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Infectivity of the components of a nuclear polyhedrosis virus of the silkworm, Bombyx mori
JOURNAL
OF INVERTEBRATH
Infectivity
PATHOLOGY19,62-65(1972)
of the Components
of a Nuclear
Bombyx
of the Silkworm, TOSHIYUIU
Laboratory
of Biochemistry,
Department
KHOSAKA
AND
of Agricultural
MICHIO
Polyhedrosis
Virus
mori HIMENO
Chemistry, Kyoto
University,
Kyoto, Japan
Received June 1, 1971 The polyhedra of the nuclear polyhedrosis virus (NPV) of Bombyz mori were dksolved under controlled dissolution with weak alkali. Five components were separated by centrifugation of the dissolved solution in a sucrose gradient. The assay of the separated components in B. mori pupae showed that two types of rods were almost equally, highly infectious, whereas the small spherical particles were much less infectious than the rods by an order of 4 log units. The infectious unit of this virus appeared to be the virus rod without the outer membrane, and the small spherical particle did not represent any infectious functional unit but was a degradation product of the viral internal substance and the outer membrane may not be essential for the virus infection in the pupal hemocoel.
According to a hypothesis first proposed by Bergold (1953) and later expanded by Bird (1959), the spherical particles are the initial subunits of a life and multiplication cycle of the NPV. Although Aizawa (1967) reported that the spherical particles were infectious, Krieg (1957), as the result of assaying the infectivity of virus particles that were obtained by dissolving the polyhedra with alkali at progressively higher pHs, reported that the rod particles were highly infectious, while the ‘Lsubunit” or spherical particles were noninfectious. Bergold (1963b) observed that virus preparations containing many rod particles without outer membranes were less infectious than those with only a few particles without membranes. According to Harrap (1970), the envelope or outer membrane was essential for the per OS infection with NPV. The virus particles with outer membranes were found in ultrathin sections, attached to the microvilli of the columnar midgut cells. In some cases only the empty outer membrane was observed attached to the microvillus, suggesting that the virus particle entered the cell without the membrane. Thus, Harrap (1970) speculated that the
The virus particles of nuclear polyhedrosis virus (NPV) of insects were 6rst isolated from polyhedra that were treated with alkali, and the large rod particles were found to be highly infectious for insects (Bergold, 1947). On the basis of electron microscopical studies, the isolated virus particles from the polyhedra of different NPV were found to consist of two kinds of rod particles and associated with them occasionally were small spherical particles, so-called “subunit” (Bergold, 1953, 1958; Krieg, 1961; Harrap and Junipar, 1966). The spherical particles within the virus rod were not observed in the thin sections of polyhedra by Morgan et al. (1956) and Bergold (1963a) or in ultrathin sections of infected tissues by Himeno et al. (1968). However, Aizawa (1967) isolated by ultracentrifugation small spherical particles 20 run in diameter from the hemolymph of NPV-infected silkworm larvae. Bergold (1963b) observed in serial sections of polyhedra some spherical embedded particles. Krieg and Huger (1969) also observed spherical particles (25-26 nm) in thin sections of NPV-infected nucleus of Galtia mellondla. Copyright Q 1972by Academic Prem. Ino.
