Viruses of Invertebrates

VIRUSES OF INVERTEBRATES Constant Vago and Max Bergoin Station de Rechercher Cytopathologiquer I.N.R.A., Montpellier-Saint-Chrirtol, France

C.N.R.S.,

I. Introduction .......................................................... ......................

248 248

A. Structure ....................................

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B. Nonoccluded Viruses. . . . . . . . . . . . . . . . . .

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. . . . . . . . . . . . 273

A. Serological Relationships .....................

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C. Titration of Virus Suspensions. . . . . . . . . . . . . . . IX. Virus Development.

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275

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D. Nonoccluded Viruses.

B. Infection of Invertebrate Tissue Cultures with Viruses from Inver280 tebrates. .......................................................... C. Infection of Tissue Cultures of Arthropod Vectors with the Viruses

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B. Insect Populations. ......

............................... B. Extraction and Formulation. . . . . . . . . . . . . . . . . . D. Biological Tests. . . . . . . . . . . . . . . . . . . 247

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288

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F. Application in the Field. ..........................................

References.. ..........................................................

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I. INTRODUCTION For a long time research on invertebrate viroses has been carried out mainly on those characterized by the formation of intracellular polyhedral and granular inclusion bodies. However, during recent years this range has been increased with the discovery of viroses with new types of inclusion bodies and of several nonoccluded viruses. I n this paper will be reviewed data and results obtained during the last five years after the period analyzed by Smith in 1962. We consider also reviews from Aizawa (1963), Aruga (1963), Bergold (1963a), Huger (1963) Tanada (1963), Smith (1963), Vago (1963a), and Martignoni (1964). No account is included in this compilation of the two particular aspects dealing with the transmission of plant viruses by arthropod vectors and the hereditary virus of Drosophila melanogaster Meigen since these have recently been reviewed, the former by Jensen (1963) ; Maramorosch (1963) ; Maramorosch and Jensen (1963) ; Smith (1965) ; the latter by Brun (1963) ; Lestrange (1963) ; Ohanessian Guillemain (1963). 11. DISTRIBUTION AMONG INVERTEBRATES The great majority of the viroses described among the invertebrates occur in the Insecta. In the Lepidoptera, newly found nuclear polyhedroses, intestinal cytoplasmic polyhedroses, and granuloses have been added to the list of inclusion body viroses, already well known. Viroses in which intracellular inclusion bodies differ from those of the former group have been described (Weiser and Vago, 1966) in Operophtera brumata L. and Acrobasis zelleri Rag. Videnova and Sengalevitch (1966) have observed viruses in Cossus cossus L. which they included also in the genus Vagoiavirus. Nonoccluded viruses have been found in the following species : Bombyx mori L. (Aizawa and Furuta, 1964), Galleria rnellonella L. (Meynadier et al., 1964), Antheraea eucalypti Scott (Grace and Mercer, 19651, Chilo sulvpressalis Walker (Fukaya and Nasu, 19661, Gonometa podocarpi (Harrap et al., 1966), and possibly Pyrausta nubilalis Hubner (Adams and Wilcox, 1965). Among the Coleoptera, Melolontha melolontha L. is affected by a new virus (Vago, 1963b; Hurpin and Vago, 1963)) called by Weiser (1965a) Vagoiavirus, which is distinguished by the formation of spindle-shaped inclusion bodies. The cockchafer suffers also from the attack of a nonoccluded virus (Krieg and Huger, 1960) and also from another

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virosis which produces a state of lethargy (Hurpin et al., 1967). Two other free viruses have been discovered : Rhabdionvirus in Oryctes rhinoceros L. (Huger, 1966) and the SIV, an iridescent type, in Sericesthis pruinosa (Dalman) (Steinhaus and Leutenegger, 1963). I n the Diptera, Kellen et al. (1966) found cytoplasmic polyhedra in the hypodermal cells and in the cells of the antenna buds, wings and legs of Culez tarsalis Coquillett.; Clark et al. (1965) described an iridescent virus, the MIV, in Aedes taeniorhyncus Wiedemann ; Weiser (1965b) observed in Aedes annulipes (Meigen) and Aedes cantans (Meigen) a nonoccluded virus. Among the Hymenoptera, Smirnoff (1961) described a new example of a nuclear polyhedrosis of intestinal cells in the sawfly Neodiprion suuinei Middleton; while in the bee, the electron microscope has revealed the etiological agent responsible for sacbrood (BrEfik et al., 1963) , and viruses associated with paralysis (Bailey et al., 1963) and “black disease” (Vago, 1964a). Recently, viral particles present in bees suffering from European foulbrood have been found to be infectious (Giauffret et al., 1966a). All these viruses are paraspherical and free in the cytoplasm. Among the Orthoptera, Meynadier (1966) purified and isolated a nonoccluded virus which produced a paralysis in Gryllus bimaculatus Geer. Henry and Jutila (1966) observed polyhedral bodies in the grasshopper Melanoplus sanquinipes. Apart from the insects, viroses have now been identified in Arachnids and in Crustacea. The acarian Panonychus citri (McGregor) is infected by a nonoccluded virus (Smith et al., 1959) and possibly Panonychus ulmi Koch (Steinhaus, 1959). Vago (1966) has isolated and studied with the electron microscope a free, paraspherical virus in the crab Macropipus depurator L., suffering from paralysis.

111. NEWTYPESOF VIROSES Most of the viral diseases described in recent years in invertebrates belong to types of viroses already known. On the other hand, cases arise of types which, because of their pathogenicity or viral characteristics, constitute groups which are not yet known among the invertebrates or even in the field of general virology. These new types will be briefly mentioned here. Spindle Viroses: Recentlr a virus disease of a Scarabeidae (Coleoptera) has been described which differs from all previously known invertebrate types (Hurpin and Vago, 1963; Vago, 196313). It develops in the fat body and blood cells of M . melolontha larvae and is characterized by the formation of cytoplasmic spindle-shaped inclusion bodies

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of 0.5 to 12 p and more spherical bodies measuring up to 20 p. I n ultrathin sections with the electron microscope these spindles show a homogeneous structure, whereas the spherules contain in an opaque matrix numerous ovoid virus particles measuring 400 X 250 mp (Vago, 196313). I n shape and structure this is a new type of invertebrate virus which has been named Vagoiavirus by Weiser (1965a). The purified virus was obtained by dissolution of the inclusion bodies. Some of the histochemical changes occurring during the disease were studied by Morris (1966). A virosis of 0. brumata described by Weiser and Vago (1966) is closely related with this Melolontha spindle virus. Oval bodies containing viral particles of the Vagoiavirus type were found in the cytoplasm and fat body. Videnova and Sengalevitch (1966) have recently observed a virosis in C. cossus (Lepidoptera) with inclusion bodies similar to type Vagoia-

vim.

Iridescent Viroses: The iridescent viruses, first observed in the dipteron Tipula paludosa Meigen have since been subjected to numerous studies. A disease of this type has been found in the coleopteron S. pruinosa (Steinhaus and Leutenegger, 1963): the fat body and the centrifuged pellet of the purified virus are both iridescent. The isolated viruses are icosahedral and measure 130 mp. They contain deoxyribonucleic acid (DNA) but differ serologically from the Tipula virus (Day and Mercer, 1964). The development of Sericesthis iridescent virus (SIV) in the cellular cytoplasm resembles that of Tipula iridescent virus (TIV), and this has also been observed in A. eucalypti cells grown in vitro (Bellett, 1965a, b; Bellett and Mercer, 1964). Clark et al. (1965) noted in some orange-tinted iridescent fat body tissue of A. taeniorrynchus larvae, spheroidal viral particles of approximately 180 mp which proved to be infective to the host and to three other mosquito species : Aedes fulvus pallens Wiedemann, Aedes vexans Meigen, and Psorophora ferox Humbold (Chapmann et al., 1966). An iridescent virosis has also been found in the Lepidoptera from Chilo suppressalis larvae showing a bluish tint; a virus of 160 mp was obtained. This multiplied in the fat body cell cytoplasm (Fukaya and Nasu, 1966), in the majority of the insect’s tissues (Mitsuhashi, 1966a), and in hemocytes grown in vitru (Mitsuhashi, 1966b). Bee Sacbrood: Although the viral nature of this disease has long been known, it is only recently that the pathogen itself has been observed. Spheroidal particles of 30 mp (BrEBk et al., 1963; BrFBk and KrBlik,

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1965) and 28 mp (Vago, 1964a; Bailey et al., 1964) have been seen with the electron microscope. BrGk and KrBlik (1965) distinguished 12 subunits on the periphery of the virus and considered the particle to be an icosahedron composed of 42 subunits. Similar particles have been seen in ultrathin sections of some nonidentified tissue (Lee and Furgala, 1965a). The same authors identified the viral nucleic acid as ribonucleic acid (RNA) (Lee and Furgala, 1965b). European Foulbrood: Although the role of various bacteria in the etiology of this disease has been studied for a long time, it was Vago (1964a) who suggested that a virus was concerned. The high infectivity of bacteria-free filtrates has recently demonstrated the presence of a virus, shown on the electron microscope to be spheroidal corpuscles 27-30 mp in diameter (Giauffret et al., 1966a). Bee Paralysis, Black Disease: These diseases cover a group of symptoms which have been attributed to such causes as faulty nutrition, hibernation, and intoxication. I n some cases the insects have difficulty in flying and their legs tremble and are paralyzed; in other cases the insects become dark in color. The infectious nature of the disease was recognized by Vecchi and Zambonelli (1961), who considered it to be of viral origin. Later, Bailey et al. (1963) demonstrated the existence of two viruses in paralyzed bees. One of these, called acute bee paralysis virus (ABPV), was an isometric particle measuring 28 mp in diameter; the other, called chronic bee paralysis virus (CBPV), was a particle of irregular shape, measuring 27 x 45 mp. Furgala and Lee (1966) observed the paraspherical particles of the ABPV in the fat body of experimentally infected bees, while similar viruses were obtained from bees suffering from “forest disease” (Vago, 1964a). Particles of the same size as those of the purified virus (Giauffret et al., 1966b) (Fig. 1) and smaller particles (Lee and Furgala, 1965c) were found in the cytoplasm of the nerve cells. Transparency Disease of Scarabaeids: A case of a cytoplasmic, spherical nonoccluded virus was observed in the coleopteron M . melolontha. Heidenreich in 1939 described a pathological condition he called “Wassersucht,” the symptoms of which were the disintegration of the fat body and a pronounced transparency of the whole body, and suggested that it might be of viral origin. This was confirmed by Krieg and Huger (1960) who found in the cytoplasm of the blood and fat cells polyhedral particles of 60 to 75 mp in diameter containing DNA. Flaccidity Disease of the Silkworm: The viral origin of a type of flaccidity known also as “Maladie des tdtes claires,” demonstrated by Paillot (1930, 1941) was confirmed by Yamazaki et al. (1960), then by Aisawa and Furuta (1964), who observed particles of 25 and 30 mp

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in diameter dispersed throughout the cytoplasm of the infected cells. The virus is susceptible to heat and to the actioii of the digestive juice of the silkworm and is neutralized with an antiserum prepared from the isolated virus (Aizawa et al., 1964). Paralysis of Crickets: Meynadier (1966) described a spheroidal nonoccluded virus of 35 mp diameter in a paralyzed Gryllus bimaculatus cricket. This was the first virosis to be described in the Orthoptera.

FIG.1. Purified virus particles from adult honeybee A p k mellifera suffering from black disease. Negative staining with potassium phosphotungstate. Magnification: X 100,000. (Original.)

Crab Paralysis: Vago (1966) observed spheroidal viral particles of 50 to 60 mp in size in the crab Macropipus depurator suffering from paralysis. The virus was obtained from the muscle, heart, and glandular tissue, and inoculation from these tissues provoked paralytic symptoms in experimental animals. Melolontha Lethargy: A pathological condition of 1 i . melolontha larvae characterized by a cessation of feeding and absence of movement was found to be transmissible by inoculation and by injection of diseased tissue. An icosahedral virus of 80 mp was isolated. It forms geometrical patterns within cytoplasmic clusters located in the fat body.

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No iridescent effect has been obseved (Hurpin et al., 1967; Vago et al., 1967). Densonucleosis of Galleria mellonella: A type of virosis with a very individual mode of action was discovered in the greater wax moth G. mellonella (Meynadier et al., 1964). The disease is very contagious and epizootic: paralysis and death occur 4 to 6 days after infection. The viral pathogen has been isolated: it is spheroidal and small in size, 2123 ma (Fig. 2) (Vago et al., 1966a) and contains DNA (Truffaut et al.,

FIG.2. Section through adipose tissue of Galleria mellonella. larva infected with densonucleosis virus. Adipose cells with hypertrophied nuclei ( n ) and a dense central mass (dm). Magnification: X 875. (From Amargier e t nl., 1965.)

