CONFORMITY OF LIGHT AND ELECTRON MICROSCOPIC STUDIES ON VIRUS PARTICLE DISTRIBUTION IN LYMPHOCYSTIS TUMOR CELLS OF FISH

CONFORMITY O F LIGHT AND ELECTRON MICRO- SCOPIC STUDIES ON VIRUS PARTICLE DISTRIBUTION

I N LYMPHOCYSTIS TUMOR CELLS O F FISH*

Roland Walker Department of Biology, Renssdaer Polytechnic Institute, Troy , N . Y .

Richard Weissenberg Philadelphia, Pa.

The characteristic skin tumors of lymphocystis disease a re due to the enormous hypertrophy of host connective tissue cells under the influence of a virus. Only the infected cells hypertrophy ; they become encapsulated and they do not multiply. Nuclei a r e proportionately enlarged, and basophilic cytoplasmic inclusions tend to form a rich network in mature cells. The pattern and texture of these inclusions differ both with age of the cell, and with host species. The disease has been reported in many species of Teleosts, and may be epidemic in Percidae, Centrarchidae, and Pleuro- nectidae. It may be fairly easily transmitted within the host species or genus, bu t rarely across family limits. Though the etiology was at first uncertain, and various parasites had been suggested as agents, the idea of a causative virus was proposed by Weissenberg (1914). The conditions of infectivity seemed to fit this hypothesis, and Weissenberg’s (1951) transfer of the disease to FunduEiLs by an ultrafiltrate of Stizostedion cells gave still stronger support. For fur ther historical and comparative discussion and references, see Weissenberg’s ( 1 9 6 5 ~ ) review in this mono- graph.

With careful cytological study of the inclusion bodies, Weissenberg (1949, 1951 & 1960) showed that in lymphocystis material fixed in osmic acid solution (method of Kopsch) , or postosmified, there was a pattern of minute osmiophil granules often associated with the inclusions as a cortical layer. He interpreted these as elementary bodies of the causative virus. This pattern was also found in his material from earlier studies, including lymphocystis cells from Acerim, Stizostedion, Lepomis, and Plewonectes. The granules were seen not only at the surface of simple inclusion bars, but in the interstices of fenestrated inclusion patterns, The granules were seen both in young cells and in mature ones, and associated both with pale inclusions and with dark, osmiophil ones. The sharp contrast provided by osmification allowed visualization of particles below the size susceptible to sure measurement by visible light. With the weaker contrast of routine fixatives and stain, the granulation was scarcely discernible. Besides the densely osmiophil granules, paler ones of similar size were often seen,

*This investigation was supported in part by U.S. Public Health Service Grant No. CA-05790-02-03 to R. Walker from the National Cancer Institute.

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376 Annals New York Academy of Sciences

sometimes apparently within the substance of the inclusion body, hence “medullary” as opposed to the “cortical” osmiophil granules. Weissenberg ( 1 9 5 1 ~ ) suggested tha t the pale “granules of the inclusion substance” might be developmental stages, o r immature elementary bodies. Weissen- berg (1949) also called attention to some larger granules and (1960) to configurations within the granular zones looking like rods, dumbbells, tetrads, etc. He speculated on their possible relationship to reproduction of elementary bodies by analogy with division or sporulation of nonviral microorganisms.

Electron microscopy of thin sections of mature lymphocystis cells of the walleye, S t i z o s t d o n (Walker, 1962), showed large numbers of 200 m p particles with capsids having hexagonal profiles or smaller pentagonal profiles (end sections). These section profiles imply the icosahedral form that is so characteristic of many virus particles. Capsomere structure was not resolved. These icosahedral viral particles might well have corresponded to Weissenberg’s osmiophil granules, but their topographic relation to the inclusion network was not clearly shown. Indeed, Weissenberg ( 1 9 5 1 ~ ) had reported tha t in Stizostpdion there was progressive dispersal of the granules with maturation of the cell.

Walker and Wolf (1962 & unpublished) have followed the development of lymphocystis cells in the bluegill, Lepowzis. Electron microscopy again shows 200 m p icosahedral particles. I n this case the topography is clear during the middle stages of development. At 10 to 13 days a f te r inoculation the particles a re characteristically gathered at the surface of the pale (non- osmiophil) inclusion bodies. Throughout the month of active development (a short cycle at 2 5 ° C . ) there a re some particles a t the surface of inclusions, but more and more virus particles become aggregated in other parts of the cytoplasm and there they may assume microcrystalloid (face cen- tered cubic) array.

