra contain nothing but virus particles. Third, the size and weight of the virus particles is so much greater than that of the polyhedron-protein mole­ cules that they can easily be separated by centrifugation. For liberation of the virus particles, the polyhedra must be dissolved in weak alkaline solutions. The concentration of alkali necessary to dissolve the polyhedra of different insects is not the same, and must first be determined. To prevent destruction of the virus particles, it is very important not to use too much alkali. There is a definite relation­ ship between the quantity of polyhedra, the volume of liquid in which they are suspended, and the amount of alkali used to dissolve them. The alkali concentration has to be just high enough to dissolve all polyhedral bodies, but not so great as to destroy the virus particles. Rather high concentrations of alkali are necessary, but it is not advisable to use buffers because if all polyhedra are dissolved, the pH will have dropped from an initial value of over 10 to about 8.5 at the most. If the final pH is higher than 8.5, then many virus particles will have lost their infectivity. This can easily be discerned by the complete change of their morphological appearance (see Section IV, Β, 1). However, in every virus suspension from polyhedra, even when prepared most care­ fully, there is a certain number of particles that have shed their develop- 416 GERNOT Η. BERGOLD mental membrane; therefore, one can always find several empty develop­ mental membranes in such preparations. The principal methods and detailed procedure for the isolation and purification of any insect virus were first described by Bergold (1947). Since then, this method for the isolation of nuclear polyhedrosis viruses is followed in principle, or with only minor changes, by virtually all insect virologists. 2. Detailed Procedure Five milligrams of polyhedra are suspended in 1 ml of 0.004 to 0.03 Μ Na2C03 plus 0.05 Μ NaCl. The polyhedra should dissolve at room temperature in about 1 to 2 hours with occasional or continuous gentle shaking. After the suspension has become somewhat opaque, it is centrifuged for about 5 minutes at about 2000 to 4000 g. If the polyhedra were not very pure, a small brownish sediment of insoluble material consisting of impurities will result. The supernatant has a bluish-white appearance, caused by the virus particles suspended in the clear poly­ hedron protein solution. If part of the sediment is white, then the alkali concentration was not high enough to dissolve all polyhedra. The super­ natant is then centrifuged for about 1 hour at 10,000 to 12,000 g in order to sediment the virus particles. These will collect in a bluish-white pellet. After discarding the supernatant, the virus pellet is resuspended in C02-free distilled water of the same volume, and centrifuged for another hour at about 10,000 to 12,000 g. The resulting clear super­ natant is discarded, and the final pellet of virus particles dissolved in one-seventh of the original volume in C02-free distilled water. The resulting suspension of pure virus particles should have a whitish-blue appearance. If the virus particles do not suspend well, then too much of the water-insoluble polyhedron protein has been carried over, resulting in precipitation. The addition of extremely small amounts of alkali will resuspend the virus particles. However, this will often cause destruction of many virus particles with consequent loss of infectivity. If the virus particles are washed further with distilled water, they become sticky and suspend less readily in distilled water. Addition of microliter amounts of 0.01 Μ Na2C03 will dissolve the polyhedron protein again and will readily resuspend the virus particles. By these methods of liberation and purification of virus particles, one can obtain about 1 to 2 mg of highly purified virus particles from about 35 mg of polyhedra (Bergold, 1947). This corresponds to about 3 to 5 percent of the weight of polyhedra. The yield depends on the species of insect from which the polyhedra were harvested. 13. NATURE OF NUCLEAR-POLYHEDROSIS VIRUSES 417 IV. STRUCTURE OF POLYHEDRA AND MORPHOLOGY OF VIRUSES A. Size and Structure of Polyhedra The size and shape of nuclear polyhedra varies considerably not only between polyhedra from different insects, but often also within polyhedra of the same species. They crystallize as dodecahedra, tetra- hedra, cubes, or forms irregular in appearance. In some species of insects, like the silkworm, B. mori, the prevailing types of polyhedra are dodec­ ahedra, whereas those of L.