1967). The lesions localized in most organs consist of dense, voluminous, Feulgen-positive masses present in the highly hypertrophied nucleus (Fig. 3) (Amargier et al., 1965). The virus is densely packed in these masses. Cell cultures of G . mellonella and B. mori have been successfully infected with the densonucleosis virus (Vago and Luciani, 1965; Vago et al., 1966b). It is possible to detect infection in larvae, nymphs, and adults by using a serum prepared from the purified virus (Giran, 1966). Flaccidity Disease of Antheraea eucalypti: A virus disease has been observed in larvae of the lepidopteron A. eucalypti. On dissection, only the midgut seemed to be attacked, but spheroidal 50 mp viral particles

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FIG.3. Ultrathin section through a nucleus of an adipose cell of a Galleria mellonella larva infected with densonucleosis virus, stained with uranyl acetate and lead citrate. NM, nuclear membrane; PZ, peripheral zone with few virus particles; IM, intranuclear electron-dense mass with virus particles closely packed. Magnification: X 21,000. (From Vago et al., 1966a.)

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were found in the hemolymph. The latter were accompanied by even smaller particles which might be liberated “cores.” I n ultrathin sections mesodaeum cell nuclei were seen to contain the virus (Grace and Mercer, 1965). Malaya Disease of Oryctes: The disease of the coleopteron 0. rhinoceros is characterized by a swelling of the abdomen and frequent diarrhea. Spherical particles of approximately 165 mp and rods of 200 x 70 mp were obtained by differential centrifugation of crushed diseased tissue. The virus multiplies principally in the nuclei of the f a t body cells, but i t also develops in the cytoplasm. The rods are formed from the dense virogenic stroma, from the fibrillar and granular stroma material, or from spherical shells which are thought to be a n immature stage of the virus. The name Rhabdionvirus has been suggested for the virus (Huger, 1966). IV. THEINCLUSION BODIES

A . Structure

It is usual to divide insect virus inclusion bodies into three types: nuclear polyhedra, cytoplasmic polyhedra , and granules, a classification which takes into consideration the shape, the intracellular and tissue distribution, and the nature of the viruses they contain. Electron microscope investigations of their ultrastructure have shown that all these inclusion bodies are built on the same model (Morgan et al., 1955; Day et al., 1956; Bergold and Suter, 1959; Smith and Hills, 1962; Bergold, 1963b, c ) ; they consist of crystals of protein molecules which form a network within which virus rods (in the case of nuclear polyhedra and capsules) or icosahedral viruses (in the case of cytoplasmic polyhedra) are enclosed. The inclusion bodies of a cytoplasmic polyhedrosis of the mosquito Culex tarsalis have recently been shown to have the same crystalline structure (Kellen et al., 1966). The structure of these three types of inclusion bodies, recently exhaustively reviewed by Bergold (1963a, 1964), Smith (1963), and Huger (1963), will not be dealt with here. Certain intestinal cytoplasmic inclusions reported by Vago (1959) in B . mori larvae have been described in several lepidopterous larvae under the name of “intestinal pseudopolyhedra” (Atger et al., 1961). Although these polyhedral inclusions, varying in size from 0.4 to 3.3 p , can be mistaken for viral cytoplasmic polyhedra, they may be distinguished from the latter, using the staining methods of Vago and Amargier (1963) which do not affect the pseudopolyhedra. Furthermore, they are insoluble in weak alkaline solutions and soluble in 0.3% acetic acid. They

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have not been found to possess any infective property, nor to contain any internal viral particle when examined with the electron microscope. A new type of viral inclusion body which does not fall into the above-mentioned categories has recently been described in larvae of M . melolontha attacked by the disease known as “spindle virosis” (Hurpin and Vago, 1963; Vago, 1963b). This is the first inclusion body virosis observed in Coleoptera. A cross-section of a diseased larva stained by

FIQ.4. Section through adipose tissue of third-instar larva of Melolontha melolontha suffering from spindle virosis. s, spherules with radial fissures ( f ) ; sp, spindleshaped inclusion. Both inclusions are often contained in “pockets” (p) surrounded by a membrane. Vago and Amargier staining. Magnification: X 875. (Original.)

the technique of Vago and Amargier (1963) revealed the presence of two types of inclusions in the cytoplasm of the fat body cells (Fig. 4): one, spindle-shaped which stained bright red; the other, larger, and ovoid which stained rose color. These two types of inclusion were often contained in “pockets” surrounded by a membrane within which a number of spindle-shaped, and a few ovoid, inclusions could be seen. The spindle-shaped inclusions (or spindles) vary in size from a few fractions of a micron in the early stages of the disease to 12 p for the giant forms. They are usually very regular in shape, with a length:

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diameter ratio of about 2. They stain dark blue with Giemsa, are unchanged or show only a light blue color with hemalum erythrosine, and appear gray-blue with hematoxylin ferric-erythrosine (Amargier et al., 1964). In addition to the perfect spindles with their two tapered extremities, arrangements which seem to be the result of the fusion of two spindles along their major axes, or of 3, 4, or more half spindles into characteristic geometrical figures were also found. The ovoid inclusions, or spherules, also vary in size and may reach 20 p a t maximum diameter. They are always definitely fewer in number than the spindles. They stain light blue with Giemsa and yellow with hemalum erythrosine and hematoxylin ferric-erythrosine. Radial fissures are always present in the central part of the spherule. These fissures, which can be seen in unstained spherules, show up very clearly in sections through the center of the spherule. Electron microscope examination of these inclusions (Vago, 196313) has shown no structure to the spindle which appears to be composed of a substance uniformly dense to the electrons. The spherule, on the other hand, contains large-sized ovoid virus particles arranged apparently in rays passing from its center to the periphery (Fig. 5 ) . There is no virus in the cortical zone of a well-developed spherule, but in the central zone, holes corresponding to the fissures seen under the light microscope may be recognized. Viruses in process of occlusion may be observed a t the periphery of spherules in course of formation. Thus it seems that the spherule is in the real sense the virus inclusion body, the role and significance of the spindles remaining a t present unknown. Weiser and Vago (1966) have recently described oval (3.5 p in length) and spindle-shaped (14 p in length) inclusion bodies in two lepidopterous larvae, 0. brumuta and A . zelleri. These inclusion bodies which are localized in the cytoplasm of infected fat body cells contain large-sized ovoid virus particles comparable with those observed in spherules of M . melolontha.

B. Physicocheinical Properties and Chemical Composition The chemical composition of the nuclear and cytoplasmic polyhedra and of the granules have been reviewed by the authors quoted above. Subsequent publications only will be dealt with here. A recent contribution to the study of the chemical composition of inclusion bodies is the discovery of silicon in the protein of the nuclear polyhedra of the lepidopteron Heliothis zea (Boddie) (Estes and Faust, 1966). According to the authors, the silicon (0.12% of the dry weight of the polyhedra) would be present as the orthosilicate ion Si044-, the negative charges of the four oxygen atoms being balanced by the posi-

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tive charges of the Fe2+ and Mg2+ cations, the presence of which has been demonstrated in the polyhedra by Holoway and Bergold (1953, 1955). These silicates would be a structural component of the protein matrix, involved in the spatial structure of the polyhedra. Faust and Adams (1966, 1967) have confirmed the presence of silicon (0.1 to 0.3% of the dry polyhedra by weight) in nuclear and cytoplasmic polyhedra

FIQ.5. Section of ovoid inclusion body, or spherule, of the spindle virosis of Melolontha melolontha. Virus particles (v) enclosed in the protein matrix with electron-dense central mass (cm) and an external coat (c). Uranyl acetate-lead citrate staining. Magnification : X 60,000. (Original.)

and in the capsules of various Lepidoptera. The silicates would play a part in the dissolution of the polyhedra. In fact, according to these authors, the dissolution in vitro of the polyhedra by treatment with dilute sodium carbonate solution or with enzymatically inactive alkaline gut juices would begin with the solubilization of the silicates. The removal of the hydrophobic properties of the polyhedra conferred by the silicates would then enable the polyhedron protein to pass into solution and liberate the viruses. I n support

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of this assumption, Estes and Faust (1966) showed that 80% of the silicon content of polyhedra was removed in solution by alkaline treatment and was no longer associated with the polyhedral protein. It is only a t this stage under in vitro conditions that the enzymic degradation of polyhedron protein would occur. The presence of ribonucleic acid (RNA) in the nuclear polyhedra of the silkworm B. mori, reported as early as 1946 by Tarasevitch, was finally confirmed by Faulkner (1962) and Aizawa and Iida (1963). Chemical and chromatographic analyses showed that RNA was an integral part of the polyhedral structure. Since it is generally accepted that the virus rods present in the nuclear polyhedra contain only deoxyribonucleic acid (DNA), the RNA must be a component of the surrounding framework. Both types of nucleic acid were found to be simultaneously present in the nuclear polyhedral bodies isolated from TABLE I NUCLEIC ACIDSCONTENTS I N SOMENUCLEAR POLYHEDRA RNA

Origin of polyhedra Bombyx mori Heliolhis zea Trichoplusia ni Spodoptera exigua

polyhedra)

(dw.

DNA (w/mg. polyhedra)

References

5.8 2.02 8.7 12.7

5.7 6.81 12.2 23.7

Faulkner (1962) Estes and Ignoffo (1965) Faust and Estes (1965) Faust (1966)

other Lepidoptera (Estes and Ignoffo, 1965; Faust and Estes, 1965; Faust, 1966). As shown in Table I, the amount of DNA and RNA varies with the type of polyhedral virus. I n B. mori larvae inoculated with the same virus suspension, the RNA contents of nuclear polyhedra vary with the silkworm strains concerned (Aizawa and Iida, 1963). Shvedtchikova and Tarasevitch (1966) have also demonstrated the simultaneous presence of RNA and DNA (8% and 0.2%, respectively) in capsules isolated from Dendrolinus sibiricus (Butler). The presence of RNA in nuclear polyhedra and the capsules obviously raises the question of its origin and role in virus replication. Although drawing attention to the differences in the base composition of inclusion body RNA and virus DNA, Faulkner (1962) considered that the RNA found in B. mori polyhedra might be “a messenger type involved in the production of either virus DNA, virus protein or the polyhedral protein.” Studying this problem, Tarasevitch et d. (1966a, b) showed specifically that the RNA was an integral component of the infective fraction of the polyhedra. For this purpose they extracted the nucleic

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acids from B. mori polyhedra with alkaline treatment and inoculated 1st instar larvae with aliquots of the centrifuged extract not treated or incubated with either ribonuclease or deoxyribonuclease. The highly infective nature of the nontreated alkaline extract was almost entirely lost after incubation with either of these enzymes. Most probably this loss of infectivity arises from the destruction of a “DNA-RNA-protein” complex responsible for infective properties of the polyhedra. These authors considered that this polyhedron RNA was identical with the infective RNA isolated from diseased B. mori by Gershenson e t al. (1963). Chemical and chromatographic analyses of cytoplasmic polyhedra from several lepidopterous species revealed the presence of RNA without any trace of DNA (Krieg, 1956; Xeros, 1956a, b; Aizawa and Iida, 1963). A detailed investigation of nucleic acid extracted by the phenol method from icosahedral and hexahedral cytoplasmic inclusion bodies of B. mori showed that the extract could be fractionated into two components by chromatography on a methylated albumin column eluted with a continuous sodium chloride gradient (Hayashi and Kawase, 1965a). The authors assumed that the first fraction eluted a t 0.2 M NaCl concentration was oligonucleotide, and the second eluted a t 0.60-0.65 NaCl was viral nucleic acid (see Section VII). This assumption has recently been confirmed by Kawase (1967) who showed that only one peak a t the concentration of 0.60-0.65 M NaCl appeared on the chromatogram of RNA preparations obtained from a purified suspension of icosahedral virus. Apparently, therefore, the nucleotide fraction is not derived from virus particles themselves but is more likely a constituent of the polyhedral matrix. The amino acid analysis of the nuclear and cytoplasmic polyhedron proteins of R. mori was carried out recently with the aid of an autoanalyzer (Kawase, 1964). Although the amino acid composition of the polyhedron protein from several nuclear polyhedra had been the subject of earlier studies (see Bergold, 1963a, 1964), this was the first report of the analysis of the amino acids of polyhedron protein of cytoplasmic polyhedra. Polyhedron proteins of nuclear and cytoplasmic polyhedra (icosahedral and hexahedral inclusions) contain the same amino acids, the most marked differences between the cytoplasmic and nuclear polyhedra being in their lysine, proline, serine, alanine, and valine contents. The amino acid composition of the polyhedron protein of the cytoplasmic polyhedra formed in the caudal portion of the midgut differed from that found in the anterior region, and showed similar patterns to that of the polyhedra formed in starved larvae. The amino acid composition of the inclusion bodies of Peridroma saucia (Hubner)