These various, somewhat uncoordinated results suggested the desirability of examining a wider range of material by light and electron microscopy in parallel.

Materials and Me thods Unfortunately, we have recently had access to fresh lymphocystis tissue

only from L e p o m i s and from mature tumors of Stizostedion. For electron microscopy small samples of such growths were fixed in Palade’s buffered osmic acid or in KMnO4 before methacrylate imbedding. Thin sections were examined with a Hitachi HU-11 electron microscope for virus structure, inclusion texture, and cellular detail. The present report includes only a small supplement to the published report on Stizostedion (Walker, 1962). Instead, the emphasis is on electron microscopic comparison with various sorts of material described by Weissenberg. For the sake of available pre-

Walker & Weissenberg : Lymphocystis Cells in Fish 377

served material we overrode the prejudice against electron microscopy of inadequately fixed tissue. Preliminary trials showed that, however inap- propriate the fixative might have been for cytoplasmic ultrastructure, the virus particles were usually stable in shape, and their topographic relations were reasonably well shown. By this compromise with technique it became possible to draw on other host species and on developmental stages, including material from some of which Weissenberg had published illustra- tions. Actually, cells from osmic acid fixation (Kopsch) or from formalin material originally postosmified were reasonably satisfactory for electron microscopy, even after reimbedding from paraffin to methacrylate. Old bottle-stored formalin specimens, postosmified shortly before methacrylate imbedding, were sometimes less satisfactory, but even they gave some valuable collateral information. Fo r general topography electron micrographs taken a t 2,500 x have been reproduced here a t ca. 4,000 x. For virus form or measurement, we include electron micrographs taken a t 10,000 x, now at 16,400 x. We have chosen to discuss material from Stizostedion v. vitreum (Mitchill) , Pleuronectes flesus L., and some from Cichlasoma syn- spilum Hubbs, to supplement the accompanying report by Weissenberg (19656).

Results FIGURE 1 shows a lymphocystis cell from a 105-day experimental infec-

tion of Stizostedion. This is one of the smaller cells, 150 p in diameter.

FIGURE 1. Small lymphocystis cell from a 105-day experimental infection of Stizostedion. Sjovall (25 per cent formalin, postosmified). n, nucleus; A-B, see text. x 416.

FIGURE 2. Mature lymphocystis cell from Stizostedion, showing a lacy network of inclusion substance, In. x 121.


FIGURE 3. Stizostedioiz lymphocystis cell with basophil inclusion net. Carnoy,

FIGURE 4. Comparable cell showing osmiophil granulation incrusting the in- hematoxylin. x 486.

clusion net. Formalin, postosmified. x 486.

Beside the large eccentric nucleus (n) is an inclusion body jus t beginning to assume complex form. For comparison, FIGURE 2 shows the lacy network of the expanded inclusion in the cytoplasm of a mature cell. The inclusion in FIGURE 1 shows many irregular regions of intense osmiophilia. The region from A to B of FIGURE 1 is given higher resolution in a drawing by Weissenberg (1949, FIGURE 7 ) in which the osmiophil pattern is composed of minute granules interpreted as elementary bodies like those of macro- viruses.

FIGURES 3 and 4 emphasize the contrast in light microscopy between the basophilic inclusion net seen with routine stains (FIGURE 3 ) , and the osmiophil granulation of a comparable cell (FIGURE 4 ) . The patterns strongly suggest tha t the basophil strands correspond to the pale cores in FIGURE 4 tha t are surrounded by osmiophil granules. Conversely, there is jus t a hint of pale granules associated with the basophilic strands in FIGURE 3.

Low-power electron micrographs of Stizostedion. lymphocystis cells (FIG- URES 5 t o 7 ) show the relative uniformity of size of the osmiophil virus particles. FIGURE 5 i s from the same fish as FIGURE 1 and, though Sjovall fixation is not ideal fo r cytoplasmic ultrastructure, i t is possible t o recog- nize microvillous cell surface ( M V ) a t the upper left, fairly homogeneous cortical cytoplasm, and the inclusion zone with a layer of virus particles incrusting the meshes of the relatively dense inclusion substance. FIGURES 6 and 7 of a Palade-fixed cell show, respectively, nucleus, cytoplasm, and inclusions ; and inclusions, cortex, and microvilli. I n these three figures the inclusion substance is without granules. It is recognizably different in texture from nearby cytoplasm, but it ranges from dark, dense texture in FIGURE 5 to pale in FIGURE 7. The icosahedral form of the particles, fur ther confirming their viral nature, will be discussed below.


