The Arthropod Viruses

THE ARTHROPOD VIRUSES Kenneth M. Smith Agricultural Research Council, Virus Research Unit, Combridge, England

1. Introduction . . . . . . . . . . . 11. Types of Virus Diseases . . . . . . . . 111. Distribution within the Arthropods . . . . . IV. Pathology . . . . . . . . . . A. External Symptoms . . . . . . . . B. Internal Symptoms . . . . . . . . . V. The Inclusion Bodies . . . . . . . . . VI. Virus Morphology, Ultrastructure, and Replication . VII. Methods of Transmission . . . . . . . A. Natural Dissemination . . . . . . . B. Artifical Methods of Transmission . . . . VIII. Cross-Transmission . . . . . . . . . IX. Latent Virus Infections . . . . . . . . X. Isolation and Purification . . . . . . . . XI. Chemical Composition . . . . . . . . XII. Serology . . . . . . . . . . . . XIII. Tissue Culture . . . . . . . . . . . XIV. Viruses Multiplying in Both Arthropods and Plants . . . . . XV. Interference between Viruses . References . . . . . . . . . . .

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I. INTRODUCTION In view of the rapid advances made in recent years in the study of viruses in general, the time seems appropriate for some description of these advances so far as the arthropod viruses are concerned. Rickettsiae, which have been described in the larvae of Tipula paludosa Meigen ( Diptera ) and Melolontha melolontha Linnaeus ( Coleoptera) , are not included. There have been two recent reviews of the viruses affecting insects (Bergold, 1958; Smith, 1959) so that emphasis is laid on developments since those dates. The virus diseases of insects have been known for many years but it is only during the last decade or so that intensive research has been carried on. At first only the inclusion-body diseases-polyhedroseswere known and studied, mainly because of the ease of diagnosis by means of the polyhedra using the optical microscope. The invention of the electron microscope has, of course, greatly enlarged the scope 195

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of virus research in general and has enabled several noninclusion diseases, comparable to the virus diseases of plants and the higher animals, to be discovered in the arthropods. The apparent distribution of viruses among the Arthropoda is very uneven, and is probably mainly an expression of our lack of knowledge. With increasing investigation viruses are being found in orders of insects, such as the Neuroptera, in which none had been previously described, and outside the Insecta altogether, as in the red spiders, or spider mites, belonging to the Arachnida. Furthermore, cross-transmission experiments have shown that virus diseases can be induced artificially in insect orders in which no naturally occurring virus diseases have yet been found. These experiments prove the old conception that all insect viruses are highly specific in their action to be erroneous. The new methods of “staining” for electron microscopy, particularly the negative staining with phosphotungstic acid (PTA) ( Brenner and Home, 1959) have added greatly to our knowledge of the morphology and ultrastructure of viruses. They also suggest that the method of replication of the insect viruses is more in line with the process of assembly characteristic of viruses from other groups rather than the “life cycle” with developmental stages suggested by some workers. The methods of isolation and purification have been improved and already two or three of the cytoplasmic viruses affecting insects have been obtained in crystalline form. Two lines of approach to the study of arthropod viruses which have not made the same rate of progress as other techniques are tissue culture and serology but, as will be indicated later, work on these two aspects is now being carried on. One of the controversial problems connected with all viruses is that of classification and nomenclature, and there is no doubt that some kind of classification is sorely needed as much with the arthropod viruses as with the viruses of plants and the higher animals. Some attempt has been made to apply the binomial system to the insect viruses, and a few genera have been erected such as Borrelinu, Bergoldia, Smithia, and so forth. The real difficulty in developing a classification of the viruses attacking the arthropods is the lack of knowledge concerning the fundamental properties of the viruses. Since morphology of the particles seems unlikely to be of much help, many of the viruses from the cytoplasmic polyhedroses appear identical under the electron microscope (and may actually be so), it will be necessary to obtain exact information on the chemical and physical properties of the viruses together with their serological relationships before a stable and lasting classification can be obtained.

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11. TYPESOF VIRUS DISEASES

The virus diseases affecting the arthropods can be arbitrarily divided into two groups, the inclusion-body diseases and those without intracellular inclusions. The first group consists of two sharply distinct types, those with polyhedral, many-sided crystals in which the virus is occluded in the affected tissues, known as polyhedroses, and those with very small crystals-granules-in the tissues, known as granuloses. These granules usually contain a single virus rod compared to hundreds in the polyhedra. The polyhedroses are divided again into two groups, the nuclear polyh,edroses in which the rod-shaped viruses multiply in the cell nuclei, and the cytoplasmic polyhedroses in which the nearspherical viruses multiply in the cytoplasm. These rather peculiar inclusion bodies, the polyhedra and granules, seem to occur only in the virus diseases of insects. The second group of diseases has no polyhedra or granules and the viruses occur freely in the tissues as in the virus diseases of plants and the higher animals. So far, few examples of this type of virus disease have been discovered, probably because of the greater difficulty of diagnosis. About five altogether are known, one in the Diptera, one in the Lepidoptera, one in the Coleoptera, and two in the mites (arachnids). In each of these five cases the virus has been characterized under the electron microscope; there are one or two others in this category, such as bee paralysis and the inherited virus of Drosophila, in which the causative agent has not been isolated. This latter disease has recently been reviewed comprehensively by L’Heritier (1958) and so will not be dealt with here. 111. DISTRIBUTION WITHIN

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ARTHROPODA

The distribution of virus diseases among the arthropods, as known at present, is very uneven. As previously pointed out, this is probably a reflection of lack of knowledge rather than a genuine restriction. By far the greatest number of virus infections, probably more than a hundred, have been recorded from the Lepidoptera; of these the most numerous are the nuclear and cytoplasmic polyhedroses. A number of grandoses have also been recorded; so far this type of virus disease has only been found in the Lepidoptera. In addition one free virus without inclusions is known. In the Hymenoptera several polyhedroses of the nuclear type have been described, and two viruses affecting the honey bee are known; of these one attacks the larvae causing the disease of “sacbrood,” the other attacks the adult insect causing “bee paralysis.”

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Three viruses have been recorded from the Diptera, two from the same species, the larvae of Tipula paludosa Meigen, the crane fly. Of these two, one causes a nuclear polyhedrosis, the other, known as the Tipula iridescent virus (TIV), has no intracellular inclusions. The third virus is the inherited virus u of Drosophila. In the Coleoptera one virus without intracellular inclusions is known, and two with a possible third, outside the Insecta, occur in the spider mites ( Arachnida) . These are all natural virus infections; in addition there is a polyhedrosis which develops in certain larval forms of the Neuroptera when inoculated with a nuclear polyhedrosis virus from a lepidopterous larva, Porthetria dispar (Linnaeus). It is not yet known whether this is a case of cross-transmission or the stimulation of a latent virus, but from the shape and appearance of the polyhedra and the vinis bundles contained therein it looks more like a genuine cross-transmission. This is further borne out by the work of Gershenson (1960), who gives more evidence that the shape of the polyhedra is decided by the virus and not the host species. Virus diseases have also been induced by inoculation with TIV in a number of insect larvae of various kinds, which have never previously been known to have a virus disease. IV. PATHOLOGY

A. External Symptoms As would be expected, since the sites of virus multiplication are not the same, the external symptoms of the nuclear and cytoplasmic polyhedroses are very different. One of the organs almost invariably attacked in the nuclear disease is the skin; this becomes extremely fragile and ruptures at a touch, liberating the liquefied body contents which consist largely of polyhedra. Caterpillars infected with this type of disease assume a characteristic position with the body attached by the abdominal feet and the head hanging downward. Before this final stage of the disease is reached the color of the larva may change somewhat, and in the case of Bombyx mori Linnaeus, the silkworm, the skin assumes a yellowish hue from which the name for the nuclear polyhedrosis of “jaundice” is derived. Caterpillars infected with a cytoplasmic polyhedrosis are smaller than normal and lag behind healthy larvae in their development. They do not show the extreme disintegration characteristic of the nuclear disease since the skin is not attacked, but frequently white patches show through the skin in the region of the mid-gut; these are the cytoplasmic poly-

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hedra which accumulate in the gut cells and sometimes in the gut lumen. At this stage of the disease polyhedra may be regurgitated or voided with the feces. The external symptoms in caterpillars affected with a granulosis virus closely resemble those of the nuclear polyhedroses; the skin is attacked and easily ruptures liberating the liquefied body contents. Diseases caused by the noninclusion viruses vary greatly in their external appearance. Larvae infected with TIV are immediately recognized by the blue or violet iridescence of the fat body which shows through the skin. In a disease of Cirphis unipunctata ( Haworth) ( Lepidoptera), infected third-instar larvae appear swollen and darker than healthy larvae. The cuticle has a waxy appearance and the mid-portion of the larva is sometimes slightly enlarged (Wasser, 1952). In bee paralysis the symptoms are somewhat indeterminate since there appear to be several disorders not due to virus infection which may closely resemble bee paralysis. The most reliable diagnostic features seem to be trembling of the body, legs, and wings, and often sprawled legs and wings. Frequently a high percentage of the bees become hairless and have dark and greasy-looking abdomens. In the disease of bee larvae known as sacbrood, the larval skin, which is normally shed during the final moult, is retained as a sac filled with a fluid containing a granular substance. The virus affecting the citrus red mite (Arachnida), like that of bee paralysis, appears to affect the nervous system, since diseased mites become paralyzed with the legs held in a stilt-like manner. In some cases diarrhea is a concomitant symptom.

