JOURNAL OF INVERTEBRATE PATHOLOGY 49, 175- 187 (1987)

Fine Structure and Distribution of Three Types of Virus-like Particles in the Sheep Blowfly, Lucilia cuprina and Associated

Cytopathic Effects

K. C. BINNINGTON, E. LOCKIE, E. HINES, AND A. C. M. VAN GERWEN

CSIRO Division of Entomology. GPO BO.Y 1700. Cunherra, Austruliu 2601

Received 14 June 1986; accepted 18 August 1986

The ultrastructure is described for three types of virus-like particles (VLP l-3) found in thin sections of the sheep blowfly Lucilia cuprinu and, in the case of one type (VLP I). in negatively stained preparations. VLP I appears to be morphologically similar to the particles of chronic bee paralysis virus and RS virus of DrosopiziIu and causes a highly characteristic vesiculation of mitochondria. VLP 2 has two forms, spherical and filamentous; these are seen mainly in the gut where the filaments proliferate in the apical parts of the ceils and resemble reovirus-like particles found in the gut of MUKU domesricu. The third type of particle (VLP 3) is intranuclear and seen only rarely. It is arrayed in quasi-crystalline inclusions which resemble inclusions reported in cells and tissues of Drosophila. 0 1987 Academic Press. Inc.

KEY WORDS: Luciliu ctrprinu; sheep blowfly; virus-like particles: ultrastructure; cytopathic ef- fects.

INTRODUCTION

During ultrastructural studies on the sheep blowfly, Lucilia cuprina, we have observed three types of virus-like particles (VLP). Those we call VLP 1 are small, membrane-bound, nonoccluded particles structurally similar to those of chronic bee paralysis virus (Lee and Furgala, 1965) and RS virus of Drosophila (Plus et al., 1975). They are found only in the cytoplasm where they are associated with cytopathic effects including a structural transforma- tion of mitochondria similar to that seen in some virus-infected plants (Binnington, 1987). The second type of nonoccluded particles which we call VLP 2 occur mainly in the gut and appear to be similar to reo- virus-like particles found in Musca by Moussa (1978). The third type which we call VLP 3 are also nonoccluded, and seen only rarely; they form arrays in the nucleus and resemble the particles of a virus found in Drosophila (Akai et al., 1967; Filshie et al., 1967; Rae and Green, 1968). In this paper we describe the ultrastructure of the three types of VLP in thin sections and the

appearance of negatively stained VLP 1. We also describe the cytopathic effects found in a variety of tissues of Lucilia con- taining VLP 1.

MATERIALS AND METHODS

Flies

Females from laboratory cultures of L. cuprina established for many years as well as some from recently colonized strains were used. These included both recently emerged unfed females and protein-fed gravid ones.

Electron Microscopy

Conventional sections. Flies were dis- sected under a blowfly Ringer solution (e.g., Hansen Bay, 1978) buffered to pH 7.2 with Mops and tissues placed in 2.5% glu- taraldehyde (0.1 M phosphate buffer, pH 7.2; 0.15 M sucrose) at room temperature for 2 hr. Tissues were then rinsed in the same buffer, postfixed in 1% osmium te- troxide (0.1 M phosphate buffer, pH 7.2: 0.15 M sucrose), and then rinsed in three 15-min changes of 30% ethanol before

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VIRUS-LIKE PARTICLES IN L/rc,i/icr c~cp~inrr 177

being stained en bloc with 1% m-any1 ace- tate in distilled water for 1 hr. Tissues, em- bedded in EponlAraldite (Mollenhauer. 1964), were sectioned, stained with uranyl acetate and lead citrate, and examined with a JEOL 1OOC electron microscope.

Negative staining of tissue en bloc. The samples were incubated in colloidal lan- thanum by adding 1% lanthanum hydroxide to the fixative as a modification of the method described by Revel and Karnovsky (1967). The processing was as outlined above, except that phosphate buffer was replaced with cacodylate buffer.

Negative staining of purified VLP 1. Ini- tial attempts to extract VLP 1 employed a method modified from that used for chronic bee paralysis virus (Bailey et al., 1968) using 5- 10 frozen flies in which VLP 1 had been observed previously in sectioned gut. Frozen flies were homogenized in: 10 ml 0.05 M KPO, buffer; 0.02% sodium DIECA (diethyldithiocarbamate); 0.1 ml diethyl ether; 0.2 ml chloroform. The extract was centrifuged for 20 set in an Eppendorf Model 5414 and the pellet discarded. The supernatant was centrifuged at 107,OOOg for 13 min. The resultant pellet was then resus- pended in buffer and stained with 1% am- monium molybdate.