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nuclear polyhedrosis, determined by the same technique, agree closely with the above-mentioned data (Van Der Geest and Craig, 1967). A few remarkable chemical properties must be mentioned in addition to the structural peculiarities of the inclusion bodies of the spindle virosis of M . melolontha described above (Bergoin et al., 1967). Both spindle and spherule are insoluble in such organic solvents as methanol, ethanol, ethyl ether, chloroform, solvent mixtures of methanol-chloroform (1-2 v/v) and alcohol-ether (3-1 v/v). They are unaffected when submitted to the prolonged action a t 38°C. of 0.25% trypsin in a 0.1 M phosphate buffer solution at p H 7, 0.25% pepsin in an HC1-KC1 buffer solution a t pH 2.2, and of the successive action of these enzymes. They dissolve very slowly in 0.1 N sodium hydroxide and are practically insoluble in 0.01 N solution. It was only by adding reducing compounds to 10-11 pH buffer solutions that the inclusion bodies would rapidly dissolve. Preliminary results indicate that they are partly protein in nature, their nitrogen content being 12%; while 14 amino acids have been separated from their hydrolyzate (6 N HC1 a t 110°C. for 24 hours) by thin-layer chromatography. It seems likely, therefore, that the action of the reducing compounds takes place a t the level of the protein disulfide bonds, in the opening of which they probably participate. V. PURIFICATION Purification techniques of viruses from invertebrates have developed during recent years with the discovery of a large number of nonoccluded viruses (see Section 111). The purification of virus suspensions has generally been achieved as the result of a trial-and-error approach and, with the exception of the viruses from polyhedroses and granuloses the purification of which has been codified, no scheme of separation useful for all viruses can be put forward. Nevertheless, repeated high- and low-speed centrifugation cycles constitute the most widely used method , purification usually being completed by means of density-gradient centrifugation,

A . Viruses with Inclusion Bodies The free virus particles of nuclear and cytoplasmic polyhedroses and granuloses may be purified from hernolymph, or from infected tissue homogenized in distilled water, by differential centrifugation after lowspeed clarification to remove coarse material from the virus suspensions. This technique is especially useful for the preparation of small quantities of virus, such as those required for tissue-culture infection (Vago and Bergoin, 1963; Vaughn and Faulkner, 1963). However, the most commonly used method for preparation of purified virus particles is to liber-

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ate them from the protein network in which they are enclosed by alkaline treatment of the inclusion bodies. These methods have recently been reviewed by Bergold (1963a, 1964), Huger (1963), and Smith (1963). Vaughn (1965) purified nuclear polyhedrosis viruses of B. mori by ion-exchange chromatography of infective supernatants from hemolymph and virus suspensions prepared by dissolving inclusion bodies in dilute Na2C03 , using cation exchange columns. The columns equilibrated with 0.01 M phosphate buffer a t pH 7.8 were loaded with 0.5 ml. of virus suspension and eluted successively with 40 ml. followed by 25 ml. of the 0.01 M phosphate buffer (fractions 1 and 2) and 30 ml. followed by 25 ml. 0.4 M KC1 (fractions 3 and 4 ) . Under these of the phosphate buffer conditions, the infective fraction of the virus suspension prepared by dissolving the polyhedra was completely eluted by the 0.01 M phosphate buffer (fractions 1 and 2). On the other hand, the infective fraction of the hemolymph supernatant was only partly eluted by the same buffer (about 50%), the remainder being eluted only when the ionic strength of the eluent was increased (mostly in fraction 3). I n addition to their use for virus purification, these results demonstrated differences in the elution pattern of the virus depending upon its origin. In the case of the spindle virosis of M . melolontha (see Section I V ) the dissolution of the spherules in order to liberate virus particles requires special treatment (Bergoin et al., 1967). The fat body tissue of infected larvae was ground in 0.2 M ascorbate-Tris buffer solution (pH 7.5) a t 4°C.and the suspension centrifuged a t 1800 g for 10 minutes. The pellet, resuspended in distilled water, was cleaned by 6 to 8 centrifugation cycles to obtain a pure suspension of spherules and spindle-shaped bodies. The alkaline solutions of varying ionic strengths used to dissolve the nuclear polyhedra and granules (0.004-0.03 M Na2COS 0.05 M NaC1) and the cytoplasmic polyhedra (2% Na2C03) had only slight action on the inclusion bodies: after 3 hours in the presence of 2% sodium carbonate the spindles were dissolved, but the spherules, although swollen, had not burst open. It was only by adding reducing compounds such as dimercaptopropanol, cysteine, or thioglycolic acid to buffer solutions at pH 10-11 that the spherules would rapidly dissolve; a t a concentration of 0.33 mg./ml. both spherules and spindles were dissolved after 15 minutes a t laboratory temperatures in a 0.1 M sodium thioglycolate 0.1 M bicarbonate solution brought to pH 10.25 with sodium hydroxide. After dissolution of spherules the suspension was centrifuged a t 1300 g for 5 minutes for clarification and virus sedimented at 16,000 g for 30 minutes. The virus pellet was suspended in distilled water and washed twice under the same conditions. This treatment gave a purified suspension of virus particles.

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B. Nonoccluded Viruses For purification of nonoccluded viruses from invertebrates, differential centrifugation techniques are generally employed. Since it is impossible to quote all the methods used, we shall confine ourselves here to the purification of one large and one small nonoccluded virus arbitrarily chosen. The icosahedral nonoccluded viruses belong to the iridescent group : TIV, SIV, MIV, CIV (see Section 111) are easy to purify because of their abundance and large size. Preparation of a very pure suspension of SIV from infected Galleria was achieved by Day and Mercer (1964) using the following method. The infected larvae were triturated in a 0.01 M borate-HC1 0.1 M NaCl buffer solution at p H 7.5. Bacterial growth was inhibited by adding 0.08% sodium azide. The suspension was clarified by centrifuging a t 1000 r.p.m. for 5 minutes. The supernatant was filtered through muslin and centrifuged a t 15,000 r.p.m. for 20 minutes. The surface of the virus pellet was generally covered with a layer of cell debris which was removed by gently washing with the buffer. The virus pellet was allowed to stand overnight a t 4°C. in buffer. A further low- and highspeed centrifugation cycle was performed and the virus pellet, resuspended in buffer, was Centrifuged in a sucrose density gradient for 20 minutes a t 15,000 r.p.m. The visible virus zone was diluted in buffer and centrifuged again a t high speed. The resulting pellet was resuspended in borate NaCl buffer and the solution dialyzed against buffer overnight a t 4°C. Tests using electron micrographs, analytical centrifugation, and electrophoresis showed the high purity of these virus suspensions. Among small-sized viruses, highly purified suspensions of densonucleosis virus were obtained by triturating fat body tissue in a 0.2 M ascorbate-Tris buffer a t p H 7.5. All operations were carried out a t +4"C. The suspension was clarified by centrifugation a t 9000 g for 30 minutes. The lipid-containing layer a t the top of the tube was discarded and the supernatant drawn off. The pellet suspended in buffer was centrifuged under the same conditions. The mixed supernatants were filtered successively through glass wool and Millipore membranes 8 p, 5 p, 1.2 and 0.45 p. The filtrate was centrifuged a t 200,000 g for 1 hour and the pellet redispersed by allowing to stand overnight in 0.01 M , p H 7.0 phosphate buffer. T o obtain a purified suspension, two or three low-(6000 g for 30 minutes) and high-(200,000 g for 1 hour) speed centrifugation cycles were required. Purification was achieved by centrifugation on a cesium chloride density gradient (Truffaut et al., 1967). Other small viruses such as acute bee paralysis virus, black disease, sacbrood virus, and European foulbrood virus are purified from emul-

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sions of infected tissue homogenates with carbon tetrachloride or Freon. The emulsion is then centrifuged and the washed viruses recovered from the supernatant (Bailey et al., 1963, 1964; Bailey and Gibbs, 1964; Bailey, 1965a; Lee and Furgula, 1965b; Giauffret et al., 1966a, c). OF VIRUSES VI. STRUCTURE

A. Viruses with Inclusion Bodies The fine structure of nuclear polyhedrosis and granulosis virus rods was recently reinvestigated. Bergold (1963a) studied the detailed structure of virus particles enclosed in polyhedra either in bundles or not, and confirmed the existence of the intimate and developmental membranes previously described in purified virus preparations. Vago (196413) noted the twisting of virus particles in bundles from G. mellonella larvae suffering from nuclear polyhedrosis, at a stage prior to the inclusion of bundles in the crystalline lattice of polyhedra. This torsion of virus particles was also seen in virus bundles of the nuclear polyhedrosis of Burathra brassicae L. after alkaline treatment of polyhedra (Ponsen et aE., 1964, 1965). It might be due either to the helical structure of the virus, or to the continuity of the intimate membrane which would form up a long tube folded a t the end of each virus particle. Bena (1963) favored the latter explanation for the nuclear polyhedrosis virus of Malacosoma alpicola Staudinger. By treating nuclear polyhedra from B. brassicae, Adoxophyes reticulana HB, and Orgyia antiqua L. with 0.1 N sodium carbonate solution for 5 to 10 minutes, Ponsen (1965) observed a progressive dissolution of polyhedron protein, disintegration of membranes enclosing virus particles, and finally dissolution of virus protein with liberation of filaments with a length and diameter corresponding to the central channel of the virus rods. He defined them as the DNA cores of the virus particles. Further investigation of the long threads associated with granulosis viruses in the larvae of Lepidoptera was carried out by Smith et al. (1964) and Smith and Brown (1965a, b) . These threads were found both in the centrifugation supernatant of diseased larvae macerated in distilled water and in ultrathin sections of infected tissue in twelve species of Lepidoptera infected with granuloses. They appeared as viruslike threads of varying lengths branched in an intricate manner. Negative staining with phosphotungstic acid suggested a composite structure for the threads with an outer coat and an inner helical core. The short thick virus rod occluded in the capsule would probably be responsible for the formation of these threads. U-shaped capsules with a removable cap a t one end enabling the rods to emerge were seen in purified suspensions of threads and in cell sections of infected fat body

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tissue. I n the process of emergence (Fig. 6 ) , the virus rod elongated in the cell cytoplasm pushing out with it the outer membrane in which it was originally enveloped in the crystal, and uncoiled to form a long thread with a smaller diameter. These long virus threads may represent an alternative replication cycle of the virus, the increased length of the rod being a means of increasing the amount of DNA (Smith and Brown, 1965a, b). They probably would break up into shorter fragments which would produce new

FIG.6. Section through two granules, side by side in the same cell of fat body tissue from a diseased Noctuidae larva. In one is the thick virus rod with its enveloping membrane; in the second crystal the virus rod is in process of emergence. Magnification X l0,OOO. (From Smith and Brown. 196513 ; courtesy of K. M. Smith.)

capsules. However, the reason for this complicated cycle involving the emergence of rods from the granule and the uncontrolled and unlimited branching of the threads is as yet unknown. The icosahedral structure for cytoplasmic polyhedrosis viruses has been proposed by Smith and Hills (19621, who showed that virus particles of cytoplasmic polyhedrosis from Antheraea pernyi Guer. Men. consisted of an inner core containing 12 large subunits and an outer protein shell made up of a large number of much smaller subunits. I n a detailed study of the cytoplasmic polyhedrosis virus of the silkworm B. mori using the negative staining technique with phosphotung-