B. Internal Symptoms In sections of larvae infected with a polyhedrosis, the first abnormality to be observed is the presence of the polyhedra. In late stages of the disease these crystals appear in enormous numbers; if the polyhedrosis is a nuclear one they can be seen packed into the cell nuclei which become greatly enlarged. They are usually found in the fat body, blood cells, and tracheae; they do not stain readily but appear somewhat translucent against the stained background. The nuclear polyhedra may be of different sizes varying from 0.2 p up to 0.7 p or 1 p ; as a rule, however, the polyhedra in one nucleus are of the same size. In some species, notably Porthetria dispar (Linnaeus), it is possible to see particles inside the polyhedra which are in fact bundles of virus rods; these are best seen by dark-field microscopy. In cytoplasmic polyhedroses the polyhedra appear similar but may be larger with a rounded outline. They differ from the nuclear polyhedra in their capacity to stain readily with Delafield or Heidenhain’s

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hematoxylin or Giemsa solution. They do not, of course, occur in the nuclei but in the cell cytoplasm of the mid-gut; later they may develop in all the cells of the alimentary canal. In some species, such as Antherueu pernyi, for example, the cytoplasmic polyhedra may vary in size from the limit of resolution of the optical microscope to 1p in diameter or more. They also occur in colonies surrounded by a membrane. The development of the polyhedra in both types of polyhedrosis has been studied by Xeros (1953). By pretreatment with normal hydrochloric acid the nuclear polyhedra were stained intensely with bromophenol blue which enabled their development to be followed. As already stated, the nucleus of an infected cell enlarges greatly, and most of the chromatin material clumps together to form an off-central, oval, chromatic mass. This is strongly Feulgen-positive, as are many granules which develop in it. The next stage is the transformation of the chromatic mass into a network while, at the same time, numbers of propolyhedra appear outside the network. It is thought that the formation of the polyhedra is an abrupt process; the propolyhedra all being formed together and subsequently growing in size, each one in step with its neighbor. As the polyhedra grow and begin to pack the nucleus the nuclear net undergoes further changes. It may expand to embrace a much greater quantity of the nucleus and also many polyhedra; at this stage there is a visible diffusion of Feulgen-positive material away from the strands of the net. Alternatively, the pore contents of the net may transform into a mass of highly refractile nonstaining globules, the Feulgen- and bromophenol blue-positive materials of the strands being dissipated in the process. In the cytoplastic polyhedroses, unlike the case of the nuclear polyhedroses, the formation of the polyhedra is a prolonged and continuous process, those forming and maturing first arising apically, and the later ones forming more basically. The formation of cytoplasmic polyhedra in the small basal cells of the mid-gut appears to be a relatively abrupt process, as with the nuclear polyhedra. The nuclear polyhedrosis of the larva of Tipula puludosu Meigen is an interesting disease and one different in many respects from any of the foregoing. It is solely a blood disease and a larva in the late stages of the disease appears white as opposed to the normal tan color. At this stage the number of blood cells is enormously increased; if a small puncture is made in the skin there is an immediate exudation of infected blood cells. The development of the disease appears to be as follows: In ultrathin sections of infected blood cells in the early stages of the disease the rod-shaped virus particles begin to appear in the center of the nucleus, which enlarges until it almost fills the cell. Next the polyhedra begin to develop; there is usually a focus point at which the virus par-

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ticles seem to gather, and it almost looks as if the virus particles themselves migrated toward this point (Fig. 1 ) . As the polyhedra increase in size and number, the number of virus rods in the center of the nucleus decreases. Finally the completed polyhedra are ranged round the periphery of the nucleus. In most polyhedroses the virus particles appear to

FIG. 1. Thin section of part of a blood cell from the larva of Tipukz puludoso Meigen affected with its nuclear polyhedrosis. Note the polyhedral body forming on the right and the apparent migration of the virus particles toward it. Magnifica-

tion:

x 12,500.

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FIG.2. Thin section of a blood cell similar to Fig. 1, but at a later stage of the disease. Note the great enlargement of the nucleus with a few virus rods still present. In the three polyhedral crystals the virus rods are regularly arranged in rows, apparently following the growth of the crystals. Magnification: x 10,000.

be occluded entirely at random, but in T. paludosa the virus rods are arranged in lines which seem to follow the shape of the crystal rather like growth rings (Fig. 2 ) . The last row of particles, as shown in Fig. 2, which have not been occluded in the crystal are stained black by the osmic acid, in contrast to the unstained virus particles inside the crystal. Figure 3 is a photomicrograph showing three stages in the destruction

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of the blood cell. In the photograph the smallest dark cell shows the enlarged nucleus and the beginning of the formation of the polyhedra. Below is a larger cell with an irregular outline due to the massed polyhedra. The third cell, which has enlarged to its maximum extent, has

FIG.3. Photomicrograph of three blood cells similar to those in Figs. 1 and 2, but not sectioned. They show three stages in the development of the disease. Top right; cell in early stage, polyhedra beginning to form; bottom right; cell enlarging, polyhedra mostly formed; top left; cell enlarged to bursting point and polyhedra discharged. Magnification: x 1500.

burst and the polyhedra are shown being discharged. There is still a ring of polyhedra round what was the periphery of the nuclear membrane; it will be seen that the shape of the “polyhedra” approximates that of a crescent or segment of an orange rather than the more usual polyhedron.

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In the granuloses the fat body seems to be the main focus of virus development, although, as the disease progresses, the blood cells, skin, and tracheae are also affected. There seems to be some doubt as to whether the virus multiplies in the nucleus or the cytoplasm or both, although granules seem to be present in the nuclei of the fat cells in the granuloses of Pieris brassicae Linnaeus and Melanchra persicariae Linnaeus (Smith and Rivers, 1956). Bergold ( 1948) and Steinhaus and Thompson (1949) have also observed granules in the cell nuclei. Tanada (1959a), working on a granulosis of the army worm Pseuduletia unipuncta (Haworth), states that the nuclei of the fat cells become hypertrophied and are apparently filled with granules. In a study of the granulosis of Choristoneuru murinuna (Hiibner) on the electron microscope Huger and Krieg (1961) report that formation of the granules takes place in both nucleus and cytoplasm. In a more recent paper Huger (1961) describes a new method of staining of the granules for optical microscopy and claims that the results obtained by this method confirm those of Huger and Krieg. Of the virus diseases without intracellular inclusions that caused by the Tipula iridescent virus (TIV) has been most studied. Here again the fat body is the initial site of virus multiplication, although the virus is capable of multiplying in almost any insect tissues such as the skin, muscles, wing buds, legs, and head. Sections of infected fat body under the optical microscope show rapid disintegration of the fat and enlargement of the cells. In an advanced stage the accumulation of virus in the fat body is so great that the virus aggregates are visible under the oil immersion lens. The pathology of this disease, however, is best observed with the electron microscope. Although TIV contains about 13%DNA ( deoxyribonucleic acid), ultrathin sections reveal that the virus is confined to the cell cytoplasm and never invades the nucleus. It is of interest to compare Figs. 2 and 4, which are electron micrographs of sections through a blood cell and fat body cell, respectively, of a larva of the same insect species, T . paludosa. In Fig. 2 is a blood cell infected with the virus of a nuclear polyhedrosis; the rod-shaped virus particles are seen only in the enlarged nucleus and in the polyhedral crystals. In Fig. 4 is a part of a fat body cell infected with TIV; the virus can be seen accumulating in large quantities in the cell cytoplasm while the nucleus is completely virus-free. The other noninclusion virus disease of which the histopathology .has been studied is the so-called “Wassersucht” of cockchafer grubs MeZoZontha spp. (Krieg and Huger, 1960). This is similar to “Heidenreich‘s disease” or “histolytic disease” of Oryctes spp. (Surany, 1960). Here again the fat body is the site of virus multiplication; in normal

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FIG.4. Thin section of part of a fat body cell from the same larval species as in Figs. 1-3, affected with the Tipuku iridescent virus. Note that the virus is confined to the cytoplasm and is beginning to orientate. Compare Fig. 2. Magnification: x 12,500.

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larvae the fat body cells contain large numbers of albuminoid spheres consisting of inhomogeneous, partly fibrous material. In virus-infected cells these albuminous spheres are apparently transformed into virogenetic stroma and give rise to virus particles. The progressive stages of

FIG.5. Whole mount of citrus red mite affected with a virus disease. Note the many crystals which are not virus but are characteristic of the infection. Magnification: x100.

decomposition of the albuminoid spheres can be observed in the light microscope and are of diagnostic importance. A peculiar internal reaction to a virus infection is found in the citrus red mite, Panonychus citri ( McGregor) ( Arachnida) . The external symptoms of this disease, paralysis of the legs and diarrhea, have already

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been mentioned. In sections of diseased mites, either stained or unstained, numerous dark round bodies occur scattered throughout the tissues and may be found also in the legs. Under the optical microscope these bodies show some internal structure and appear to be crystalline. They are best seen in whole mounts of diseased mites which have been cleared and mounted in balsam or DePex medium. When viewed by polarized light the crystals become highly birefringent (Figs. 5 and 6 ) .

FIG.6. The same as Fig. 5, but photographed by polarized light. Magnification:

x 100.

The exact nature of these crystals, which have only been observed in the diseased mites, is at present obscure. At first it was thought that they might be virus crystals, but examination of thin sections under the electron microscope showed that this was not so. It is just possible that virus particles could be occluded in the crystals as, owing to the small size of the particles, about 30 mp, they are not easily resolved when embedded in a matrix. On the other hand, the crystals may be a product of altered metabolism caused by the disease. Nothing is known at present

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of their chemical composition but they are extremely hard and are unaffected by Carnoy fixation, dehydration in ethanol, or clearing in xylol.

V. THE INCLUSION BODIES As we have seen already the term “inclusion bodies” refers to the nuclear and cytoplasmic polyhedra and the so-called granules. Much of the available information is related to the nuclear polyhedra since these have been known for many years. The most obvious difference between the nuclear and cytoplasmic polyhedra is in their reaction to weak alkali and to staining. Exposure to weak sodium carbonate for a few minutes dissolves the nuclear polyhedra completely leaving the rod-shaped virus particles within a membrane. Similar treatment of the cytoplasmic polyhedra dissolves out the spherical virus particles but only partially dissolves the crystals, leaving behind a matrix honeycombed with holes, the sockets in which the virus particles had rested; there is no membrane. Cytoplasmic polyhedra pick up stains such as Delafield’s or Heidenhain’s hematoxylin much more readily than nuclear polyhedra. In a mixed suspension of both types, fixed by mild heating on a slide and stained with Giemsa solution, the cytoplasmic polyhedra stain readily and are easily distinguished from the unstained nuclear polyhedra. In the nuclear polyhedrosis of a dipterous insect, Tipula paludosa Meigen, the polyhedra behave toward alkalies in a very different manner from the nuclear and cytoplasmic polyhedra of the Lepidoptera. These rather peculiarly shaped polyhedra, which may be rectangular or shaped like a segment of an orange, are negatively birefringent and appear to be genuine crystals. Their behavior in the presence of different reagents is most unusual and differentiates them sharply from either of the other two groups of polyhedra. They are resistant to trypsin and to dilute and weak acids and alkalis. In 1 N sodium hydroxide they elongate to six or more times their length, becoming first biconvex spindles and then elongating into crescents or wormlike shapes. At about three times their normal length this elongation is completely reversible, and in water, at pH 5.8, they return to their original size and shape. After such treatment, however, the polyhedra are “activated; in other words, they now respond in a similar manner in ammonia, 1-12% sodium carbonate and hydrochloric acid, pH 1-4, but not to 1 N hydrochloric acid or 25%sodium carbonate. The elongation and contraction or return to normal shape, which take place along the same axis, can be repeated indefinitely in these solutions and take place as rapidly as the solutions can be alternated. The polyhedra are not usually dissolved even after half an hour in