Turning to Pleuronectes, FIGURES 8 and 9 (like 3 and 4 for Stizostedion) contrast the pattern of basophilic and osmiophilic substance. I n the inclusion zone they a re reciprocal. Electron micrographs at 4,100 x (FIGURES 10 and 11) again show the multiplicity of osmiophil granules, but these Fig- ures a re at opposite ends of the range of texture of the central substance (in both it would be basophilic). FIGURE 10 shows the densest inclusion texture, while FIGURE 11 shows the palest, loosest (brown versus clear types; Weissenberg, 1951a, p. 610). In FIGURE 10, at PG and elsewhere, pale granules a re visible closer to the dense inclusion core than a re the dark particles. A t the point indicated the plane of section clearly shows the pale granules separate from the inclusion core. However, tangential sections showing overlap might have suggested tha t the pale granules were an integral par t of the inclusion core. In FIGURE 11 the central masses of

FIGURES 5-7. Electron micrographs (EM), 4,050 x , of Stizostedion lymphocystis cells. Osmiophil virus particles incrusting bars of inclusion substance. For these and all other electron micrographs, the line is 1 p . Ca - capsule of cell; Cd - capsid of virus particle; Cy - cytoplasm; E R - endoplasmic reticulum; In - inclusion body or substance; Nd - nucleoid of virus particle; Nu - nucleus of cell ; PG - pale granule; V P - virus particle.

FIGURE 5. EM of 105-day cell. Formalin, postosmified. FICIJRE 6. EM of nuclear membrane, cytoplasm, and inclusion with incrusting

FIGURE 7. Similar Palade preparation, but with paler inclusion, and showing virus particles. Palade fixation.

microvillous cell surface.


FIGURES 8-13. See legends at bottom of next page.


virus particles are in fenestrations of the clear inclusion net where there is “new surface” folded in. The arrows in FIGURE 11 point to configurations previously described by Weissenberg (1960, figure 12 B,d ; 1965a, figure 4,b) in the same Pleuronectes material, as “paired rods” of the type surrounded by a clear zone.

The formalin material of Ciclilasomn available to us was not good for ultrastructure ; nevertheless there are several noteworthy points. The es- sential absence of microvilli (FIGURE 13) seems to hold for cells of all sizes. A few mitochondria are in the peripheral cytoplasm. Inclusion substance may be either dark or clear; in FIGURE 13 the texture of the clear inclusion appears very much like that of the nucleus (not shown) except for the absence of a limiting membrane, as is also true of early inclusions in Lepomis . This lobed, pale inclusion (FIGURE 13, I n ) , surrounded by a thin layer of osmiophil virus particles ( V P ) , probably represents the type of inclusion described by Weissenberg (1965b, Figure 17) from the peripheral region of advanced Cichlasoma cells.

In FIGURE 12 some small crystalloid arrays of virus particles are visible. Such arrays are much commoner in this Cichlusoma material than in Stizo- t ed ion or Pleuronectes. Lepomis also shows considerable crystalloid array of virus particles in mature lymphocystis cells (Walker & Wolf, 1962).

The arrow points to a special configuration like the “paired dumbbells” discussed by Weissenberg (1960) in Plpuronectes. The present electron micrograph (FIGURE 12, 4,lqO x) shows that the osmiophil globes of the dumbbells are essentially sim’ilar to those virus particles in adjacent crystalloid array. On the basis of this Cichlasoma section, Weissenberg ( 1 9 6 5 ~ ) has advanced the opinion that a relation may exist between pairing of osmiophil rods and dumbbells and the arrangement of virus particles in the rows of crystalloid array.

FIGURES 14 to 18, all at 16,400 x, show the characteristic osmiophil virus particles of three species and under different conditions of fixation. FIGURE 14 shows a strongly osmified preparation of Stizostedion lymphocystis after formalin-osmic treatment. No internal texture shows in the particles, but even if a trifle shrunken or distorted, most of the particles show hexa-

FIGURES 8-13. FIGURES 8 and 9 a r e Plei~ronectes lymphocystis cells, showing inclusion zone and cortex. x 492. FIGURES 10 and 11 are Pleuronectes inclusions. EM, x 4,100. FIGURES 12 and 13 are Cichlasowta lymphocystis cells. Formalin, postosmified. EM, x 4,100.

