- Email: [email protected]
Cytopathology of the soybean looper, Pseudoplusia includens, infected with the Pseudoplusia includens icosahedral virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
45,
16-23
(1985)
Cytopathology of the Soybean Infected with the Pseudoplusia Yu-CHAN
CHAO,
Looper, Pseudoplusia inchdens, in&dens lcosahedral Virus1~2~3
S. Y. YOUNG,
AND K. S. KIM
Departments of Entomology and Plant Pathology, University of Arkansas, Fayetteville. Arkansas 72701 Received February 10, 1984; accepted June 12. 1984 Ultrastructural responses of soybean looper cells of various tissues infected with Pseudoplusia in&dens icosahedral virus (PIIV), a newly characterized RNA virus [Y. C. Chao, H. A. Scott, and S. Y. Young (1983) /. Gen. Viral. 64, 1835-18381. were studied in situ. Most cells of fat body and epidermis consistently contained virus particles and associated cytopathic structures. Virus particles, corresponding to those of purified PIIV in morphology and size, always occurred in the cytoplasm either in membrane-bound virogenic stroma and/or freely in the ground cytoplasm. Virogenic stroma, which appeared to be distinct from those induced by other insect viruses, consisted of electron-dense matrix material, in which virus particles were embedded, and membranous vesicles, 70 or 80 nm in diameter, containing nucleic acid-like tibrils. Virus particles in virogenic stroma occurred as hexagonally arranged crystalline arrays made up primarily of homogeneously dense particles, while those in the ground cytoplasm were dispersed randomly and had an electron-lucent central core. Extremely large numbers of virus particles were also located in noncellular cuticle layers of the integument. $0 IY85 Academic Prer>. Inc. KEY WORDS: Pseudoplusia inchdens: P. includens icosahedral virus; cytopathology: electron microscopy.
other larvae that appear healthy, and the infection does not cause pronounced external symptoms (Chao et al., 1983). Thus, the pathogenesis and cytopathic effects induced by this virus are of great interest. Cytopathology induced by morphologically similar viruses of insects, Nl3V group, has not been described. The occurrence of virus-like particles in various forms and associated cytopathic changes observed in cells of various tissues of soybean looper infected with PIIV are described here.
INTRODUCTION Pseudoplusia in&dens icosahedral virus (PIIV) is a newly characterized RNA virus with an average diameter of 40 nm (Chao et al., 1983). Although some physicochemical similarities exist between PIIV and insect viruses in the Nudaurelia capensis p virus (NPV) group (Greenwood and Moore, 1982; Juckes, 1970; Reinganum et al., 1978), such as similar particle morphology and size, they are unrelated serologically, and the molecular weight of the polypeptides of these viruses are significantly different (Chao et al., 1983). PIIV is an entity isolated from soybean looper by injecting larvae with extract from
MATERIALS
Soybean looper larvae used in this study were from a laboratory culture of P. inchderzs originally collected from Rapides Parish, Louisiana, and were reared on a semisynthetic diet (Burton, 1969). Fourth-stage larvae were inoculated by injecting 1.0 ~1 of a suspension containing 400-500 pg/ml PIIV into the hemocoel (Chao et al., 1983). Seven days after inoculation, larvae were
t Published with the approval of the Director, Arkansas Agriculture Experiment Station. ? Use of a trade name does not imply endorsement or guarantee of the product or the exclusion of other products of similar nature. 3 This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement 82 CRSR-2-1000.
16 0022-201 l/85 $1.50 Copyright All tights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
AND METHODS
CYTOPATHOLOGY
fixed by injecting modified Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0) into the hemocoel using a 25-gauge needle. After injection, the larvae were dissected in a Petri dish containing the same fixative. Hemocytes were collected by cutting an anal proleg and bleeding directly into a test tube containing fixative. After 2 hr of fixation, the test tube was centrifuged at 20,000 g for 1 hr. The supernatant was discarded and the pellet, composed mainly of hemocytes, was treated as other tissues. The different tissues dissected, such as integument, midgut, brain, fat body, and hemocytes, were transferred into separate vials containing fresh fixation and held for approximately 2 hr. After washing with buffer solution, the tissues were postfixed in 1% 0~0, in the same buffer for 2 hr, and bulk stained in 0.5% aqueous uranyl acetate overnight at 4°C. Tissues were then dehydrated in an ethanol series and propylene oxide before embedding in Spurr’s low-viscosity medium @purr, 1969). A total of five infected larvae were dissected and prepared for electron microscopical observation, and two uninoculated larvae, prepared identically, were used as the control. Thin sections cut with a glass knife were double stained with 2% aqueous uranyl acetate and lead citrate before examining in a JEOL 100 CX electron microscope. RESULTS
The particles and/or associated cytopathic changes described in this paper were absent in control cells, but were consistently observed in most tissues from all infected larvae studied except in midgut epithelium and nervous tissues. Based on the degree and frequency of occurrence of cytopathic changes, the most susceptible tissues appeared to be the fat body, epidermis, and tracheal epithelial cells. Some cytopathic changes were observed in visceral muscle and hemocytes. The following detailed description of cytopathic effects is
