Observations on the morphogenesis and structure of a hemocytic poxvirus in the midge Chironomus attenuatus

Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved

J. ULTRASTRUCTURE RESEARCH 40, 581-598 (1972)

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Observations on the Morphogenesis and Structure of a Hemocytic Poxvirus in the Midge Chironomus attenuatus DONALD B. STOLTZ and MAX D. SUMMERS

The Cell Research Institute, The University of Texas, Austin, Texas 78712 Received February 24, 1972 An account is given of the ultrastructure of a poxvirus infecting larvae of a midge, Chironomus attenuatus. The virus appears to replicate only in hemocytes, and is of the occluded type. Immature virions are formed in association with an amorphous virogenic stroma, portions of which are sequestered into the developing particles. Further maturation of virions occurs either free in the cytoplasm or during (or after) occlusion within the crystalline matrix of polyhedra. A new intermediate in the development of cores from nucleoids is described. In addition, the presence of a unit membranelike structure in the poxvirus core is indicated. A possible nucleation site for the deposition of polyhedron protein is described. It is becoming apparent that insects are commonly subject to poxvirus infections. The insect poxviruses differ, however, from those affecting vertebrates in that they are without exception occluded within the crystalline matrices of large proteinaceous bodies termed polyhedra; this property is shared by three other large classes of insect viruses, namely, the granulosis, and nuclear and cytoplasmic polyhedrosis viruses (35). By virtue of the fact that they are occluded, such viruses are relatively much more stable under normal environmental conditions, and thus have considerable potential in biological control. The insect poxviruses represent the most recently discovered group of occluded insect viruses, the first being described as recently as 1963 (38). Since then, poxviruses have been found in 14 additional insect species and transmitted to at least 3 others (Table I). The present report constitutes the first known instance of poxvirus in a dipteran species from N o r t h America. The disease differs from a similar one described in a European chironomid (18, 22) in that in the present instance only hemocytes appear to be infected. MATERIALS AND METHODS Virus-infected fourth-instar Chironomus attenuatus Walker (Chironomidae) larvae were collected during the summer months from the Houston "bayou." Larvae were immersed in cold 3.5% glutaraldehyde in cacodylate buffer (0.05 M, pH 7.0), and the integuments

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TABLE I THE PRESENTLY KNOWN INSECT POXVIRUSES Host order

Lepidoptera

Host species

Amsacta moorei (transmitted to Estigmene acrea) Operophtera brumata Cossus cossus

Coleoptera

Diptera

Orthoptera

Choristoneura biennis (transmitted to C. fumiferana and C. pinus Euxia attxiliaris Dermolepida albohirtum Melolontha melolontha Figulus sublaevis Othnonius batesi Phyllopertha horticola Camptochironomus (Chironomus) tentans Chironomus luridus Chironomus attenuatus Chironomus plumosus Melanoplus sanguinipes

References

19, 31 44 42 8; J. C. Cunningham personal communication. Suner, in press. Goodwin, unpublished 6, 38 40 17 41 43 18, 22 Present study. Stoltz, unpublished 21

were ripped open to expose tissues to the fixative. After 1 hour, the material was rinsed in buffer, and postfixed for an additional hour in 2 % OsO4 in the same buffer. Larvae were dehydrated in an ethanol series and embedded in Epon. Thick sections, made for purposes of orientation and preliminary identification of tissues, were stained with 1% toluidine blue in borax and examined with a phase contrast microscope. Thin sections were stained with uranyl acetate and lead citrate, and examined with either a Siemens Elmiskop I or a Hitachi H U 11E electron microscope.

OBSERVATIONS AND DISCUSSION Symptomatology Diseased fourth- (and occasionally, third-) instar larvae are identified by the presence of irregularly distributed white masses just b e n e a t h the integument. Infected larvae appear n o r m a l i n size and, as far as can be determined, are as active as u n infected larvae. M o s t infected larvae nevertheless appear to die of the disease in the late f o u r t h instar. Fro. 1. Phase-contrast micrograph of poxvirus-infected hemocytes. The dark spots in the hemocytes are polyhedra. The point of attachment of a hemocyte aggregate (H) to a muscle fiber is indicated by an arrowhead. M, muscle fibers. Fro. 2. Virus-infected hemocyte (H) under the basement membrane (bin) of fat body (F). Spherical, immature virions are present in the cytoplasm of the hemocyte. FI6. 3. Portion of hemocyte aggregate near a muscle fiber (M). The nuclear chromatin of the infected hemocytes is marginated. Polyhedra are present in the cytoplasm. Note that the polyhedra within individual cells are morphologically similar.