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1 N sodium hydroxide at 2OOC. In a solution of equal parts of 1 N sodium hydroxide and 1 N potassium cyanide, the response is speeded up enormously and the polyhedra elongate even further to reach their maximum length in 1% minutes. They dissolve completely in 2d minutes. The polyhedra give a positive reaction with xanthoproteic and ninhydrin tests, and stain with bromophenol blue after treatment for 15 minutes with 1 N hydrochloric acid at 6OOC. Their substance is Feulgenpositive throughout, and each polyhedron has intensely staining granular bands at about % L, intervals. Tests for sulfur have been negative. With such resistant polyhedra as these it is not possible to observe the virus bodies within by the methods applicable to the other types of polyhedra (Smith and Xeros, 1954). However, as will be seen from Fig. 2, it has been possible by means of thin sections of the polyhedra to demonstrate by electron microscopy the presence of a rod-shaped virus which resembles superficially the rod-shaped virus particles of other nuclear polyhedroses. An important question in the relationship between the polyhedral crystals and the virus is how far the crystalline shape of the polyhedra is directed by the infecting virus rather than by the properties of the host cell. It was pointed out some years ago by Smith that the polyhedra in the nuclear polyhedrosis of Panaxiu dominula ( Linnaeus ) , the cream spot tiger moth, were always rectangular in shape. The polyhedra in T . paludosu, the crane fly, are also of a characteristic shape and appearance, but these polyhedra, as we have already seen, differ in many ways from the polyhedra in other orders. If it could be proved that a particular type of polyhedra was characteristic of a particular virus this fact would be of the greatest use in cross-inoculation studies, which are discussed in Section VIII. Gershenson (1960) considers that he has established that the characteristic differences in the shape of nuclear polyhedra depend not on the species peculiarities of the host but exclusively on the specific properties of the virus-each virus causes the formation of polyhedra of one definite shape. For example, the nuclear polyhedrosis virus of the mulberry silkworm causes the polyhedra to be rhombododecahedra; that of the nun moth, tetrahedra; and of the pine moth, hexahedra. Gershenson points out the difficulties sometimes met with in ascertaining the exact crystal shape by the general pattern visible under the microscope. He suggests the use of a staining method whereby only the angles and edges of the polyhedra are stained, the bulk of the crystal remaining unstained. The method is first to mordant the polyhedra with 5% aqueous solution of phenol and then stain with carbolfuchsin. Penetration of the stain always starts from the edges and angles; by regulating

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the duration of the mordant treatment and of the staining the stain can be confined to those regions. By the use of this method it is possible to determine the crystallographic structure in difficult cases. Occasionally polyhedra of an aberrant shape appear among the other polyhedra of normal shape and this seems to be due to a mutation in the virus. Gershenson (195s) infected a cell nucleus with two types of nuclear polyhedra-the normal virus and the mutant obtained from it -and found that polyhedra of two sharply differing types develop, some with the shape characteristic of the original virus and some the shape of the mutant virus. Since both were formed under the uniform conditions of the same nucleus it would seem that the change in shape is associated more with a change in the structure of the substance of the polyhedra themselves rather than with conditions of crystallization. No account is included here of the physicochemical properties of the inclusion bodies, since these have been extensively reviewed by Bergold ( 1958).

VI. VIRUSMORPHOLOGY, ULTRASTRUCTURE, AND REPLICATION Broadly speaking, the viruses affecting the Arthropoda are either rod-shaped or polyhedral (near-spherical) . The clover wound-tumor virus, one of those multiplying in both plants and insects also appears to be polyhedral in shape. The viruses of the nuclear polyhedroses and of the granuloses are almost without exception rod-shaped. The actual material of the virus particle is enclosed within a membrane, the intimate membrane. Surrounding this, but a slight distance from it, is a second membrane, sometimes called the developmental membrane. As a rule there is only one virus rod inside the second membrane but in some species, notably, Porthetriu dispar, there may be three or more (Fig. 7 ) . The average size of the virus rods is about 300 mp by 40-50 mp. The virus particles from the nuclear polyhedroses and from the granuloses are all very similar in appearance, but in the latter a head capsule has been observed at either end of the intimate membrane, the purpose of which is not known (Smith and Hills, 1962b). Recently Kaesberg (1956) suggested that the small plant viruses were not really spherical but icosahedra with twenty sides. He demonstrated this on the electron microscope by means of shadow analysis. A particular type of polyhedron, if it is regular, should cast a specific shadow, if it is cast with a specified orientation with respect to the position of the vertices of the polyhedral particle. The difficulty of applying the “double-shadow” technique to the very small plant viruses is due to the smallness of shadow detail in comparison with the roughness of the

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FIG.7. High-resolution electron micrograph of a section through part of a nuclear polyhedron from Hemerobius stigma Steph. ( Neuroptera) inoculated with a nuclear polyhedrosis virus from Porthetria dispar (Linnaeus). Note that the virus rods occur in bundles within the second membrane and that they do not interrupt the crystalline lattice. Compare Fig. 12. Magnification: X200,OOO.

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film upon which the shadows are cast. In the Tipula iridescent virus (TIV), however, a large particle of uniform size ( 130 mp in diameter) is available, and this is extremely suitable for shadow analysis. When air-dried the particles have an appearance that suggests a polyhedral morphology. Following freeze-drying the TIV particles are uniformly hexagonal in contour. Both single- and double-shadowing have been applied to the TIV particle, and a comparison made of the shapes of shadows cast by a model icosahedron. These experiments show fairly unequivocally that the TIV particle is an icosahedron (Williams and Smith, 1958). Electron micrographs of air-dried particles of a virus from a cytoplasmic polyhedrosis from Antheraea mylitta Drury showed that they had a six-sided contour (Smith, 1958). The double shadow technique was then applied to frozen-dried particles of this virus and, in spite of the difficulties that are inherent in the application of this technique to very small viruses, there seems little doubt that the virus is an icosahedron. Further evidence for this is suggested from the shape of the socket in the polyhedral crystal which contained the virus (Hills and Smith, 1959 ) . One of the most interesting and important aspects of virology is the method of replication of viruses. We know it is not comparable to the reproduction of microorganisms but consists essentially of a process of assembly or biosynthesis. It is here that an intimate knowledge of the substructure of virus particles is of importance since from this may be deduced how the various parts are assembled during the replication of the virus. A great deal of information on the ultrastructure of viruses has been made available by the negative-staining technique with phosphotungstic acid ( Brenner and Horne, 1959). In earlier electron micrographs of the spherical viruses using the conventional shadow technique it was sometimes possible, if the resolution was good, to observe that the surface of the particle was irregular and bumpy. Now, however, with negative staining it is possible actually to count these “knobs” on the surface or, in other words, the protein subunits of the outer coat. X-ray diffraction studies of the tobacco mosaic virus particle (TMV) showed that the protein subunits of the TMV particle form a helix, and “helix” seems to be the key word in the architecture of the rod-shaped viruses. In a study of a nuclear polyhedrosis of a lepidopterous lama, Day et al. (1958) state that the virus rods show evidence of dense axial material suggesting that they may consist of hollow protein rods possibly with DNA near the axis just as TMV consists of hollow protein rods with RNA (ribonucleic acid) near the axis.

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Three types of insect-virus particle have been studied with the negative-staining technique by Smith and Hills (1962b); these are from a nuclear polyhedrosis, a granulosis, and a free virus, the Tipula iridescent virus. So far as the rod-shaped viruses are concerned, it has not yet been possible to observe the actual assembly of new particles by high-resolution electron microscopy. Some observations on the ultrastructure are, however, relevant here; there appears to be a difference in the fine structure of the rods from the nuclear polyhedroses and the granuloses, respectively. In the breakdown of the virus rods from the nuclear polyhedroses the first stage in the liberation of the contents of the intimate membrane is the peeling-off of the outer capsule. The capsules break in the center and fold backward, thus forming two spheres still joined in the middle (Fig. 8). These finally break apart and are thought to be the same as Bergold‘s spherical subunits, which he said were discharged from the intimate membrane ( Bergold, 1958). The intimate membrane is then exposed; it measures about 20A in thickness, and has a slightly different structure at either end. At very high magnification the contents of the intimate membrane give the appearance of a wide-spaced helix; these contents are discharged from either end of the intimate membrane and appear to uncoil as they flow out. This helix is considered to be in part nucleic acid (DNA), and differs markedly from Bergold’s subunits (Fig. 9 ) . The morphological detail of the virus rod extracted from the granulosis disease differs in a slight respect from the foregoing. The intimate membrane shows a close-packed helical structure, but whether this is formed by the membrane or its contents is not yet clear. It has not, however, been possible to resolve this helix on empty intimate membranes. Although the actual assembly of the parts forming a new rod have not yet been observed, the presence in both types of virus of a helical structure similar to that observed in the rod of TMV strongly suggests a similar method of assembly. Krieg (1961) has also made a study on the electron microscope of the virus rods from nuclear polyhedroses and considers that they show a helical structure. Based on his former observations of a central hole in the subunits he has proposed a model for rod-shaped insect viruses similar to that made by Franklin and Klug for tobacco mosaic virus. In considering the ultrastructure of the rod-shaped viruses we have seen that the key word is helix and we might suggest that in the contemplation of the “spherical” viruses the corresponding keyword might be “icosahedron.” The negative-staining technique has entirely changed the type of electron micrograph which can now be obtained of the

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FIG. 8. The virus from the nuclear polyhedrosis of Bombyx niori (Linnaeus), the silkworm, treated with weak alkali. Note ( A ) the capsule breaking in the center and folding backward; ( B ) the two spheres thus formed; ( C ) the contents of the intimate membrane which at high magnification appear to be helical, in some cases a protrusion at the end is visible; ( D ) the empty intimate membrane. Magnification: x30,OOO.

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small viruses, especially those from plants. As it is now possible to count the protein subunits on the face of the particle, the number of morphological units can be found by adding some combination of the numbers 12, 20, 30, and 60, or a multiple of 60 appropriate for the particular clustering arrangement ( Klug and Caspar, 1960). In the spherical viruses it is almost possible to deduce from the picture of the subunit arrangement on the surface of the particle what is the total number of subunits by counting the number on the edges, but the knowledge of the internal detail is dependent on X-ray diffraction studies. Thus, in the adenovirus there are 6 subunits on the edges giving

FIG. 9. High-resolution electron micrograph of the virus rod from a granulosis disease of Pieris bmssicae Linnaeus. Note the apparent helical structure of the rod. Magnification: x400,OOO.

a total of 252; there appears to be no virus described as yet with 5 subunits on the edge with a total of 162 subunits. Next comes tomato bushy stunt virus with 4 along the edges and 92 subunits; a newly described virus from a species of red spider (Arachnida) appears to have 3 and 42 subunits, respectively; the virus of turnip yellow mosaic has 2 subunits along the edge, with 1 in the middle giving a total of 32; this virus appears not to be a true icosahedron. Finally there is the bacteriophage ax-174 with 2 along the edge and a total of 12 subunits. It is thus possible to form a sequence of these very small viruses by counting the protein subunits; this emphasizes how similar these viruses may be in their make-up, even though they come from such totally dissimilar hosts. In the foregoing sequence the hosts consist

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of man himself, two species of plants, a member of the arthropods, and a bacterium. When we come to consider a large icosahedron like that of the Tipula iridescent virus (TIV), which measures 130 mp, the number of subunits is correspondingly greater. Examination of the outer protein coat of TIV by negative staining on the electron microscope shows it to be composed of 812 protein subunits together with lipids, which apparently help bind the subunits into a rigid structure. The subunits

FIG.10. High-resolution electron micrograph of a particle of the Tipula iridescent virus, negatively stained with PTA. Note the protein subunits which are hollow and hexagonal when viewed end-on. Magnification: x480,OOO.