OF
PIIV
INFECTION
17
representative of those observed primarily in fat body and epidermis of integument unless otherwise stated. The particles were isometric spheres of 35-40 nm in diameter, which corresponded to those of purified PIIV (Chao et al., 1983). These particles, assumed to be PIIV, were always cytoplasmic regardless of the type of tissue, and occurred either in large membrane-bound virogenic stroma or freely in the ground cytoplasm (Figs. 1, 2). Virogenie stroma consisted of electron-dense ground substances, forming the matrix of stroma in which virus particles were embedded, and membranous vesicles, 70-80 nm in diameter, containing fibrils which were often aligned along the inside of the bounding membrane (Figs. 1, 2, 3). The vesicle membranes were often continuous with the bounding membranes of the stroma, indicating that they are formed by the invaginations of the stroma membrane (Fig. 2). Virus particles in infected cells were of two morphological types: one with an electron-lucent central cavity surrounded by an electron-dense wall (lucent particles, hereafter), and the other with solid electrondense spheres (dense particles, hereafter) (Figs. 2, 3). Virus particles in virogenic stroma occurred usually as hexagonally arranged crystalline arrays, primarily of dense particles (Figs. 2, 3). Although doughnut-shaped, lucent particles also occurred in the stroma, their arrangement was less well-defined than that of the dense particles (Fig. 2). These lucent particles, which appeared slightly larger in size than dense particles, were apparently released into the ground cytoplasm through disrupted areas of bounding membrane of virogenic stroma (Fig. 3). Virions in the ground cytoplasm were, therefore, predominantly lucent particles, and were randomly dispersed singly or as irregular aggregates of various forms and sizes (Fig. 3). Three cytopathic features in cells depicted possible different stages of infection. The first feature was the appearance of
18
CHAO,
YOUNG,
AND
KIM
FIG. 1. Low magnification of a section through the cytoplasm of a fat body cell exhibiting virus particles (VP) in membrane-bound virogenic stroma (A,B) and in the ground cytoplasm. L. lipid globule; M, mitochondria; Bar, 1000 nm. FIG. 2. A higher magnification of the virogenic stroma A from Fig. 1 showing the structural details. Two types of virus particles, dense (Dp) and lucent (Lp), are apparent. The dense particles, which occurred as hexagonally arranged crystalline arrays, are embedded in electron-dense matrix substance. The lucent particles. which appear somewhat larger and less orderly arranged than dense particles, are morphologically identical to those (Lp) in the ground cytoplasm. Fibril-containing membranous vesicles (Fv) aligned along the inside of the bounding membrane are also shown. Bar. 500 nm. FIG. 3. A higher magnification of the virogenic stroma B from Fig. 1 exhibiting compartmentalized “lobes” with different morphology. The right lobe (1) contains crystallized dense particles embedded in electron-dense matrix, while the left lobe (2) contains only a few randomly distributed lucent particles (small arrowheads) in light matrix. An area of disrupted bounding membrane (between large arrowheads), through which a group of virus particles is apparently released (arrow). is also shown. Bar. 500 nm.
CYTOPATHOLOGY
membranous cavities enclosing fibril-containing vesicles that were structurally identical to those associated with virogenic stroma (Fig. 2). The vesicles were usually aligned along the inner surface of the cavity membrane, which was often continuous with them (Fig. 4). The second feature was the appearance of virogenic stroma and virus particles dispersed in the ground cytoplasm (Fig. 1). The third was extremely large amounts of virus particles which occupied most areas of the cytoplasm (Figs. 5, 6). Some of these particles in the cytoplasm were often arranged into nonmembranous crystalline arrays (Fig. 5). Although the nuclei of some such cells were often completely surrounded by a pool of virus particles, they never occurred in the nucleoplasm (Fig. 5). In some cells, especially those of epidermal cells of the integument and hemocytes, virus particles were often observed in membranous bodies which appeared to be lysosomes (Fig. 7). Those bodies could easily be distinguished from virogenic stroma because they did not exhibit the electron-dense matrix and the virus particles were not arranged in a crystalline array (Figs. 2, 3). In addition, the bodies often contained loose concentric membranes exhibiting myelinic figures, which are common structures of insect lysosomes (Moussa, 1978; Kim, 1981; Kitajima and Gamez, 1983). High concentrations of virus particles were also observed in the cuticle layers of the integument (Fig. 8). The particles were distributed evenly throughout the cuticle, which consisted of multilayers of lamellae (Fig. 8). Although some particles were aligned in rows embedded in the matrix of chitin-protein microlibrils of the lamellae (Smith, 1968), no crystallized particles were observed (Fig. 8). Epidermal cells underneath the virus-containing cuticle were always filled with cytoplasmic particles, suggesting that the particles in the cuticle were derived from infected epidermal cells (Fig. 8).