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Cytopathology An examination of toluidine blue-stained thick sections showed that poxvirus inclusion bodies appeared to be restricted to hemocytes. Infected hemocytes were usually found in large aggregates, either attached, or in close proximity, to other tissues such as the tracheal epithelium, epidermis, muscle, nerve, and fat body (Fig. 1). The association between hemocytes and non-infected tissues is often a close one, and the adjoining cell boundaries may not be readily visualized at low magnification, even with an electron microscope. It is possible, therefore, that some authors may have misidentified such aggregates of hemocytes as lobes of infected fat body, etc., in other poxvirus infections of insects. Mitoses were not seen in the hemocyte aggregates. Thus far, the following tissues have been examined in the electron microscope: hemocytes, fat body, epidermis, tracheal and midgut epithelia, and abdominal muscle and nerves. As was expected on the basis of light microscopic observations, only hemocytes appeared to support replication of the virus. While polyhedra and virus cores were occasionally detected in the fat body, no virogenic stroma or immature forms of the virus have been found in that tissue; this suggests that no virus replication as such occurs in the fat body. Virus-related structures may gain entrance to the fat body as a result of partial lysis of tissues in the region of fat body-hemocyte contact. As seen in the electron microscope, the nature of the close association between hemocyte aggregates with other tissues such as fat body and muscle is somewhat more clear. The aggregates are in all cases composed of relatively small cells; hemocyte plasma membranes are distinct, and do not fuse either with each other or with membranes of other tissues. In many cases, hemocytes apparently can penetrate underneath the basement membrane of other tissues (Fig. 2). Only in the fat body, however, do hemocytes actually appear to penetrate into intercellular channels. Many characteristic features of the disease are apparent even at very low magnification in electron micrographs (Fig. 3). The most obvious sign of cytopathology is the presence of cytoplasmic virus-containing polyhedra 1, up to about 6/~m in diameter. 1 These are not polyhedra in the strict geometric sense, The term is retained here simply to denote a probable analogy, in both structure and function, with the more obviously polyhedral inclusion bodies of cytoplasmic and nuclear polyhedrosis virus infections of insects. F~a. 4. Nuclear inclusions type A and B of a virus-infected cell are indicated. The spherical type B inclusions are associated with, and may arise from, a laminar precursor (L); the peripheral structure of type B inclusions is identical to that of a polyhedron (P; and inset). FIas. 5-7. Concentric cytoplasmic lamellae. Repeating units in these complexes appear to be identical in structure to the limiting polyhedron "membrane", as indicated by the rectangle in Fig. 6. Lamellae are shown separated by deposits of electron-dense material in Fig. 5. Fig. 7 shows probable fusion of the lamellae (L) with the edge of a polyhedron (P). Evidence of periodic substructure within lamellae can be seen in the area circled in Fig. 6 (and see inset).

