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An in vivo study of intracytoplasmic membranous structures associated with chronic stunt virus infection in granular hemocytes of Amyelois transitella
J O U R N A L O F U L T R A S T R U C T U R E RESEARCH
79, 158-164 (1982)
An in Vivo Study of Intracytoplasmic Membranous Structures Associated with Chronic Stunt Virus Infection in G ran ular Hemocytes of Amyelois transitella 1 D A R L E N E F . H O F F M A N N AND W I L L I A M R . K E L L E N
Stored-Product Insects Research Laboratory, USDA, Agricultural Research Fresno, California 93727 Received May 5, 1981, and in revised form October 23, 1981 The fine structure of cytoplasmic, membranous structures associated with chronic stunt virus infection was studied in granular hemocytes of larvae of Amyelois transitella. Although nuclei of infected hemocytes apparently were not affected, viroplasmic bodies, the assumed sites of viral assembly, often filled the cytoplasm of these cells. Early stage viroplasmic bodies were bounded by single envelopes and contained finely granular, electron-dense viroplasms in which progeny virions formed aggregations or paracrystalline arrays, In mature viroplasmic bodies, viral aggregations and arrays were enveloped and segregated by multilayered membranous structures. Irregular, vesicular bodies occupied the electron-lucent areas between aggregations. Prior to cell lysis, membranous structures within viroplasmic bodies disintegrated in close association with secondary, membrane-bound bodies that were probably autophagic elements.
Chronic stunt virus (CSV) was originally described (Kellen and Hoffmann, 1981) from the cytoplasm of granular hemocytes of larvae of Amyelois transitella, a serious lepidopterous pest of almonds and walnuts in California. In laboratory tests, neonatal larvae usually succumbed to infection within a few days; however third- or fourth-instar larvae that initially acquired infection frequently developed chronic disease that caused retarded growth and delayed mortality. The virus is 38 nm in diameter, isometric, and contains single-stranded RNA. Hillman et al. (1981) reported the physicochemical characterization of CSV and tentatively assigned the pathogen to the caliciviruses. Caliciviruses have a distinctive morphology and several unique physicochemical characteristics which distinguish them from the other members of the Picornaviridae (Burroughs et al., 1978; Schaffer, 1979). CSV is the first calicivirus reported from an arthropod. In our initial report (Kellen and Hoff-
mann, 1981) on the host-pathogen relationships of this disease, we noted that aggregations and paracrystalline arrays of viral particles were usually enclosed in complex, membranous structures. These cytolysome-like viroplasmic bodies o c c u r r e d throughout the cytoplasm and contained internal multilaminar membranous systems, vesicles, and areas of finely granular, electron-dense viroplasm. Viroplasmic bodies were the assumed sites of viral assembly and storage. The purpose of this study is to provide additional information on the ultrastructure of membranous structures associated with the viroplasmic bodies in the cytoplasm of infected hemocytes of A. transitella.
1 Lepidoptera: Pyralidae. 158 0022-5320/82/050158-07502.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS
Host. Infected larvae used in this study were from a diseased laboratory culture that was routinely reared on a bran diet contaminated with semipurified virus. Methods for preparing viral suspensions and maintaining diseased cultures were reported previously (Kellen and Hoffmann, 1981). Electron microscopy. Chronically infected larvae used for electron microscopy were between 60 and 85 days old. Infected granular hemocytes reported here were examined in situ, usually in the larval head cap-
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES sule. Small pieces of larval, tissue, including intact head capsules, were dissected in cold (4°C) 2.5% glutaraldehyde in phosphate buffer at pH 7.3 with 0.25 M sucrose. Specimens were postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Spurr's medium (Spurr, 1969). Thin sections were cut with a diamond knife on a Sorvall 2 MT-2B ultramicrotome and stained with lead citrate and uranyl acetate. Sections were examined with an Elmiskop 1A operated at 80 kV. OBSERVATIONS
Although chronically diseased larvae used in this study were over 60 days old, many of their hemocytes had signs of early or mixed stages of infection. We assumed, therefore, that new populations of susceptible hemocytes were continually produced by hemopoietic organs during larval development (Hoffmann et al., 1980). In our culture, the newly formed hemocytes were subject to viral invasion because the larvae were maintained on contaminated diet; moreover, autoinfection of new hemocytes could have resulted from virions released from associated lysed, infected cells. The earliest forms recognized in the morphogenesis of viroplasmic bodies were intracytoplasmic, membrane-bound struct u r e s that c o n t a i n e d finely granular, electron-dense viroplasms (Figs. 1 and 2). Isolated and scattered viral particles sometimes occurred in the matrix material (Fig. 1). Irregular, electron-lucent patches appeared in the viroplasms as developing virions coalesced to form aggregations and paracrystalline arrays (Fig. 2). Presumed mature viroplasmic bodies contained scattered, dense masses of granular viroplasms that were often closely associated with large accumulations of virions and internal multilayered membranous structures (Figs. 1-3). Early and mature forms of viroplasmic bodies frequently occurred together (Figs. 1 and 2). Multilayered membranes appeared to en2 Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by USDA and does not imply approval to the exclusion of other products that may also be suitable.