OF
PIIV
19
INFECTION
Tracheal elements were scattered throughout the larvae and appeared in most tissue sections. The tracheal epithelial cells which surrounded the tracheal elements were usually heavily infected. Large areas of the cytoplasm were often replaced by highly concentrated masses of virus particles (Fig. 6). The basal laminae bordering these tracheal epithelial cells and adjacent tissues were also penetrated by virus particles. DISCUSSION
The consistent presence of virus-like particles comparable in morphology and size to those of purified PIIV (Chao et al., 1983), and the absence of such particles in uninoculated control tissue suggests that the particles observed are indeed the infectious entity of PIIV replicated in infected cells. This suggestion is further strengthened by the fact that the particles were associated with the structure that appeared to be virogenic stroma, which have been known in many cases to be sites of virus replication and/or assembly (Maramorosch, 1977; Matthews, 1982). The most striking feature of the virogenic stroma, in addition to virus particles, was the fibril-containing vesicles which were formed by invaginations of bounding membrane of the stroma. Although some variations occurred, vesicles similar to those observed in this study have been reported in cells infected with other insect viruses (Granados, 1973; Kim, 1980), other animal viruses (Dalton and Haguenau, 1973), and plant viruses (DeZoeten et al., 1972; Esau and Hoefert, 1972; Kim, 1977). There is also evidence that the fibrils in the vesicles contain nucleic acids which are involved in virus replication (DeZoeten et al., 1972; Matthews, 1973), which suggests that such vesicles are induced by virus infection and are involved in the processes of virus biogenesis. The membranes of fibril-containing vesicles of this study were clearly formed by invaginations of the bounding membrane of virogenic stroma. These virus-induced ves-
20
CHAO.
YOUNG,
AND
KIM
FIG. 4. Fib&containing vesicles (Fv), aligned along the inner surface of membranous cavities (MC). in the cytoplasm of an epidermal cell. The membrane of some vesicles is continuous with that of the cavities (unlabeled arrows). These vesicles are structurally identical to those associated virus-containing virogenic stroma shown in Fig. 2. Bar. 500 nm. FIG. 5. A heavily infected epidermal cell with a large portion of the cytoplasm filled with virus particles (VP.). No virus particles, however, appear in the nucleoplasm (NJ. Nm, nuclear membrane: bar, 500 nm. Inset: Crystalline arrays of virus particles in the cytoplasm without a bounding membrane. Bar, 500 nm.
icles have been demonstrated to be derived from the membranes of various cell organelles such as rough endoplasmic reticulum (Esau and Hoefert, 1972), nuclear envelope (DeZoeten et al., 1972), both endoplasmic reticulum and nuclear envelope (Kim, 1977,
1980), chloroplast (Matthews, 1973), and mitochondria (Garzon et al., 1978; Hatta and Ushiyama, 1973; Hatta et al., 1983; Russo and Martelli, 1982). It seems, depending on virus-host combinations, that any given membranous cell organelle has
CYTOPATHOLOGY
OF PIIV INFECTION
21
FIG. 6. A tracheal epithelial cell enclosing four tracheal elements (T). Large portion of the cytoplasm is tilled with virus particles (VP). Bar, 500 nm. FIG. 7. Virus particles enclosed in lysosomes. Multilayered, loose concentric membranes (arrowhead) are apparent. M, mitochondria; bar, 500 nm. FIG. 8. High concentrations of virus particles occurring throughout the cuticle layers of the integument. Some particles are aligned in rows (arrowheads) embedded in the microfibrils of the cuticle lamellae, but no crystallized particles occur. The cytoplasm of an epidermal cell (Ep) adjacent to the virus-containing cuticle filled with virus particles (VP) is also shown. Bar, 500 nm.
the capability to increase or produce such membranes in response to virus infection. It is of interest to note that vesicles structurally indistinguishable to those observed in this study have been demonstrated to originate from rough endoplasmic reticulum and nuclear envelope in insect cells (Coleoptera) infected with a reovirus (Kim, 1980).
The occurrence of two types of particles, electron-lucent and -dense, centered in virogenic stroma indicates morphogenesis of virus particles. Apparently, the dense particles, which were mostly in hexagonal arrays embedded in the ground matrix material of virogenic stroma, gradually became loosened from the arrays and eventually were released into the ground cytoplasm.
22
CHAO.
YOUNG,
In these processes the particles became slightly larger or swollen due, perhaps, to the loss of tightness of the crystalline arrangement, resulting in the appearance of lucent particles. This explanation is supported by the fact that the majority of loose particles in the ground cytoplasm was the lucent particles. In addition, particles in cuticle layers, which are extracellular and therefore must be derived from an intracellular replication site, are all loose and lucent particles. It has been reported that some other viruses of insects (Murphy et al., 1970) and plants (Kim, 1977) having isometric morphology also appear to have an electron-lucent center when examined in situ. When infected cells are overpopulated with released particles, the virus particles apparently become crystallized (Fig. 5). as in the cases of other isometric insect viruses (Garzon et al., 1978; Murphy et al., 1970). These crystallized particles are. however, unlike those in virogenic stroma, not membrane bound or embedded in the ground material. The presence of masses of virus particles dispersed throughout the cuticular layers of the integument is of interest because such have rarely been reported in infections by other insect viruses. The cuticle is noncellular; therefore, the particles in it must have been transported from nearby cells where they were replicated. Other than the presence of virus particles, many of the epidermal cells exhibited no sign of degeneration or lysis. It is conceivable that the cell membranes of epidermal cells become disrupted due to heavy infection, and masses of virus particles were released into the cuticle through disrupted membranes. In this process, some cuticle-digesting enzymes, such as protease and chitinase, known to be synthesized in epidermal cells (NobleNesbitt, 1963; Locke, 1966), could also be released and act on the cuticular microfibrils, making the passage of virus particles easier. Alternatively, it is also possible that released virus particles were incorporated with the cuticle precursor material and, when new layers of cuticle formed virus
AND KIM
particles became embedded in them. A similar suggestion was made by Mussen and Furgala (1977), who observed sacbrood virus particles in the cuticle of honey bee larvae infected with sacbrood virus. Those particles, however, occurred as random clusters in severely damaged cuticle, while the PIIV particles were evenly distributed throughout seemingly undamaged cuticle layers. REFERENCES BURTON, R. L. 1969. “Mass Rearing of the Corn Earworm in the Laboratory.” U.S. Dept. Agr., Agr. Res. Serv. 33-134, Washington, D.C. CHAO, Y. C., SCOTT, H. A., AND YOUNG. S. Y. III. 1983. An icosahedral RNA virus of the soybean looper (Pseudoplusia in&dens). J. Gen. Viral.. 64, 1835-1838. DALTON, A. J., AND HAGUENAU, F. 1973. “Ultrastructure of Animal Viruses and Bacteriophages”. Academic Press. New York. DEZOETEN, G. A., GAARD, G.. AND DIEZ. E B. 1972. Nuclear vesiculation associated with pea enation mosaic virus-infected plant tissue. Virology. 48, 638-647.