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In addition, several changes are apparent in the gross nuclear ultrastructure of infected cells, though the virus itself is always cytoplasmic. Most obvious changes include a margination of the chromatin (Fig. 3) and the appearance of two different kinds of nuclear inclusions (Fig. 4), here designated for convenience types A and B. Type A nuclear inclusions are found in the nuclei of almost all infected cells and, more rarely, in the cytoplasm. The inclusions are small and irregular in shape; similar inclusions have been described in C. luridus infected with a poxvirus (22). The less commonly found type B inclusions are more electron dense than the A-type, and more regular in outline. They appeared occasionally to develop as bulbous outpocketings of laminar material (L, Fig. 4). The distinctive peripheral structure of type B inclusions consists of an electron-dense line separated from the matrix of the inclusion by an electron-lucent band of approximately 250 A in width. This limiting structure is identical in appearance to that surrounding all polyhedra. The matrices of types A and B nuclear inclusions resemble in appearance that of "light" and " d a r k " polyhedra (described below), respectively. Accumulations of concentric lamellae, similar to those associated with nuclear type B bodies, were often seen in the cytoplasm (Fig. 5) and, rarely, in the nucleus. Occasionally, such lamellae are seen to be concentrically disposed about polyhedra (Figs. 6 and 7); what may be a partially tangential section of a polyhedron, shown in Fig. 7, reveals apparent continuity of structure between lamellae and polyhedron. The correspondence between the structure of lamellae and that of the limiting " m e m b r a n e " of polyhedra is particularly clear in Fig. 6. Regular periodic substructural detail is present in such lamellae (Fig. 6, inset). Deposits of electron dense material may appear within the lamellae (Fig. 5); this material is identical in appearance to the matrix of B-type nuclear inclusions and to the more electron-dense peripheral matrix (Fig. 6) of polyhedra. What appear to be similar lamellae are also

Fio. 8 (a-d) Stages in the development of the immature poxvirion represented by incomplete viral shells. Note the increase in degree of curvature with increase in shell size. The virogenic stroma (S) shown in (a) and (c) consists of an amorphous fibrogranular matrix with interspersed vesicular elements; the latter have the appearance of unit membranes at higher magnifications. Developing shells appear to selectively sequester only amorphous, flocculent material from the stroma, as shown in (c); i.e., the vesicular elements are not sequestered. The shell in (d) is connected to membrane; this particular particle is considered somewhat abnormal, since very little stroma has been sequestered. Scales are given in micrometers, or fractions thereof. (e) Immature poxvirion. The unit membrane which constitutes the inner component of the shell is easily seen. Figs. 8 a-e are all at equivalent magnifications. FIG. 9. (a-c) Tangential or oblique sections of portions of the shell of the immature poxvirion. Faint lattice lines running in the directions of the arrows can be seen. FIG. 10. (a) Poxvirus shell showing an apparent double trilaminar membrane aspect (circled). (b) Poxvirus shell showing faint suggestion of external subunits (arrowheads). Fins. 11-13. Development of electron-dense nucleoids. Note that the nucleoids pull away from the inner surface of the shell. The unit membrane of the shell is particularly clear in Fig. 13 (arrowhead).

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a feature of p o x v i r u s - i n d u c e d c y t o p a t h o l o g y in C. luridus larvae (22). L a m e l l a e are discussed further below, in relation to the nucleation a n d g r o w t h of p o l y h e d r a .