measure 85 by 140 A and are hollow and hexagonal when viewed end-on (Fig. 10). They are arranged to form an icosahedron, each side being an equilateral triangle. There is one outstanding phenomenon which has been observed in the replication of many spherical or near-spherical viruses from both plants and animals: that is the appearance of large numbers of empty protein shells of the same size as the virus. This phenomenon was first observed by Markham and Smith (1949) with the plant virus causing turnip yellow mosaic. These empty shells, called “top-component” because they form the top layer during ultracentrifugation, do not con-

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tain RNA and are therefore not infectious. Somewhat similar empty shells have been described by Horne and Nagington (1959) in studies on the structure and development of poliovirus. The highest count of empty shells that they recorded was 11-14% 5% hours after infection. Careful study of the empty shells of TIV suggests very strongly that they are stages in the reproduction of the virus. This is borne out by the following facts: First, empty, or partially empty, shells occur predominantly in the early stages of the disease. In some sections, indeed, practically no mature particles are visible but large numbers of empty shells occur. Second, what appear to be stages in the development of the contents of the particle can be seen. In ultrathin sections of the shells threads can be seen radiating out from the center; the next apparent stage is the build-up of spherical accumulations in the center of the particle which is thought to contain the DNA. As development proceeds the space between the spheres and the inner surface of the protein subunit shell fills up and becomes the mature virus particle (Smith and Hills, 1962a). VII. METHODS OF TRANSMISSION

A. Natural Dissemination The commonest method of spread of the insect viruses is undoubtedly by the ingestion of contaminated food materials. The polyhedra and granules greatly aid this process by their persistence, their comparative resistance to weather conditions, and their long retention of viable virus. Added to this is the fact that in some cases the semiliquid cadavers of larvae, which have died of a granulosis virus, seem to have a strong attraction for healthy caterpillars of the same species. In most cases for infection to take place it seems necessary for the virus to be actually ingested; mere contact between diseased and healthy larvae is not enough. A certain amount of virus spread is probably brought about by the action of parasites; this is almost equivalent to an injection since the virus is borne on the ovipositor of the parasite and is carried into the body of the larva during the process of egg-laying. As an example of this Thompson and Steinhaus (1950) showed that adults of the parasite Apanteles sp. could transmit virus to three successive larvae at hourly intervals. The polyhedra of certain nuclear diseases can pass unchanged through the gut of a carnivorous bug, Rhinoceris annulatus Linnaeus, and of the bird, Erithacus rubecula Linnaeus (Franz et al., 1955); this, of course, helps the spread of the virus.

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Another, and more important, means of natural dissemination is by hereditary transmission. This occurs with both types of polyhedroses and with the granuloses. It is a commonplace that certain individuals of lepidopterous larvae reared in the laboratory under virus-free conditions frequently develop a virus disease which then spreads through the remainder of the stock. There are many well-documented instances of this transovarial dissemination which was first suggested as occurring in the silkworm Bombyx mmi by Conte ( 1907) and Bolle (1908). Larvae of Telea polyphemus Cram. have been observed to die of a nuclear polyhedrosis within 48 hours of emergence from the egg (Smith and Wyckoff, 1951). Sometimes larvae of a certain species fail to emerge from the egg and subsequent examination reveals the presence of large numbers of polyhedra ( Rivers, personal communication). As regards transovarial transmission, the question has been raised from time to time as to whether the virus is inside the egg or merely adsorbed as a contamination on the outside. In the latter case, the young larva would become infected when it ate its way out of the shell. Under these circumstances there would be a delay of at least 10 days before the disease would take effect; it is reasonable therefore to suppose that in those cases where the larvae died within a few hours of emergence, or died without emerging, genuine hereditary transmission had taken place. Furthermore, there are numerous cases where surface-sterilization of the egg by NaOH, HCl, formaldehyde, and trichloroacetic acid has failed to produce virus-free progeny. Rivers reports of experiments with a granulosis and the eggs of Pieris brassicae Linnaeus, in which the shell was entirely removed so that the embryo was exposed; in spite of this a proportion of the resulting larvae were infected with the granulosis virus. In an experiment on the use of a nuclear polyhedrosis to control the larvae of the sawfly Neodiprion sertifer (Geoffroy) on pine trees, the virus was introduced into an area where it had not previously existed. At the end of the season a few female sawflies were collected and brought back to the laboratory where the eggs were collected and placed under virus-free conditions. A high proportion of the larvae resulting from these eggs died of a polyhedrosis. There is much other evidence of a similar nature, and it is now an established fact that transovarial transmission of many insect viruses does take place. It is interesting to find that similar hereditary transmission takes place with several of the leafhopper-transmitted plant viruses which have been shown to multiply within their insect vectors. This subject is discussed at greater length in Section XIV.

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B . Artificial Methods of Transmission It is sometimes difficult to infect lepidopterous larvae with a virus by the usual method of feeding them with contaminated food material. In such cases the larvae can be induced to regurgitate; the drop of regurgitated fluid is then quickly replaced by a drop of purified virus which is sucked up by the insect. About 20%infection with TIV was achieved in the larvae of P . brassicae by this method. By far the most successful method of artificial transmission is by injection. The preparation of suitable needles for this purpose has been described by Maramorosch ( 1951) and Martignoni ( 1955). These consist essentially of glass capillary tubes cemented to hypodermic needles which may be either intact or cut off 6 mm. from their bases. Maramorosch used a paste of glycerin and litharge for cementing the glass and metal together but Martignoni found sealing wax adequate. In a more recent paper Martignoni (1959) suggests a more elaborate needle for microinjection which will stand heat sterilization. There are two procedures which are described in detail in his paper. Very good results, however, can be achieved by the use of the finest steel hypodermic needles, about 0.01 or 0.02 ml. being injected at a time. Success depends a good deal on the purity and concentration of the virus inoculum. The best results are obtained after centrifuging the virus on a sucrose gradient; the death rate due to extraneous bacterial infection can be greatly reduced by the addition to the inoculum of 200 I.U. of penicillin and 200 I.U. of streptomycin per milliliter, respectively. Spraying a virus suspension onto adult bees has been shown to spread the disease of bee paralysis (Burnside, 1945), the virus presumably entering by the spiracles. This is one of the few examples where the virus attacks the adult insect. VIII. CROSS-TRANSMISSION For a number of years the opinion has been strongly held by some insect virologists that these viruses are species-specific and that crosstransmission does not occur. The chief difficulty in resolving the problem of cross-transmission lies in the lack of a reliable means of virus identification. The obvious method, which is by means of serological relationships, is at present lacking. However, there are other methods available; one of these is the fact, pointed out by Gershenson (1960) and others, that the shape of the polyhedral crystal is a function of the virus and not of the host. By comparing the shape of the polyhedra in the source insect with those produced in the inoculated insect, it would then be

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possible to say, with fair accuracy, whether a genuine cross-transmission had taken place. Identification of the exact crystallographic shape of the polyhedra can be aided by a special staining technique which picks out the edges of the crystals (Gershenson, 1960). Another method is to use a virus which has such distinctive qualities that it cannot be mistaken in whatever host insect it occurs. The Tipula iridescent virus (TIV) is particularly suitable for this purpose since, as we shall see later, it has unmistakable characteristics. It is thus important when attempting cross-transmission to use a polyhedrosis virus with strongly characteristic polyhedra. It sometimes happens that in a given disease a few aberrant polyhedra will appear. These are thought to be due to a virus mutation; when inoculated into fresh insects of the same species the polyhedra produced are all of the aberrant shape (Gershenson, 1960; Aruga et al., 1961). This type of virus is especially suitable for cross-transmission work. So far as the nuclear polyhedroses are concerned Gershenson (personal communication) has suggested that there may exist a group specificity where one virus is transmissible among members of the same genus. It is certainly the case that the nuclear polyhedrosis of Aglais urticae ( Linnaeus ) is intertransmissible among the vanessid butterflies, including Vanessa atalanta (Linnaeus) , V. cardui (Linnaeus), and Nymphalis io (Linnaeus), the polyhedra in each species being identical. Similarly with the cytoplasmic polyhedroses of larvae belonging to the Arctiidae, the tiger moths, apparently the same virus with like polyhedra is intertransmissible between Arctia caia ( Linnaeus ) , A. villica ( Linnaeus), Phragmutobia fuliginosa ( Linnaeus ) , Parasemia plantaginis ( Linnaeus), and Euphagia quadripunctaria (Poda) . In discussing cross-transmission of the cytoplasmic polyhedroses, however, the possibility must be borne in mind that the viruses causing this type of disease may be one and the same. In the granulosis disease of the pierids the virus is intertransmissible in Pieris brassicae (Linnaeus), P . rapaa (Linnaeus), and P . nupi (Linnaeus), and there seems no reason to suppose that more than one virus is involved. Cross-transmission, however, is not confined to related groups, and, if we accept the criterion of similar polyhedra indicating similar viruses, a most striking example of the cross-transmission of a nuclear polyhedrosis is the infection of larvae of Hemerobius spp. (Neuroptera) from larvae of Porthetria dispar (Linnaeus) ( Lepidoptera). In this case not only were the polyhedra identical, but also the arrangement of the bundles of virus rods inside the polyhedra (Fig. 11) (Sidor, 1960; Smith et al., 1959b ) .

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FIG. 11. Photomicrograph of polyhedra from Hemerobius stigma. Steph. ( Neuroptera), which had been inoculated with a nuclear polyhedrosis virus from a caterpillar of P. dispar. Note the virus bundles in the polyhedra indicated by arrows. Magnification: x 1500.

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An interesting point, and one which supports the conception of a true cross-transmission of the nuclear virus from P. dispar to the larva of Hemerobius spp., is the apparently identical crystalline lattice of the polyhedra in the two insects (compare Fig. 7 with Bergold, 1958, p. 80). The most interesting examples of cross-transmission, and the most extensive, however, have been achieved by the second method of differentiation, i.e., by using a virus which itself has unmistakable characteristics. This is the TIV so-called because of the iridescence produced by the microcrystals of virus forming in the living insect and in pelleted virus. Recent work (Smith et al., 1961) has shown that the host range of this virus is very wide. Using the microinjection technique, successful transmission was obtained to the larvae of seven species of Diptera, in addition to the original host Tipula paludosa Meigen, eleven species of Lepidoptera, and three species of Coleoptera. All the infected insects showed the characteristic blue or violet iridescence of the fat body, and the virus particles were the usual icosahedra and were present in vast quantities. The fat body was the initial site of multiplication in each type of insect, but the virus also multiplied in the skin, muscles, wing-buds, legs, and head. The concentration of virus was very high in the legs and wing-buds of the pupae of P . dispar, causing them to atrophy and to show brilliant iridescence. For successful cross-transmission it is important to have the virus in high concentration and free from contaminating microorganisms. It was found that some species of insect required higher virus concentrations than others to become infected. For example, the larvae of Sphinx ligustri (Linnaeus) were easily infected with a dose of 8 x 10:’ virus particles but not with one of 8 X lofi particles which gave 100% infection with the larvae of P . brassicae. To infect the larvae of the silkworm Bombyx mori (Linnaeus) a dose of TIV similar to that used for S . ligustri was given.