ESAU, K., AND HOEFERT, L. L. 1972. Development of infection with beet western yellows virus in the sugarbeet. Virology, 48, 724-738. GARZON. S.. CHARPENTIER. G.. AND KUKSTAK, E. 1978. Morphogenesis of the Nodamura virus in the larvae of the Lepidoptera Galleria me//one//a CL.). Arch. Viral., 56, 61-76. GRANADOS, R. R. 1973. Insect poxviruses: Pathology, morphology, and development. Misc. P/r&l. Entomol.
Sot.
Amer.,
9, 73-94.
GREENWOOD,L. K., AND MOORE, N. E 1982. The NLCdaurelia group of small RNA-containing viruses of insects: Serological identification of several new isolates. J. Invertehr. Pathol.. 39, 407-409. HATTA, T., FRANCKI. R. I. B.. AND GRIVELL. C. J. 1983. Particle morphology and cytopathology of galisoya mosaic virus. J. Gen. Viral.. 64, 687-692. HATTA. T., AND VSHIYAMA. R. 1973. Mitochondrial vesiculation associated with cucumber green mottle mosiac virus-infected plants. J. Gen. Viral., 21, 9-17.
JUCKES. I. R. M. 1970. Viruses of the pine emperor moth. Bull. S. Afr. Sot. Plant Pathol. Microbial., 4, 18. KIM, K. S. 1977. An ultrastructural study of inclusions and disease development in plant cells infected by cowpea chlorotic mottle virus. J. Gen. Viral., 35, 535-543.
KIM. K. S. 1980. Cytopathology of spotted cucumber beetle hemocyte containing virus-like particles. J. Im,ertebr. Pathol.. 36, 292-301.
CYTOPATHOLOGY KIM, K. S. (Ceratoma
1981. Ultrastructure of bean leaf beetle rrifurcata) hemocytes and their phagocytic activities on tobacco mosaic virus, a nonbeetle-transmitted virus. J. Ultrastruct. Res., 75, 300-313. KITAJIMA, E. W., AND GAMEZ, R. 1983. Electron microscopy of maize rayado fino virus in the internal organs of its leafhopper vector. Intervirology, 19, 129-134. LOCKE, M. 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect. Calpodes e&legs (Lepidoptera:Hesperiidae). J. Morphol., 118, 461-494. MARAMOROSCH, K. (ed.). 1977. “The Atlas of Insect and Plant Viruses.” Academic Press, New York. MATHEWS, R. E. E 1973. Induction of diseases by viruses with special reference to turnip yellow mosiac virus. Annu. Rev. Plant Pathol., 11, 147-170. MATTHEWS, R. E. F. 1982. Classification and nomenclature of virus. Intervirology, 17, l-200. MOUSSA, A. Y. 1978. A new cytoplasmic inclusion virus from Diptera in the Queensland fruitfly. Dams
23
OF PIIV INFECTION tryoni Pathol.,
(Frogg) (Diptera: Tephritidae).
J. Invertebr.
32, 77-87.
MURPHY, F. A., SCHERER, W. F., HARRISON. A. K.. DUNNE, H. W., AND GRAY, G. W., JR. 1970. Characterization of nodamura virus, an arthropod transmissible picornavirus. Virology, 40, 1008- 1021. MUSSEN, E. C., AND FURGALA, B. 1977. Replication of sacbrood virus in larval and adult honey bees, Apis
rnellifera.
J. Invertebr.
Pathol.,
30, 20-34.
NOBLE-NESBITT, J. 1963. The cuticle and associated structures of Podura aquatica at the moult. Quart. J. Microsc. Sci., 104, 369-391. REINGANUM, C., ROBERTSON, J. S., AND TINSLEY, T. W. 1978. A new group of RNA viruses from insects. J. Gen. Viral., 40, 195-202. Russo. M., AND MARTELLI, G. P. 1982. Ultrastructure of turnip crinkle- and saguaro cuctus virus-infected tissues. Virology, 118, 109- 116. SMITH, D. S. 1968. “Insect cells. Their Structure and Function.” Oliver and Boyd, Edinburgh. SPURR, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. UItrastruct. Res., 26, 31-43.