Virus morphogenesis and structure A hypothetical sequence of m o r p h o g e n e t i c events involved in the f o r m a t i o n of the C. attenuatus p o x v i r i o n is given in Figs. 8-16. It is clear t h a t several different discrete structural entities occur d u r i n g the course of virus m o r p h o g e n e s i s . Immature virus. I n the scheme a d o p t e d here, the i m m a t u r e virion (Fig. 8 e) is defined as a spherical structure containing a m o r p h o u s material; the latter is sequestered f r o m a virogenic s t r o m a (Fig. 8 a a n d c) in association with which i m m a t u r e particles are formed. The first obvious virus-related structures to a p p e a r are i n c o m p l e t e shells, usually situated on the p e r i p h e r y of the s t r o m a (Fig. 8 a - c ) ; such shells a p p e a r to consist of an inner unit m e m b r a n e m o d i f i e d by the a p p o s i t i o n of w h a t a p p e a r s to be a layer of p o o r l y defined subunits or spicules. D e v e l o p i n g shells are occasionally c o n t i n u o u s with u n m o d i f i e d 1 m e m b r a n e (Fig. 8 d), as other a u t h o r s have o b s e r v e d (6, 12, 20). It has been previously n o t e d t h a t the degree of curvature of the developing vaccinia virus shell a p p e a r s to progressively increase as the shell enlarges (3); this also is the case in the p r e s e n t study (Fig. 8 a - d ) . Sections tangential to the surface of the i m m a t u r e shell m a y reveal the presence of very faint lattice lines (Fig. 9); these are m o r e clearly seen in Fig. 15 of de H a r v e n a n d Y o h n ' s p a p e r (13). Such lattice lines are m o s t likely a p r o d u c t of r a n d o m hexag o n a l p a c k i n g of the surface subunits; the statistical p a c k i n g of subunits on m e m 1 This is meant to infer only that such membrane does not show evidence of an apposed layer of subunits. Fro. 14. (a-i) Development of mature viral cores from the "rectangular nucleoid." (a) Rectangular nucleoids (N). (b and c) Appearance of layer of subunits (arrowheads) at opposite ends of long axes of rectangular nucleoids. Note concomitant loss of subunits from areas of the virus shell; the junctions of shell and unmodified membrane are indicated by arrows. (d) Subunit layer of core almost complete. The subunit layer has entirely disappeared from the virus shell, leaving only a trilaminar unit membrane (arrowhead). (e) Virus particle containing a mature core (C). (f) Immature particle (/) and particle containing a mature core (C) are shown side by side. Note that the envelope of the corecontaining particle has lost its layer of subunits and no longer exhibits a rigid spherical curvature. (g) Stages in the development of cores from rectangular nucleoids. The particle with the mature core (C) has assumed a more elliptical shape; the core is slightly biconcave. N, nucleoid. (h) Particle with a mature core (C). The core subunits are more clearly visible (arrowheads). (i) Section interpreted as being tangential to an edge of a mature core, similar to that shown in (h). Alternatively, a fusion of core membranes has occurred. The linear structure appears to consist of two closely appressed unit membranes, with a layer of subunits (arrowheads) on either side. Fits. 15 and 16. Mature virus particles. Trilaminar elements (arrowheads) similar to unit membranes are present near the periphery of the cores. FIG. 17. Lateral body (arrow). C, core. Fit. 18. Occluded virion with bulges (arrowheads) opposite the concavities of the core (C). Lattice lines of the polyhedron are visible. Cores of such particles are likely to be considerably more biconcave that those of nonoccluded virions.

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brane surfaces has been discussed by Caspar (9). Lattice lines were less obvious in the present study, probably because of the fact that the subunits themselves were poorly visible, as seems to be the case for the insect poxviruses in general. Perfect hexagonal packing of such subunits would result in the formation of sheets or cylinders rather than spheres (virus): such structures have been clearly shown to occur in cells infected with several other poxviruses (4, 10, 13, 16, 29, 30, 32), but were not seen in the present study. The lack of definition of the layer of subunits in the case of the insect poxviruses has led to a certain amount of confusion concerning the structure of the immature virion. In particular, this layer may appear not only to be well separated from the unit membrane of the shell, but also to have itself the trilaminar structure of a unit membrane (compare Fig. 10 a and b). Indeed, it has been proposed by some that the shell of the immature poxvirion consists of two concentric unit membranes (19). A similar problem in interpretation has also been encountered in the case of an icosahedral cytoplasmic deoxyribovirus, mosquito iridescent virus (MIV). Sectioned M I V particles contain two structures which are morphologically identical to typical unit membranes (36); negative staining, however, shows that the outer " m e m b r a n e " may in fact correspond to an icosahedral lattice of subunits (Stoltz, in preparation). Obviously, great care must be exercised in the interpretation of micrographs of the larger, more complex viruses. In summary, it seems most reasonable at present to assume that all immature poxviruses share a common shell structure, consisting of a single unit membrane and a r a n d o m hexagonal surface lattice of subunits. Intermediate and mature forms. The morphogenesis of cores begins with the appearance of an electron dense nucleoid at one point on the periphery of the enclosed material derived from the virogenic stroma (Figs. 11-13). At this stage, the nucleoid has no particular form, but appears to be more compact than the surrounding stroma. The striations detected in nucleoids of several other poxviruses (11, 23, 27-29, 32) were not seen in the present study. The nucleoid, with adjacent stroma, is usually detached from the shell membrane (Figs. 12 and 13). Most investigators seem to agree that nucleoids contain viral D N A , but disagree as to their mode of origin. In particular, uncertainty exists as to whether a complete FIG. 19. Polyhedron occluding both mature (my) and immature virions (iv). Note that none of the completely occluded vMons have the morphology of immature particles. FI~. 20. Polyhedron occluding only mature virions. The indicated particles illustrate cores which are markedly biconcave when sectioned transversely (a) to the thin (flattened) axis, and roughly square (b) if cut parallel to that axis. Note that the core of occluded particle a is more markedly biconcave than those of the more peripheral, newly occluded particles. FIo. 21. Occlusion of trilaminar vesicular profiles. F~G. 22. Occlusion of irregular, unmodified membrane (presumably shell precursor) containing what appears to be material derived from virogenic stroma. A normal mature virion is also being occluded. FIG. 23. Polyhedra in two different cells showing variation in number of occluded virions.