FIGURE 8. Basophil inclusion net. Formalin, hematoxylin. FIGURE 9. Pale inclusion net incrusted with osmiophil granules. Formalin,

FIGURE 10. Dark inclusion substance, virus particles, and pale granules. FIGURE 11. Clear inclusion substance and virus particles. (Arrows) see text.

FIGURE 12. Virus particles in crystalloid array. (Arrow) see text. FIGURE 13. Clear inclusion with virus particles at i ts surface.

postosmified.

Formalin, postosmified.


FIGURES 14-18. E M of lymphocystis virus, x 16,400. See legends at bottom of next page.


gonal profiles. One pale granule, PG, is essentially full size, yet not sharply enough delineated to be a well-formed empty capsid. The more delicate Palade fixation and osmification of FIGURE 15 shows a clear distinction between polygonal capsid profile and the central nucleoid. The latter may show a tangled filamentous texture (Walker, 1962). The pale line between capsid and nucleoid is not a gap; in empty capsids this is darker than the central space. Around the capsid is a faint shadow of denser texture than the cytoplasmic matrix. This mantle seems to keep the capsids spaced apart, even in the closest pack. Measurement of capsid diameter a t about 200 to 220 m p is more precise than an earlier estimate of 375 m p (Weissenberg, 1949).

FIGURES 16 and 17, still a t 16,400 x, seem to show Pleuronectes virus particles significantly smaller than those of either Stizostedion or Cichla- soma; perhaps 130 to 150 m p instead of 200 to 220 m p in diameter. The smaller size of Pleuronectes virus particles was seen so often as to be con- sidered characteristic. We have, however, a more recent specimen from the Irish Sea (formalin fixation), large-celled, with dark inclusions and very few virus particles. Those which do show have a dense nucleoid ( ? ) of about 130 mp, surrounded by a pale membrane (capsid?) about 200 m p across. It is difficult to believe that the more commonly seen Pleuronectes virus particle is only a nucleoid (why then icosahedral?) ; and besides, the particles are often center-spaced a t less than 200 mp, so we cannot postulate an invisible capsid.

Again pale granules may be seen in these Pltwronectes cells, and a t PG in FIGURE 17 a tangential section may account for the appearance of con- tinuity of pale granules with the substance of the dark inclusion core. This pattern has been seen in formalin-osmic and Sjovall preparations of both PEezronectes and Stizostedion and was interpreted by Weissenberg as developmental stages of the elementary bodies. However, pale granules have not been clearly recognized in Palade or KMnOA preparations of Stizosted- ion or Lepomis, and in these an alternative “developmental stage” may be seen (Walker & Wolf, unpublished). At the surface of inclusion bodies, besides complete particles and a few empty capsids, a certain number of segmentally incomplete (half-formed ? ) capsids may be seen, with icosahedral angles already recognizable. The formed segment shows both the osmio-

FIGURE 14. Stizostedion. Formalin, postosmified. Hexagonal 200 mp profiles of capsids: pale granule.

FIGURE 15. Stizostedion. Palade. Virus particles show capsids and nucleoids ; endoplasmic reticulum is sparse.

FIGURES 16 and 17. Pleuronectes. Smaller virus particles. FIGURE 16 has clear inclusion substance. Formalin, postosmified. FIGURE 17 has dark inclusion substance ; hexagonal virus profiles and pale granules.

FIGURE 18. Cich lasom. Formalin storage, postosmified. Hexagonal 200 ma virus profiles in crystalloid array.


phi1 shell and the paler lining, bu t is empty of nucleoid. The gap in capsid structure is generally turned toward the inclusion substance. The two clear pictures suggesting different mechanisms of virus particle maturation a re not due to species difference since both pictures a re seen in Stixostedion. (See the discussion below. )

FIGURE 18 of Cich~asoma virus supplements FIGURE 12 to the extent of suggesting the icosahedral form of the particles in crystalloid array, and indicating a n approximate size of 200 mp.