OF INVERTEBRATE
PATHOLOGY
45,
16-23
(1985)
Cytopathology of the Soybean Infected with the Pseudoplusia Yu-CHAN
CHAO,
Looper, Pseudoplusia inchdens, in&dens lcosahedral Virus1~2~3
S. Y. YOUNG,
AND K. S. KIM
Departments of Entomology and Plant Pathology, University of Arkansas, Fayetteville. Arkansas 72701 Received February 10, 1984; accepted June 12. 1984 Ultrastructural responses of soybean looper cells of various tissues infected with Pseudoplusia in&dens icosahedral virus (PIIV), a newly characterized RNA virus [Y. C. Chao, H. A. Scott, and S. Y. Young (1983) /. Gen. Viral. 64, 1835-18381. were studied in situ. Most cells of fat body and epidermis consistently contained virus particles and associated cytopathic structures. Virus particles, corresponding to those of purified PIIV in morphology and size, always occurred in the cytoplasm either in membrane-bound virogenic stroma and/or freely in the ground cytoplasm. Virogenic stroma, which appeared to be distinct from those induced by other insect viruses, consisted of electron-dense matrix material, in which virus particles were embedded, and membranous vesicles, 70 or 80 nm in diameter, containing nucleic acid-like tibrils. Virus particles in virogenic stroma occurred as hexagonally arranged crystalline arrays made up primarily of homogeneously dense particles, while those in the ground cytoplasm were dispersed randomly and had an electron-lucent central core. Extremely large numbers of virus particles were also located in noncellular cuticle layers of the integument. $0 IY85 Academic Prer>. Inc. KEY WORDS: Pseudoplusia inchdens: P. includens icosahedral virus; cytopathology: electron microscopy.
other larvae that appear healthy, and the infection does not cause pronounced external symptoms (Chao et al., 1983). Thus, the pathogenesis and cytopathic effects induced by this virus are of great interest. Cytopathology induced by morphologically similar viruses of insects, Nl3V group, has not been described. The occurrence of virus-like particles in various forms and associated cytopathic changes observed in cells of various tissues of soybean looper infected with PIIV are described here.
INTRODUCTION Pseudoplusia in&dens icosahedral virus (PIIV) is a newly characterized RNA virus with an average diameter of 40 nm (Chao et al., 1983). Although some physicochemical similarities exist between PIIV and insect viruses in the Nudaurelia capensis p virus (NPV) group (Greenwood and Moore, 1982; Juckes, 1970; Reinganum et al., 1978), such as similar particle morphology and size, they are unrelated serologically, and the molecular weight of the polypeptides of these viruses are significantly different (Chao et al., 1983). PIIV is an entity isolated from soybean looper by injecting larvae with extract from
MATERIALS
Soybean looper larvae used in this study were from a laboratory culture of P. inchderzs originally collected from Rapides Parish, Louisiana, and were reared on a semisynthetic diet (Burton, 1969). Fourth-stage larvae were inoculated by injecting 1.0 ~1 of a suspension containing 400-500 pg/ml PIIV into the hemocoel (Chao et al., 1983). Seven days after inoculation, larvae were
t Published with the approval of the Director, Arkansas Agriculture Experiment Station. ? Use of a trade name does not imply endorsement or guarantee of the product or the exclusion of other products of similar nature. 3 This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement 82 CRSR-2-1000.
16 0022-201 l/85 $1.50 Copyright All tights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
AND METHODS
CYTOPATHOLOGY
fixed by injecting modified Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0) into the hemocoel using a 25-gauge needle. After injection, the larvae were dissected in a Petri dish containing the same fixative. Hemocytes were collected by cutting an anal proleg and bleeding directly into a test tube containing fixative. After 2 hr of fixation, the test tube was centrifuged at 20,000 g for 1 hr. The supernatant was discarded and the pellet, composed mainly of hemocytes, was treated as other tissues. The different tissues dissected, such as integument, midgut, brain, fat body, and hemocytes, were transferred into separate vials containing fresh fixation and held for approximately 2 hr. After washing with buffer solution, the tissues were postfixed in 1% 0~0, in the same buffer for 2 hr, and bulk stained in 0.5% aqueous uranyl acetate overnight at 4°C. Tissues were then dehydrated in an ethanol series and propylene oxide before embedding in Spurr’s low-viscosity medium @purr, 1969). A total of five infected larvae were dissected and prepared for electron microscopical observation, and two uninoculated larvae, prepared identically, were used as the control. Thin sections cut with a glass knife were double stained with 2% aqueous uranyl acetate and lead citrate before examining in a JEOL 100 CX electron microscope. RESULTS
The particles and/or associated cytopathic changes described in this paper were absent in control cells, but were consistently observed in most tissues from all infected larvae studied except in midgut epithelium and nervous tissues. Based on the degree and frequency of occurrence of cytopathic changes, the most susceptible tissues appeared to be the fat body, epidermis, and tracheal epithelial cells. Some cytopathic changes were observed in visceral muscle and hemocytes. The following detailed description of cytopathic effects is