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stic acid, Hosaka and Aizawa (1963, 1964) suggested that it consisted of a shell of two concentric icosahedral layers and a central inner core. Each layer has 12 morphological subunits, localized at 12 vertices of the icosahedron. The subunits of the outer shell were hollow pentagonal discs 200 A in external diameter, from which a hollow projection composed of apparently segmented tubes protrudes. The subunits of the inner shell were probably tubular and connected with the outer shell subunits by tubular structures about 100 in length. Because of its size and structure the virus of the spindle disease of M. melolontha represents a new type of virus from an invertebrate. After shaking or homogenizing fragments of fat body tissue of infected larvae with phosphotungstic acid, Vago and Croissant (1964) observed ovoid particles of about 370 X 250 mp with rounded extremities. The virus coat was formed by subunits of 22 f 2 mp, which in some cases seemed to form a linear structure with regular intersections resembling those of vaccinia or of contagious pustular dermatitis virus particles (Nagington and Horne, 1962). Bergoin et al. (1967) also found this structure in viruses released from spherules by alkaline treatment in a reducing solution (see Section V). The virus coat can be recognized in ultrathin sections of the spherule (Fig. 5 ) , the central zone being occupied by a dense reniform mass.

a

B . Nonoccluded Viruses Some 15 nonoccluded viruses pathogenic to various arthropods have been identified over the last four years. Compared with the 5 nonoccluded viruses described earlier, this figure both emphasizes the progress recently made in this field of invertebrate virology and confirms the predictions of Smith (1963). I n many cases, however, even if the viruses have actually been observed, only their general form and size are known: it is notable that the study of their ultrastructure has been undertaken only rarely. Three iridescent viruses with cytoplasmic affinities and related to Tipula iridescent virus (TIV) were described under the names of: Sericesthis iridescent virus (SIV) (Steinhaus and Leutenegger, 1963) ; mosquito iridescent virus (MIV) (Clark et al., 1965) ; Chilo iridescent virus (CIV) (Fukaya and Nasu, 1966). These are large viruses (130 to 180 mp in diameter), hexagonal in outline, and consisting of a central electron-dense core assumed to contain nucleic acid and a less dense outer coat. The icosahedral structure of these viruses already demonstrated for TIV (Williams and Smith, 1958) was confirmed for SIV by Mercer and Day (1965) using the metal-shadowing technique. Viruses of this group can form aggregates of closely packed and orientated particles responsible for the iridescence of the infected tissues.

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Sections of infected tissue or virus pellet, however, show that the virus particles within the crystals are not generally in actual contact (Fig. 7), but are separated from each other by a regular interparticle space of low density (Day and Mercer, 1964; Xeros, 1964; Mercer and Day,

FIG.7. Section of an iridescent pellet of SIV particles, stained with uranyl acetate, showing well-formed hexagonal profiles and central core containing DNA. Note regularity of arrangement of virus particles and of interparticle gap. Magnification: X 160,000. (From Day and Mercer, 1964; courtesy of M. F. Day.)

1965; Fukaya and Nasu, 1966; Mitsuhashi, 1966b). TIV or SIV airdried particles are about 1300 A in diameter, while the center-to-center distance of neighboring particles in crystals formed in centrifugation pellet is 1700 A for TIV (Williams and Smith, 1957) and 1900 A for SIV (Mercer and Day, 1965). Williams and Smith (1957) suggested that this ((open” space between

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particles was occupied by a surface coat invisible in electron micrographs, while Klug et al. (1959) considered that each particle was surrounded by a water shell immobilized by “long-range forces.” Mercer and Day (1965) studied the structure of the surface layer of SIV particles, in particular by combining it with homologous antibody, or polyethyleneimine (a polymer having a positive surface charge opposite to the surface charge of SIV particles). These experiments demonstrated the presence of a diffuse outer coat about 500 A thick and having the same geometric shape as the dense particre itself. This coat invisible on electron micrographs may be a mucopolysaccharide gel in character, which keeps the particles 1000 A apart in the hydrated crystal. The authors were not able to confirm the presence of the 812 protein subunits (capsomeres) described on the surface of TIV particle (Smith and Hills, 1962).

The virus particles of the disease in M. melolontha known as “lethargy” consist of a central electron-dense core of about 70 mp diameter representing a nucleocapsid surrounded by a slightly opaque substance 65 mp in thickness (Vago et al., 1967). They are closely packed within intracytoplasmic ovoid clusters of 2 to 6 p average diameter and often grouped into geometrical figures. Center-to-center distance of adjacent particles is in this case 200 mp. I n the cluster an electron-dense, undifferentiated zone with homogeneous or thready structure can be observed. The progressive transition of this zone toward that containing virus particles suggests that it may consist of a virogenic stroma. Furthermore, structureless, often elongated, and highly electron-dense masses are present a t the periphery or within clusters, the significance of which, however, is unknown (Fig. 8) (Vago et al., 1967). Sacbrood virus of the larval honeybee Apis mellifera Linnaeus has the typical axis symmetry 5:3:2, as shown by BIEak and Krhlik (1965) using the rotation technique of particles negatively stained with neutral potassium phosphotungstate. The authors distinguished 12 subunits a t the periphery of the virus particle and suggested that its surface was an icosahedron formed from 42 subunits. Lee and Furgala (1965b) observed groups of virus particles within cytoplasmic vesicles of cells from infected larvae. In negatively stained suspensions, they identified three forms of virus particles each 28 1 mp in diameter: one, uniformly electron-dense ; a second, showing an electron-dense exocentric core 19-23 mp;and a third, empty particles with 3.6-mp thick walls. In the case of the hereditary 0-virus of Drosophila, “glovefinger” elements 140 X 70 mp were observed in ultrathin sections of ovarian cysts. These appeared to be formed by a helical nucleocapsid, surrounded by an inner coat and an outer layer of radiating spicules (Berkaloff et al.,

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FIG.8. Section of fat body tissmc of hlelolontha melolontha suffering from lethargy disease, stained with uranyl acetate and lead citrate. Virus particles are closely packed within intracytoplasmic clusters. Note regular arrangement of virus particles in some clusters. z, undifferentiated zone, with homogeneous or thready structure ; m, highly electron-dense masses at the periphery of clusters. Magnification: X 130,000.(From Vago et al., 1967.)

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1965). This virus is morphologically related to the viruses of vesicular stomatitis and rabies (Berkaloff et al., 1966; Ohanessain and Echalier, 1966; Plus, 1966; Printz, 1966). The other nonoccluded viruses, the structure of which has not yet been studied, are either spheroidal or irregular in form.

PROPERTIES AND CHEMICAL VII. PHYSICOCHEMICAL OF VIRUSES COMPOSITION Bergold (1963a, 1964), Smith (1963), and Huger (1963) have reported in detail on the physicochemical properties and chemical analyses of insect viruses. Only the most recent work will be considered here.

A . Physicochemical Properties There are increasing numbers of data on temperature sensitivity of viruses and their resistance to the action of chemical agents. It has been found that isolated virus particles of the cytoplasmic polyhedra from B. mori suspended in distilled water retained their virulence after 18 days at -15"C., +5"C., and +25"C., but were inactivated after 3 days a t 25°C. in 0.1% formalin (Hukuhara and Hashimoto, 1966a). Acute bee paralysis virus was inactivated by heating to 75°C. for 10 minutes (Bailey and Gibbs, 19641, and that isolated from European foulbrood a t 60" for 30 minutes (Giauffret et al., 1966a). Nonoccluded viruses of honeybee were not affected by carbon tetrachloride or Freon used for their purification (see Section V) , Virus particles of silkworm flaccidity were destroyed at 100°C. for 10 minutes, but retained some of their virulence when kept a t 80°C. for the same period (Aizawa et al., 1964). The virus was rapidly inactivated by certain disinfectants such as chlorinated lime y400, 3% antiformin, 2% formaldehyde, and 7.5% HC1. It did, however, retain a certain infectivity level after 8 hours in 0.1% HgClz and in 3% cresol soap. A dilute solution of SIV was entirely inactivated a t 60°C. for 5 minutes or 50°C. for 30 minutes, but it resisted 50°C. for 10 minutes (Day and Mercer, 1964). The addition of MgClz considerably increased the thermal instability of this virus. SIV was very resistant to ether and chloroform, suggesting the absence of a lipid fraction essential for the maintenance of its infectivity. It also retained its infectivity after exposure to either pH 3.0 or 10.5 for 3 hours. Thermolability, resistance to organic solvents and to a large range of pH are properties which SIV shares with the adenovirus group (Day and Mercer, 1964). Aqueous suspensions of the nonoccluded virus of the citrus red mite, P . citri were inactivated by 2 hours a t 50"C., or 30 minutes a t 65"C.,

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but withstood 5 weeks a t 25°C. (Gilmore and Munger, 1963). Freezing and thawing of an aqueous solution inactivated the virus, whereas frozen glycerin suspensions and freeze-dried or vacuum-desiccated diseasedmite material retained their infectivity for several months. The a-virus of Drosophilu is inactivated in a 40% sucrose solution; hence this sugar cannot be used for its density gradient purification (Plus, 1962). Sedimentation coefficients of several viruses have recently been reported: for SIV particles it was 2200 S (Day and Mercer, 1964), a value similar to that given by Weber et al. (1963) for TIV; for virus particles isolated from icosahedral cytoplasmic polyhedra of B. mori it was about 400 S (Hukuhara and Hashimoto, 1966a; Kawase, 1967), and about 160 S for sacbrood and acute bee paralysis virus particles (Bailey et al., 1964).

B. Chemica 1 Composition Some recent data have been gathered about virus chemical components and several detailed analyses of the nucleic acids have been performed. Chemical analysis of a standard sample of SIV containing 6.01 X loll particles per milliliter showed 14.8 mg. protein/ml.; 0.243 mg. phosphorus; 2.65 mg. total nitrogen; 2.86 mg. DNA (17.6% of the dry weight of the virus) (Day and Mercer, 1964). The amino acid analysis of purified viruses of nuclear and cytoplasmic (hexahedral and icosahedral) polyhedroses of B. mori using an autoanalyzer, showed a close similarity in qualitative composition of these three viruses, as in the case of their polyhedron proteins (see Section IV) (Kawase, 1964). The pattern of amino acid composition of the two cytoplasmic types of virus particles was similar, while the nuclear polyhedrosis virus had less histidine, arginine, and glutamic acid and more threonine and alanine. The type of nucleic acids has been determined for several nonoccluded viruses: virus particles of the mite P . citri contained 10.6% of ribonucleic acid (Estes and Faust, 1965); bee sacbrood virus and the virus of G. podocarpi (Lepidoptera) are both RNA types (Lee and Furgala, 1965b; Harrap et al., 1966) ; SIV and densonucleosis virus particles contained DNA (Day and Mercer, 1964; Truffaut et al., 1967). One of the most interesting aspects of virus chemistry is probably the study of biophysical and biological properties of their native nucleic acid. Such research has been carried out on several insect viruses. Hayashi and Kawase (1965a, b) and Hayashi et al. (1965) have studied the viral nucleic acids of the two types of cytoplasmic polyhedra of B. mori (see Section IV). The two nucleic acids identified and designated IPB-

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RNA and HPB-RNA (the prefixes I P B and H P B being derived from icosahedral and hexahedral polyhedra) produce viscous precipitates in cold ethanol after extraction by the phenol method and are very closely related to each other, Their elution pattern on a methylated albumin column was similar, both being eluted a t 0.60-0.65 M NaCl concentration. Their base composition was of the AU type with A/U and G/C ratios close to unity, suggesting that each consisted of a double-stranded RNA of high molecular weight. They had the same sedimentation coefficient szo, = 17 S a t high concentration and two peaks of 14 S and 17 S a t low concentration. A detailed study of biophysical and biological properties of a viral nucleic acid from an invertebrate has been carried out by Onodera e t al. (1965) on the nuclear polyhedrosis virus of B. mori. The authors extracted infective viral DNA by mild alkaline treatment of purified virus particles and fractionated i t on a methylated albumin column eluted with a continuous sodium chloride gradient. The infective fraction eluted between 0.60 and 0.75 M NaCl in 0.05 M phosphate buffer solution a t pH 6.7 gave a typical nucleic acid absorption curve with maximum absorption at 258 mp, minimum a t 232 mp, and Azeo/Azso ratio of 2.0. The sedimentation coefficient of the viral DNA in 0.2 M KC1-0.01 M phosphate buffer at p H 7.0 was szo, = 13.1 S. This DNA showed a sharp melting curve with a melting temperature T, = 86'437°C. and a net tendency to renaturation when it was cooled a t room temperature. Its C) ratio of 1.38 and A / T and base composition showed a (A T ) / ( G G/C ratios both equal to unity. The guanine-cytosine content of the viral DNA obtained from base analysis was 41.9%,which was close to the 40.7% calculated from melting temperature according to Marmur and Doty (1962). The heat-denatured DNA treated with 1.8% formaldehyde a t 37°C. for 24 hours showed a hyperchromicity of 10%. All these properties are characteristic of a double-stranded DNA. The molecular weight of this DNA was estimated using Doty's equation (Doty et al., 1958) to be 1.8 X lo8, while the equation of Burgi and Hershey (1963) gave 2.2 x lo6.The native DNA was infectious when inoculated a t 0.4 and 0.2 pg. per pupa, whereas a t 10 times this dose viral DNA treated with pancreatic deoxyribonuclease lost its infectivity. The other detailed analysis of nucleic acid was performed for the densonucleosis virus by Truffaut e t al. (1967). They showed that the viral nucleic acid was a double-stranded DNA. Its typical melting curve, its lack of reaction with formaldehyde a t 37"C., and the increase in its apparent density after thermal or alkaline denaturation all supported this contention. The melting temperature of native DNA was T, = 845°C.; its sedimentation coefficient szo, was 17.7 S; its G C percentage was equal to 3 7 4 0 mole 7%. Using the equation described by Eigner and

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Doty (1965) for double-stranded DNA of higher molecular weights than 4 x 106 daltons, the molecular weight of the nucleic acid of densonucleosis virus would be 5 x 106 daltons, a value which seems high in comparison with the size of the virus particle. VIII. SEROLOGY The discovery in recent years of a large number of viruses affecting invertebrates and the improvements in their purification techniques have encouraged the development of serological studies which up to recently had been confined to nuclear polyhedroses and granuloses viruses (Smith, 1962; Bergold, 1963a, 1964). The main purposes of these investigations were the comparison of antigen relationships between different viruses and research into multiplication sites of viral antigens and the titration of virus suspensions.