IX. LATENTVIRUSINFECTIONS Latent virus infections occur in man and the higher animals, plants, and insects; in the latter the phenomenon is frequently encounteredwhole populations or stocks of insects may have a latent virus infection. It is now well-established that certain factors can stimulate a latent infection into virulence. These factors are varied in nature and have been called “stressors” by Steinhaus (1958). These stressors may be loosely grouped under various headings, such as temperature and humidity, overcrowding, unsuitable food, application of chemicals, and inoculation with a foreign virus. All the stressors probably work in the

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same way by reducing the vitality of the insects and thus rendering them more susceptible to infection; no doubt the concentration and condition of the latent virus infection also play a part. Examples of such stimulation can easily be given, but for a more comprehensive review of the subject the reader is referred to Bergold (1958). It has been shown by Kurisu (1955) and Aruga (1957) that a cytoplasmic polyhedrosis can be induced in larvae of the silkworm Bombyx mori by subjecting them to low temperatures; the writer has found that low temperatures and high humidity can provoke a polyhedrosis in the larvae of Cerura vinula (Linnaeus), the puss moth. A similar effect of environmental conditions on virus latency is shown by some experiments with the larvae of the clothes moth Tineola bisselliella (Hummel). A stock of these larvae had been grown for many years at a research station where they had been kept under rigidly controlled temperature conditions and fed on sterilized food; under these conditions no virus disease had ever been observed. Some of the same stock were brought to Cambridge and reared under virus-free conditions and fed on the same sterilized food; they were, however, maintained under conditions of varying temperature and humidity. Within a short time a nuclear polyhedrosis developed and spread rapidly through the stock. A fresh batch of larvae from the same outside source kept in a controlled temperature chamber has remained virus-free for a period of years. Overcrowding frequently causes the development of a virus disease, especially when caterpillars are grown in captivity. This is particularly true of the granulosis disease which attacks the caterpillar of the small white butterfly Pieris rapae (Linnaeus) (Fig. 12). In Cambridge it is practically impossible to rear the progeny of wild butterfiies of this species collected in the district because they all succumb to a granulosis, but this apparently does not happen under field conditions. As insect collectors know very well, it seems only necessary to breed a given species of caterpillar for a certain period after which virus disease frequently develops in the stocks ( Smith, 1952). In some cases unsuitable food will provoke a latent virus infection but this does not always happen. Various workers have reported the onset of polyhedrosis in larvae of B . mori by changing the food plant from mulberry to Scorzonera hispunica Linnaeus (Ripper, 1915) or to Maclura aurantiaca Nutt. (Vago, 1951,1953). On the other hand, Tanada (1956) was unable to induce the appearance of virus disease in an armyworm (Pseudaletia unipunctata) by changes in the food plant. Much work has been carried out on the stimulation of latent infections by the application of chemicals, and indeed Yamafuji and his

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colleagues claim the artificial production of polyhedrosis in the silkworm B. mori by means of hydroxylamine, potassium nitrite, barium hydroxide, etc. For a complete list of Yamafuji’s publications the reader is referred to Bergold (1958, page 115). Krieg (1955) attempted to provoke polyhedrosis in B. mori by feeding with various chemicals. He used NH,OH( 1N ), KNO, ( 0 . 3 3 N ) ,

FIG. 12. High-resolution electron micrograph of a thin section through three granules from a granulosis disease of Pieris rapae ( Linnaeus ) , showing the crystalline lattice. Note presence of only one virus rod in each granule, one cut transversely, the other longitudinally. Compare Fig. 7. Magnification: x 110,000.

NaF( O.Ol%),and thioglycolic acid ( 1%)but obtained only negative results. Krieg suggests that in countries like Japan, Italy, and southern France, where silkworms have been grown for many years, all the stocks carry a latent virus infection, as opposed to countries where silkworm growing is more recent. This would account for the successful stimulation of polyhedroses in the former countries. In some cases it is possible to stimulate a latent polyhedrosis virus into activity by inoculation with a polyhedrosis virus from another

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species. This has been demonstrated with larvae of the winter moth Operqvhteru brumata (Linnaeus) and of the pine looper Bupalus piniarius (Linnaeus). In each case, however, inoculation with a nuclear polyhedrosis virus invoked the development of a cytoplasmic polyhedrosis, and this seems to be what usually happens; the writer has not observed the reverse phenomenon, i.e., stimulation of a nuclear polyhedrosis by inoculation with cytoplasmic polyhedra. However, Krieg ( 1957) states that application of cytoplasmic polyhedra from Dusychiru pudibundu ( Linnaeus ) ( Lepidoptera) apparently stimulated into virulence a nuclear polyhedrosis in Neodiprion sertifer (Geoffroy), the European sawfly (Hymenoptera). There is practically no information on the state of the virus when in a condition of latent infection. Ring zones and small polyhedra have been observed in healthy stocks of B. mori larvae which had shown no indication of virus infection for several years ( Roegner-Aust, 1949). and cytoplasmic polyhedra have been observed in the alimentary canal of a butterfly, Argynnis dia, and of the hawk moth, Sphinx ligustri. In their experiments with two strains of a cytoplasmic virus in the silkworm B . mori, Aruga et ul. (1961) suggest that one virus may be in an occult state in which it does not interfere with the second inoculated virus until stimulated into action by cold treatment. The term “occult” has been suggested by Andrewes (1958) as indicating a condition of latency in which the virus particles have not been observed and in which their exact state is not known.

X. ISOLATION AND PURIFICATION In attempting to isolate the viruses from the insect polyhedroses and granuloses it must be remembered that besides the necessity of eliminating the normal cell components there is the additional complication of removing the inclusion-body protein. The first step is to isolate the inclusion bodies from the insect host; this is best done by grinding up the whole insects and filtering through fine muslin into boiled distilled water. By means of a short cycle of spins on an ordinary angle centrifuge a pure suspension of the inclusion bodies can be obtained. To liberate the virus particles from the polyhedra it is necessary to dissolve the latter in dilute alkali; this is a highly critical operation since an incorrect alkali concentration will also dissolve out the virus particles. The procedure for the purification of the rod-shaped viruses from the nuclear polyhedroses and the granuloses is given in detail by Bergold (1958) and so will not be repeated here. The isolation of the virus from the cytoplasmic polyhedra is a more difficult process, since the near-spherical viruses of this type of disease

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tend to dissolve in dilute alkali more readily than the polyhedral crystal. A method for the purification of this type of virus has recently been worked out ( Hills and Smith, 1959). There are two methods for extracting the polyhedra from cytoplasmic polyhedroses which have been recommended in the past but are now known to be unsatisfactory. One method was to open the larvae and remove the guts, which were allowed to drain into a flask; the other was to grind up the larvae and extract the polyhedra by differential centrifugation. Under the conditions of these two methods it was found that the polyhedra showed severe etching and deformation of the edge while all the virus in the sockets near the crystal surface was lost. A more successful method is to open the larvae and plunge them immediately into distilled water and then extract the polyhedra; this gives undamaged polyhedra with the individual virus particles on the surface of the crystal covered with a layer of protein. The following is the method used for extracting the virus from cytoplasmic polyhedra from Antherueu mylittu Drury. Dissolve 50 mg. of polyhedra in 1.0 ml. of 2%sodium carbonate solution for 30 seconds. The bulk of the liquid is increased to 30 ml. by the addition of doubledistilled and boiled water and centrifuged at 38,000 r.p.m. for 30 minutes. The pellet is suspended in distilled water and cleaned by centrifugation at 10,000 r.p.m. for 15 minutes, The supernatant is reduced in bulk and subjected to gradient centrifugation through sucrose; a clearly defined band is obtained. The band is removed, increased in bulk to 5 ml. with water, and centrifuged for 30 minutes at 36,000 r.p.m. The pellet thus obtained is pure virus. The conditions described above were optimum for that particular species and it was necessary to experiment further to find the best conditions for extracting the virus from the cytoplasmic polyhedra of other species. For the isolation of the virus from a number of other species the optimum conditions are as follows: 0.5% sodium carbonate for 1.25 minutes for the polyhedra from Arctiu caju (L.); 1.0%for 2 minutes for Antheraea pernyi Gukrin-Mknhville; 1.5 minutes at 1.0%for Evanessa untiopa (Linnaeus); and 1 minute at 1.0%for Pyrurneis curdui (Linnaeus) and Calophsiu h u l a (Hufnagel). In each case the pellet was pure virus. There is not much information available on the method of puri6cation of the insect viruses from noninclusion-body diseases because only a few have been described so far. Probably the easiest of all viruses to isolate and purify is the Tipula iridescent virus (TIV). The purification is aided by the large size of the virus particles and by the absence of particles of similar size in the tissues of the diseased host. To obtain the

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purified suspension the larvae are cut up in distilled water and allowed to stand for 24 hours; occasional shaking aids liberation of the virus. The suspension is then clarified by low-speed centrifugation and the virus obtained essentially pure by two cycles of high- and low-speed centrifugation. If necessary the virus can be further purified by centrifugation in a sucrose gradient. When centrifuged down the virus crystallizes spontaneously and the pellets have fascinating optical properties. By transmitted light the pellet appears an orange or amber color, but by reflected light it has an iridescent turquoise appearance. Within the pellet may be seen small regions reflecting the incident light quite brilliantly, giving the entire pellet the appearance of an opal ( Williams and Smith, 1957). When dealing with very small arthropods infected with a free virus such as a virus disease of the citrus red mite Pnnonychus citri ( McGregor) the following procedure was adopted (Smith et al., 1959a). An aqueous suspension of the diseased mites, about 40 mg., was ground up in a hand homogenizer and spun on a clinical centrifuge to clear debris. The supernatant was then centrifuged on a Model L Spinco at 36,000 r.p.m. (110,000 9 ) . The pellet was taken up in 1 ml. distilled water and given a low-speed spin to clear debris. The supernatant was then run in a Model E Spinco centrifuge until the peak was about halfway down. The Spinco was stopped and the supernatant removed; it was next mixed with potassium phosphotungstate and sprayed onto the grids for electron microscopy. An alternative method is to suspend the pellet, after sedimenting on the model L Spinco, and purify on a sucrose gradient.