62
INFECTIVITY
OF AN INSECT
virus particles without out.cr membranes were incapable of infecting the cell. On the other hand, Bird (1959) suggested that the outer membrane did not play a decisive role in virus infection, since rod particles wit,hout membranes remained infect,ious. In a previous report, Iihosalta et al. (1971) showed that the -XI’V of B. nrori consisted enveloped by two of an internal matrix layers, the outer and inner membranes, that were highly organized structures. The “subunit” described above was considered to be a degradation product of the const.ituents of the internal matrix within the virus rod. We have presented our study on the relative infectivit,y of the separate viral components of XPV obtained from the larvae of B. mori. The results indicate that the infectious units of NPV of B. nlori are rod particles without the outer membrane in pupal hemocoel. MATERIALS
liw
METHODS
The procedures for the preparation and purification of polyhedra and for the isolation and purification of the virus particles from t,he polyhedra were similar to t,hose described previously (Khosaka et al., 1971) except that a constant condition, 0.01 M Na2C0, containing 0.05 M NaCl, for 1 hr at Z’C, was used for the isolation of virus part.icles from t,he polyhedra. Sucrose gl,arlient centrifugation. The separation of viral components and the gradient. fract,ionations ivere carried out as described by Khoslta et al. (1971). For further separation, the opalescent bands visible in the gradients were separately removed, and each band was ccntrifugcd at high speed. The sediment was suspended in ~/15 sodium phosplzat,e buffer (I’S), pH 7.2, and then subjected to another centrifugation in a sucrose gradient,. Injectivity assay. In each series of assays, t,he fractions with corresponding viral componems were pooled and adjusted, with M/G PB, pH 7.2, to obtain equal concentrat,ion at ODz,, unit. The materials t.o be as-
63
VIRCS
sayed were centrifuged for 15 min at 6,000 rpm at 2°C to remove the bacterial contaminants. The supernatants were carefully removed and then subjected to lo-fold serial dilution with the sterilized buffer described above. A 0.025 ml sample of inoculum was injected into each of the hemococls of groups of pupae, which were then incubated at 28°C. Observations were made daily foi symptoms of virus disease, which usually appeared 5-7 days after inoculation. The observations \verc discontinued when the pupae developed to adults. A pupa was considered to be infected with virus when polyhedra were observed in the hemolymph with the light microscope. RESULTS
As reported previously (Rhosaka et, al., 1971), the viral components from nlkalidissolved polyhedra could be separated into five bands by sedimentation in a sucrose gradient. Electron microscopical st,udies rcvealed that the lowest and next to the lolvest bands from the bottom of the gradient. consisted of rod particles with and without the outer membrane, respectively. The uppermost band contained small spherical particles which were heterogeneous in size and shape. In addition, two kinds of empt,y membranes were found as two bands in the middle of the sucrose gradient. In order to compare the infect,ivity associated with the fractions containing the rods, empty membranes, and small spherical particles, the assays were carried out, on groups of pupae which had developed from larvae reared on mulberry leaves. In the cont.rol experiment, none of the pupae injected 1vit.h M/G PB, pH 7.2, deveioped virus disease. The results presented in Table 1 clearly demonstrated that the rods were highly infectious, while the fraction of small spherical particles was not only less infectious than thr rods by an order of 4 log units, but also was slightly less infectious than the fraction of empty membranes.
64
KHOSAKA
These observat,ions indicate that the rods are the infectious units and that the small spherical particles are not infectious. The spherical particles are apparently degradation products of rod particles as shown by electron microscopical studies (Khosaka et al., 1971). In an attempt to test the relative infectivity of the two types of rods, the rods were purified with a second cycle of centrifugation in a sucrose gradient, but a complete separation was not obtained. Electron microscopical examination revealed that the band of either type of rods was always contaminated with a small proportion of the other rod type. The virus rods found in the two bands did not show any significant difference in infectivity (Table 2) when assayed with TABLE
1
COMPARISON OF TNFECTNITIES OF ROD PARTICLES, EMPTY MEMBRANES, AND INTERNAL VIRAL COMPONENT TO PUPAE OF Bombyx mori
Assay material
Rod particles
Dimtion
100 lo10-Z 10-a lo-4
Empty
membrane
10-S l(rU 100 10-l IF 1(ra 10-4
Spherical
particles
lo-5 100 10-l lcF 10-s 10-d
No. of pupae wdESeasedm
percent infectivity
13 13 13 13 8 2 0 13 13 4 0 0 0 7 2 0 0 0
100 106 100 100 62 15 0 106 100 31 0 0 0 54 15 0 0 0
5 Thirteen pupae were tested at each dilution of the assay material. Each pupa was injected with 0.025 ml of inoculum and incubated at 28°C for over 10 days. Virus disease usually developed in 5-7 days.