A. Serological Relationships This aspect of serology has been examined by a number of workers.

It has been shown that TIV and SIV particles have three antigens detectable by gel-diffusion tests, but none is common to both viruses (Day and Mercer, 1964). Furthermore, these two viruses are not related serologically to adcnovirus 5. There is no serological relationship between CIV and TIV nor between CIV and SIV (Fukaya and Nasu, 1966). Strains of acute bee paralysis virus (ABPV), isolated from different bee colonies, were found to be serologically identical (Bailey and Gibbs, 1964). The same was true of sacbrood virus (SBV) (Bailey et al., 1964). On the other hand, during infectivity neutralization tests with absorbed antisera ABPV and CBPV particles were shown to be serologically distinct (Bailey, 1965a). Antisera prepared against ABPV and CBPV neutralized the infectivity of extracts of bees suffering from black disease or Waldtrachtlcranlcheit (Bailey, 1965b). These results support the hypothesis of Giauffret et al., (1966c), according to which certain forms of these diseases are in fact manifestations of the same original viral entity. As opposed to this, no serological relationship could be discovered by gel-diffusion tests, precipitation tests in tubes, or neutralization of infectivity tests between SBV and ABPV particles in spite of the morphological resemblance between these two viruses (Bailey et al., 1964). Virus particles of icosahedral and hexahedral cytoplasmic polyhedra of B. mori are serologically indistinguishable by complement fixation and gel-diffusion tests (Hukuhara and Hashimoto, 1966a). These results confirm the close resemblance between these two virus strains (see Section VII). On the other hand, there is no serological affinity between these viruses and that of the nuclear polyhedrosis of the same insect. The serological specificity of viral antigens seems to be independent of

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the specificity of viruses themselves and does not appear to be modified by the development of the virus in a host different from the original one. Thus, nuclear polyhedrosis virus of B. mori adapted to G . mellonella by successive passages was neutralized by antiserum against silkworm nuclear polyhedrosis, indicating that its serological specificity had not been modified by this adaptation (Aizawa, 1962). However, it is not neutralized by antiserum against silkworm cytoplasmic polyhedrosis virus. Likewise, TIV particles from different lepidopterous larvae are serologically identical with those from T. paludosa. (Smith et al., 1961; Oliveira and Ponsen, 1966). Hukuhara and Hashimoto (1966b) observed that TIV particles showed no serological relationship with nuclear and cytoplasmic polyhedroses viruses of B. mori which, however, is also susceptible to this virus.

B. Detection of Site of Viral Antigen Multiplication The serological properties of viruses have also helped investigations of certain aspects of pathogenesis. Some light has been shed on the process of nuclear polyhedra formation in B. mori using the fluorescent antibody technique with two antisera : one, against purified nuclear polyhedrosis virus particles; the other against polyhedron protein (Krywienczyk, 1963). Fluorescence first appeared in the cytoplasm of infected cells prior to the intranuclear formation of polyhedra, which suggests that synthesis of the polyhedron protein takes place in this part of the cell. Intracytoplasmic fluorescence tended to decrease progressively as the first polyhedra appeared and it disappeared when the nuclei were filled with polyhedra. With the same technique, Bellett and Mercer (1964) were able to follow multiplication of SIV particles in A . eucalypti cell cultures. Three days after infection with SIV a t 25"C., cytoplasmic foci of fluorescence were noted in a few cells stained with fluorescent antibody. Antigen multiplication was indicated by an increase in size and intensity of fluorescence of the cytoplasmic foci which had filled practically all the cytoplasm after 5 days. By combining techniques of antibody fluorescence and autoradiography with incorporation of tritiated thymidine, Bellett (196510) demonstrated that viral DNA was probably synthesized in the cytoplasmic foci which contained the antigen. Oliveira and Ponsen (1966) carried out a similar study with hemocytes of P . brassicae larvae inoculated with TIV. The first fluorescent zones due to the presence of viral antigen appeared on the third day in the cytoplasm of hemocytes of larvae reared a t room temperature. Densonucleosis virus was detected by the gel-diffusion technique in G . mellonella larvae in the first stage of infection, as well as in apparently healthy pupae and adults (Giran, 1966).

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C . Titration of Virus Suspensions Counting the cells stained by fluorescent antibody has been employed as a method of titration SIV particles in cell cultures of A . eucalypti (Bellett, 1965a,b). The latent period of induction of SIV in cell cultures was about 4 days after inoculation. The production and liberation of infective virus continued until the eighth day. Measuring the precipitation end points, Bailey and Gibbs (1964) noted that extracts from different parts of bees infected with acute bee paralysis virus contained the same amounts of viral antigen. Passive hemagglutination appears to be the most sensitive serological technlque so far employed for titration of insect viruses (Cunningham et al., 1966). The authors, using passive hemagglutination tests with tanned rabbit erythrocytes and hemagglutination inhibition tests, obtained an antiserum against TIV actually capable of detecting as little as 0.26 pg. of virus. Attempts to obtain direct hemagglutination of goose eiythrocytes by TIV between pH 4.2 and 8.0 solutions a t 37" or 4" C. failed. IX. VIRUSDEVELOPMENT Earlier investigations supplied information particularly on development of polyhedrosea and granuloses viruses, and this has been more fully examined recently because a considerable divergence of opinion on the evidence available exists. Among the recently discovered viruses only a few cases of intracellular del-e!opment have been studied and these are mostly for the iridescent viroses and the densonucleosis.

A . Nuclear Polyhedrosis Viruses Vago and Croissant (1963) showed that in B. mori cell cultures a t the beginning of pathogenesis short, almost coccoid, rods, which later lengthen and have a simple membrane, appear in the abundant granular zones surrounding the nucleolus and generally in the vicinity of the chromatin. Each rod is subsequently enveloped in an individual coat. The grouping of virus rods and their inclusion in a developmental membrane was followed hour by hour. According to Bird (1964a), the first sign of infection of Choristoneura fumiferana Clemens in the cell is the accumulation of highly stained corpuscles in a fine filamentous network. These corpuscles coalesce to form the rods of virus. The latter then group together in bundles dispersed throughout the nucleus and are orientated in the same direction as they become attached to the newly formed membranes. These are double and progressively enclose the virus completely.

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I n the case of nuclear inclusion body virosis of Tipula the occlusion of the virus rods is carried out in concentric lines following the contour of the crystal (Smith, 1962).

B. Cytoplasmic Polyhedrosis Viruses In this group of viroses, Wittig et al. (1960) observed a large number of virus particles in the cytoplasm of mid-gut epithelial cells of Colias philodice Boisduval. The occlusion of virus polyhedra is brought about by a progressive accumulation of polyhedron protein a t the periphery of virus particles. Smith (1963) noted virus particles in small depressions scattered over the surface of polyhedra which he considered to be the latest to be occluded. Bird (1965) observed masses made up largely of empty capsids in the cytoplasm of intestinal cells of Orgyia leucostigma J. E. Smith. The virus cores seemed to be in the vicinity of these masses and from their shape and their affinity for stain were probably related to the ribosomes. The assembly of the virus was apparently completed only just before its occlusion into a polyhedron. The protein appeared to pass through the voluminous masses of virus particles and would crystallize to form the polyhedron. Each polyhedron continued to grow in size, the largest being found near to the striated border of the intestinal epithelium cells (Fig. 9 ) . This author (Bird, 1966) observed more or less the same type of virus development as for cytoplasmic polyhedrosis of C. fumiferana, except that here he did not find the masses of incomplete viral particles belonging to the early infection stages. Xeros (1966) showed that in ten cytoplasmic polyhedroses of Lepidoptera, proteins and RNA content of the virogenic stromata increased as they developed. As in the case of nuclear polyhedroses, elaboration of virus particles a t the expense of stromata precedes the formation of polyhedral inclusion bodies which progressively incorporate the virus particles as they mature.

C. Granulosis Viruses One constant cytopathological character of granuloses is the formation of a strongly stained network in the highly hypertrophied nucleus and in the cytoplasm where the reticulation is more open. Huger (1960) found that in C. fumiferana the network was first Feulgen-positive, but as the granules developed it became Feulgen-negative. Electron microscope investigations (Huger and Krieg, 1960, 1961; Huger, 1963) have thrown further light upon these changes. The typical network suspected to be of nuclear origin would probably release virus rods and protein particles

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during its disintegration. The granules would form by a progressive deposition of protein material around each rod. Since the network containing DNA finally expands across thc whole cell, virogenesis would not likely be limited to either nucleus or cytoplasm. Bird (1963, 1964a) considers that the formation of virus particles would begin in the nucleus and would continue in cytoplasm in a material which is probably of nuclear origin. Each rod is progressively enveloped in a membrane while it is not unusual to see a granule beginning to form a t the extremity of a rod. Smith et al. (1964) and Smith and Brown (1965a,b) examining purified suspensions or using thin sections of infected tissue, noted that the thick short rod within the granule is thought to emerge as a result of the raising of a removable cap at one end of the crystal. $et free into the cytoplasm, the rod branches often in a right-angled direction.

D . Nonoccluded Viruses 1. Iridescent Viroses

Those authors who have worked on the various stages Of formation of Tipula iridescent virus (TIV) in natural and in experimental hosts have put forward more or less contradictory hypotheses. According to Smith and Hills (1962), empty preformed membranes would become progressively filled with nucleic acid beginning from the center. This opinion is supplemented by the views of Xeros (1964), who observed the presence of virogenic zones producing fibrils of nucleoprotein. At the same time open virus membranes appeared to arise from microvesicles and began filling up with nucleoproteins to form the body of the virus. According to Bird (1962), spherical bodies would appear from a fibrous matrix and aggregate to form the dense central body of the virus. The coat would form later and always from the residue of this fibrous matrix. 2. Densonucleosis

I n the fat body of infected G. inellonella larvae the nucleus loses all its internal structure, hypertrophies, and becomes filled by a homogeneous zone. This zone forms dense, voluminous, Feulgen-positive masses which consist of virus corpuscles joined together by a noncrystalline protein substance (Amargier et d., 1965; Vago et al., 1966a). 3. Malaya Disease

According to research, which up t o now has not been very extensive, the lengthened particles which are regarded as the mature form of the

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FIG.9. Section through a mid-gut cell of Orgyia leucostigma infected with cytoplasmic polyhedrosis virus showing the formation of polyhedra. S, material

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virus may arise from a fibrillar or granular material, from filaments, or from immature spherical stages. Furthermore, the virus seems to develop both in the nucleus and in the cytoplasm (Huger, 1966).