A higher concentration of VLP 1 was ob- tained by extracting larger numbers of flies (70-100) from cultures known to have an infection rate of at least 50%. The flies were homogenized in 10 ml of blowfly Ringer and spun for 5- 10 min in an Eppen-

dorf Model 5414. The supernatant was passed through a coarse filter and then through a 0.45 km filter. The filtrate was stained with 1% neutral sodium phospho- tungstic acid, 1% uranyl acetate or 1% am- monium molybdate.

RESULTS

VLP I

Morphology. Two types of VLP I are seen in sections, oval and filamentous which may be up to 1000 nm (Fig. 1). In transverse section the filaments are seen to be arranged in an hexagonal array (inset, Fig. 1). Filaments are seen only rarely in negatively stained whole mounts of puri- fied particles (Fig. 2) with oval forms being much more common; in some cases the oval forms have a small protrusion from one end (Fig. 3) similar to those seen on particles of chronic bee paralysis virus (Lee and Furgala, 1965).

VLP 1 have a membranous envelope in- distinguishable from other cellular mem- branes, a putative nucleocapsid in the form of an electron-dense ring and an electron- lucent core (Fig. 4). Invaginations of cell membranes including those of the endo- plasmic reticulum and the outer nuclear membrane are frequently seen in infected cells (Fig. 5) and filamentous forms appear to be extreme examples of the invagina- tions (Fig. 1). Images of oval forms similar to those seen in whole mount negatively stained preparations are seen in extraccl-

FIG. I. Corpus allatum containing oval (double arrows) and filamentous forms (closed arrow) of VLP I. Mitochondria are characteristically vesiculated (TM). Inset shows an array of filaments in transverse section. X 50,000. Inset, x 40.000.

FIG. 2. Negatively stained filaments of VLP 1. x 150,000. FIG. 3. Negatively stained oval form of VLP I with a small protrusion at one end (arrow).

x 150800. FIG. 4. Sections of oval forms of VLP I in the corpus allatum. x 180,000. FIG. 5. Distended tubule of smooth endoplasmic reticulum tSER) with VLP I and invaginations of

the membrane (arrowheads). x 70.000. FIG. 6. Section of oval forms of VLP I in extracellular space of a corpus allatum infiltrated with

colloidal lanthanum during fixation. x 144,000.

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VIRUS-LIKE PARTICLES IN Lucitiu cuprinrr 179

lular spaces of corpora allata infiltrated with colloidal lanthanum (Fig. 6). From negatively stained preparations, VLP 1 are estimated to be ca. 20 x 28 nm. Positively stained sectioned profiles are larger, ca. 28 x 38 nm.

Cytopathological effects associated with VLP 1. The mitochondria of infected cells are invariably transformed into vesicle- containing structures analagous to those seen in some virus-infected plants (Bin- nington, 1987). The matrix of some mito- chondria, more electron dense than normal, is displaced by concentrically ar- ranged vesicles attached to the outer mito- chondrial membrane (Binnington, 1987). The vesicles are attached to the outer mito- chondrial membrane at sites occupied by regularly arranged deposits of electron- dense material and this part of the mito- chondrion is often invaginated (inset, Fig. 7). This configuration has been termed a “rosette” in virus-affected plant mitochon- dria (Harrison et al., 1970; Hatta and Ushiyama, 1973).

Prevalence of infection. A number of highly inbred strains held in separate cages in a constant temperature room were found to contain VLP 1 at an infection rate of at least 50%. There was a lower prevalence of VLP 1 in recently colonized strains which may indicate that VLP 1 is rare or absent in field populations. Examination of flies by sectioning is labor intensive and detecting the presence of VLP 1 in field populations of Lucilia will require a more convenient method of assay. VLP 1 were present both

in protein-fed and sugar-fed females. No VLP have been detected in ultrastructural studies of larvae (n = ca. 40) from the same strains of L. cuprina (Binnington, un- publ. data).