OF
PIIV
INFECTION
17
representative of those observed primarily in fat body and epidermis of integument unless otherwise stated. The particles were isometric spheres of 35-40 nm in diameter, which corresponded to those of purified PIIV (Chao et al., 1983). These particles, assumed to be PIIV, were always cytoplasmic regardless of the type of tissue, and occurred either in large membrane-bound virogenic stroma or freely in the ground cytoplasm (Figs. 1, 2). Virogenie stroma consisted of electron-dense ground substances, forming the matrix of stroma in which virus particles were embedded, and membranous vesicles, 70-80 nm in diameter, containing fibrils which were often aligned along the inside of the bounding membrane (Figs. 1, 2, 3). The vesicle membranes were often continuous with the bounding membranes of the stroma, indicating that they are formed by the invaginations of the stroma membrane (Fig. 2). Virus particles in infected cells were of two morphological types: one with an electron-lucent central cavity surrounded by an electron-dense wall (lucent particles, hereafter), and the other with solid electrondense spheres (dense particles, hereafter) (Figs. 2, 3). Virus particles in virogenic stroma occurred usually as hexagonally arranged crystalline arrays, primarily of dense particles (Figs. 2, 3). Although doughnut-shaped, lucent particles also occurred in the stroma, their arrangement was less well-defined than that of the dense particles (Fig. 2). These lucent particles, which appeared slightly larger in size than dense particles, were apparently released into the ground cytoplasm through disrupted areas of bounding membrane of virogenic stroma (Fig. 3). Virions in the ground cytoplasm were, therefore, predominantly lucent particles, and were randomly dispersed singly or as irregular aggregates of various forms and sizes (Fig. 3). Three cytopathic features in cells depicted possible different stages of infection. The first feature was the appearance of
18
CHAO,
YOUNG,
AND
KIM
FIG. 1. Low magnification of a section through the cytoplasm of a fat body cell exhibiting virus particles (VP) in membrane-bound virogenic stroma (A,B) and in the ground cytoplasm. L. lipid globule; M, mitochondria; Bar, 1000 nm. FIG. 2. A higher magnification of the virogenic stroma A from Fig. 1 showing the structural details. Two types of virus particles, dense (Dp) and lucent (Lp), are apparent. The dense particles, which occurred as hexagonally arranged crystalline arrays, are embedded in electron-dense matrix substance. The lucent particles. which appear somewhat larger and less orderly arranged than dense particles, are morphologically identical to those (Lp) in the ground cytoplasm. Fibril-containing membranous vesicles (Fv) aligned along the inside of the bounding membrane are also shown. Bar. 500 nm. FIG. 3. A higher magnification of the virogenic stroma B from Fig. 1 exhibiting compartmentalized “lobes” with different morphology. The right lobe (1) contains crystallized dense particles embedded in electron-dense matrix, while the left lobe (2) contains only a few randomly distributed lucent particles (small arrowheads) in light matrix. An area of disrupted bounding membrane (between large arrowheads), through which a group of virus particles is apparently released (arrow). is also shown. Bar. 500 nm.
CYTOPATHOLOGY
membranous cavities enclosing fibril-containing vesicles that were structurally identical to those associated with virogenic stroma (Fig. 2). The vesicles were usually aligned along the inner surface of the cavity membrane, which was often continuous with them (Fig. 4). The second feature was the appearance of virogenic stroma and virus particles dispersed in the ground cytoplasm (Fig. 1). The third was extremely large amounts of virus particles which occupied most areas of the cytoplasm (Figs. 5, 6). Some of these particles in the cytoplasm were often arranged into nonmembranous crystalline arrays (Fig. 5). Although the nuclei of some such cells were often completely surrounded by a pool of virus particles, they never occurred in the nucleoplasm (Fig. 5). In some cells, especially those of epidermal cells of the integument and hemocytes, virus particles were often observed in membranous bodies which appeared to be lysosomes (Fig. 7). Those bodies could easily be distinguished from virogenic stroma because they did not exhibit the electron-dense matrix and the virus particles were not arranged in a crystalline array (Figs. 2, 3). In addition, the bodies often contained loose concentric membranes exhibiting myelinic figures, which are common structures of insect lysosomes (Moussa, 1978; Kim, 1981; Kitajima and Gamez, 1983). High concentrations of virus particles were also observed in the cuticle layers of the integument (Fig. 8). The particles were distributed evenly throughout the cuticle, which consisted of multilayers of lamellae (Fig. 8). Although some particles were aligned in rows embedded in the matrix of chitin-protein microlibrils of the lamellae (Smith, 1968), no crystallized particles were observed (Fig. 8). Epidermal cells underneath the virus-containing cuticle were always filled with cytoplasmic particles, suggesting that the particles in the cuticle were derived from infected epidermal cells (Fig. 8).