X. I n Vitro PATHOGENESIS Up to ten years ago the study of invertebrate viruses was confined to in vivo examinations using histological and cytological methods and experimental infection procedures. In recent years there has been a progressive improvement in invertebrate tissue culture technique, and thus new possibilities are offered for in vitro work on viral pathogenesis. I n fact, the successful infection of tissue culture by a specific virus enables the development cycle of the virus to be followed, thus facilitating the observation of its intracellular affinities a t the ultramicroscopic level. Virology today has a t its disposal a large range of cultures prepared from species belonging to the most widely differing invertebrate groups, and particularly to the insects. A . Invertebrate Tissue Cultures Over the last few years there have been many publications describing the production of various invertebrate cell cultures, but too often these have consisted of descriptions only of simple temporary survival of cells. To be successful, however, in vitro virology must have the support of authentic cell cultures showing regular and prolonged cell multiplication. The main tissue and organ cultures obtained since 1960 are as follows. Among the insects are the Diptern Aedes aegypti L. (Vago and Flandre, 1963; Grace, 1966; Peleg, 1966), Culex tarsalis (Ball and Chao, 19601, Drosophila melanogaster Meigen (Horikawa and Sugahara, 1960a,b; Horikawa and Fox, 1964; Echallier et al., 1965); the Homoptera cicadellid vectors Cicadella viridis L., Macrosteles sexnotatus Fall, Philaenus spumarius L. (Vago and Flandre, 1963), Agallia constricts, Macrosteles fascifrons StH1. (Hirumi and Maramorosch, 1963, 1964a,b, c) , Nephotettix cincticeps Uhler (Mitsuhashi, 1965a) ; the Heteroptera Triatoma infestans Klug and Rhodnius prolixus StB1. (Vago and Flandre, 1963); the Lepidoptera Anosia plexippus L. (Hirumi and Maramorosch, 1964d), Antheraea eucalypti Scott (Grace, 1962a,b), Antheraea pernyi Guer. (Vago and Croissant, 1963), Chilo suppressalis Walker forming the capsids of virus particles; VC, cores of virus particles visible in polyhedra; VCI, virus particles being incorporated into a polyhedron; VB, material forming the cores of virus particles or ribosomes. Magnification: X 90,000. (From Bird, 1965; courtesy of F. T. Bird.)

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(Mitsuhashi, 196513), G. mellmella (Sen Gupta, 1961 ; Vago and Chastang, 1962a,b; Vago and Flandre, 1963), L. dispar (Vago and Chastang, 1962a; Vago and Bergoin, 1963; Vago and Flandre, 1963), Samia Cynthia (Drury) (Hartzell, 1961), in addition to B. mori; the Dictyoptera Blabera fusca Brunner (Landureau, 1965) and Blatella germanica L. (Ting and Brooks, 1965). Among the ticks are Dermacentor marginatus Sulzer, Dermacentor pictus Herinann (RehGek, 1962) and Hyaloma dromedarii Koch (RehMek, 1965a). Among the Crustacea are one lobster and several species of crab (Pianfetti et al., 1962). Among the Mollusca are Helix aspersa Muller (Flandre and Vago, 1963), Helix pomatia L., Planorbina glabratus Say and Pomatiopsis lapidaria Say (Burch and Cuadros, 1965), two species of oyster (Vago and Chastang, 1960b) and the common Octopus vulgaris Lamarck (Necco and Martin, 1963a,b). Finally, among the Platyhelminthes are survival and regeneration of three species of planarians (Chandebois, 1963; Sengel, 1963). Fibroblasts are generally chosen for cultures required for viral contamination because they are the most easily prepared and are often susceptible to various viruses. Blood cells may be cultivated but with inconsistent success, while the viability of cultures of intestinal epithelial cells is still difficult to maintain for any lengthy period. The infective agent is prepared in various ways: Usually the hemolymph removed from a diseased animal is diluted with the culture medium. I n the case of the nuclear polyhedrosis of G. mellonella, Heitor (1963) has defined the stage a t which the free viruses are most abundant in the hemolymph. Recently cell cultures were infected also by purified viruses of densonucleosis (Vago et al., 1966b) and of Sericesthis iridescent disease (SIV) (Bellett and Mercer, 19f.54). Observation techniques are generally the same as those applied in vertebrate virology, sometimes with adaptations for invertebrate tissues. For the demonstration of inclusion bodies a specific stain has often been used (Vago and Amargier, 1963). Little work has been carried out on examination of infected culture tissue by electron microscopy (Grace, 1962b; Vago and Croissant, 1963). In the viral infection of arthropod vector tissue cultures the virus is recognized by serological and contamination methods used for vertebrate tissue cultures (RehAc'ek, 1965b). B. Infection of Invertebrate Tissue Cultures with Viruses from Invertebrates Numerous results have already been obtained with practically all virus groups described in the invertebrates.

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1 . Nuclear Polyhedroses Viruses

This is the type of virus most used for culture infection. The cells most frequently employed are the ovarian sheath fibroblasts and the hemocytes of the lepidoptera B. mori, L. dispar, A . pernyi, G. mellonella, P. saucia, Mamestra brassicae L. (Vago and Chastang, 1960b; Martignoni and Scallion, 1961; Sen Gupta, 1963a,b; Vago and Bergoin, 1963; Vago and Croissant 1963; Vaughn and Faulkner, 1963; Faulkner and Vaughn, 1965). Nuclear hypertrophy, the progressive development of polyhedra, and the disintegration of the tissues have been observed. Some of these experiments showed that the cells were receptive in vitro to viruses of which they were not the spontaneous hosts. 2. Cytoplasmic Polyhedroses Viruses

An infection was accidentally discovered in a fibroblast culture of A . eucalypti (Grace, 1962b). Vago and Bergoin (1963) infected ovarial fibroblasts of L. dispar with the cytoplasmic polyhedrosis virus of the same species; characteristic cytopathogenic action took place in the absence of nuclear changes. 3. Granuloses Viruses

Ovarial sheath fibroblasts of I,. dispar were contaminated with infectious material from P . brassicae (Vago and Bergoin, 1963). Although no virus of this type is known in Lymantria, the granules which formed in the culture were identical in shape and size with those observed in Pieris.

4. Densonucleosis Virus This virus, of a new type, discovered a few years ago, and obtained from diseased G. mellonella larvae, has infected fibroblast cultures of this species as well as of B. mori which is not its natural host (Vago and Luciani, 1965). More recently, such cultures have been successfully infected with a suspension of the purified virus in Bm 22 medium (Vago et al., 1966b). The in vitro cytopathogenic action is comparable with that described in naturally infected tissue. The nuclear lesions do not resemble any of the cellular changes provoked by other viruses of invertebrates in tissue culture. 6. Sericesthis Iridescent Virus (SIV)

This virus isolated from the coleopteron Xericesthis (Steinhaus and Leutenegger, 1963) was introduced in a purified form into ovarian cell

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cultures of A . eucalypti. The elaboration of DNA in the cytoplasmic granules was observed with fluorescence and historadiographic techniques. I n electron microscopy, the viral particles were often seen to be grouped (Bellett and Mercer, 1964). The optimal temperatures for antigen production and the titer of the virus in the infected cells were determined (Bellett, 1965a) ; the quantitative aspects of the infection of the same cultures by SIV were then analyzed (Bellett, 1965b). 6. Chilo Iridescent Virus (CIV) .

Another type of iridescent virus pathogenic for the lepidopteran Pyralidae C . suppressalis (Fukaya and Nasu, 1966) was used for multiplication experiments with in vitro cultivated hemocytes of its host (Mitsuhashi, 196613). The medium to which had been added a suspension of CIV obtained from the fat tissue of diseased larvae was introduced into the culture. The cells first formed groups and after 10 days iridescence appeared. The electron microscope revealed numerous CIV particles in the cytoplasm.

C. Infection of Tissue Cultures of Arthropod Vectors with the Viruses they Transmit Problems relating to the biological transmission of viruses pathogenic to animals and plants have recently been studied in vitro because of the current possibilities of keeping alive and of culturing the tissues of several arthropod vectors.

1. Development of Vertebrate Viruses Among the group A arboviruses, the West Nile virus is propagated in the imaginal discs of the A. aegypti mosquito. (Peleg and Trager, 1963a,b). Eastern equine encephalomyelitis (EEE) virus has been kept alive only in cultures of fat body and hypodermis of the D. pictus and Zxodes ricinus ticks, and to a less degree of D. marginatus (RehhEek and Pesek, 1960). These two viruses as well as the Sindbis and Semliki viruses have been multiplied in vitro in monocellular layers of epidermal and central ganglion tissue of the tick H . dromedarii (keh&?ek,1965b). In group B arboviruses, multiplication of European tick-borne encephalitis (TBE) virus has been observed in D. marginatus and H y a Zomma asiaticum P. Sch. and E. Schl. fibroblast cultures (RehhFek, 1963). The following viruses which are transmitted under natural conditions by ticks multiply well in H . dromedarii fibroblasts cultures: West Nile

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virus, European tick-borne encephalitis virus, louping ill virus, Kyasanur Forest disease virus, Russian spring summer encephalitis virus, Powassan virus, Omsk hemorrhagic fever virus. However, yellow fever virus, St. Louis encephalitis virus, and Japanese B encephalitis virus, which are carried by mosquitoes, show only a feeble development in the same cultures (RehGek, 1965b).

2.Development of Plant Viruses Rice dwarf disease virus (RDV) has been extracted from the fat tissue of contaminated females of N . cincticeps by homogenization and centrifugation and introduced into the culture medium. Granules invaded the cytoplasm until the cell was destroyed. Nine days after infection particles of 30 and 70 m p are found and are considered to be forms of RDV (Mitsuhashi, 1 9 6 5 ~ ) .

XI. ECOLOGY AND DISSEMINATION The conditions which influence the natural and experimental establishment and development of viroses have been the subject of much research because of the practical questions involved.

A . Climatic and Nutritional Factors

It is generally considered that high temperatures accelerate the course of a viral disease, but in the laboratory some insects are found to be more susceptible to virus when reared a t low temperatures (Franz, 1961). The polyhedrosis and granulosis of Agrotis segetum Schiff and Haderia sordida Bkh. can develop simultaneously in an insect, but the former becomes dominant a t the higher and the latter a t the lower temperature (Shvetzova, 1964). At a high temperature and in the absence of adequate aeration, humidity can provoke or favor the symptoms of polyhedrosis in L. dispar (Wallis, 1962). Nevertheless, relative humidities of 25, 75, and 100% have no effect upon the mortality rate in groups of G. mellonella larvae treated with a Borrelinavirus (Pimentel and Shapiro, 1962). Nutrition has long been known to be an important factor in insect viral susceptibility. Pimentel and Shapiro (1962) achieved a higher polyhedrosis mortality in G. wbellonella larvae kept on a high protein medium. Prolonging the period without food did not increase the incidence of the polyhedrosis. B. Insect Populations The condition of the insect population appears to have a considerable influence upon the development of viroses (Grison, 1964). Wellington (1962) observed that in high density populations of Malacosoma pluviale

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the sluggish colonies were more important centers of nuclear polyhedrosis multiplication and propagation than the active colonies. The observation previously made that an epizootic polyhedrosis infection coincided with the period of most intense insect invasion was confirmed by Vasiljevic (1961) for L. dispar, by Jahn (1962) for Lyda hypotrophica Htg., by Jahn and Sinreich (1963) for Himeria pennaria L. and by Tashiro and Beavers (1966) for P . citri. Vasiljevic (1961) considers that crowding in I,. dispar caterpillars may be regarded as a preliminary condition for a physiological change, and for the stimulation of the virus toward epizootic proportions. Tanada (1961), Jaques (1962a), and Gilmore and Munger (1965) confirmed that an increase in population density was associated with an increase in viral infection. Jacques considers that the stressor role exerted by crowding in this case is less of a determinant than the many individual contacts which occur.

C. Composite Injections Under natural conditions the simultaneous action of a granulosis and a nuclear polyhedrosis favored the development of epizootic infections in Pseudaletia unipuncta Haworth (Tanada, 1961, 1965). The simultaneous development of a nuclear polyhedrosis, a granulosis and a microsporidian infection gave rise to an epizootic in the same species (Tanada and Chang, 1962). Niklas (1960) considered that in the same cockchafer population a mixed viral, rickettsia, protozoan, and perhaps bacterial infection increased the individual pathogenic potentialities shown by each of these microorganisms when acting alone. I n an insect suffering from a mixed viral infection, the viruses may show antagonism or interference reactions. Aruga et al. (1963a) observed interference between several strains of cytoplasmic polyhedroses in the silkworm. Aruga et al. (1963b) noted that the virus of the cytoplasmic polyhedrosis of Dendrolimus spectabilis (Butler) interfered with the virus of the cytoplasmic polyhedrosis of B. inori when both were fed to 5th instar silkworm larvae. A synergism seems to exist between the cytoplasmic polyhedroses viruses of B. mori and Colias eurythema Boisduval when they were fed simultaneously to 2nd instar silkworm larvae. However, in the Colias larvae the Colias virus interfered with the infection caused by the virus of Bombyx (Tanada and Chang, 1964). D . Dissemination The spread of a viral disease in an insect population under natural conditions is a function of all the factors which ensure or favor the transmission and dissemination of the pathogen.