AND
HIMENO TABLE
RELATIVE
2
INFECTIVITY OF Two TYPES OF VIRUS ROD PARTICLES No. of
Assay material
Dilution
?rzT diseaseda
Rod particles with outer membrane
100 lo-’ 10-z 10-a lo-4 lo0 l(r’ lO-2 10-S lo-’
5 5 5 1 0 5 5 5 2 0
Rod particles without outer membrane
Percent infectivity
100 100 100 20 0 100 100 100 40 0
Li Five pupae whose larvae had been reared on a semisynthetic medium were tested with the assay material at each dilution. Each pupa was injected with 0.025 ml of inoculum and kept at 28°C for over 10 days.
aseptically reared pupae. This indicates that the infective unit of this virus is the rod particle wit.hout the outer membrane. DISCUSSION
As predicted from a study on the structure of this virus (Khosaka et al., 1971), the small spherical particles, which are heterogeneous in size and shape, are much less infectious than the rods by an order of 4 log units. The infectivity of the spherical fraction is not significant as an infectious unit, and it will come from the contamination of the rod particles. Moreover, since the molecular weight of DNA extracted from the virus particles is over 2 X lo7 as determined by electron microscopical examination (Onodera et al., unpublished), t.he small spherical part.icles wit,h a diameter about 20 nm could not, contain a DNA molecule of such a size. The one spherical particle is apparently not, an infective unit. It was shown previously that the fraction of empty membranes also contaminated the slender rods and the spherical particles (Khosaka et al., 1971).
INFECTIVITY
OF AN INSECT VIRUS
The infectivity depended on the contaminants. Although the complete separation of the two types of rods, with and without outer membrane, could not be accomplished, the relative infectivity of the two rods is almost identical as shown in Table 2. This seems to suggest that the outer membrane does not have a critical function during the process of virus infection in the pupal hemocoel. On the other hand, the outer membranes may be essential for infection through the midgut as concluded by Harrap (1970). Observations of thin sections of polyhedra (Morgan et al., 1955; Bergold, 1963a) show that in some cases, such as in the NPV of Porthetria d&par, Lymantria monacha, and Laphygma frugipercla, there are often groups of slender rods enclosed within one outer membrane, and in other cases, such as in the NPV of B. mori, the majority of the slender rods occur singly within an outer membrane, but at times up to seven rods may occur within one outer membrane. These observations also suggest that the outer membrane may not be essential for virus infectivity in pupal hemocoel. Our study suggests that the infectious unit of NPV of B. mori is the rod particle without the outer membrane in pupal hemocoel. The authors wish to thank Professor K. Onodera (Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University) and Professor Y. Tanada (Division of Entomology, University of California) for hi useful advice and discussion and also acknowledge Dr. K. Hayashiya (Faculty of Textile Science, Kyoto University of Industrial Arts and Textile Science) for kindly providing the semisynthetic medium for rearing the silkworm. REFERENCES AIZAWA, K. (1967). *Mode of multification of the nuclear-polyhedrosis virus of silkworm. J. Sericult. BEBQOLD, G.
Sci. Jap.,
H. polyeder-Virus
36,3Zi-331.
(1947). Die Isolierung des und die Natur der Polyeder.
2. NatuTfOTsch., f?b, 122-143.
BERGOLD, G. H.