XI. CHEMICAL COMPOSITION Bergold (1958) has dealt at length with the chemical composition of the rod-shaped viruses, so that only some recent work on the chemistry of the cytoplasmic viruses is briefly mentioned here. All the rod-shaped viruses affecting insects have so far been found to contain deoxyribonucleic acid ( DNA ) , while some near-spherical viruses from cytoplasmic polyhedroses contain ribonucleic acid (RNA). It would have been interesting if this had proved to be a fundamental difference between rodshaped and near-spherical viruses; however, as we shall see later, the Tipula iridescent virus (TIV), a cytoplasmic virus of polyhedral shape, contains DNA exclusively. Xeros (1962) has studied the nucleic acid content of the homologous nuclear and cytoplasmic polyhedrosis viruses. Chromatographic and electrophoretic analysis of the nucleic acid content of the cytoplasmic polyhedra from Laothoe populi (Linnaeus ) showed that they

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contained 0.9%RNA and no DNA. Chromatographic analysis of cytoplasmic polyhedra from a further five species showed the presence in them of similar quantities of RNA and the absence of DNA. The TIV, which has the interesting and unique property of forming crystals spontaneously both in vivo and in vitro, contains between 12 and 13%DNA but no RNA, although it multiplies exclusively in the cytoplasm. Analyses of TIV carried out by Thomas (1961) indicate that it contains 12.4%DNA and 5%lipid, most of which is phospholipid. The remainder of the virus, 82.4%,appears to be protein including some phosphoprotein. The DNA contains only adenine, guanine, thymine, and cytosine, no methylcytosine or 5-hydroxymetliylcytosine, and is apparently similar to other insect virus DNA. Thomas and Williams (1961) have studied the localization of DNA and protein in the TIV particle by means of enzymatic digestion and electron microscopy. De-embedded sections of TIV particles fixed in ethanol were incubated with pepsin at pH 1.8, trypsin at pH 7.7 and DNase (deoxyribonuclease) at pH 7.7. The outer shell of the particles but not the inner core was removed by the action of pepsin. Conversely, the inner core, but not the outer shell, was removed by the action of trypsin and DNase in combination, but not by either enzyme acting alone. Thomas and Williams consider this to mean that the outer shell of the particle is protein in nature and the inner core is nucleoprotein. Chemical analyses showed that the cores contain 8996 of the whole virus phosphorus but only 35% of the nitrogen, while the outer shells contain only 5%of the phosphorus but 63%of the nitrogen. In a series of experiments Gershenson et al. (1961) present evidence that insect cells infected with a virus of DNA-type (nuclear polyhedrosis ) produce, instead of the RNA normal for them, a modified type which is capable of inducing a DNA-virus when introduced into a susceptible host. Put briefly, their experiments were as follows, RNA was isolated by a modification of the phenol method from healthy fifth-instar larvae or young pupae of Bombyx mori (Linnaeus), RNA was similarly isolated from larvae which had been infected with their own nuclear polyhedrosis by injection. The RNA was dissolved in pyrophosphate or phosphate buffer at pH 7.0 and purified by centrifugation (20,00025,000 g ) at O04OC. Solutions of RNA from healthy or virus-infected larvae or pupae were injected intralymphally into healthy fifth-instar larvae or pupae of B. mori. The results of these experiments were that out of 200 larvae injected with RNA from healthy larvae only 1 developed a nuclear poly-

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hedrosis, whereas out of 894 larvae injected with RNA from virus-infected larvae 383 (44%)developed a nuclear polyhedrosis. Gershenson and his co-workers have tested a number of possible alternative explanations. In order to be certain that the effect observed with RNA from infected larvae was not a result of accidental presence of virus in the preparations, the following experiment was carried out. Purified polyhedra from B . mori were added to healthy pupae during homogenization and RNA was isolated from the homogenate. This RNA failed to induce nuclear polyhedrosis, showing that the virus had been eliminated during the isolation of the RNA. Furthermore, the high-speed centrifugation alone is sufficient to remove the virus. To ascertain that it was the RNA itself that was the infectious element and not some other components, solutions of the RNA were treated with RNase (ribonuclease); such solutions almost lost their infectivity, 10 diseased out of 191 larvae. RNA solutions, on the other hand, treated with DNase gave 37 nuclear polyhedroses out of 80 larvae injected. Solutions of RNA from infected insects become practically noninfectious after standing for 24 hours at room temperature, whereas the infectivity of virus suspensions is unaffected under those conditions. One of the greatest pitfalls in this type of experimental work is the possibility of latent virus infections which may be stimulated into virulence by a variety of conditions (see Section IX). Gershenson considers that this eventuality may be met by the use of a mutant strain of virus with polyhedra of a characteristic shape. Such a strain of virus with hexagonal polyhedra was isolated from a larva of Antheraea pernyi; from a number of pupae infected with this virus the RNA was isolated and injected into healthy larvae of A . pernyi. Some of these larvae developed a nuclear polyhedrosis which was characterized by the presence of the hexagonal polyhedra instead of the tetragon-tritetrahedal type of the normal virus. In other words, the shape of the polyhedra is used as a marker to pinpoint the actual virus. If this work is confirmed it will become evident that genetic information can be transmitted not only from DNA to RNA but also in the reverse direction. This would confirm the suggestion of Stent (1958) that in some stages of multiplication of DNA viruses the process is controlled by RNA (and protein) without the participation of DNA. XII. SEROLOGY As we have already mentioned, the investigation of the serological relationships of the insect viruses has been much neglected and it is only recently that a start has been made. This is a fruitful field of study and should yield information which would be useful in many ways.

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The number of apparently distinct viruses would almost certainly be reduced and light would probably be thrown onto the question as to how far the large number of cytoplasmic polyhedrosis viruses are one and the same. A sound system of nomenclature and classification also needs to be based on, among other things, the serological relationships of the different viruses. It would be interesting also to ascertain if there existed any kind of relationship between some of the insect viruses and those viruses which are capable of multiplying in both insects and plants (see Section XIV). Most of the serological work done so far relates to the rod-shaped viruses of the nuclear polyhedroses and granuloses; this is understandable enough since they are so much easier to isolate and purify than the cytoplasmic polyhedrosis viruses. Krywienczyk, MacGregor, and Bergold ( 1958) have shown that viruses from the nuclear polyhedroses and the granuloses belong to two serologically distinct groups, since no cross-reaction occurs between them, and this was to be expected since they are distinct in other ways. They also showed that there was a strong cross-reaction between viruses from closely related hosts, but with polyhedrosis viruses from less closely related hosts there was only a weak cross-reaction. In a further paper (Krywienczyk and Bergold 196Oa) the serological relationships of seventeen insect viruses from four continents were investigated by the complement-fixation technique. On the whole, the results support the grouping of the viruses into polyhedroses and granuloses, although one granulosis virus from Recurvaria milleri Busck gives detectable cross-reaction with six polyhedrosis viruses. The polyhedrosis viruses from the Hymenoptera, two in number, give strong cross-reactions only within their own group and are probably closely related if not identical. In a paper published shortly after the above, Krywienczyk and Bergold (1960b) consider that when intact virus is injected into guinea pigs, antibodies are formed mainly against the membranes. They suggest that serological studies of intact insect viruses so far reported are in reality studies of the antigenic structures of the virus membranes, and not of the virus nucleoprotein. It would therefore be of interest to study the serological relationships of the DNA from the different nuclear polyhedrosis viruses and also between the DNA and RNA viruses. The same workers (Krywienczyk and Bergold, 1960c) have studied the serological relationship between the inclusion body proteins of Lepidoptera and Hymenoptera. They find that the proteins belong to three serologically distinct groups. Two of these groups, the nuclear polyhedron proteins and the granuloses proteins from Lepidoptera, are

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the same as established for the corresponding viruses. The granulosis protein from R . milleri, however, showed, like its virus, a higher crossreactivity with polyhedron proteins than with the other granulosis proteins. The third group is represented by the nuclear polyhedron proteins of the sawflies ( Hymenoptera). Stobbart (unpublished, and in Smith et al., 1961) has carried out complement fixation and gel diffusion tests on TIV from three different hosts in the Lepidoptera Porthetria dispar ( Linnaeus ) , Pieris brassicae (Linnaeus) and Nymphalis io (Linnaeus). From these tests the TIV from all three hosts appears identical, Additional tests carried out with TIV from its natural host, the fly larva Tipula paludosa Meigen, showed the virus to be the same. XIII. TISSUECULTURE Trager (1935) was one of the pioneers in the tissue culture of insect cells. Using the hanging-drop method and a physiological saline he was able to cultivate the epithelial cells of the silkworm ovary ( Bombyx mori), and obtained development of the nuclear polyhedra of that insect. Wyatt (1956) improved upon this technique by adding 10% of the insect hemolymph and modifying the culture medium. To reduce the activity of the tyrosinase, the hemolymph for culture medium was heated for 5 minutes at 6OOC. and the coagulated protein removed. Wyatt formulated a physiological solution containing cations and amino acids as they occur normally in silkworm hemolymph. In both hangingdrop and small tube cultures, use of this medium brought about increased cell number and longer life of cultures. Vago (1959a) has carried out similar experiments using the ovarian fibroblasts of B . mori and of Galleria mellonellu. These were inoculated with the supernatant from hemolymph centrifugates from the larvae of B. mori, G. mellonellu, Porthetria dispar, S . pavonia, and Antheraea pernyi infected with polyhedroses. Different types of lesions were observed in the cells which appeared to be most advanced in cells from the same host species and less so in different species. Some work based on the use of diapause pupae of the silkmoth, Philosamia aduena, as a source of ovarian tissue for explants, and of hemolymph for inclusion in the culture medium has recently been published (Jones and Cunningham, 1960). The medium used consisted essentially of salts, sugars, and organic acids based on the work of Wyatt ( 1956), lactalbumin hydrolyzate (Difco), TC yeastolate (Difca), and pupal hemolymph, the last being preheated before addition to the medium. Streptomycin and penicillin were also added to the medium at 100 I.U.and 50 I.U. per milliliter respectively.

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In the preparations incubated at 2 5 O C . , cells began to migrate from the explants on about the third day, and from the fourth to ninth days the cellular outgrowths spread extensively. The medium was changed every fifth day. The cultures remained healthy for up to 4 weeks, and in appearance they resembled primary chick fibroblast cultures. What seems to be the successful use of some simple and readily reproducible techniques for the tissue culture of insect cells and their subsequent infection with virus has been reported from China (Zan-Yin et al., 1959). The nutrient solution was composed of 90% Trager’s solution A and 101& healthy silkworm hemolymph; its initial pH was 6.7. Trager’s solution A, prepared according to formula A of Trager (1935), was used as the basic constituent of all media and also for washing the tissues and suspending the cells throughout the experiments. For trypsinized cell cultures the trypsin solution was prepared from Bactotrypsin ( 1:250) at a concentration of 0.25% by weight using Trager’s solution A as diluent. Penicillin and streptomycin were added to the nutrient solution to final concentrations of 100 I.U. and 100 pg. per milliliter, respectively. By these methods male and female gonad, muscle, trachea, intestine, and silk gland tissue cultures were successfully obtained by using modified hanging-drop and stationary test-tube techniques. Monolayer tissue cultures could be prepared from both suspended and trypsinized cells. Cultivation of the virus of the nuclear polyhedrosis of B. mori in monolayer tissue cultures caused cytopathic changes of the cells and the formation of polyhedra in the cell nuclei was observed. Subcultures of male and female gonad cells have been passed through 22 successive generations. With subcultures and the use of dehydrated silkworm serum, the authors state that experimental work can now be carried on all the year round without the necessity of raising silkworms. Studies on the multiplication in vitro of a nuclear polyhedrosis v i r u s in insect amoebocytes have recently been described by Martignoni and Scallion (1961). They used a slightly different culture medium containing 15%homologous insect blood plasma and 15%fetal bovine serum. The hemocytes of sixth-instar larvae of Pmidroma saucia (Hiibner) were cultured on cover glasses in 60-mm. Petri dishes with 4 ml. of this medium. After 20-24 hours incubation at 25OC. in a gas mixture of 1%oxygen, 3$ carbon dioxide, and 96%nitrogen, a continuous monolayer of single cells and polynucleated syncitia is formed. The cultures were inoculated with the supernatant from a centrifuge of nuclear polyhedra and incubated for 3-4 hours at 2 5 O C . in the gas

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mixture described above. Five days after infection numerous polyhedra could be observed in the nuclei. Using uninociilated cultures of ovarian tissues from the Tussock moth (Dasychira sp.), Grace ( 1958) observed the development of polyhedra, showing the presence of an unsuspected virus. In the use of tissues from apparently healthy insects the ever-present possibility of latent virus infections must be borne in mind. Although the study of insect viruses by means of tissue-culture techniques is far behind similar studies with viruses of the higher animals, the brief account given above shows that a start has now been made and the way opened for obtaining some much needed information on the modes of replication of the insect viruses.