159
velop and segregate viral aggregations in mature viroplasmic bodies (Fig. 3). However, the internal membranous structures disintegrated in older viroplasmic bodies, thus releasing free virions from the aggregations. Also, many amorphic, vesicular bodies formed between masses of virions; these structures later appeared to form autophagic elements that assisted in the destruction of internal viroplasmic membranes, thereby leading to the final release of virions to the cytoplasmic matrix of the host cell. DISCUSSION
We interpreted the different forms of membrane-bound structures within infected hemocytes of A. transitella as being stages in the morphogenesis of viroplasmic bodies associated with assembly of CSV. Many of the bodies had complex, internal, membranous elements that appeared similar to those reported from other insects infected with small, isometric viruses. For example, there have been several reports on viroplasmic membranes associated with the replicative cycle of wound tumor virus (WTV), a reovirus in the leafhopper vectors, Agalliopsis novella and Agallia constricta. Granados et al. (1967) noted that loose aggregations or small arrays of WTV were located in electron-dense, lysosomelike inclusion bodies in the cytoplasm of the gut epithelial cells of A. novella. Clusters of virions also occurred in defined bodies and in tubule-like structures in fat cells. Similarly, the cytoplasm of A. constricta hemocytes was reported to be sometimes entirely replaced by WTV viroplasms, microcrystals, and associated tubular structures. These relationships are similar to those of CSV in A . transitella hemocytes, except that the extensive development of tubular formations and structures, as reported by Hirumi et al. (1967), was not observed. Shikata and Maramorosch (1967) reported that particles of WTV in A. constricta fatbody tissue were engulfed within multi-
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membranous structures and that small accumulations of virions occurred in electrondense structures surrounded by double membranes. Myelin-like layers were observed at the periphery of dense viral aggregations; however, there were no membranes around typical viroplasmic matrices. Membranes appeared to have surrounded viral microcrystals while forming. Our observations indicate that CSV assembly and storage probably occur within a common membrane-bound viroplasmic body. Viroplasms initially form in the structure; later, accumulations of viral particles become surrounded by secondary, multilayered membranous envelopes. A 37- to 40-nm virus-like particle reported from hemocytes of the bean leaf beetle, Cerotoma trifurcata, formed crystalline arrays in association with granular and laminated structures (Kim and Scott, 1978). Viral particles occurred in the ground cytoplasm and in cytoplasmic vacuoles (viroplasmic bodies). Some of the reported granular matrices (viroplasms) were appressed to the vacuolar membranes. In contrast, a 70- to 75-nm particle in hemocytes of the spotted cucumber beetle, Diabrotica undecimpunctata, was associated with large accumulations of membranous vesicles in the perinuclear space and cisternae of the endoplasmic reticulum (Kim, 1980). Finally, Steiger et al. (1969) reported that
arrays of virus-like particles were embedded in membrane-bound, "lysosome-like bodies" in the cytoplasm of glial cells of the ant, Formica lugubris. The synthesis of both viral protein and viral RNA has been shown to be associated with cytoplasmic, membranous structures (Caliguiri and Tamm, 1969, 1970; Girard et al., 1967; Sreevalsan, 1970). We assume that the structures associated with CSV replication and assembly in A. transitella serve a similar function. Thus, the proliferation of intracytoplasmic membranes has been commonly associated with response to infection by small R N A viruses. In many respects CSV viroplasmic bodies and related membranes appear as if they may have been derived from an initial cellular defense mechanism to isolate and contain loci of viral invasion. Nuclear changes have been among the earliest cytopathic effects observed in calicivirus infections. Hackett (1961), reporting on cellular changes caused by vesicular exanthema of swine virus (VESV), noted an accumulation of chromatin threads and granules in nuclei and an increase in the size and staining intensity of nucleoli of infected pig kidney tissue culture cells. Also, Love and Sabine (1975) observed changes involving all nuclear components of primary kitten kidney cell cultures infected with feline calicivirus (FCV). In contrast,