Tissue distribution and cytopathology. VLP 1 have been detected in the following tissues of L. cuprina females: corpus al- latum, corpus cardiacum, aorta, esophagus (including esophageal cuticle and muscle), gut, Malpighian tubules, tracheal cells, so- matic muscle, oenocytes of the fat body, and perineurial cells of the thoracic gan- glion.

(a) Corpus allaturn-Oval forms occur in the basal lamina and extracellular spaces as well as intracellularly where they are seen in tubules of smooth endoplasmic retic- ulum (SER), the predominant organelle of the corpus allatum (Figs. 8, 9). No VLP 1 are seen in nuclei but large numbers are seen within distensions of the inner and outer nuclear membranes. In some cases the distensions are adjacent to patches of peripheral heterochromatin suggesting a possible interaction between VLP 1 and chromatin (Fig. 10). Tubules of SER which contain VLP 1 are often grossly distended by the particles and lie in close apposition to one another thus forming narrow channels of unstructured cytoplasm (Fig. 8); in advanced stages of infection large areas of the cytoplasm assume this homo- geneous unstructured appearance (Fig. 11). Some packets of VLP 1 contain closely packed particles. are more electron-dense, and resemble lysosomes (Fig. 12). VLP 1

FIG. 7. Transformed mitochondria (TM) in corpus allatum infected with VLP I, The vesicles have a peripheral position and are attached to the outer mitochondrial membrane at sites occupied by elec- tron-dense material (arrows). Tubules of SER which contain VLP 1 are closely associated with the transformed mitochondria. Inset shows “rosette” configurations (asterisks). x 24,500. Inset x 19.000.

FIG. 8. Corpus allatum with tubules of SER distended by VLP 1 and surrounded by cytoplasm with a homogeneous appearance. Note the oval forms of VLP I in extracellular space (ES) between the basal lamina and the plasma membrane. x 59.000.

FIG. 9. Corpus allatum with connections (arrow) between narrow and dilated tubules of SER con- taining VLP 1. Note the relatively electron-lucent spheres (arrowheads) which are associated with microtubules and may represent capsids of VLP I. x 46.500.

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VIRUS-LIKE PARTICLES IN LLIL.;/~u (.l@~ 181

were notably absent from nerve terminals which are seen frequently in sections of the corpus allatum.

(b) Corpus cardiacum/aorta-VLP 1 are seen frequently in these tissues but as in the corpus allatum, nerve cells are not in- fected, distribution of the particles being limited to glial cells.

(c) Esophagus-In many infected flies epidermal cells are hypertrophied com- pared with controls and contain a profusion of VLP 1 (Figs. 13, 14). Oval forms are contained within cisternae of rough endo- plasmic reticulum and in extracellular spaces (Fig. 13). They are also present in the cuticular lining of the esophagus (Figs. 13. 15).

(d) Gut-In some flies gut tissue is se- verely damaged by VLP 1. Endoplasmic reticulum is the major organelle infected and drastically altered mitochondria oc- cupy abnormally homogeneous cytoplasm (Fig. 17).

(e) Muscle-VLP 1 are seen in esopha- geal, gut (Fig. IS), and somatic muscles (Fig. 16).

(f) Tracheal cells--In flies containing VLP 1, tracheal cells (Fig. 18) are invari- ably infected and in some flies trachea are the only tissues in which VLP 1 are seen.

(g) Fat body-No VLP 1 were seen in fat-containing cells but they were present in oenocytes contiguous with fat body tissue.

(h) Nervous system-No VLP 1 have been seen in nerve cell bodies, central axons, or peripheral axons. However, they

are frequently seen in glial cells of the corpus cardiacurn and were present also in glia of the thoracic ganglion (Fig. 19).

Replication of VLP 1. There are a number of features of infected cells sugges- tive of virus replication: relatively elec- tron-lucent spheres, ca. 30 nm in diameter which either form aggregations or are asso- ciated with microtubules may be capsids (Figs. 9, 21) and membrane invaginations may indicate sites where nucleocapsid components become enveloped. Fibrillar material within mitochondrial vesicles is structurally similar to material in vesicles of transformed plant mitochondria thought to be RNA (Di Franc0 et al., 1984). Spatial associations of VLP 1, putative capsids, and dense material on the surface of trans- formed mitochondria (Fig. 2 1) invite specu- lation that these associations are related to replication of VLP 1, but at present there is no evidence for this.