65
(1953). The multiplication of insect viruses. In Symp. Sot. Gen. Microbid., 2. 276-m. BERGOLD, G. H. (1958). Viruses of insects. In “Handbuch der Virusforschung” (C. Hallauer and K. F. Meyer, eds.), Vol. 4, pp. 60-142. Springer, Vienna. BERQOLD, G. H. (1963a). Fine structure of some insect viruses. J. Insect Pathol., 5, lll128. BERGOLD, G. H. (1963b). The nature of nuclearpolyhedrosis viruses. In “Insect Pathology, An advanced treatise” (E. A. Steinhaus, ed.), Vol. 1, pp. 413456. Academic Press, New York. BIRD, F. H. (1959). Polyhedrosis and granulosis viruses causing single and double infections in the spruce budworm, Choristoneura fum.iferana Clemens. J. Inuertebr. Palhol., 1, 406430. HARRAP, K. A., AND JUNIPAR, B.E. (1966). The internal structure of an insect virus. virology, 29, 175-178. HARRAP, K. A. (1970). Cell infect,ion by nuclear polyhedrosis virus. Virology, 42, 311-318. HIMENO, M., Y~SUDA, S., KROSAKA, T., ASD ONODERII, K. (1968). The fine structure of nuclear-polyhedrosis virus of the silkworm. J. Invertebr. Pathol., 11, 516-519. KHOSAKA, T., HIM~NO, M., .~ND ONODERA, K. (1971). Separation and structure of components of nuclear polyhedrosis virus of the silkworm. J. ViToZ., 7, 267-273. KRIEG, A. (1957). Veer Aufbau und Vermehrungsm6glichkeiten von stabchenformigen Insekten-Viren. I., 2. Natzlrforsch., 12b, 124l121. E(RIEQ, A. (1961). ifber Aufbau und die Vermehrungsmijglichkeiten von stlbchenformigen Insekten-Viren. II., Z. Naturforsch., 16b, 115117. KRIEG, A., .~ND HUGER, A. H. (1969). New ultracytological findings in insect nuclear polyhedrosis. J. znvertebr. Pathol., 13, 272-279. MORGAN, C., BERGOLD, G. H., MOORE, D. H., AND Ross, H. M. (1955). The macromolecular parscrystalline lattice of insect viral polyhedral bodies demonstrated in ultrathin sections examined in the electron microscope. J. Biophys. Biochem. Cytol., I, 189-W. MORGAN,C.,BERQOLD, G.H., ASD Rosn,H.M. (1956). Use of serial sections to delineate the structure of Porthetria &spar virus in the electron microscope. J. Biophys. Biochem. Cytol., 2, 23-28.
OF INVERTEBRATH
Infectivity
PATHOLOGY19,62-65(1972)
of the Components
of a Nuclear
Bombyx
of the Silkworm, TOSHIYUIU
Laboratory
of Biochemistry,
Department
KHOSAKA
AND
of Agricultural
MICHIO
Polyhedrosis
Virus
mori HIMENO
Chemistry, Kyoto
University,
Kyoto, Japan
Received June 1, 1971 The polyhedra of the nuclear polyhedrosis virus (NPV) of Bombyz mori were dksolved under controlled dissolution with weak alkali. Five components were separated by centrifugation of the dissolved solution in a sucrose gradient. The assay of the separated components in B. mori pupae showed that two types of rods were almost equally, highly infectious, whereas the small spherical particles were much less infectious than the rods by an order of 4 log units. The infectious unit of this virus appeared to be the virus rod without the outer membrane, and the small spherical particle did not represent any infectious functional unit but was a degradation product of the viral internal substance and the outer membrane may not be essential for the virus infection in the pupal hemocoel.