FIG. 1. Portion of the cytoplasm of an infected granular hemocyte of Amyelois transitella showing several associated viroplasmic bodies (VPB). The bodies at top and lower left are bounded by single membranes (open arrows) that enclose finely granular, electron-dense viroplasms (VP). Two bodies at center and right of figure are bounded by multiple membranes (arrows) and contain highly ordered arrays of mature virions (V); center VPB has an irregular arrangement of internal membranous structures. Unassociated developing viral particles are also present (arrowheads). G, hemocyte granules; M, mitochondrion; R, ribosomes. Scale bar = 0.5/zm. FIG. 2. Section through a portion of the cytoplasm of an infected granular hemocyte showing four viroplasmic bodies (VPB) bounded by membranous envelopes (arrows). Electron-dense granular viroplasms (VP) are associated with adjacent lucent areas and aggregations of viral particles (V); lower body contains portion of a viral microcrystal. Several unassociated viral particles (arrowheads) are present in the lucent area of the upper body. M, mitochondrion; R, ribosomes; RER, rough endoplasmic reticulum. Scale bar = 0.5/~m. FXG. 3. Section showing portion of a viroplasmic body surrounded by a single membrane and bordered by a~acent hemocyte cytoplasm (broad arrows). Aggregations of virus (V) and viroplasms (VP) bounded by internal, multilayered membranous structures (narrow arrows) are evident throughout the body. A zone of tightly stacked membranes (arrowhead) is closely associated with viroplasm. Loosely associated developing virus (open arrow) and a scattering of ribosomes (R) are also present. Scale bar = 0.5/zm.
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES
161
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HOFFMANN AND KELLEN
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES
163
164
HOFFMANN AND K E L L E N
nuclei of A . transitella cells infected in vivo with CSV showed no effects in any of the stages examined. REFERENCES BURROUGHS, J. N., DOEL, T. R., SMALE, C. J., AND BROWN, F. (1978)J. Gen. Virol. 40, 161-174. CALIGUIm, L. A., AND TAMM, I. (1969) Science 166, 885-886. CALIOUIRI, L. A., AND TAMM, I. (1970) Virology 42, 100-111. GIRARD, M., BALTIMORE, D., AND DARNELL, J. E. (1967) J. Mol. Biol. 24, 59-74. GRANADOS, R. R., HIRUMI, H., AND MARAMOROSCH, K. (1967) J. Invertebr. Pathol. 9, 147-159. HACKETT, A. J. (1961) Virology 15, 102-112. HENRY, J. E., AND OMA, E. A. (1973) J. Invertebr. Pathol. 21, 273-281. HILLMAN, B., MORRIS, T. J., KELLEN, W. R., HOFFMANN, D. F., AND SCHLEGEE, D. E. (1981) J. Gen. Virol., in press.
HIRUMI, H., GRANADOS, R. R., AND MARAMOROSCH, K. (1967) J. Virol. 1, 430--444. HOFFMANN, J. A., ZACHARY, D., HOFFMANN, D., BREHELIN, M., AND PORTE, A. (1980) in GUPTA, A. P. (Ed.), Insect Hemocytes, pp. 29-66, Cambridge Univ. Press, London. KELLEN, W. R., AND HOFFMANN, D. F. (1981) J. Invertebr. Pathol. 38, 52-66. KIM, K. S. (1980) J. Invertebr. Pathol. 36, 292-301. KIM, K. S., AND SCOTT, H. A. (1978) J. Invertebr. Pathol. 31, 77-83. LOVE, D. N., AND SABINE, M. (1975) Arch. Virol. 48, 213-228. SCHAFFER, F. (1979) Compr. Virol. 14, 249-284. SHIKATA,E., AND MARAMOROSCH, K. (1967) J. Virol. 1, 1052-1073. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-43. SREEVALSAN, Z. (1970) J. Virol. 6, 438-444. STEIGER, U., LAMPARTER, H. E., SANDRI, C., AND AKERT, K. (1969)Arch. Ges. Virusforsch. 26, 271282.
