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Fine structure of lymphocystis virus of fish
DISCUSSION
AND
PRELIMINARY
REPORTS
503
ules at the surface of inclusions, and paler granules in the inclusion substance itself (presumably in the Feulgen-positive zone). He interpreted the osmiophile granules as elementary bodies of the virus, and the ACKNOWLEDGMENTS paler granules as developmental stages. In The author wishes to acknowledge the financial view of the inherent limitations of light miassistance provided by the United Fund of Austin croscopy it has seemed desirable to recxand Travis County, Cancrr Research Fund-Grant, amine the osmiophile granules. for a portion of this work. He is indebted to Mrs. Mature lymphocystis warts were obMary Lou Gammans and Mrs. Betty S. Gibson tained during the April spawning run of for their tcrhnical assistance. Stizostedion at, the New York State HatchREFERENCES ery at Constantia on Lake Oneida. Lym1. MOLLENIIAUER, H., J. Biophysic. Biochem. Cyphocystis cells were fixed in buffered ostol.6, 431-35 (1959). mium tetroxide and sectioned for electron 2. GLAUERT, A. M., and GLAUERT, R. H., J. Biomicroscopy. Virus particles are abundant in physic. Biochem. Cytol. 4, 191-94 (1958). the cytoplasm (Fig. l), especially in the 3. DALTON, A. J., Anat. Record 121, 281 (1955). inclusion zone, which is, howcrer, not 4. MILLONIG, G., J. Biophysic. Biochem. Cytol. sharply delimited. Virus was not seen in 11,73639 (1961). the nucleus, nor outside the giant ~11s. The 6. BERNHARD, iv., Came?" Rcseurch 20, 712-27 particles are completely embedded in the (1960). cytoplasmic matrix and are not signifi6. MOORE, D. H., “Tumors Induced by Viruses: cantly related to the endoplasmic reticulum. Ultrastructural Studies” (A. J. Dalton and F. Haguenau, eds.), pp. 113-150. Academic The latter is sparse, and ribosomes arc Press, New York, 1962. found scat,tered rat,her than restricted to 7’. IJcl~~, J. H., J. Riophysic. Biochem. Cytol. 2, the reticular membranes. Mitochondria are 799-801 (1956) also not closely related to the denser areas NORTOK G. MCDUFFIE, JR. of virus accumulation. The condition of Clayton Foundation Biochemical Institute mitochondria and cndoplasmic ret,iculum l,~'ti7,wsity of Texas might imply relatively slow metabolism and Austin 12, Texas synthesis, and it should be noted that the Received July 23, 1962 great cell size and virus quantity are the result, of months of growth. The present maFine Structure of Lymphocystis Virus of Fish terial includes no developmental stages. In Lymphocystis disease of the pike perch these Stixostedion cells virus part,icles have Stizostedion vitreum (Mitchill) is essen- not yet, been seen in crystalline array, tially similar to that of various other fresh- though in the sunfish (Lepomis; unpublished) there is occasionally an elegant water and marine fish species. Skin warts lattice. are composed of enormously hypertrophied The virus particles are large and polyconnective tissue cells, up t,o a millimeter in hedral (Figs. l-3). There is a sharply osdiameter. In each, the nucleus is propormiophile capsule (capsid) about 200 rnp in tionately enlarged and the cytoplasm condiameter (180-220 nip). The capsid is about tains a rich network of Feulgen-positive 12 mp thick, and there is a 12-rnp pale space inclusion bodies. The cells are heavily ensurrounding the ljO-mp nucleoid. The latter capsulated, but after a growth period of appears as a ball of osmiophile threads several months they slough out, presumably roughly 10 1~~ thick. The commonest capsid releasing virus. Weissenberg described the are hexagonal ; a frw smaller prodisease in European perches and flounders profiles (1) and has described developmental stages files are pentagons. These are compatible in Stizostedion (2). Virus was implicated in with sections of regular icosahedra. However capsomere arrangement is not clear the disease by transfer experiments (S-5) and by microscopy of the inclusion bodies. enough to verify the three-dimensional Weissenberg (6) showed osmiophile gran- form; and capsomere number is uncertain. mammary tumors fixed wit,h KMn04 should provide more information on relations hetween cellular membrane systems and the milk agent.
504
DISCUSSION
AND
PRELIMINARY
REPORTS
FIGS. l-3. Virus particles in the cytoplasm of giant lymphocystis cells of Stizostedion. Palade fixation, methacrylate embedding. The line is a micron. Magnification: Fig. 1, X 30,000; Figs. 2 and 3, X 60,000.