VLP 2

These particles are seen mainly in the gut and occur in greater numbers in older flies. They differ from those of VLP 1 in being larger and not ensheathed by a membrane and occur in two forms: pleomorphic fila- ments which generally occupy the apex of gut cells (Figs. 22, 23) and spheres (inset top left Fig. 23).The spheres are scattered throughout the gut cells and seen occasion- ally in trachea. The reovirus-like particles described by Moussa (1978) in M. dotnes- tica also have a filamentous form found in the apex of gut cells and are of similar di-

FIG. 10. VLP 1 are often seen within distended nuclear membranes of the corpus allatum. In some cases the distensions appear to be spatially related to peripheral clumps of heterochromatin (arrows). x 11,000.

FIG. 11. Large area of unstructured cytoplasm (asterisk) in the corpus allatum containing trans- formed mitochondria (TM) and bordered by tubules of SER rich in VLP 1. x 18,500.

FIG. 12. Highly electron-dense, lysosome-like packets of VLP 1 (arrows) in the corpus allatum. x 17,500.

FIG. 13. Hypertrophied esophagus (cf. Fig. 14) heavily infected with VLP 1. VLP 1 are present within the epidermal cells (EC), the cuticle (C), and extracellular space (ES). x8000.

FIG. 14. Normal esophagus. C, cuticle; EC. epidermal cells; M. muscle. x 26,000.

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VIRUS-LIKE PARTICLES IN Llrcificr cuprim 183

mensions to VLP 2 of Lucifia. The width of the filamentous forms (and the diameter of the spheres) is ca. 60 nm. Within VLP 2 fil- aments there are dense areas ca. 60 nm in diameter which resemble spherical forms (Fig. 23, inset top left Fig. 23). These more electron-dense areas contain small, roughly spherical structures ca. 18 nm in diameter (inset bottom right Fig. 23). The signifi- cance of these structures is unknown but one possibility is that they may be periph- eral capsomeres analogous to those de- scribed by Moussa (1978) in Musca reo- virus-like particles. Apparently hollow spheres, ca. 35 nm in diameter which are often associated with VLP 2 may be nu- cleocapsids (Fig. 24). Although occupying a considerable volume of cytoplasm, par- ticularly in aged females (Fig. 22), VLP 2 does not appear to have any cytopathic ef- fects. Mitochondria and other organelles appear normal.

VLP 3

Seen very infrequently in the L. cuprina cultures examined, and only in the nucle- us, VLP 3 are moderately electron-dense spheres with a diameter of ca. 40 nm. They are arranged into nonoccluded, quasi-crys- talline inclusions (Fig. 2.5) which resemble those described in cultured cells of An- theraea, Aedes, and Drosophila (Filshie et al., 1967) and in tissues of Drosophila (Akai et al., 1967; Rae and Green, 1968).

DISCUSSION

Of the three types of virus-like particles which we have observed in L. cuprina, VLP 1 appears to be the most significant; it is present in large numbers disseminated throughout the fly tissues and is seen in a high percentage of laboratory cultured flies. Tissues containing VLP 1 have pro- found ultrastructural alterations with mem-

FIG. 15. Higher magnification image showing VLP 1 within endoplasmic reticulum (ER) and the cuticle (C) of the esophagus. x 26,500.

FIG. 16. Somatic muscle containing VLP within membranous packets and in extracellular space (arrows). Note the vesiculated mitochondria (TM). x 13.500.

FIG. 17. Gut. severely affected by VLP 1 (arrows) and containing barely recognizable transformed mitochondria (TM). x 15,000.

FIG. 18. Trachea and muscle surrounding the midgut. containing packets of VLP 1 (arrows) and transformed mitochondria (TM). x 7000.

FIGURES ON OVERLEAF

FIG. 19. Neural lamella (NL)/perineurial (PN) sheath of the thoracic ganglion with oval and tila- mentous forms of VLP 1 present in perineurial glial cells which contain transformed mitochondria (TM). x 24,000.

FIG. 20. Electron-lucent spheres (arrows) are sometimes present in large numbers adjacent to transformed mitochondria and may indicate sites of replication of nucleocapsids. x 26,000.

FIG. 21. Higher magnification of possible nucleocapsids (arrows) associated with a transformed mitochondrion. The cytoplasmic side of the outer mitochondrial membrane has particularly well de- veloped electron-dense deposits (arrowheads) and these appear to be contained within a further mem- brane (M). x 64,500.