OF
PIIV
19
INFECTION
Tracheal elements were scattered throughout the larvae and appeared in most tissue sections. The tracheal epithelial cells which surrounded the tracheal elements were usually heavily infected. Large areas of the cytoplasm were often replaced by highly concentrated masses of virus particles (Fig. 6). The basal laminae bordering these tracheal epithelial cells and adjacent tissues were also penetrated by virus particles. DISCUSSION
The consistent presence of virus-like particles comparable in morphology and size to those of purified PIIV (Chao et al., 1983), and the absence of such particles in uninoculated control tissue suggests that the particles observed are indeed the infectious entity of PIIV replicated in infected cells. This suggestion is further strengthened by the fact that the particles were associated with the structure that appeared to be virogenic stroma, which have been known in many cases to be sites of virus replication and/or assembly (Maramorosch, 1977; Matthews, 1982). The most striking feature of the virogenic stroma, in addition to virus particles, was the fibril-containing vesicles which were formed by invaginations of bounding membrane of the stroma. Although some variations occurred, vesicles similar to those observed in this study have been reported in cells infected with other insect viruses (Granados, 1973; Kim, 1980), other animal viruses (Dalton and Haguenau, 1973), and plant viruses (DeZoeten et al., 1972; Esau and Hoefert, 1972; Kim, 1977). There is also evidence that the fibrils in the vesicles contain nucleic acids which are involved in virus replication (DeZoeten et al., 1972; Matthews, 1973), which suggests that such vesicles are induced by virus infection and are involved in the processes of virus biogenesis. The membranes of fibril-containing vesicles of this study were clearly formed by invaginations of the bounding membrane of virogenic stroma. These virus-induced ves-
20
CHAO.
YOUNG,
AND
KIM
FIG. 4. Fib&containing vesicles (Fv), aligned along the inner surface of membranous cavities (MC). in the cytoplasm of an epidermal cell. The membrane of some vesicles is continuous with that of the cavities (unlabeled arrows). These vesicles are structurally identical to those associated virus-containing virogenic stroma shown in Fig. 2. Bar. 500 nm. FIG. 5. A heavily infected epidermal cell with a large portion of the cytoplasm filled with virus particles (VP.). No virus particles, however, appear in the nucleoplasm (NJ. Nm, nuclear membrane: bar, 500 nm. Inset: Crystalline arrays of virus particles in the cytoplasm without a bounding membrane. Bar, 500 nm.
icles have been demonstrated to be derived from the membranes of various cell organelles such as rough endoplasmic reticulum (Esau and Hoefert, 1972), nuclear envelope (DeZoeten et al., 1972), both endoplasmic reticulum and nuclear envelope (Kim, 1977,
1980), chloroplast (Matthews, 1973), and mitochondria (Garzon et al., 1978; Hatta and Ushiyama, 1973; Hatta et al., 1983; Russo and Martelli, 1982). It seems, depending on virus-host combinations, that any given membranous cell organelle has
CYTOPATHOLOGY
OF PIIV INFECTION
21
FIG. 6. A tracheal epithelial cell enclosing four tracheal elements (T). Large portion of the cytoplasm is tilled with virus particles (VP). Bar, 500 nm. FIG. 7. Virus particles enclosed in lysosomes. Multilayered, loose concentric membranes (arrowhead) are apparent. M, mitochondria; bar, 500 nm. FIG. 8. High concentrations of virus particles occurring throughout the cuticle layers of the integument. Some particles are aligned in rows (arrowheads) embedded in the microfibrils of the cuticle lamellae, but no crystallized particles occur. The cytoplasm of an epidermal cell (Ep) adjacent to the virus-containing cuticle filled with virus particles (VP) is also shown. Bar, 500 nm.
the capability to increase or produce such membranes in response to virus infection. It is of interest to note that vesicles structurally indistinguishable to those observed in this study have been demonstrated to originate from rough endoplasmic reticulum and nuclear envelope in insect cells (Coleoptera) infected with a reovirus (Kim, 1980).
The occurrence of two types of particles, electron-lucent and -dense, centered in virogenic stroma indicates morphogenesis of virus particles. Apparently, the dense particles, which were mostly in hexagonal arrays embedded in the ground matrix material of virogenic stroma, gradually became loosened from the arrays and eventually were released into the ground cytoplasm.
22
CHAO.
YOUNG,
In these processes the particles became slightly larger or swollen due, perhaps, to the loss of tightness of the crystalline arrangement, resulting in the appearance of lucent particles. This explanation is supported by the fact that the majority of loose particles in the ground cytoplasm was the lucent particles. In addition, particles in cuticle layers, which are extracellular and therefore must be derived from an intracellular replication site, are all loose and lucent particles. It has been reported that some other viruses of insects (Murphy et al., 1970) and plants (Kim, 1977) having isometric morphology also appear to have an electron-lucent center when examined in situ. When infected cells are overpopulated with released particles, the virus particles apparently become crystallized (Fig. 5). as in the cases of other isometric insect viruses (Garzon et al., 1978; Murphy et al., 1970). These crystallized particles are. however, unlike those in virogenic stroma, not membrane bound or embedded in the ground material. The presence of masses of virus particles dispersed throughout the cuticular layers of the integument is of interest because such have rarely been reported in infections by other insect viruses. The cuticle is noncellular; therefore, the particles in it must have been transported from nearby cells where they were replicated. Other than the presence of virus particles, many of the epidermal cells exhibited no sign of degeneration or lysis. It is conceivable that the cell membranes of epidermal cells become disrupted due to heavy infection, and masses of virus particles were released into the cuticle through disrupted membranes. In this process, some cuticle-digesting enzymes, such as protease and chitinase, known to be synthesized in epidermal cells (NobleNesbitt, 1963; Locke, 1966), could also be released and act on the cuticular microfibrils, making the passage of virus particles easier. Alternatively, it is also possible that released virus particles were incorporated with the cuticle precursor material and, when new layers of cuticle formed virus
AND KIM
particles became embedded in them. A similar suggestion was made by Mussen and Furgala (1977), who observed sacbrood virus particles in the cuticle of honey bee larvae infected with sacbrood virus. Those particles, however, occurred as random clusters in severely damaged cuticle, while the PIIV particles were evenly distributed throughout seemingly undamaged cuticle layers. REFERENCES BURTON, R. L. 1969. “Mass Rearing of the Corn Earworm in the Laboratory.” U.S. Dept. Agr., Agr. Res. Serv. 33-134, Washington, D.C. CHAO, Y. C., SCOTT, H. A., AND YOUNG. S. Y. III. 1983. An icosahedral RNA virus of the soybean looper (Pseudoplusia in&dens). J. Gen. Viral.. 64, 1835-1838. DALTON, A. J., AND HAGUENAU, F. 1973. “Ultrastructure of Animal Viruses and Bacteriophages”. Academic Press. New York. DEZOETEN, G. A., GAARD, G.. AND DIEZ. E B. 1972. Nuclear vesiculation associated with pea enation mosaic virus-infected plant tissue. Virology. 48, 638-647.