According to a hypothesis first proposed by Bergold (1953) and later expanded by Bird (1959), the spherical particles are the initial subunits of a life and multiplication cycle of the NPV. Although Aizawa (1967) reported that the spherical particles were infectious, Krieg (1957), as the result of assaying the infectivity of virus particles that were obtained by dissolving the polyhedra with alkali at progressively higher pHs, reported that the rod particles were highly infectious, while the ‘Lsubunit” or spherical particles were noninfectious. Bergold (1963b) observed that virus preparations containing many rod particles without outer membranes were less infectious than those with only a few particles without membranes. According to Harrap (1970), the envelope or outer membrane was essential for the per OS infection with NPV. The virus particles with outer membranes were found in ultrathin sections, attached to the microvilli of the columnar midgut cells. In some cases only the empty outer membrane was observed attached to the microvillus, suggesting that the virus particle entered the cell without the membrane. Thus, Harrap (1970) speculated that the
The virus particles of nuclear polyhedrosis virus (NPV) of insects were 6rst isolated from polyhedra that were treated with alkali, and the large rod particles were found to be highly infectious for insects (Bergold, 1947). On the basis of electron microscopical studies, the isolated virus particles from the polyhedra of different NPV were found to consist of two kinds of rod particles and associated with them occasionally were small spherical particles, so-called “subunit” (Bergold, 1953, 1958; Krieg, 1961; Harrap and Junipar, 1966). The spherical particles within the virus rod were not observed in the thin sections of polyhedra by Morgan et al. (1956) and Bergold (1963a) or in ultrathin sections of infected tissues by Himeno et al. (1968). However, Aizawa (1967) isolated by ultracentrifugation small spherical particles 20 run in diameter from the hemolymph of NPV-infected silkworm larvae. Bergold (1963b) observed in serial sections of polyhedra some spherical embedded particles. Krieg and Huger (1969) also observed spherical particles (25-26 nm) in thin sections of NPV-infected nucleus of Galtia mellondla. Copyright Q 1972by Academic Prem. Ino.
62
INFECTIVITY
OF AN INSECT
virus particles without out.cr membranes were incapable of infecting the cell. On the other hand, Bird (1959) suggested that the outer membrane did not play a decisive role in virus infection, since rod particles wit,hout membranes remained infect,ious. In a previous report, Iihosalta et al. (1971) showed that the -XI’V of B. nrori consisted enveloped by two of an internal matrix layers, the outer and inner membranes, that were highly organized structures. The “subunit” described above was considered to be a degradation product of the const.ituents of the internal matrix within the virus rod. We have presented our study on the relative infectivit,y of the separate viral components of XPV obtained from the larvae of B. mori. The results indicate that the infectious units of NPV of B. nlori are rod particles without the outer membrane in pupal hemocoel. MATERIALS
liw
METHODS
The procedures for the preparation and purification of polyhedra and for the isolation and purification of the virus particles from t,he polyhedra were similar to t,hose described previously (Khosaka et al., 1971) except that a constant condition, 0.01 M Na2C0, containing 0.05 M NaCl, for 1 hr at Z’C, was used for the isolation of virus part.icles from t,he polyhedra. Sucrose gl,arlient centrifugation. The separation of viral components and the gradient. fract,ionations ivere carried out as described by Khoslta et al. (1971). For further separation, the opalescent bands visible in the gradients were separately removed, and each band was ccntrifugcd at high speed. The sediment was suspended in ~/15 sodium phosplzat,e buffer (I’S), pH 7.2, and then subjected to another centrifugation in a sucrose gradient,. Injectivity assay. In each series of assays, t,he fractions with corresponding viral componems were pooled and adjusted, with M/G PB, pH 7.2, to obtain equal concentrat,ion at ODz,, unit. The materials t.o be as-
63
VIRCS
sayed were centrifuged for 15 min at 6,000 rpm at 2°C to remove the bacterial contaminants. The supernatants were carefully removed and then subjected to lo-fold serial dilution with the sterilized buffer described above. A 0.025 ml sample of inoculum was injected into each of the hemococls of groups of pupae, which were then incubated at 28°C. Observations were made daily foi symptoms of virus disease, which usually appeared 5-7 days after inoculation. The observations \verc discontinued when the pupae developed to adults. A pupa was considered to be infected with virus when polyhedra were observed in the hemolymph with the light microscope. RESULTS
As reported previously (Rhosaka et, al., 1971), the viral components from nlkalidissolved polyhedra could be separated into five bands by sedimentation in a sucrose gradient. Electron microscopical st,udies rcvealed that the lowest and next to the lolvest bands from the bottom of the gradient. consisted of rod particles with and without the outer membrane, respectively. The uppermost band contained small spherical particles which were heterogeneous in size and shape. In addition, two kinds of empt,y membranes were found as two bands in the middle of the sucrose gradient. In order to compare the infect,ivity associated with the fractions containing the rods, empty membranes, and small spherical particles, the assays were carried out, on groups of pupae which had developed from larvae reared on mulberry leaves. In the cont.rol experiment, none of the pupae injected 1vit.h M/G PB, pH 7.2, deveioped virus disease. The results presented in Table 1 clearly demonstrated that the rods were highly infectious, while the fraction of small spherical particles was not only less infectious than thr rods by an order of 4 log units, but also was slightly less infectious than the fraction of empty membranes.