79, 158-164 (1982)
An in Vivo Study of Intracytoplasmic Membranous Structures Associated with Chronic Stunt Virus Infection in G ran ular Hemocytes of Amyelois transitella 1 D A R L E N E F . H O F F M A N N AND W I L L I A M R . K E L L E N
Stored-Product Insects Research Laboratory, USDA, Agricultural Research Fresno, California 93727 Received May 5, 1981, and in revised form October 23, 1981 The fine structure of cytoplasmic, membranous structures associated with chronic stunt virus infection was studied in granular hemocytes of larvae of Amyelois transitella. Although nuclei of infected hemocytes apparently were not affected, viroplasmic bodies, the assumed sites of viral assembly, often filled the cytoplasm of these cells. Early stage viroplasmic bodies were bounded by single envelopes and contained finely granular, electron-dense viroplasms in which progeny virions formed aggregations or paracrystalline arrays, In mature viroplasmic bodies, viral aggregations and arrays were enveloped and segregated by multilayered membranous structures. Irregular, vesicular bodies occupied the electron-lucent areas between aggregations. Prior to cell lysis, membranous structures within viroplasmic bodies disintegrated in close association with secondary, membrane-bound bodies that were probably autophagic elements.
Chronic stunt virus (CSV) was originally described (Kellen and Hoffmann, 1981) from the cytoplasm of granular hemocytes of larvae of Amyelois transitella, a serious lepidopterous pest of almonds and walnuts in California. In laboratory tests, neonatal larvae usually succumbed to infection within a few days; however third- or fourth-instar larvae that initially acquired infection frequently developed chronic disease that caused retarded growth and delayed mortality. The virus is 38 nm in diameter, isometric, and contains single-stranded RNA. Hillman et al. (1981) reported the physicochemical characterization of CSV and tentatively assigned the pathogen to the caliciviruses. Caliciviruses have a distinctive morphology and several unique physicochemical characteristics which distinguish them from the other members of the Picornaviridae (Burroughs et al., 1978; Schaffer, 1979). CSV is the first calicivirus reported from an arthropod. In our initial report (Kellen and Hoff-
mann, 1981) on the host-pathogen relationships of this disease, we noted that aggregations and paracrystalline arrays of viral particles were usually enclosed in complex, membranous structures. These cytolysome-like viroplasmic bodies o c c u r r e d throughout the cytoplasm and contained internal multilaminar membranous systems, vesicles, and areas of finely granular, electron-dense viroplasm. Viroplasmic bodies were the assumed sites of viral assembly and storage. The purpose of this study is to provide additional information on the ultrastructure of membranous structures associated with the viroplasmic bodies in the cytoplasm of infected hemocytes of A. transitella.
1 Lepidoptera: Pyralidae. 158 0022-5320/82/050158-07502.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS
Host. Infected larvae used in this study were from a diseased laboratory culture that was routinely reared on a bran diet contaminated with semipurified virus. Methods for preparing viral suspensions and maintaining diseased cultures were reported previously (Kellen and Hoffmann, 1981). Electron microscopy. Chronically infected larvae used for electron microscopy were between 60 and 85 days old. Infected granular hemocytes reported here were examined in situ, usually in the larval head cap-
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES sule. Small pieces of larval, tissue, including intact head capsules, were dissected in cold (4°C) 2.5% glutaraldehyde in phosphate buffer at pH 7.3 with 0.25 M sucrose. Specimens were postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in Spurr's medium (Spurr, 1969). Thin sections were cut with a diamond knife on a Sorvall 2 MT-2B ultramicrotome and stained with lead citrate and uranyl acetate. Sections were examined with an Elmiskop 1A operated at 80 kV. OBSERVATIONS
Although chronically diseased larvae used in this study were over 60 days old, many of their hemocytes had signs of early or mixed stages of infection. We assumed, therefore, that new populations of susceptible hemocytes were continually produced by hemopoietic organs during larval development (Hoffmann et al., 1980). In our culture, the newly formed hemocytes were subject to viral invasion because the larvae were maintained on contaminated diet; moreover, autoinfection of new hemocytes could have resulted from virions released from associated lysed, infected cells. The earliest forms recognized in the morphogenesis of viroplasmic bodies were intracytoplasmic, membrane-bound struct u r e s that c o n t a i n e d finely granular, electron-dense viroplasms (Figs. 1 and 2). Isolated and scattered viral particles sometimes occurred in the matrix material (Fig. 1). Irregular, electron-lucent patches appeared in the viroplasms as developing virions coalesced to form aggregations and paracrystalline arrays (Fig. 2). Presumed mature viroplasmic bodies contained scattered, dense masses of granular viroplasms that were often closely associated with large accumulations of virions and internal multilayered membranous structures (Figs. 1-3). Early and mature forms of viroplasmic bodies frequently occurred together (Figs. 1 and 2). Multilayered membranes appeared to en2 Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by USDA and does not imply approval to the exclusion of other products that may also be suitable.