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genome is sequestered into the closed particle before nucleoid formation, or whether D N A , etc., might in fact enter after shell closure. In the lack of definitive evidence for either of these theories, the simplest alternative (i.e., the former) is perhaps preferable. Clearly, developing virus particles sequester, in a selective way (Fig. 8 c), material derived f r o m a stroma which almost certainly consists, at least in part, of viral D N A (33). It seems most likely that the developing particle would acquire a complete genome at this time. It is even possible that viral shell membranes m a y be directly involved in the segregation of progeny genomes into developing virions; the shell m e m b r a n e would thus have a functional role analogous to that suggested for the bacterial mesosome (26). Assuming, then, that the immature poxvirion in fact contains a complete genome, it becomes reasonable to propose that the nucleoid results f r o m a condensation of some of the previously sequestered stroma. Other than nucleoids, structural intermediate forms in the development of poxviruses remain undefined, simply because they have not as yet been detected, at least with any degree of confidence. It is possible that intermediate forms are so evanescent as to be rarely detectable. In the case of C. attenuatus poxvirus, an obvious intermediate in the development of cores, which presumably arise f r o m nucleoid precursors, is the "rectangular 1 nucleoid" (Fig. 14a), a previously undescribed entity. While this structure has a definite form, unlike what may be referred to as " i m m a t u r e " nucleoids (e.g., as in Figs. 11-13), it lacks the differentiated peripheral structure of the mature core (see below). The rectangular nucleoid is formed while the immature virion is still spherical in form: that is, before loss of the subunit layer from the shell. Transformation of the nucleoid, f r o m an irregular to a roughly rectangular form, occurs freely in the lumen of the virus particle, without any apparent attachment to or association with the shell membrane. The nature of the changes involved in the development of definitive cores f r o m what we assume to be their immediate precur1 The term "rectangular" is obviously used here in a very loose sense. Such nucleoids would be truly rectangular if the corners were sharp rather than rounded.

FIo. 24. Three adjacent hemocytes showing variation in size, shape, and electron density of viruscontaining polyhedra. Arrows indicate probable sequence of development of immature (IP) to more mature polyhedra (MP). The central matrix of the latter gradually becomes less electron dense. Immature polyhedra have less regular outlines, presumably because they are still involved in occluding virus. Compare also the polyhedra shown in Figs. 4 or 23 with that shown in Fig. 20; these micrographs are reproduced from negatives of approximately equal density, and printed under identical conditions. FIG. 25. Polyhedron showing variation in electron density of matrix immediately adjacent to occluded virions. See also the particles indicated in Fig. 20. FIG. 26. Budding poxvirion at plasma membrane of infected hemocyte. Remnants of the subunit layer of the shell are indicated (arrowhead). C, core. FI~. 27. Enveloped virions in intercellular spaces, after budding through plasma membranes. The host cell membrane (arrow) is separated from the outer viral membrane on one particle.