XIV. VIRUSESMULTIPLYINGIN BOTH ARTHROPODSAND PLANTS The fact that some viruses have the power of multiplication, both in the plant host in which they cause a recognizable disease and in the insects which transmit them from plant to plant (vectors), has only been generally accepted within recent years. At the moment unequivocal evidence of such multiplication is restricted to certain viruses with leafhopper (Jassidae) vectors, although it is also possible that one or two viruses may multiply in aphid vectors. The first circumstantial evidence bearing on this problem came from the work of Kunkel (1926), who showed that there was a delay in the development of infective power of at least 10 days, after feeding on an aster plant with “yellows,” before the aster leafhopper (Macrosteles fascifrons S a l ) became infective to a healthy susceptible plant. Kunkel interpreted this delay as an “incubation period while the virus developed in the insect; at the time this interpretation was not accepted by many workers. In 1933 a Japanese worker, Fukushi, demonstrated that the virus of the dwarf disease of rice was transmitted to the progeny of the leafhopper vector (Nephotettix apicalis var. cincticeps), but only if the female was viruliferous (virus-bearing). This means, of course, that the v i r u s passed by way of the egg, but not by the sperm. In 1940 Fukushi showed that the virus was passed to the sixth generation. The virus was retained for at least 374 days and infections were obtained in about 1200 rice plants by 82 infective leafhoppers derived from a single viruliferous female. Black (19%) camed out similar experiments to those of Fukushi with the virus of clover club leaf which he has shown to be transmitted through the egg of the leafhopper vector, Agalliopsis novella Say. From a pair of viruliferous leafhoppers the breeding was carried out through 21 generations over a period of 5 years. The insects were fed

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throughout on virus-immune lucerne plants without loss of infectivity. Black has calculated that, if multiplication of the virus is not assumed, the dilution of the original virus in the parent insect exceeded lo-'*. For a more complete account of the evidence for the multiplication of viruses in both plant and leafhopper the reader is referred to review articles by Black ( 1959) and Maramorosch (1959). In the case of one or two aphid-transmitted plant viruses there is a fairly long delay in the development of infective power, called an incubation period in the leafhoppers, after feeding on a source of virus. It is possible, therefore, that in these cases there may be multiplication of the virus in the aphid. Stegwee and Ponsen (1958) developed a technique for inoculating the virus of potato leafroll into its vector, the aphid Myzus persicue Sulz. They succeeded in getting a number of serial transfers and consider that the amount of dilution involved is good evidence of multiplication. In a recent paper Stegwee and Peters (1961) describe a method of isolating the leafroll virus from the aphids by means of density gradient centrifugation. Since it has now been proved that multiplication of certain viruses takes place in both insect and plant, it would be expected that there would be some effect of such multiplication on the insect. For many years workers have sought in vain for some evidence of a disease in the insect vector, and it is only recently that possible manifestations of disease have been found. Littau and Maramorosch (1956, 1958) describe cytological changes which occur in aster leafhoppers carrying the virus of aster yellows. It has been established that these changes occur in the nuclei of fat body cells of male leafhoppers 18 to 28 days after acquiring the virus by feeding or injection. In viruliferous insects the nuclei tended to be stellate rather than rounded and the cytoplasm was reticulate instead of homogeneous with many vacuoles. Another case of the deleterious effect of a plant virus upon its leafhopper vector has been described by Jensen (1958, 1959a,b). Vectors of peach yellow leafroll, a strain of Western X-disease, virus were found to live only half as long as virus-free individuals of the same species, Callodonus rnontunus. Maramorosch (1960) suggests that it would be of interest if inoculation of insects with hemolymph from viruliferous individuals could be carried out. This would eliminate the possibility that shortened life was due to a toxic substance acquired from virus-infected plants. This possibility, however, is largely discounted by the fact that of the insects feeding on the viius-diseased plants only the active transmitters had the shortened life, while nontransmitting individuals lived for the normal period. In studies on the wheat striate mosaic and its leafhopper vector,

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Delphucodes pallucida Fabricius, Watson and Sinha ( 1959) found that many embryos from a viruliferous female died at a late stage of their development and only about one-third as many nymphs were derived from infective as from virus-free females. At first this was ascribed to the action of the virus causing a disease in the progeny, but now a doubt has arisen as to whether a lethal gene derived from inbreeding may not be the cause of death. Not much is known of the morphology of the leafhopper-transmitted viruses owing to the fact that most of them are not mechanically inoculable to plants. However, the invention by Brakke (1951) of the sucrose gradient centrifugation technique enabled several of this type of virus to be isolated and purified (Black, 1955; Brakke et al., 1951, 1954). The purification and crystallization of the wound-tumor virus of sweet clover was achieved and an electron micrograph of a section through a crystal made by Vatter and Whitcomb is shown in Black's review (1959). The virus appears to be spherical, or near-spherical, measuring about 60 mp in diameter; this is large compared to most of the other near-spherical plant viruses. Two other leafhopper-transmitted viruses have been shown to be rod-shaped and about the same size; one of these is the potato yellow dwarf virus, which is more or less rod-shaped but appears to be malleable (Brakke et al., 1951). The other is one which Herold d. al. (1960) have studied in corn (maize), probably the virus of corn stunt. By means of ultrathin sections they have demonstrated the presence of rod-shaped particles in the cytoplasm around the nucleus. Electron micrographs of pur%ed preparations show the virus particles to be very regular in size and shape and to have a highly differentiated structure with two limiting membranes and a dense, rod-shaped core in the center. The particles have dimensions of 242 mp in length and 48 mp in diameter, and they are considered to be very similar in appearance to the rod-shaped viruses of the nuclear polyhedroses of insects. By means of ultrathin sections of infected leafhoppers Fukushi et al. ( 1960) have succeeded in demonstrating by electron microscopy the virus of rice dwarf within the insect vector N . cincticeps. The virus appears to be spherical and measures 40-60 mp in diameter. This is the first time a plant virus has been visualized within its insect vector. By means of sucrose gradient centrifugation Black (1955) and his co-workers have developed antisera to several leafhopper-borne viruses. In 1954 Black and Brakke showed that the supernatants from ultracentrifuged extracts of plants and leafhoppers infected with woundtumor virus (WTV) contained an antigen which was smaller than the v i r u s itself, but reacted specifically with WTV antiserum. This sub-

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virus, or soluble antigen, appears in both plants and insects during early stages of infection with WTV (Whitcomb and Black, 1959) and an increase in the titer of the wound-tumor soluble antigen (WTSA) precedes the appearance and development of the mature infective virus particle. From this work it is now possible to estimate the proportions of infected insects either by serological analysis of single insects or by obtaining the WTSA titer of mass extracts (Whitcomb and Black, 1961). It has been suggested from time to time that plant viruses which multiply in both plant and insect vector might have originated as a symptomless virus infection of the insect which had become adapted to a plant host. Maramorosch (1955) has discussed this and emphasized that the insect host range is narrow and the plant host range quite broad; he considered that this, together with the lack of symptoms in the insect, suggested long adaptation in the arthropod host. However, as Black (1959) has pointed out, evidence is now accumulating that in certain cases, at all events, the insect vectors are themselves diseased. Black (1953) showed that if the WTV was maintained for many years by grafting in plants it lost its affinity for the insect and could no longer be transmitted by it. The work of Black, Wolcyrz, and Whitcomb (1958) suggests that the vectorless strain of WTV does not multiply in its vector and so cannot be transmitted by it. This loss of affinity for its insect vector in a fairly short time might be evidence that the adaptation to the insect was of comparatively recent origin. The same phenomenon has been demonstrated with the thrips-transmitted virus of tomato spotted wilt and with certain aphid-borne viruses. What information there is on the morphology of the leafhoppertransmitted viruses suggests that they are rather larger than the other plant viruses with different vectors. In the case of the virus causing corn stunt there seems to be a superficial resemblance to the rod-shaped virus particles of the nuclear polyhedroses. BETWEEN VIRUSES XV. INTERFERENCE

The interactions between two or more viruses in the same plant, and the cross-immunity often exhibited by like viruses, have been known for many years and the subject has been reviewed in this series by Bennett (1953). Slight evidence seems now to be forth-coming of somewhat similar occurrences in insects affected by two viruses at the same time. It is of course no unusual occurrence for an insect to be infected by two viruses, but there is less knowledge of the interactions between the two. Caterpillars have been found infected with a nuclear and a cytoplasmic polyhedrosis (Smith et al., 1953; Bird and Whalen, 1954), a nuclear polyhedrosis and a granulosis virus (Tanada, 1959b),

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a cytoplasmic polyhedrosis and a granulosis (Vago, 1959b), and the writer has observed on several occasions the larvae of Tipula paludosa Meigen infected simultaneously with its nuclear polyhedrosis and the cytoplasmic iridescent virus. In the case of the armyworm Pseudaletia unipunda (Haworth) (Tanada, 1959) infected with a nuclear polyhedrosis and a granulosis, both viruses attack the fat body and the multiplication of the granulosis virus may be restricted by the more rapid invasion of the fat tissue by the virus of the nuclear polyhedrosis. Tanada considers that there is an increased virulence (synergism) in the remains of a larva infected with both viruses, and he finds that heat-inactivated granulosis virus is still capable of enhancing the virulence of unheated nuclear polyhedrosis virus, but that this does not hold for the reverse procedure. Bird (1959) has described a type of cellular immunity in a case of double infection of the spruce budworm Choristoneura fumiferana Clemens with a nuclear polyhedrosis and a granulosis. Prior infection by one virus interferes with infection by the second. In general it appears that a cell will accept either virus but not both. Adjacent cells are frequently infected with different viruses but usually blocks of cells are infected with the same virus. Both viruses multiply in the same type of tissue but opinion seems to be divided as to whether the granulosis viruses multiply in the nucleus or the cytoplasm. Bird considers they are cytoplasmic viruses but if they do invade the nucleus it is understandable that multiplication would be difficult with a nuclear polyhedrosis virus already in possession. Aruga et al. (1961) have observed interference between two cytoplasmic polyhedrosis viruses of the silkworm Bombyx mori ( Linnaeus ) , both of which multiply in the cytoplasm of the same mid-gut cells, when the two viruses were fed to the larvae at various time intervals. The feeding of one type of virus at intervals after cold treatment indicated that the latent virus infection which was activated by the cold treatment also interfered with the inoculated virus. In conclusion an interesting case of interference between two plant viruses in their leafhopper vector may be briefly stated. There exist two strains of the aster yellows virus, known respectively as aster yellows and Californian aster yellows. By the use of differential host plants Kunkel (1955) was able to show that a leafhopper which fed first on a plant with aster yellows became viruliferous for that virus and was unable to transmit Californian aster yellows when transferred to a plant with that virus. The same phenomenon occurred when the procedure was reversed. Kunkel points out that these experiments prove only that leafhoppers, infective for one virus, do not transmit the other. They have

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not shown that both viruses may not be picked up by the insect. Presumably, however, the virus first acquired is multiplying inside the insect and so the available multiplication sites are occupied, leaving no opportunity for the second virus to reproduce itself. Possibly with the development of Black‘s serological technique more light will eventually be thrown on this problem. ACKNOWLEDGMENT The author wishes to acknowledge support from grant No. E-3400 of the U.S. Public Health Service. All illustrations are from the Agricultural Research Council, Virus Research Unit, Cambridge.