ESAU, K., AND HOEFERT, L. L. 1972. Development of infection with beet western yellows virus in the sugarbeet. Virology, 48, 724-738. GARZON. S.. CHARPENTIER. G.. AND KUKSTAK, E. 1978. Morphogenesis of the Nodamura virus in the larvae of the Lepidoptera Galleria me//one//a CL.). Arch. Viral., 56, 61-76. GRANADOS, R. R. 1973. Insect poxviruses: Pathology, morphology, and development. Misc. P/r&l. Entomol.
Sot.
Amer.,
9, 73-94.
GREENWOOD,L. K., AND MOORE, N. E 1982. The NLCdaurelia group of small RNA-containing viruses of insects: Serological identification of several new isolates. J. Invertehr. Pathol.. 39, 407-409. HATTA, T., FRANCKI. R. I. B.. AND GRIVELL. C. J. 1983. Particle morphology and cytopathology of galisoya mosaic virus. J. Gen. Viral.. 64, 687-692. HATTA. T., AND VSHIYAMA. R. 1973. Mitochondrial vesiculation associated with cucumber green mottle mosiac virus-infected plants. J. Gen. Viral., 21, 9-17.
JUCKES. I. R. M. 1970. Viruses of the pine emperor moth. Bull. S. Afr. Sot. Plant Pathol. Microbial., 4, 18. KIM, K. S. 1977. An ultrastructural study of inclusions and disease development in plant cells infected by cowpea chlorotic mottle virus. J. Gen. Viral., 35, 535-543.
KIM. K. S. 1980. Cytopathology of spotted cucumber beetle hemocyte containing virus-like particles. J. Im,ertebr. Pathol.. 36, 292-301.
CYTOPATHOLOGY KIM, K. S. (Ceratoma
1981. Ultrastructure of bean leaf beetle rrifurcata) hemocytes and their phagocytic activities on tobacco mosaic virus, a nonbeetle-transmitted virus. J. Ultrastruct. Res., 75, 300-313. KITAJIMA, E. W., AND GAMEZ, R. 1983. Electron microscopy of maize rayado fino virus in the internal organs of its leafhopper vector. Intervirology, 19, 129-134. LOCKE, M. 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect. Calpodes e&legs (Lepidoptera:Hesperiidae). J. Morphol., 118, 461-494. MARAMOROSCH, K. (ed.). 1977. “The Atlas of Insect and Plant Viruses.” Academic Press, New York. MATHEWS, R. E. E 1973. Induction of diseases by viruses with special reference to turnip yellow mosiac virus. Annu. Rev. Plant Pathol., 11, 147-170. MATTHEWS, R. E. F. 1982. Classification and nomenclature of virus. Intervirology, 17, l-200. MOUSSA, A. Y. 1978. A new cytoplasmic inclusion virus from Diptera in the Queensland fruitfly. Dams
23
OF PIIV INFECTION tryoni Pathol.,
(Frogg) (Diptera: Tephritidae).
J. Invertebr.
32, 77-87.
MURPHY, F. A., SCHERER, W. F., HARRISON. A. K.. DUNNE, H. W., AND GRAY, G. W., JR. 1970. Characterization of nodamura virus, an arthropod transmissible picornavirus. Virology, 40, 1008- 1021. MUSSEN, E. C., AND FURGALA, B. 1977. Replication of sacbrood virus in larval and adult honey bees, Apis
rnellifera.
J. Invertebr.
Pathol.,
30, 20-34.
NOBLE-NESBITT, J. 1963. The cuticle and associated structures of Podura aquatica at the moult. Quart. J. Microsc. Sci., 104, 369-391. REINGANUM, C., ROBERTSON, J. S., AND TINSLEY, T. W. 1978. A new group of RNA viruses from insects. J. Gen. Viral., 40, 195-202. Russo. M., AND MARTELLI, G. P. 1982. Ultrastructure of turnip crinkle- and saguaro cuctus virus-infected tissues. Virology, 118, 109- 116. SMITH, D. S. 1968. “Insect cells. Their Structure and Function.” Oliver and Boyd, Edinburgh. SPURR, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. UItrastruct. Res., 26, 31-43.