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sors, the rectangular nucleoids, is uncertain. Some micrographs, however, suggest that peripheral differentiation of the rectangular nucleoid begins at opposite ends of the structure, with the addition of subunits (Fig. 14b-d, and g). The mature poxvirus core (Fig. 14e-h) is a more flattened, roughly rectangular structure, with a peripheral structure which in fact closely resembles the shell of the immature virion. The core peripheral structure, like that of the shell, consists of both a trilaminar component and a layer of subunits (Figs. 14e, h, and i; 15-17). The precise nature of the trilaminar component is unknown. While it does resemble in appearance a unit membrane, it does not appear to originate directly from the shell membrane, even though the earliest stages of nucleoid formation occur in close proximity to the shell (3, 23). If a unit membrane is indeed present, it would appear to originate de novo, as does the shell membrane (•2); if so, poxvirions may contain more enzymes than are now known to be present (34). Peripheral core subunits have been very clearly demonstrated by Easterbrook for vaccinia virus (15); that author also remarked on the similarity of the core peripheral structure to the shell of the immature poxvirion. The "flat sac" intermediate in core morphogenesis, proposed by de Harven and Yohn (13), may instead represent a tangential section of the edge of a core and, as such, it would be analogous to the structure shown here in Fig. 14i. Concomitant with the appearance of definitive cores are changes in the structure of the virus shell or envelope. First, the layer of subunits is progressively lost (Fig. 14b-g); second, the rigid spherical curvature of the shell changes to a more elliptical outline (Fig. 14f and g). The two events would seem to be causally related. The structure of the mature virion (Figs. 15 and 16) is similar to that proposed for some poxviruses (32), but differs from certain others. In particular, mature Yaba and Shope fibroma poxvirions appear occasionally to acquire an additional external unit membrane, the source of which is inapparent in micrographs. In the case of Shope fibroma virus, observations of a double external membrane are rare, and are not considered to be significant (32). The mature C. attenuatus poxvirion is considered to be structurally similar to the particles illustrated in Fig. 8 of reference 32 (Shope fibroma) and Fig. 13 of reference 30 (Yaba). Not all poxviruses possess biconcave cores (7, 17). The presence of tubular structures in the nucleoid of several poxviruses has been well documented (7, 17, 19, 23, 28); however, these were not seen in the present study. Lateral bodies were occasionally observed on one or both sides of the cores (Fig. 17), but were usually poorly visible in this study. The presence of lateral bodies, even where these are not clear, is nevertheless assumed to be directly related to the frequently observed bulges in the viral membrane opposite the core concavities (Fig. 18). Lateral bodies may be somewhat labile, since in many cases the space between the core and envelope is relatively electron-lucent (Fig. t4 g and h); on the

HEMOCYTIC POXVIRUS OF CHIRONOMUS

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other hand, the visualization of lateral bodies may depend primarily on the plane of section of virus particles. Cores, but not nucleoids, were occasionally seen free in the cytoplasm of both hemocytes and fat body cells; their relationship to morphogenetic or infection processes is unknown at present.

Occlusion Both mature and immature particles may undergo occlusion within polyhedra (Fig. 19). Generally, however, polyhedra first begin to appear only after the formation of some mature virions, and most polyhedra occlude primarily mature particles (Fig. 20); this may simply be due to the fact that both polyhedra and mature particles, unlike the immature forms, are not situated close to virogenic stroma. Occasionally, unmodified vesiculate or irregular membranous profiles are occluded (Figs. 21 and 22); the membranous profiles shown in Fig. 22 are interpreted as aberrant virus shell precursors, similar to those formed in the presence of rifampin (20). Membranes of normal cytoplasmic elements and organelles, however, are not occluded. Specificity of recognition is therefore suggested to reside in the viral shell membrane, but not in the external subunits. Our observations suggest that further morphological transitions of some C. attenuatus poxvirions may occur after occlusion within polyhedra. For example, almost all completely occluded virions possess definitive cores: there are few if any immature particles inside most polyhedra (Figs. 19 and 20), even though there may be several in the process of being occluded on the polyhedron edge, and none in polyhedra which have apparently ceased occlusion (Fig. 4). The majority of occluded virions possess cores which are more markedly biconcave and more flattened than those of nonoccluded particles. Similar conclusions have been reached in studies of some other insect poxviruses (5, 21). Virus particles are randomly oriented within polyhedra, unlike some other insect poxviruses (7, 17). Several types of polyhedra can be distinguished: for example, some polyhedra have only a few, or are completely devoid of, occluded virions, whereas others contain a large number of particles (compare Figs. 20 and 23); the situation is similar to that observed in the case of the so-called "spheroidosis" type of insect poxvirus infection (17, 39). The fixation of (and/or infiltration of plastic into) virions in some polyhedra is poor, even though virus particles in nearby polyhedra may be very well preserved. On the basis of electron density, many polyhedra are readily classed as being either "light" (Figs. 4 and 23) or "dark" (Figs. 19 and 20). Light polyhedra always have more regular outlines, and for the most part seem to have ceased occlusion of virus. Many polyhedra show variable amounts of central light and peripheral dark matrix (Fig. 24), suggesting a gradual transformation of dark to light polyhedra. Interestingly, lattice lines are usually obvious only in the