Outside the osmiophile capsid, and not included in the 200-rnp diameter, there is a faint zone of denser material. These outer envelopes tend to hold the closest osmiophile capsids about 40 rnp apart; i.e., each has an effective thickness of 20 rnp though it shows no outer limiting line. There may be faint radial texture in this zone (Fig. 2) or coarser radial structure (Fig. 3). The
radial condensations may be more obvious toward adjacent c.apsids (Fig. 2). Aside from this loose and random linking of adjacent particles, the present material gives little support for Weissenberg’s emphasis (‘7) on bipolar rods, dumbbells, tetrads, and paired rods. This study has shown that in Stizostedion the lymphocystis giant cells do indeed con-
DISCUSSION
AND
PRELIMINARY
tain characteristic virus particles, identical with Weissenberg’s osmiophile granules. The particles are 200 rnp in diameter and have polyhedral capsids surrounding nucleoids, presumably of DN-protein. Studies in progress include ultrastructural comparison of the related lymphocystis virus and giant cells in other species of fish, developmental studies in the sunfish, and ultrastructural studies of a virus tumor also found in Stizostedion from Lake Oneida (8, 9). ACKNOWLEDGMENTS I want to thank Dr. Richard Weissenberg for much stimulating discussion, Dr. John R. Greeley and other members of the New York State Conservation Department for material, the New York State Health Department Laboratories for the use of a Siemens Elmiskop I, and especially Dr. James H. McAlear for guidance in electron microscopy. Supported in part by N.I.H. grant C-5790. REFERENCES I. WEISSENBERG,
Wiss.
R.,
Sitzber.
kgl.
preuss.
R., Zoologica 24, 245-254 K., Biol. Sp-isy Vysokk Skoly 7, l-14 (1928) (Biol. Abstr. 5). WEISSENBERG, R., Anat. Record 111, (1951). WOLF, K., Virology 18, 219-256 (1962). WEISSEKBERG, R., Cancer Research 9, (1949). WEISSENBERG, R., Arch. ges. Virusforsch. 253-266 ( 1960). WALKER, R., N. Y. State Conservationist 28-29 ( 1957 ) . WALKER, R., Am. Zoologist 1, 395-396
2. WEISSENBERG,
3. RA&~N, Brno
4. 5. 6. 7. 8.
9.
Akad.
Jg. 1914, 792-804.
ROLAND
Department Rensselaer Troy, New Received
166-167
537-542
10, 12 (2), (1961). WALKER
of Biology Polytechnic Institlcte York August 13, 19@?
Immunofluorescent Transformation
(1939). Zve’rol.
Tracing Experiments
of
Polyoma with
BHK
Virus
in
21 Cells
When mouse or hamster cells are exposed in vitro to polyoma virus, the cell cultures may be transformed. They acquire new growth characteristics and the ability to produce tumors when injected into animals of their respective species (1, 2). Sys-
REPORTS
505
terns for assaying the transformation of hamster cells by polyoma virus were devised by Stoker and MacPherson (3) and Stoker and Abel (4), who emphasize the low proportion of transformed cells, even at high virus-cell ratios. This is not accounted for by genetic differences in the cell population (5, 6), and there must be other limiting factors. The nature of the association between virus and cell in the course of transformation is unknown. It may be possible to demonstrate that cells which are not transformed never take up virus or that interaction of virus and cell fails in some other detectable way. Accordingly, we applied the fluorescent’ antibody tcchniquc, which has already been used to study the sequential processes of infection at high multiplicities (7, 8) in order t.o discover the distribution of virus antigen in the cells of the transformation system. Following Stoker and Abel (4)) we used the same polyoma virus strain and clone of BHK 21 cells and followed their method precisely at a ratio of lo9 virus plaque-forming units (PFU) to lo6 cells, which gives transformation rates between 1% and 4%. For fluorescent-staining, noninfected and infected cells were plated over glass coverslips at lo6 cells per 60-mm plate. Five or 6 hours later, virus was washed off three times in buffered saline, medium was replenished, and incubation was continued. Sample coverslips were removed at intervals for st.aining. hcetone fixation at room temperature and indirect staining with various rabbit antipolyoma sera followed by fluorescein or R.B.200 [Chadwick et al. (9)] conjugates of antirabbit globulin were very successful, but direct staining with R.B.200-conjugated antipolyoma serum was also possible. The usual criteria of specificity were upheld, namely, staining of infected cells by immune serum, lack of staining of noninfected cells by immune serum, lack of staining of infected cells by preimmune or heterologous sera, and, in the direct test, specific blocking by nonconjugated immune serum. In addition, the amount of staining was proportional to the input of polyoma virus. There were two significant results. Early