159
velop and segregate viral aggregations in mature viroplasmic bodies (Fig. 3). However, the internal membranous structures disintegrated in older viroplasmic bodies, thus releasing free virions from the aggregations. Also, many amorphic, vesicular bodies formed between masses of virions; these structures later appeared to form autophagic elements that assisted in the destruction of internal viroplasmic membranes, thereby leading to the final release of virions to the cytoplasmic matrix of the host cell. DISCUSSION
We interpreted the different forms of membrane-bound structures within infected hemocytes of A. transitella as being stages in the morphogenesis of viroplasmic bodies associated with assembly of CSV. Many of the bodies had complex, internal, membranous elements that appeared similar to those reported from other insects infected with small, isometric viruses. For example, there have been several reports on viroplasmic membranes associated with the replicative cycle of wound tumor virus (WTV), a reovirus in the leafhopper vectors, Agalliopsis novella and Agallia constricta. Granados et al. (1967) noted that loose aggregations or small arrays of WTV were located in electron-dense, lysosomelike inclusion bodies in the cytoplasm of the gut epithelial cells of A. novella. Clusters of virions also occurred in defined bodies and in tubule-like structures in fat cells. Similarly, the cytoplasm of A. constricta hemocytes was reported to be sometimes entirely replaced by WTV viroplasms, microcrystals, and associated tubular structures. These relationships are similar to those of CSV in A . transitella hemocytes, except that the extensive development of tubular formations and structures, as reported by Hirumi et al. (1967), was not observed. Shikata and Maramorosch (1967) reported that particles of WTV in A. constricta fatbody tissue were engulfed within multi-
160
HOFFMANN AND K E L L E N
membranous structures and that small accumulations of virions occurred in electrondense structures surrounded by double membranes. Myelin-like layers were observed at the periphery of dense viral aggregations; however, there were no membranes around typical viroplasmic matrices. Membranes appeared to have surrounded viral microcrystals while forming. Our observations indicate that CSV assembly and storage probably occur within a common membrane-bound viroplasmic body. Viroplasms initially form in the structure; later, accumulations of viral particles become surrounded by secondary, multilayered membranous envelopes. A 37- to 40-nm virus-like particle reported from hemocytes of the bean leaf beetle, Cerotoma trifurcata, formed crystalline arrays in association with granular and laminated structures (Kim and Scott, 1978). Viral particles occurred in the ground cytoplasm and in cytoplasmic vacuoles (viroplasmic bodies). Some of the reported granular matrices (viroplasms) were appressed to the vacuolar membranes. In contrast, a 70- to 75-nm particle in hemocytes of the spotted cucumber beetle, Diabrotica undecimpunctata, was associated with large accumulations of membranous vesicles in the perinuclear space and cisternae of the endoplasmic reticulum (Kim, 1980). Finally, Steiger et al. (1969) reported that
arrays of virus-like particles were embedded in membrane-bound, "lysosome-like bodies" in the cytoplasm of glial cells of the ant, Formica lugubris. The synthesis of both viral protein and viral RNA has been shown to be associated with cytoplasmic, membranous structures (Caliguiri and Tamm, 1969, 1970; Girard et al., 1967; Sreevalsan, 1970). We assume that the structures associated with CSV replication and assembly in A. transitella serve a similar function. Thus, the proliferation of intracytoplasmic membranes has been commonly associated with response to infection by small R N A viruses. In many respects CSV viroplasmic bodies and related membranes appear as if they may have been derived from an initial cellular defense mechanism to isolate and contain loci of viral invasion. Nuclear changes have been among the earliest cytopathic effects observed in calicivirus infections. Hackett (1961), reporting on cellular changes caused by vesicular exanthema of swine virus (VESV), noted an accumulation of chromatin threads and granules in nuclei and an increase in the size and staining intensity of nucleoli of infected pig kidney tissue culture cells. Also, Love and Sabine (1975) observed changes involving all nuclear components of primary kitten kidney cell cultures infected with feline calicivirus (FCV). In contrast,