REFERENCES Andrewes, C. H. (1958). Symposium Latency k. Masking in Viral 6. Rickettsia1 Infections, p. 196. Burgess, Minneapolis. Aruga, H. ( 1957). Nippon Sanshigaku Zasshi 26, 279-283 (English summary). Aruga, H., Hukuhara, T., Yoshitake, N., and Ayudhya, I. N. (1961). J. Insect Pathol. 3, 81-92.

Bennett, C. W. (1953). Advances in Virus Research 1, 40-65. Bergold, G. H. (1948). Z. Naturforsch. 3b, 33-42. Bergold, G. H. (1958). In “Handbuch der Virusforschung” (C. Hallauer and Meyer, eds.), 60-127. Springer, Vienna. Bird, F. T. (1959). J. Insect Pathol. 1,406-430. Bird, F. T., and Whslen, M. M. (1954). Can. J . Zool. 32, 82-86. Black, L. M. (1950). Nature 166, 852. Black, L. M. (1953). Phytopathology 43,9-10. Black, L. M. (1955). Phytopnthology 45, 208-216. Black, L. M. ( 1959). In “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. 2, pp. 157-185. Academic Press, New York. Black, L. M., and Brakke, M. K. ( 1954). Phytopathology 44,482. Black, L. M., Wolcyrz, S., and Whitcomb, R. F. (1958). Abstr. 7th Intern. Congr. Microbiol., Stockholm, 1958. Bolle, J. (1908). Z. landwirtsch. Versuchsw. Deut-Oesterr. 11, 279-280. Brakke, M. K. (1951). J. Am. Chem. SOC. 73,1847. Brakke, M K., Black, L. M., and Wyckoff, R. W. G. (1951). Am. J . Botany 38, 332.

Brakke, M. K., Vatter, A. E., and Black, L. M. (1954). Brookhaven Symposia in Biol. 6, 137-156. Brenner, S., and Horne, R. W. (1959). Biochim. et Biophys. Acta 34, 103. Burnside, C. E. ( 1945). Am. Bee J . 85,354-355,363. Conte, A. (1907). Compt. rend. 36 Session Assoc. Franc. Avance. sci. pp. 622-623. Day, M. F., Farrant, J. L., and Potter, C. (1958). 1. Ultrastruct. Research 2, 227-238. Franz, J., Krieg, A., and Langenbuch, R. (1955). 2. Ppanzenkrankh. u. Pfinzenschutz 62, 721-726. Fukushi, T. (1933). Proc. Imp. Acad. (Tokyo) 9,457. Fukushi, T. (1940). J. Fac. Agr. Hokkaido Univ. 45, 83. Fukushi, T., Shikata, E., Kimura, I., and Nemoto, M. (1960). Proc. Japan Acad. 36, 352-357. Gershenson, S. M. (1956). Doklady Akad. Nauk S.S.S.R. 100, No. 6, 1199.

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Gershenson, S. M. (1960). Problems Virol. ( U.S.S.R.) (English Translation) 5, 109-113. Gershenson, S . M., Kok, I. P., Vitas, K. I., Dobrovolskaia, G. N., and Skuratovskaia, 1. N. ( 1961).VIth Intern. Congr. Biochem., Sect. 4, Biochem.Viruses, Moscow, 1961. Grace, T. D. C. (1958). Science 128,249. Herold, F., Bergold, G. H., and Weibel, J. ( 1960). Virology 12,335-347. Hills, G . H., and Smith, K. M. (1959). J. Insect Pathol. 1, 121-128. Horne, R. W., and Nagington, J. (1959). J. Mol. Biol. 1,333438. Huger, A. ( 1961). J. Insect Pathol. 3, 338-341. Huger, A., and Krieg, A. (1961). J. Insect Pathol. 3, 183-196. Jensen, D. D. ( 1958). Phytopathology 48, 394. Jensen, D. D. (1959a). Virology 8, 164-175. Jensen, D. D. ( 1959b). Pan-Pacific Entomologist 35, 65-82. Jones, B. M., and Cunningham, I. (1960). Nature 187,1072-1074. Kaesberg, P. (1956). Science 124, 626. Klug, A., and Caspar, D. L. D. (1960). Advances in Virus Research 7, 225-325. Krieg, A. ( 1955). Naturwissenschaften 42, 589-590. Krieg, A. (1957). Arch. ges. Virusf. 7,212-219. Krieg, A. ( 1961). 2. Naturforsch. 16b, 115-117. Krieg, A., and Huger, A. (1960). J. Insect Pathol. 2, 274-288. Krywienczyk, J., and Bergold, G. H. ( 1960a). Virology 10,308-315. Krywienczyk, J., and Bergold, G. H. ( 1960b). Virology 10, 549-550. Krywienczyk, J., and Bergold, G. H. ( 1 9 6 0 ~ )J.. Immunol. 84, 404-408. Krywienczyk, J,, MacGregor, D. R., and Bergold, G. H. ( 1958). Virology 5, 476480. Kunkel, L. 0. (1926). Am. J. Botany 13, 646. Kunkel, L. 0. ( 1955). Advances in Virus Research 3,251. Kurisu, I. ( 1955). Bull. Sericult. Expt. Sta. Kumamoto 6, 48-56 ( I n Japanese). L’Heritier, Ph. ( 1958). Advances in Virus Research 5, 195-245. Littau, V. C., and Maramorosch, K. ( 1956). Virology 2, 128. Littau, V. C., and Maramorosch, K. ( 1958). Phytopathology 48, 263. Maramorosch, K. ( 1951). Nature 167, 734. Maramorosch, K. (1955). Am. 1. Botany 42, 676. Maramorosch, K. ( 1959). “Volume in Homage to Savulescu” (Ed. Rumanian Acad. ) pp. 421442. Maramorosch, K. ( 1960). Protoplama 52, 457-466. Markham, R., and Smith, K. M. ( 1949). Parasitology 39, 330. Martignoni, M. E. (1955). Science 122,764. Martignoni, M. E. (1959). J. Insect Pathol. 1, 294-296. Martignoni, M. E., and Scallion, R. J. (1961). Nature 190, 1133-1134. Ripper, M. (1915). 2. landwirtsch. Versuchsw. Deut-Oesterr. 1915, 12-15. Roegner-Aust. S. (1949). Ztschr. Angew. Entom. 31, 3-37. Sidor, C. (1960). Virology 10, 551. Smith, K. M. (1952). Biol. Revs. Cambridge Phil. SOC. 27,347457. Smith, K. M. (1958). Virology 5, 168-171. Smith, K. M. (1959). I n “The Viruses” (F. M. Burnet and W. M. Stanley, eds.), Vol. 3, pp. 369-392. Academic Press, New York. Smith, K. M., and Hills, G. J. (1962a). 11th Intern. Entomol. Congr., Vienna, 1960. Smith, K. M., and Hills, G. J. (192613). 5th Intern. Congr. Electron Microscopy, Philadelphia 2, 1. Smith, K. M., and Rivers, C. F. ( 1956). Parasitology 46, 235-242. Smith, K. M., and Wyckoff, R. W. G. (1951). Research 4, 148-155.

240

KENNETH M. SMITH

Smith, K. M., and Xeros, N. (1954). Nature 173, 866. Smith, K. M., Wyckoff, R. W. G., and Xeros, N. (1953). Parasitology 42, 287289. Smith, K. M., Hills, G . J., Munger, F., and Gilmore, J. E. (1959a). Nature 184, 70. Smith, K. M., Hills, G. J., and Rivers, C. F. (195913). J. Insect Pathol. 1, 431-437. Smith, K. M., Hills, G . J., and Rivers, C. F. (1961). Virology 13, 233-241. Stegwee, D., and Peters, D. (1961). Virology 15, 202-203. Stegwee, D., and Ponsen, M. B. (1958). Entomol. Erptl. et. Appl. 1, 291-300. Steinhaus, E. A. (1958). Proc. Intern. Congr. Entomol., Montreal, 1956 4, 725. Steinhaus, E. A., and Thompson, C. G. (1949). Science 110,276-278. Stent, G. H. (1958). Advances in Virus Research 5, 95-149. Surany, P. (1960). S. Pacific Comm. Tech. Paper No 128,62 p p . Tanada, Y. (1956). J. Econ. Entomol. 49, 52-57. Tanada, Y. (1959a). J. Insect Pathol. 1, 197-214. Tanada, Y. (195913). J. lnsect Pathol. 1,215-231. Thomas, R. S. (1961). Virology 14, 240. Thomas, R. S., and Williams, R. C. (1961). J. Biophys. Biochem. Cytol. 11, 15-28. Thompson, C. G., and Steinhaus, E. A. (1950). Hilgardia 19, 411-445. Trager, W. (1935). J. Erptl. Med. 61, 501. Vago, C. (1951). Rev. can. biol. 10, 299-308. Vago, C. (1953). Rev. ver Ci soie 5, 73-81. Vago, C. ( 1959a). Entomophaga 4, 23-36. Vago, C. (195913). J. Insect Pathol. 1,75-79. Wasser, H. B. (1952). J. Bacteriol. 64, 787-792. Wasser, H. B., and Steinhaus, E. A. (1951). Virginia J. Sci. [N.S.] 2, 91-93. Watson, M. A., and Sinha, R. C. (1959). Virology 8, 139-163. \Vhitcomb, R. F., and Black, L. M. ( 1959). Phytopathology 49, 554. \Vhitcomb, R. F., and Black, L. M. ( 1961). Virology 15, 136-145. Williams, R. C., and Smith, K. M. (1957). Nature 179, 119-120. Williams, R. C., and Smith, K. M. (1958). Biochim. et Biophys. Acta 28, 464-469. Wyatt, S. S. ( 1956). J . Gen. Physiol. 39, 841-852. Xeros, N. ( 1953). Nature 172, 309. Xeros, N. ( 1962). Biochim. et Biophys. Act4 55, 176-181. Zan-Yin, G., Tsui, N. L., and Un, Z. T. (1959). Act4 Virol. (Prague) Suppl. 3. 55-60 ( English edition).