64
KHOSAKA
These observat,ions indicate that the rods are the infectious units and that the small spherical particles are not infectious. The spherical particles are apparently degradation products of rod particles as shown by electron microscopical studies (Khosaka et al., 1971). In an attempt to test the relative infectivity of the two types of rods, the rods were purified with a second cycle of centrifugation in a sucrose gradient, but a complete separation was not obtained. Electron microscopical examination revealed that the band of either type of rods was always contaminated with a small proportion of the other rod type. The virus rods found in the two bands did not show any significant difference in infectivity (Table 2) when assayed with TABLE
1
COMPARISON OF TNFECTNITIES OF ROD PARTICLES, EMPTY MEMBRANES, AND INTERNAL VIRAL COMPONENT TO PUPAE OF Bombyx mori
Assay material
Rod particles
Dimtion
100 lo10-Z 10-a lo-4
Empty
membrane
10-S l(rU 100 10-l IF 1(ra 10-4
Spherical
particles
lo-5 100 10-l lcF 10-s 10-d
No. of pupae wdESeasedm
percent infectivity
13 13 13 13 8 2 0 13 13 4 0 0 0 7 2 0 0 0
100 106 100 100 62 15 0 106 100 31 0 0 0 54 15 0 0 0
5 Thirteen pupae were tested at each dilution of the assay material. Each pupa was injected with 0.025 ml of inoculum and incubated at 28°C for over 10 days. Virus disease usually developed in 5-7 days.
AND
HIMENO TABLE
RELATIVE
2
INFECTIVITY OF Two TYPES OF VIRUS ROD PARTICLES No. of
Assay material
Dilution
?rzT diseaseda
Rod particles with outer membrane
100 lo-’ 10-z 10-a lo-4 lo0 l(r’ lO-2 10-S lo-’
5 5 5 1 0 5 5 5 2 0
Rod particles without outer membrane
Percent infectivity
100 100 100 20 0 100 100 100 40 0
Li Five pupae whose larvae had been reared on a semisynthetic medium were tested with the assay material at each dilution. Each pupa was injected with 0.025 ml of inoculum and kept at 28°C for over 10 days.
aseptically reared pupae. This indicates that the infective unit of this virus is the rod particle wit.hout the outer membrane. DISCUSSION
As predicted from a study on the structure of this virus (Khosaka et al., 1971), the small spherical particles, which are heterogeneous in size and shape, are much less infectious than the rods by an order of 4 log units. The infectivity of the spherical fraction is not significant as an infectious unit, and it will come from the contamination of the rod particles. Moreover, since the molecular weight of DNA extracted from the virus particles is over 2 X lo7 as determined by electron microscopical examination (Onodera et al., unpublished), t.he small spherical part.icles wit,h a diameter about 20 nm could not, contain a DNA molecule of such a size. The one spherical particle is apparently not, an infective unit. It was shown previously that the fraction of empty membranes also contaminated the slender rods and the spherical particles (Khosaka et al., 1971).