FIG. 22. Filamentous forms of VLP 2 (arrows) in the apex of a midgut cell. With this type of infection cell organelles including mitochondria (M) appear normal. L, gut lumen: MV. microvilli. x 15,500.

FIG. 23. Higher magnification image of filamentous and spherical (inset. top left of picture) forms of VLP 2 showing substructure (arrows) within the filaments. Inset at bottom right of picture shows smaller spherical structures (arrows) which may represent capsomeres of VLP 2. x 92.500. lnset (top left) x 170,000. Inset (bottom right) ~275,000.

FIG. 24. Possible nucleocapsids (arrows) free in the cytoplasm adjacent to VLP 2. x 71.500. FIG. 25. Arrays of VLP 3 in the nuceus of a midgut cell. x 43,000.

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VIRUS-LIKE PARTICLES IN Luciiicc c~uprirur 185

186 BINNINGTON ET AL.

brane systems and mitochondria being drastically changed. The changes in mito- chondria are so characteristic as to be diag- nostic of infection with VLP 1 and are remarkably similar to vesiculated mito- chondria in some virus-infected plants (Binnington, 1987). Vesiculation of mito- chondria of animal cells does not appear to have been reported previously. However, cytopathic vacuoles (CPV-I), demonstrated in chicken cells infected with Semliki Forest virus, and thought to be derived from Golgi (Grimley et al., 1968, 1972; Friedman et al., 1972), bear some resem- blance to the transformed mitochondria of Lucilia. There is evidence that CPV-I type vacuoles are involved in replication of RNA of Semliki Forest virus (Grimley et al., 1968, 1972; Friedman et al., 1972) and a role in nucleic acid replication has been suggested for vesiculated plant mitochon- dria (e.g., Di Franc0 et al., 1984).

Though the resemblance of VLP 1 and other small, nonoccluded viruses is merely in their shape, there are few viruses with particles of this shape. Longworth (1978) in his review of small isometric viruses of in- vertebrates discussed two viruses with par- ticles which were oval rather than icosahe- dral, chronic bee paralysis virus and RS virus of Drosophila. Ultrastructural de- scriptions of the particles of the latter two viruses in tissues are less extensive than for VLP 1 of Luciliu but images of the particles of chronic bee paralysis virus from thin sections (Lee and Furgala, 1965) appear to be quite similar to the oval forms of Lucilia VLP 1. Negative staining confirms the morphological similarities between VLP 1, the particles of chronic bee paralysis virus (oval and filamentous forms) and RS virus (oval forms) of Drosophila (Lee and Fur- gala, 1965; Plus et al., 1975).

VLP 1 is not associated with paralysis of flies and this difference between it and chronic bee paralysis virus may be related to the absence of VLP 1 from nerve cells of Lucilia. The marked cytopathic effects as- sociated with VLP 1 suggest that it should

increase mortality rates in Lucilia cultures. Although many of the infected flies investi- gated were not overtly sick, studies cur- rently being undertaken to transmit VLP 1 to a “virus-free” strain indicate that infec- tion does increase mortality (Binnington, unpubl. data) but a better understanding of the pathogenicity of VLP 1 must await comparisons between the virus-free strain and naturally infected ones.

VLP 2 is much larger than VLP 1 but its size has not yet been determined by the more precise method of negative staining. The evidence indicates that VLP 2 is sim- ilar to the reovirus-like particle described by Moussa (1978) in M. domestica. In Musca there was a higher mortality rate in colonies containing the particles than in those that did not (Moussa, 1978) but so far we have no evidence for VLP 2 being pathogenic in L. cuprina.

It is interesting to speculate that VLP 3 of Lucilia may be related to the “arrayed VLP” described in Drosophila cells and tissues (Akai et al., 1967; Filshie et al., 1967; Rae and Green, 1968). Such particles in Drosophila have been implicated in the transformation of cells into tumourous lines (Akai et al., 1967; Gateff et al., 1984) and may be related to retroviruses (Shiba and Saigo, 1983). Furthermore it has recently been shown that the RNA of the Droso- phila VLP is homologous to that of tran- sposable elements of Drosophila (Shiba and Saigo, 1983).

ACKNOWLEDGMENTS

We are grateful to Dr. A. Gibbs and Mr. P. Chris- tian, Research School of Biological Sciences, Austra- lian National University for helpful advice.

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