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more electron-dense material (compare Figs. 6 and 18), suggesting that a disappearance of definite lattice lines accompanies the loss in electron density of polyhedra. Variation in electron density of polyhedron matrix is also evident in the immediate vicinity of occluded varions (Figs. 18, 20, and 25); generally, matrix surrounding newly occluded virions appears relatively less dense.

Nucleation and growth of polyhedra Little is known about these processes, which remain a matter for speculation only. While it is clear that the polyhedra formed during the course of several kinds of insect viroses have a distinct peripheral structure or zone (2, 22, 37), it is not known whether this type of structure plays any role in the initiation or cessation of deposition of polyhedron protein. In the present study, a distinctive peripheral structure (see Figs. 4, 6, 19, and 20) is apparently present at all developmental stages of poxvirus polyhedra, including the period during which the occlusion of virus occurs. The structure therefore is probably not involved in the limitation of polyhedron size. Preliminary observations suggest rather that the deposition of polyhedron protein may be initiated on the (inner) surface of this structure; in this regard, Fig. 5 is particularly suggestive. The polyhedra of nuclear polyhedrosis viruses (NPV) also appear to nucleate on definite structures, which may be derived from unit membrane derivatives (37); this is particularly clear in the case of a nuclear polyhedrosis of Rhynchosciara angelae, in which polyhedra grow from the inner nuclear membrane into the interior of the nucleus (Stoltz, unpublished observations). The present study, however, does not implicate any involvement of unit membranes per se in the formation of poxvirus polyhedra. It is of interest to note that within any given cell all polyhedra appear to be at a similar stage of development and exhibit similar morphology (see, e.g., Figs. 3 and 24). This is also the case, though less strictly, in virus particle morphogenesis. This suggests that virus-directed syntheses and assembly processes are not continuous throughout the infectious cycle, but may be coordinated in such a way as to ensure maximum occlusion of virions within polyhedra.

Release of Virions Virus particles are apparently released into the extracellular environment either through lysis of infected cells or by means of budding through the plasma membrane (Figs. 26 and 27). Whether host plasma membrane-enveloped or unenveloped particles, or both, are involved in the intercellular spread of infection is unknown. The present study, together with other recent reports (1, 14), suggest that budding is a general mechanism of poxvirus release.

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Comments

It is now becoming apparent that poxviruses are widespread among the insects. Alone among the occluded insect viruses, they have counterparts in the vertebrates. This immediately suggests the possibility that the vertebrate poxviruses may have had their origin in the insect viruses. The possible relatedness of vertebrate and invertebrate poxviruses is readily testable by modern methodology. The one obvious difference between the two groups of poxviruses is the fact that only the insect poxviruses are occluded in protein crystals, or polyhedra. While certain vertebrate poxviruses may undergo occlusion (24, 25), the matrix of the inclusion body is noncrystalline. The formation of both polyhedra in invertebrates and inclusion bodies in vertebrates is thought to be a viral genetic function, but it is not yet known whether any host-dependent factors influence the expression of such functions. The fact that not all vertebrate poxviruses are occluded suggests that the function is not essential for infectivity per se. The same argument undoubtedly applies to the invertebrate poxviruses; polyhedra must nevertheless be regarded as useful vehicles for the efficient delivery of virus via the oral route. In any case, it seems reasonable to predict that a non occluded insect poxvirus will eventually be found in nature, or else be inducible by mutagenic agents. This will facilitate investigation, of the sort already attempted with some vertebrate poxviruses (25), into the genetic basis of the occlusion process. This study was supported by Public Health Service Grant No. AI-09765 from the National Institute of Allergy and Infectious Diseases. D. B. S. is a Canadian National Research Council Postdoctoral Fellow. REFERENCES

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