INFECTIVITY
OF AN INSECT VIRUS
The infectivity depended on the contaminants. Although the complete separation of the two types of rods, with and without outer membrane, could not be accomplished, the relative infectivity of the two rods is almost identical as shown in Table 2. This seems to suggest that the outer membrane does not have a critical function during the process of virus infection in the pupal hemocoel. On the other hand, the outer membranes may be essential for infection through the midgut as concluded by Harrap (1970). Observations of thin sections of polyhedra (Morgan et al., 1955; Bergold, 1963a) show that in some cases, such as in the NPV of Porthetria d&par, Lymantria monacha, and Laphygma frugipercla, there are often groups of slender rods enclosed within one outer membrane, and in other cases, such as in the NPV of B. mori, the majority of the slender rods occur singly within an outer membrane, but at times up to seven rods may occur within one outer membrane. These observations also suggest that the outer membrane may not be essential for virus infectivity in pupal hemocoel. Our study suggests that the infectious unit of NPV of B. mori is the rod particle without the outer membrane in pupal hemocoel. The authors wish to thank Professor K. Onodera (Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University) and Professor Y. Tanada (Division of Entomology, University of California) for hi useful advice and discussion and also acknowledge Dr. K. Hayashiya (Faculty of Textile Science, Kyoto University of Industrial Arts and Textile Science) for kindly providing the semisynthetic medium for rearing the silkworm. REFERENCES AIZAWA, K. (1967). *Mode of multification of the nuclear-polyhedrosis virus of silkworm. J. Sericult. BEBQOLD, G.
Sci. Jap.,
H. polyeder-Virus
36,3Zi-331.
(1947). Die Isolierung des und die Natur der Polyeder.
2. NatuTfOTsch., f?b, 122-143.
BERGOLD, G. H.
65
(1953). The multiplication of insect viruses. In Symp. Sot. Gen. Microbid., 2. 276-m. BERGOLD, G. H. (1958). Viruses of insects. In “Handbuch der Virusforschung” (C. Hallauer and K. F. Meyer, eds.), Vol. 4, pp. 60-142. Springer, Vienna. BERQOLD, G. H. (1963a). Fine structure of some insect viruses. J. Insect Pathol., 5, lll128. BERGOLD, G. H. (1963b). The nature of nuclearpolyhedrosis viruses. In “Insect Pathology, An advanced treatise” (E. A. Steinhaus, ed.), Vol. 1, pp. 413456. Academic Press, New York. BIRD, F. H. (1959). Polyhedrosis and granulosis viruses causing single and double infections in the spruce budworm, Choristoneura fum.iferana Clemens. J. Inuertebr. Palhol., 1, 406430. HARRAP, K. A., AND JUNIPAR, B.E. (1966). The internal structure of an insect virus. virology, 29, 175-178. HARRAP, K. A. (1970). Cell infect,ion by nuclear polyhedrosis virus. Virology, 42, 311-318. HIMENO, M., Y~SUDA, S., KROSAKA, T., ASD ONODERII, K. (1968). The fine structure of nuclear-polyhedrosis virus of the silkworm. J. Invertebr. Pathol., 11, 516-519. KHOSAKA, T., HIM~NO, M., .~ND ONODERA, K. (1971). Separation and structure of components of nuclear polyhedrosis virus of the silkworm. J. ViToZ., 7, 267-273. KRIEG, A. (1957). Veer Aufbau und Vermehrungsm6glichkeiten von stabchenformigen Insekten-Viren. I., 2. Natzlrforsch., 12b, 124l121. E(RIEQ, A. (1961). ifber Aufbau und die Vermehrungsmijglichkeiten von stlbchenformigen Insekten-Viren. II., Z. Naturforsch., 16b, 115117. KRIEG, A., .~ND HUGER, A. H. (1969). New ultracytological findings in insect nuclear polyhedrosis. J. znvertebr. Pathol., 13, 272-279. MORGAN, C., BERGOLD, G. H., MOORE, D. H., AND Ross, H. M. (1955). The macromolecular parscrystalline lattice of insect viral polyhedral bodies demonstrated in ultrathin sections examined in the electron microscope. J. Biophys. Biochem. Cytol., I, 189-W. MORGAN,C.,BERQOLD, G.H., ASD Rosn,H.M. (1956). Use of serial sections to delineate the structure of Porthetria &spar virus in the electron microscope. J. Biophys. Biochem. Cytol., 2, 23-28.