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Even if under rearing conditions a rapid propagation of the disease is made possible by a heavy concentration of population over a small area, under natural conditions it is far less probable that the larvae will consume leaves to which dead larvae adhere (Vago, 1964~). Then again, the chances that a virus will infect the largest number of individuals will depend as much upon the diversity and the effectiveness of all the factors of transmission and dissemination of the pathogen within the biotope as upon the degree of virulence of the pathogen itself (Tanada, 1963). 1. Abiotic Spreading Agents

Wind and rain are recognized as the most important climatic factors affecting the spread of the nuclear polyhedrosis of the sawfly N . swainei (Smirnoff, 1961). The nuclear polyhedroses of Trichoplusia ni (Hubner) are disseminated by rain and heavy dews (Hofmaster, 1961). Rain washes the virus, carries it toward the soil, and distributes it over the trees, as was shown by Bird (1961a) with the nuclear polyhedrosis of Diprion hercyniae (Hartig) which was seen to spread more rapidly down than up the trees. 6. Biotic Spreading Agents

Infected larvae carry the virus during their migrations from one branch or tree to another. Smirnoff (1961) observed the movement of N . swainei larvae during a biological control experiment with the nuclear polyhedrosis of the species. Third and fifth instar infected larvae traveled as far as 200 yards on the ground seeking fresh food. The spread of the virus was ensured by the contamination of the foliage with both excrement and dead larvae. In dense laboratory populations, transmission of the nuclear polyhedrosis of Tricoplusia ni (Hubner) (Jaques, 1962b) and of nonoccluded virus of P . citri (Gilmore and Munger, 1965) was effected by the ingestion of contaminated food and by contact between the insects. Less is known about the spread of viruses by species to which they are not pathogenic. I n entomophagous insects the contamination of the laying apparatus a t the moment of oviposition has been observed by several writers. The tachinid fly Drino bohemica Mesnil will lay impartially on healthy, diseased, or even dead larvae of D . hercyniae although it does show a preference for moving larvae (Bird, 1961a). Under laboratory conditions the granulosis virus can be transmitted from infected to healthy P . brassicae larvae by Apanteles glomeratus L. (David, 1965). It is suspected that certain predators, necrophagous scavengers, invertebrates, and vertebrates act as mechanical spreaders of viral ma-

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terial. Certain insects, particularly among the wasps which feed on infected larvae of N . swainei, can transfer the virus from tree to tree (Smirnoff, 1961). The dipteron Sarcophaga aldrichi Parker can act as an important distributing agent for the nuclear polyhedrosis of Malacosoma disstria (Hubner) (Stairs, 1965). Virus dissemination by the excrement of insect predators has recently been observed. Vago et al. (1966~)have established that the Ephippigeridae are natural distributors of the virosis of L. dispar on which they prey, The inclusion bodies retain a large part of their infectiveness after passing through the alimentary canal and their elimination with the excreta. The nuclear polyhedroses and the densonucleosis viruses pass through the alimentary canal of the predator Mantis religiosa L. (Bergoin, 1966a). A new aspect of the problem of viral spread was witnessed during research work on the role of scavengers. Bergoin (196613) has described the passage of the nuclear polyhedroses viruses through the alimentary canal of Acheta domesticus L. and has shown by tests on young B. mori larvae that their virulence was preserved after ejection with the feces. The same author (Bergoin, 1966a) made a detailed study of the dissemination of nuclear and cytoplasmic viruses by orthoptera and dictyoptera; particularly A . domesticus, G . bimaculatus, and B. fueca. The role played by birds is already recognized and has been confirmed by establishing that the nuclear and cytoplasmic polyhedroses viruses and the nonoccluded virus of flacherie pass through the alimentary canal of the thrush Turdus cardis Temm. without any loss of virulecce (Ayuaawa and Yusa, 1965). Finally, the possibility of a nonmechanical transmission of a virus in the tissues of the vector, such as occurs with certain plant viruses, cannot be ruled out, although up to now no one has published anything on this subject. XII. SPECIFICITY AND RESISTANCE Insect viruses have been increasingly observed to behave with varying degrees of pathogenicity toward species which are not their spontaneous hosts. The problems concerning this specificity have been reviewed by Gershenson (1964). Stairs (1960) has recently found that the C . fumiferana nuclear polyhedrosis virus can infect a species of the same genus Choristoneura pinus Freeman. The pliable nature of virulence is also shown by the researches of Smirnoff (1963) in which he adapted a borrelinavirus pathogenic to Trichiocampus viminalis (Fallen) to the species Trichiocampus irregularis (Dyar) by successive passages through this host.

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The viruses of the nuclear polyhedroses of Malacosoma americanum Fabricius, M . alpicola, and Malacosoma pluviule (Dyar) are also infective for the species M . disstria (Fig. 10) (Stairs, 1964a). There is an affinity of the cytoplasmic polyhedrosis virus of Thaumetopoea wilkinsoni Tams toward the species Thauinetopoea pityocampa Schiff. and of the virus of T h . pityocampa toward T . wilkinsoni (Biliotti et al., 1962). Infection may also be intergeneric : Lambdina fiscellaria somniaria (Hulst) and Lambdina fiscellaria lugubrosa (Hulst) have been found to be susceptible to the nuclear polyhedroses of Orgyia pseudotsugata McDunnough and moderately to those of Caripeta divisata Walker.

I

I

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I 6

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7

I

l

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8 9 1 0

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Incubation Period (Days)

FIQ.10. Mortality-time relationship for native MuZucosomn viruses and virus from M . ulpicolu a t concentration 10" polyhedra/ml. (From Stairs, 1965; courtesy of G. R. Stairs.)

Caterpillars of T . pityocampa are susceptible to the virus of the cytoplasmic polyhedrosis of Arctia caja L., under laboratory conditions as well as in forest experiments. These results may be of interest, because A. caja larvae are easier to rear throughout the year in the laboratory than those of T. pityocampa, and the risk of skin irritation from handling the processionary caterpillar does not exist with the caterpillars of A . caja (Sidor, 1965). Aizawa (1962) succeeded in adapting the nuclear polyhedrosis virus of B. mori to the wax moth G . mellonella after a series of passages through the body of the latter insect. Several authors have recognized the weak specificity of the Tipula iridescent virus. TIV was successfully used by Bird (1961b) for infecting G . mellonella; Smith et al. (1961) for infecting 7 species of Diptera, 11

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of Lepidoptera, and 3 of Coleoptera; while Gershenson (1964) confirmed its infectious nature for the representatives of these three orders. The idea that viruses may vary in virulence is suggested by the work of Ossowski (1960a) , in which he used concentrations of a nuclear polyhedrosis virus for the biological control of Kotochaliu junodi (Heyl.) . H e found that viral material obtained from larvae gathered from a locality a certain distance away from the zone of application produced a higher mortality than material derived from larvae harvested in the experimental area. He concluded that the virus existed in different strains of varying virulence. David and Gardiner (1965a) , on the other hand, working with equal concentrations, could find no difference in virulence between a granulosis virus extracted from P. brassicae larvae imported from the Canary Islands and the same virus obtained from an English stock of Pieris. Work of Smirnoff (1961) on N . swuinei and Veber (1964) on G. mellonella has shown that an increase in virulence resulted from successive passages of a virus through several generations of the host insect. A biotic factor which is still far from clear but which appears to be important is the natural resistance of insects to viral infection. The existence of races of insects resistant to viral infection has been observed under both natural and laboratory conditions. Ossowski ( 1960a) found differences in the susceptibility of various natural populations of K. junodi to nuclear polyhedrosis. Martignoni and Schmid (1961) witnessed a similar phenomenon with the nuclear polyhedrosis of Phryganidia californica Packard, an indigenous insect, whereas two populations of recently introduced Pieris rapae L. showed no difference in their susceptibility to the granulosis virus. These data would indicate that some insect populations can develop a virus resistance after a long association with that virus. Under laboratory conditions the larvae of a strain of P. brassicae, primarily found to be resistant to granulosis as compared with another stock, still retained their resistance 4 years later (David and Gardiner, 1960, 1965a). Aruga (1963) reported that in the silkworm the tetravoltine and polyvoltine races were more resistant than the univoltine and bivoltine.

XIII. BIOLOGICAL CONTROL WITH VIRUSES A. Building Up the Virus Stocks The accumulation of viruses belonging to most of the types described in the invertebrates may now be obtained in vitro on cultured invertebrate tissues and organs. This technique is, however, not yet suited to large-scale viral production for the purposes of biological control.

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When large quantities of viral material are necessary they can sometimes be obtained by harvesting insects which had died from viruses in the field, either as a result of natural epizootic infections, or following the viral treatment of specific zones. This method is of value only for certain species, such as the gypsy moth L. dispar, in which the epizootic attack by a polyhedrosis often coincides with the maximum pullulation of the species (Vasiljevic, 1961, 1964a). This harvesting procedure has, however, been abandoned for several years (Ossowski, 1960a; Atger and Chastang, 1961), viruses being supplied primarily from large insect-rearing enterprises which are of two kinds: rearing on plants, and rearing on artificial media. 1 . Insect Rearing on Plants

For the pine processionary caterpillar T. pityocampa, Grison et al. (1959) and Grison (1960) collected infected insects under natural conditions and also infected them with virus during rearing. Some 200,000 caterpillars a t the commencement of the 5th instar were infected per 0s by spraying the foliage with an aqueous 1 gm./liter suspension of a powder titrating over 7 billion polyhedra per gram. The extraction from a 5th instar caterpillar should be about 1.5 billion polyhedra. Smirnoff (1964a) collected the larvae of the sawfly N . swainei from highly infested zones, for preference in July, when the 2nd instars seemed to be most highly receptive to viral infection. He sprayed a suspension of intestinal nuclear polyhedrosis virus a t a concentration of 1 million polyhedra/ml. each time the food supply was renewed. After extraction and purification, he obtained a suspension of 50 to 100 million polyhedra/ml. Where a complete rearing of the insect is practiced, the whole development cycle takes place in closed rooms. Gilmore and Munger (1963) reared healthy colonies of the citrus red mite P . citri a t 27°C. and 60 t o 70% relative humidity. The mites were sprayed with an aqueous suspension (2 ml./citrus) of virus isolated from the species. The diseased mites were gathered 8 to 12 days after contamination. Vago and Atger (1961) multiplied Borrelinavirus in pupae which are immobile, nonfeeding, storable forms. The larvae of various lepidoptera were infected just prior to pupation. Using this method, they obtained over 30 gm. of pure polyhedral inclusion bodies of B. mori and L. dispar. 9.Insect Rearing on Artificial Media With this technique insects may be reared throughout the whole year under regular control and with easy accessibility. Ignoffo (1964a) reared T. ni larvae on a semisynthetic medium (Ignoffo, 1963). The amount of external contamination of the eggs was reduced with sodium hypo-

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chlorite. The insects used came from strains reared for 30 generations on a semisynthetic medium. A suspension of 5 x lo6 polyhedra/ml. was sprayed onto the food and diseased larvae were collected after 4 to 5 days under rearing conditions a t 23°C. According to the author these rearing methods, the medium, and the infection procedure could serve as a basis for the commercial production of the virus for purposes of biological control, The same author (Ignoffo, 1965a,b) also reared H. zea and Heliothis virescens (Fabricius) larvae on a semisynthetic medium to which antibiotics had been added and he infected each rearing box with 0.2 ml. of a suspension containing 1.8 x lo7 polyhedra/ml. The multiplication coefficients of inclusion bodies in experimentally infected larvae of the last instar varied from 2000 to 5000.