Discussion and Concliisions It is clear t ha t the uniform small osmiophil granules of lymphocystis

cells tha t Weissenberg studied by light microscopy do indeed correspond to icosahedral virus particles of electron microscopy, as judged by their osmiophilia and by comparison of their topographic relation to inclusion bodies a t different stages and in different species. Weissenberg’s “pale granules of the inclusion substance” a re clearly recognizable in electron micrographs. Their possible status as a stage in virus maturation is com- plicated by an alternative structural pattern seen a f te r Palade fixation : segmentally incomplete capsids with the incomplete segment toward the inclusion substance.

The concept of inclusion body o r inclusion substance in lymphocystis cells must be understood in context. The striking cytoplasmic pattern of many osmified cells (e.g., FIGURES 4 and 9 ) includes both a pale core network and the incrusting or cortical virus particles. The pale core or me- dulla in these and other cells (FIGURES 7, 11, and 13) would be Feulgen positive, as would the dark inclusion core of FIGURES 5 or 10. This central core of the inclusion (inclusion substance) may safely be interpreted as the replicating pool of viral DNA or DN-protein (Walker, 1965).

The method of segregating units of this genetic material into individual protein capsids during virus maturation is not yet clear. It may be that the paler granules of FIGURES 10 and 17 represent nucleoids condensing from the surface of the DNA core, and tha t the darkly osmiophil particles of the outer layer have had protein capsids added. The alternative picture may merely indicate that capsid formation is not dependent on the presence of nucleoids (well-formed, empty, and noninfective capsids a re known in many other viruses) and that DNA material is received into the capsids shortly before their completion. If these a re not contradictory concepts, the two pictures would have to represent a relative shift in timing of the two proc- esses of nucleoid segregation and capsid formation. I n the first case, nucleoid formation may be essentially complete before capsid protein is added ; in the second, capsid formation may be well started before the genetic unit separates from the DNA of the inclusion core.


The icosahedral virus particles have the same size range (about 200 mp) in three species, while in Pleuronectes they seem to be significantly smaller.

Aggregation of mature virus particles elsewhere than at inclusion sur- faces may lead to crystalloid array in Cichlnsonia or in Lepomis. Such array is rare in Stizostedion and Pleuronectes.

Acknowledgments We wish to thank Sharon Chowdhuri, Richard Sano, and Patricia Bishko

for technical assistance. We renew our thanks, expressed in previous pa- papers, to many people who have helped us in gathering the lymphocystis material now reexamined; and to N. V. Craven, Superintendent, Lanca- shire and Western Sea Fisheries Joint Committee, Preston, Lancs., for further Pleuronectes material.

Refercnces WALKER, R. 1962. Fine structure of lymphocystis virus of fish. Virology 18:

503-505. WALKER, R. 1965. Viral DNA and cytoplasmic RNA in lymphocystis cells of fish.

Ann. N.Y. Acad. Sci. This Annal. WALKER, R. & K. WOLF. 1962. Virus a r ray in lymphocystis cells of sunfish.

Am. Zoologist 2: 566. WEISSENBERG, R. 1914. Uber infektiose Zellhypertrophie bei Fischen (Lympho-

cystiserkrankung) . Sitzber. kgl. preuss. Akad. Wiss. Jg. 1914: 792-804. WEISSENBERG, R. 1949. Studies on lymphocystis tumor cells of fish. I. Cancer

Res. 9: 537-542. WEISSENBERG, R. 1 9 5 1 ~ . Studies on lymphocystis tumor cells of fish. 11. Cancer

Res. 11: 608-613. WEISSENBERG, R. 1951b. Positive result of a filtration experiment supporting the

view tha t the agent of the lymphocystis disease of fish is a t rue virus. Anat. Record 111: 166-167.

WEISSENBERG, R. 1960. Some remarkable osmiophilic structures of the inclusion bodies in the lymphocystis virus disease of the European flounder. Arch. Ges. Virusforsch. 10: 253-266.

WEISSENBERG, R. 1 9 6 5 ~ . Fif ty years of research on the lymphocystis virus disease of fish (1914-1964). Ann. N.Y. Acad. Sci. This Annal.

WEISSENBERG, R. 19656. Morphological studies on the lymphocystis tumor cells of a cichlid from Guataniala, Cichlnsoma synsp'lum Hubbs. Ann. N.Y. Acad. Sci. This Annal.

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CONFORMITY OF LIGHT AND ELECTRON MICROSCOPIC STUDIES ON VIRUS PARTICLE DISTRIBUTION IN LYMPHOCYSTIS TUMOR CELLS OF FISH