AND
PRELIMINARY
REPORTS
503
ules at the surface of inclusions, and paler granules in the inclusion substance itself (presumably in the Feulgen-positive zone). He interpreted the osmiophile granules as elementary bodies of the virus, and the ACKNOWLEDGMENTS paler granules as developmental stages. In The author wishes to acknowledge the financial view of the inherent limitations of light miassistance provided by the United Fund of Austin croscopy it has seemed desirable to recxand Travis County, Cancrr Research Fund-Grant, amine the osmiophile granules. for a portion of this work. He is indebted to Mrs. Mature lymphocystis warts were obMary Lou Gammans and Mrs. Betty S. Gibson tained during the April spawning run of for their tcrhnical assistance. Stizostedion at, the New York State HatchREFERENCES ery at Constantia on Lake Oneida. Lym1. MOLLENIIAUER, H., J. Biophysic. Biochem. Cyphocystis cells were fixed in buffered ostol.6, 431-35 (1959). mium tetroxide and sectioned for electron 2. GLAUERT, A. M., and GLAUERT, R. H., J. Biomicroscopy. Virus particles are abundant in physic. Biochem. Cytol. 4, 191-94 (1958). the cytoplasm (Fig. l), especially in the 3. DALTON, A. J., Anat. Record 121, 281 (1955). inclusion zone, which is, howcrer, not 4. MILLONIG, G., J. Biophysic. Biochem. Cytol. sharply delimited. Virus was not seen in 11,73639 (1961). the nucleus, nor outside the giant ~11s. The 6. BERNHARD, iv., Came?" Rcseurch 20, 712-27 particles are completely embedded in the (1960). cytoplasmic matrix and are not signifi6. MOORE, D. H., “Tumors Induced by Viruses: cantly related to the endoplasmic reticulum. Ultrastructural Studies” (A. J. Dalton and F. Haguenau, eds.), pp. 113-150. Academic The latter is sparse, and ribosomes arc Press, New York, 1962. found scat,tered rat,her than restricted to 7’. IJcl~~, J. H., J. Riophysic. Biochem. Cytol. 2, the reticular membranes. Mitochondria are 799-801 (1956) also not closely related to the denser areas NORTOK G. MCDUFFIE, JR. of virus accumulation. The condition of Clayton Foundation Biochemical Institute mitochondria and cndoplasmic ret,iculum l,~'ti7,wsity of Texas might imply relatively slow metabolism and Austin 12, Texas synthesis, and it should be noted that the Received July 23, 1962 great cell size and virus quantity are the result, of months of growth. The present maFine Structure of Lymphocystis Virus of Fish terial includes no developmental stages. In Lymphocystis disease of the pike perch these Stixostedion cells virus part,icles have Stizostedion vitreum (Mitchill) is essen- not yet, been seen in crystalline array, tially similar to that of various other fresh- though in the sunfish (Lepomis; unpublished) there is occasionally an elegant water and marine fish species. Skin warts lattice. are composed of enormously hypertrophied The virus particles are large and polyconnective tissue cells, up t,o a millimeter in hedral (Figs. l-3). There is a sharply osdiameter. In each, the nucleus is propormiophile capsule (capsid) about 200 rnp in tionately enlarged and the cytoplasm condiameter (180-220 nip). The capsid is about tains a rich network of Feulgen-positive 12 mp thick, and there is a 12-rnp pale space inclusion bodies. The cells are heavily ensurrounding the ljO-mp nucleoid. The latter capsulated, but after a growth period of appears as a ball of osmiophile threads several months they slough out, presumably roughly 10 1~~ thick. The commonest capsid releasing virus. Weissenberg described the are hexagonal ; a frw smaller prodisease in European perches and flounders profiles (1) and has described developmental stages files are pentagons. These are compatible in Stizostedion (2). Virus was implicated in with sections of regular icosahedra. However capsomere arrangement is not clear the disease by transfer experiments (S-5) and by microscopy of the inclusion bodies. enough to verify the three-dimensional Weissenberg (6) showed osmiophile gran- form; and capsomere number is uncertain. mammary tumors fixed wit,h KMn04 should provide more information on relations hetween cellular membrane systems and the milk agent.
504
DISCUSSION
AND
PRELIMINARY
REPORTS
FIGS. l-3. Virus particles in the cytoplasm of giant lymphocystis cells of Stizostedion. Palade fixation, methacrylate embedding. The line is a micron. Magnification: Fig. 1, X 30,000; Figs. 2 and 3, X 60,000.