FIG. 1. Portion of the cytoplasm of an infected granular hemocyte of Amyelois transitella showing several associated viroplasmic bodies (VPB). The bodies at top and lower left are bounded by single membranes (open arrows) that enclose finely granular, electron-dense viroplasms (VP). Two bodies at center and right of figure are bounded by multiple membranes (arrows) and contain highly ordered arrays of mature virions (V); center VPB has an irregular arrangement of internal membranous structures. Unassociated developing viral particles are also present (arrowheads). G, hemocyte granules; M, mitochondrion; R, ribosomes. Scale bar = 0.5/zm. FIG. 2. Section through a portion of the cytoplasm of an infected granular hemocyte showing four viroplasmic bodies (VPB) bounded by membranous envelopes (arrows). Electron-dense granular viroplasms (VP) are associated with adjacent lucent areas and aggregations of viral particles (V); lower body contains portion of a viral microcrystal. Several unassociated viral particles (arrowheads) are present in the lucent area of the upper body. M, mitochondrion; R, ribosomes; RER, rough endoplasmic reticulum. Scale bar = 0.5/~m. FXG. 3. Section showing portion of a viroplasmic body surrounded by a single membrane and bordered by a~acent hemocyte cytoplasm (broad arrows). Aggregations of virus (V) and viroplasms (VP) bounded by internal, multilayered membranous structures (narrow arrows) are evident throughout the body. A zone of tightly stacked membranes (arrowhead) is closely associated with viroplasm. Loosely associated developing virus (open arrow) and a scattering of ribosomes (R) are also present. Scale bar = 0.5/zm.
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES
161
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HOFFMANN AND KELLEN
CHRONIC STUNT VIRUS MEMBRANOUS STRUCTURES
163
164
HOFFMANN AND K E L L E N
nuclei of A . transitella cells infected in vivo with CSV showed no effects in any of the stages examined. REFERENCES BURROUGHS, J. N., DOEL, T. R., SMALE, C. J., AND BROWN, F. (1978)J. Gen. Virol. 40, 161-174. CALIGUIm, L. A., AND TAMM, I. (1969) Science 166, 885-886. CALIOUIRI, L. A., AND TAMM, I. (1970) Virology 42, 100-111. GIRARD, M., BALTIMORE, D., AND DARNELL, J. E. (1967) J. Mol. Biol. 24, 59-74. GRANADOS, R. R., HIRUMI, H., AND MARAMOROSCH, K. (1967) J. Invertebr. Pathol. 9, 147-159. HACKETT, A. J. (1961) Virology 15, 102-112. HENRY, J. E., AND OMA, E. A. (1973) J. Invertebr. Pathol. 21, 273-281. HILLMAN, B., MORRIS, T. J., KELLEN, W. R., HOFFMANN, D. F., AND SCHLEGEE, D. E. (1981) J. Gen. Virol., in press.
HIRUMI, H., GRANADOS, R. R., AND MARAMOROSCH, K. (1967) J. Virol. 1, 430--444. HOFFMANN, J. A., ZACHARY, D., HOFFMANN, D., BREHELIN, M., AND PORTE, A. (1980) in GUPTA, A. P. (Ed.), Insect Hemocytes, pp. 29-66, Cambridge Univ. Press, London. KELLEN, W. R., AND HOFFMANN, D. F. (1981) J. Invertebr. Pathol. 38, 52-66. KIM, K. S. (1980) J. Invertebr. Pathol. 36, 292-301. KIM, K. S., AND SCOTT, H. A. (1978) J. Invertebr. Pathol. 31, 77-83. LOVE, D. N., AND SABINE, M. (1975) Arch. Virol. 48, 213-228. SCHAFFER, F. (1979) Compr. Virol. 14, 249-284. SHIKATA,E., AND MARAMOROSCH, K. (1967) J. Virol. 1, 1052-1073. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-43. SREEVALSAN, Z. (1970) J. Virol. 6, 438-444. STEIGER, U., LAMPARTER, H. E., SANDRI, C., AND AKERT, K. (1969)Arch. Ges. Virusforsch. 26, 271282.