B. Extraction and Formulation The first step is always to release the polyhedra by crushing the dead insects in water, The mixture of tissue and inclusion bodies is subjected to a series of filtrations through cloth or nylon followed by differential centrifugations and drying of the resulting paste (Vasiljevic, 1964a). The conditioning and storage problems are primarily those involved in the production of satisfactory inclusion body powders. The polyhedra deposit or suspension is fixed either on powdered bentonite (Grison et al., 1959; Grison, 1960; Smirnoff, 1964a) or on lactose (Ignoffo, 1964b, 1 9 6 5 ~ )Tablets . of nuclear polyhedra of L. dispar and of granules of P. brassicae have been successfully prepared (Vago et al., 1961). Field applications of combinations of various chemical insecticides and insecticide adjuvants containing viral suspensions have given varying results. Wetting agents such as Colloidal X-77 (Elmore, 1961; Tanada and Reiner, 1962; Tanada, 1964), Triton x-100 (Getzin, 1962; Ignoffo, 1964a; Ignoffo et al., 1965; Ignoffo and Montoya, 1966), and detergents like Tween 20 (Jaques, 1961, 1962a) seem to play an important role in obtaining better control, On the whole, the insecticides tested were found to be compatible with the nuclear polyhedrosis viruses, as in the case of ethyl parathion with the T. ni virus (Hofmaster and Ditman, 1961; Getzin, 1962; Wolfenbarger, 1965); on the other hand, methyl parathion was found to produce a 30% reduction in the mortality of Heliothis larvae normally obtained by a pure aqueous solution of polyhedra (Ignoffoand Montoya, 1966).

C . Preservation of Infectivity After isolation, purification, and conditioning of the viral material, the next problem is the preservation of infectivity until it is actually required for biological control.

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291

Working with nonoccluded viruses, Gilmore and Munger (1965) dried virus-infected mites P . citri in the presence of anhydrous CaClz for 2 hours and stored them a t -23°C. in the presence of a desiccant. Preserved thus, the material did not show any appreciable loss of infectivity after 6 months. Protein inclusion bodies of polyhedroses and of granuloses retain their infectivity for periods of up to several years. Vago et al. (1961) carried out virulence tests on nuclear polyhedra of L. dispar and on granules of the P. brassicae virosis prepared in tablet form, and found that the tablets showed no appreciable loss of virulence in experiments varying up to 5 years’ duration. Furthermore, the viruses contained in the polyhedra and granules were able to survive at temperatures which would be lethal to viruses without inclusion bodies (Hall, 1961, 1963). As regards suspensions of polyhedra, Atger and Chastang (196lj report that the cytoplasmic polyhedra of T. pityocampa virosis kept their infective properties for at least 3 years. Neilson and Elgee (1960) found that the polyhedra of D. hercyniae preserved and tested in suspension lost virulence during storage and became inactive a t the end of 12 years. Morris (1963) observed only slight mortality of 0. pseudotsugata caterpillars in a tree plot which had received a treatment with 7-yearold nuclear polyhedra.

D. Biological Tests Data gathered over recent years from biological tests have been leading toward the establishment of standard values for the comparison of the viral products used by different authors. The last International Colloquium on Insect Pathology, Wageningen, 1966, devoted a symposium to the study of achieving uniformity of test methods. 1. Nuclear Polyhedroses

According t o Grigorova (1962) the most effective concentration under laboratory conditions and using L. dispar nuclear polyhedra, was in the region of 45-50 million polyhedra/ml. of water. The larva is most susceptible to infection during its early stages. In experiments on the same virosis Vasiljevic (196413) confirmed that the longer the eggs were kept in a state of artificial hibernation, the more susceptible were the resulting larvae to infection. In addition to the LDao (lethal dose 50) increasingly greater use is being made of the LT6o (lethal time 50) criterion. This indicates the time taken by a given dose to kill 50% of the animals tested. Using nuclear polyhedra of L . fiscellaria, Morris (1962) established an LD,,, over the whole larval life of 3000 polyhedra/larva, and showed that this increased with larval age. A large number of treated animals reached the adult stage when the dose was weak or given toward

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the end of the larval phase. Ignoffo (1964b), using T. ni nuclear polyhedra, found an LDeoof 237 polyhedra/ml. and an LTBO at 25,000, 1,500, 500, and 300 polyhedra/ml. of medium of 3.7, 4.5, 5, and 8 days, respectively. Furthermore, 75% of the total larval mortality occurred during the 3rd and 4th instars. Ignoffo (1965~)reared H . zea larvae on a semisynthetic medium (Ignoffo, 1965b), the surface of which he contaminated with polyhedral inclusion bodies of nuclear polyhedra of the same species; he obtained an LDeoof 32 polyhedra/mm.2 of medium and LTBo a t 29,200, 292, 145, 58, and 29 polyhedra/mm.2 of 3.8, 3.8, 4.3, 5.3, and 6.5 days respectively. 2. Granuloses

By oral administration of a suspension of suitably diluted granulosis virus to 4th instar caterpillars of Zeiraphera diniana Guenee, Benz (1964) found that a single virus particle was capable of killing a caterpillar after a minimum of 13 days; but that, following oral application, only 1 out of 1,000 to 10,000 viral units actually managed to infect the caterpillar. The duplication of the DNA probably required 5 hours at the most. 3. Nonoccluded Vimses Gilmore and Munger (1963) showed that to infect P. citri with nonoccluded virus, which attacks this species, the limit of dilution of acarid viral material was 1 mg./400 ml. of water. They also reported (1965) that the denser the mite population on each lemon, the more pronounced were the symptoms of the disease.

E . Pathogenicity to Mammals The increasing emphasis on the industrial production of insect viruses calls for the elaboration of tests to ascertain their potential danger to vertebrates in general and to mammals in particular. This subject has featured on the programs of conferences and symposia held a t international level during the last few years. White mice and guinea pigs have been exposed to the nuclear polyhedrosis virus of H . zea and H . virescens, administered in the form of inclusion bodies, free viral particles, and polyhedra proteins, by inhalation, per os, and by intravenous, intradermal, intraperitoneal, and intracerebral injection. From a total of 155 virus-treated animals with 75 controls, only 1, a guinea pig, died and that was of an acute pneumonia (Ignoffo and Heimpel, 1965). Heimpel (1966) treated white mice and guinea pigs with a suspension of nuclear polyhedra of the T . ni virosis, per os, by inhalation and by intravenous and intracerebral in-

VIRUSES O F INVERTEBRATES

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jection. Most of the subjects under test, as well as the controls, put on weight during their 2 months under observation, with the exception of one which died from a nonviral cause. The author concluded that the virus was harmless to the mammals under examination.

F . Application in the Field 1. Methods

There are four main ways in which viral preparations may be disseminated in the field: by the application of sprays and dusts; by the introduction of infected individuals into natural populations ; by the introduction of the virus through the agency of other organisms and parasites; and by the application of a combination of viral material and insecticides. This variety of control methods involves a variety of techniques and equipment. Whereas in early experiments the infective material used was simply the pulverized infected larvae rich in inclusion bodies, today, powders or liquids are used which will preserve their infective properties for long periods (see Sections XIII, F,2, a and b). All the methods of agricultural application have been used: spraying and dusting by hand and with equipment carried on the back for small areas ; powerful traction equipment, aircraft (Smirnoff et al., 1962; Bird, 1964b) and helicopter (Grison et al., 1959; Grison, 1960) spreading which is particularly useful over large forest tracts. The activity of a virus in the field is tied in the first place to the factors which determine its ability to spread throughout the insect population to be controlled. Dispersion is assured partly by climatic and physical factors such as wind and rain, and partly by the activity of contaminated insects, their parasites, and predators. Transmission by egg laying has been observed by Bird (1961b) and Smirnoff (1962, 1964b) in sawflies of the genera Diprion and Neodiprion. Sniirnoff considers this to be one of the factors in the reduction of insect populations (See Section XI, D). The effectiveness of the treatment is also dependent upon the development of those stages of the insect which are most susceptible to the spread of the virus. Generally, the earlier larval instars are less resistant to the pathogen than the later ones. In N . szcainei (Smirnoff, 1961, 1964a), Neodiprion sertifer (Geoffroy) (Bird, 1964b), L. dispar (Ruperez, 1964), M . disstria (Stairs, 1964b), H . zea, and H . virescens (Ignoffo, 1965c), application is recommended shortly after the hatching of the eggs ti0 that maximum epizootic infection will occur toward the end of the larval stage. Jaques (1964) has described the persistence in the field of the nuclear polyhedrosis of T. ni, its virulence against the insect persisting for 95

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weeks. Applications of suspensions of nuclear polyhedra of K . junodi (Ossowski, 1960b), N . sertifer (Bird, 1964b), and Heliothis (Ignoffo et al., 1965), and of cytoplasmic polyhedra of T. pityocampa (Burgerjon and Grison, 1965) adhered well to the foliage and were not easily carried away by natural or simulated rainfall. When submitted to open-air conditions and particularly to frost, the granulosis virus of P. brassicae was still present on cabbage leaves 4 months after its application and was capable of killing 1st instar larvae feeding upon them (David and Gardiner, 1966). 2. Results

a. Nuclear Polyhedroses. i. Lepidoptera. Ossowski (1960~)has successfully controlled K. junodi with the application a t the beginning of the larval stage of suspensions containing 500 and 1000 polyhedra/mm.*. Elmore (1961) obtained 100% mortality with T . ni. Stairs and Bird (1962) used against C . fumiferana suspensions of the nuclear polyliedrosis and the granulosis pertaining to the species, either alone or in combination. Morris (1963) obtained up to 90% mortality with 0. pseudotsugata larvae treated with a suspension containing 100 million polyhedra/ml. In some species susceptibility to the virus changes particularly rapidly during the larval stage. This is the case with M . disstria: the same suspension a t lo7 polyhedra/ml. applied to the lst, 3rd, and 4th instars resulted in 92%, 1476, and 0% mortality, respectively (Stairs, 1964

b) .

Ignoffo et al. (1965) obtained effective control over H . zea on sorghum, wheat, and cotton with doses of 10l2, 6 X loll, and 6 X 1011 PIB’s (polyhedral inclusion bodies) /acre, respectively ; and over H . virescens on cotton with 6 X 10l1 PIB’s/acre. Rollinson et al. (1965) reduced a population of L. diapar by using a suspension of 2.7 X lo8 polyhedra/ml. ii. Hymenoptera. Bird and Burck (1961) hand-sprayed aqueous suspensions of 1 million polyhedra/ml. against D. hercyniae: only a small area was initially treated, the virus spreading rapidly with the reappearance of epiaootics each year. Bird (1964b) treated pine forests by aircraft delivering suspensions of 200,000 to 5 million polyhedra/ml., the latter concentration producing a mortality of over 80% in N . sertifer populations. Good results Were also obtained by Rivers (1964) against this species with a suspension of 60,OOO polyhedra/ml. applied a t the rate of 2 liters/hectare. Smirnoff (1961) noted the best results against h’. swainei with concentrations between 500,000 and 3 million polyhedra/ml., and found that higher concentrations of inclusion bodies did not accelerate the

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development of the disease. By selecting a particularly active strain of the same virus a mortality of 100% was obtained a t a concentration of 2 million polyhedra/ml. (Smirnoff, et al., 1962). b. Cytoplasmic Polyhedroses. It was found necessary to use the helicopter for the control of the pine processionary caterpillar T . pityocampa in the vast forests of France. Treatment averaged 1,200 billion polyhedra/hectare and was followed by a high mortality spread over all larval instars. Furthermore, 90% of the survivors were infected with the virus and the disease pursued its course in the caterpillars buried in the soil for pupation (Grison et al., 1959; Grison, 1960). I

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c. Granuloses. The very high virulence of certain granuloses which had already been observed by Paillot encouraged several workers to try them for microbiological control purposes. Their inclusion bodies are easily purified and can be kept for long periods in dry state or in aqueous suspensions. Spraying a suspension of granules onto cabbage plants gave rise to epizootics in a population of P . rapae; only a few larvae reached the 5th instar and very few pupated (Wilson, 1960). Stairs and Bird (1962) obtained promising results against C . fumiferana. Finally, preliminary tests of the persistence of the virus of P. brassicae under natural conditions indicate that it may eventually be useful for biological control measures (David, 1965; David and Gardiner, 1966). d. Nonoccluded Viruses. The initial trials of nonoccluded viruses for

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CONSTANT VAGO AND MAX BERGOIN

biological control were carried out with the P . citri virus, on the one hand, and with the densonucleosis virus of G. mellonella, on the other. Gilmore (1965) carried out field experiments on orange trees infected with the citrus red mite, P. citri; the virus was transmitted either by spraying with viral suspensions or by the introduction of laboratorycontaminated mites. Epizootic infections developed which kept down the mite populations to levels which varied with the treatment given (Fig. 11). Lavie et al. (1965) showed that the densonucleosis virus exerted a pathogenic action on the greater wax moth G. mellonella in beehives. The rapidity of spread of the disease was a function of the density of the Galleria population. The virus is effective in hives inhabited by bees, but the degree of efficacy is difficult to assess.

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