Outside the osmiophile capsid, and not included in the 200-rnp diameter, there is a faint zone of denser material. These outer envelopes tend to hold the closest osmiophile capsids about 40 rnp apart; i.e., each has an effective thickness of 20 rnp though it shows no outer limiting line. There may be faint radial texture in this zone (Fig. 2) or coarser radial structure (Fig. 3). The
radial condensations may be more obvious toward adjacent c.apsids (Fig. 2). Aside from this loose and random linking of adjacent particles, the present material gives little support for Weissenberg’s emphasis (‘7) on bipolar rods, dumbbells, tetrads, and paired rods. This study has shown that in Stizostedion the lymphocystis giant cells do indeed con-
DISCUSSION
AND
PRELIMINARY
tain characteristic virus particles, identical with Weissenberg’s osmiophile granules. The particles are 200 rnp in diameter and have polyhedral capsids surrounding nucleoids, presumably of DN-protein. Studies in progress include ultrastructural comparison of the related lymphocystis virus and giant cells in other species of fish, developmental studies in the sunfish, and ultrastructural studies of a virus tumor also found in Stizostedion from Lake Oneida (8, 9). ACKNOWLEDGMENTS I want to thank Dr. Richard Weissenberg for much stimulating discussion, Dr. John R. Greeley and other members of the New York State Conservation Department for material, the New York State Health Department Laboratories for the use of a Siemens Elmiskop I, and especially Dr. James H. McAlear for guidance in electron microscopy. Supported in part by N.I.H. grant C-5790. REFERENCES I. WEISSENBERG,
Wiss.
R.,
Sitzber.
kgl.
preuss.
R., Zoologica 24, 245-254 K., Biol. Sp-isy Vysokk Skoly 7, l-14 (1928) (Biol. Abstr. 5). WEISSENBERG, R., Anat. Record 111, (1951). WOLF, K., Virology 18, 219-256 (1962). WEISSEKBERG, R., Cancer Research 9, (1949). WEISSENBERG, R., Arch. ges. Virusforsch. 253-266 ( 1960). WALKER, R., N. Y. State Conservationist 28-29 ( 1957 ) . WALKER, R., Am. Zoologist 1, 395-396
2. WEISSENBERG,
3. RA&~N, Brno
4. 5. 6. 7. 8.
9.
Akad.
Jg. 1914, 792-804.
ROLAND
Department Rensselaer Troy, New Received
166-167
537-542
10, 12 (2), (1961). WALKER
of Biology Polytechnic Institlcte York August 13, 19@?
Immunofluorescent Transformation
(1939). Zve’rol.
Tracing Experiments
of
Polyoma with
BHK
Virus
in
21 Cells
When mouse or hamster cells are exposed in vitro to polyoma virus, the cell cultures may be transformed. They acquire new growth characteristics and the ability to produce tumors when injected into animals of their respective species (1, 2). Sys-
REPORTS
505
terns for assaying the transformation of hamster cells by polyoma virus were devised by Stoker and MacPherson (3) and Stoker and Abel (4), who emphasize the low proportion of transformed cells, even at high virus-cell ratios. This is not accounted for by genetic differences in the cell population (5, 6), and there must be other limiting factors. The nature of the association between virus and cell in the course of transformation is unknown. It may be possible to demonstrate that cells which are not transformed never take up virus or that interaction of virus and cell fails in some other detectable way. Accordingly, we applied the fluorescent’ antibody tcchniquc, which has already been used to study the sequential processes of infection at high multiplicities (7, 8) in order t.o discover the distribution of virus antigen in the cells of the transformation system. Following Stoker and Abel (4)) we used the same polyoma virus strain and clone of BHK 21 cells and followed their method precisely at a ratio of lo9 virus plaque-forming units (PFU) to lo6 cells, which gives transformation rates between 1% and 4%. For fluorescent-staining, noninfected and infected cells were plated over glass coverslips at lo6 cells per 60-mm plate. Five or 6 hours later, virus was washed off three times in buffered saline, medium was replenished, and incubation was continued. Sample coverslips were removed at intervals for st.aining. hcetone fixation at room temperature and indirect staining with various rabbit antipolyoma sera followed by fluorescein or R.B.200 [Chadwick et al. (9)] conjugates of antirabbit globulin were very successful, but direct staining with R.B.200-conjugated antipolyoma serum was also possible. The usual criteria of specificity were upheld, namely, staining of infected cells by immune serum, lack of staining of noninfected cells by immune serum, lack of staining of infected cells by preimmune or heterologous sera, and, in the direct test, specific blocking by nonconjugated immune serum. In addition, the amount of staining was proportional to the input of polyoma virus. There were two significant results. Early