Detection and Localization of Aleutian Disease Virus and its Antigens in vivo by Immunoferritin Technique M. S. Shahrabadi and H. J. Cho ABSTRACT
On demontra la presence de particules d'aspect viral et d'un diametre d'environ 22 nm, dans Tissues from mink infected with aleutian des macrophages de la rate, des ganglions disease virus were examined by the electron lymphatiques mesenteriques et dans les cellumicroscope for the presence of virus par- les de Kupffer dans du foie, de dix i 13 jours ticles. Virus-like particles, measuring 22 nm apres l'infection. Ces particules occupaient in diameter, were observed in macrophages of ordinairement des vacuoles cytoplasmiques des spleen, mesenteric lymph node and in Kupffer macrophages et les cellules de Kupffer et on cells in liver of mink ten to 13 days after in- les retrouvait aussi de temps en temps a l'infection. The virus-like particles were usually terieur du noyau. Les cellules des visons tepresent in vacuoles inside the cytoplasm of moins ne recelaient pas de telles particules. macrophages and Kupffer cells and, occa- Afin d'etablir une relation entre la presence de sionally, similar particles were observed in- ces particules et celle du virus de la maladie des side the nucleus. Cells from uninfected mink Iles Aleoutiennes, on examina les tissus infecdid not contain such patricles. To correlate tes a l'aide de la technique 'a l'immunoferritine. the existence of these virus-like particles with On demontra ainsi la presence de ce virus dans the presence of aleutian disease virus antigen les vacuoles cytoplasmiques des macrophages in infected cells, tissues were processed for de la rate, des ganglions lymphatiques mesenimmunoferritin technique. It was found that teriques et du foie des visons infectes; on reaaleutian disease virus antigen was present lisa aussi que la localisation du virus corresin vacuoles inside the cytoplasm of cells from pondait it celle des particules qui ressemblaient the infected spleen, lymph node and liver, and it un virus et dont le diametre atteignait 22 that the location was similar to that of the 22 nm. On decela aussi de l'antigene viral, sous nm virus-like particles. In addition, some viral la forme de materiel cytoplasmique granuleux. antigen was also detected as cytoplasmic Le noyau de certaines cellules infectees congranular material. The nuclei of some cells tenait egalement le virus de la maladie des also contained aleutian disease virus antigen. Iles Ale'outiennes. La distribution de ce virus The pattern of aleutian disease virus antigen i l'interieur des cellules infectees corresponwas similar to the distribution of virus-like dait 'a celle des particules ressemblant 'a un particles in cells of infected tissue. It is sug- virus. II semble par consequent que le virus gested that virus replication occurs inside the se multiplie 'a l'interieur du noyau et s'accunucleus with subsequent accumulation of mule ensuite dans les vacuoles cytoplasmivirus in the vacuoles of the cytoplasm. ques.
RESUME
Cette experience visait particules virales, 'a l'aide electronique, dans certains atteints de la maladie des
'a rechercher des de la microscopie organes de visons Iles Aleoutiennes.
*Department
of Medical Bacteriology, University of Alberta, Edmonton, Alberta T6G 2E1 (Shahrabadi) and Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta TlJ 3Z4 (Cho). Slbmitted November 19, 1976.
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October, 1977
INTRODUCTION Aleutian disease (AD) is a common persistent viral infection of mink which causes serious economic loss to commercial mink ranches throughout the world. The disease is characterized by generalized plasmacytosis, hypergammaglobulinemia and immunologically-mediated lesions (8, 9, 22). The
435
progress of the disease is "slow", although the initial replication of the aleutian disease virus (ADV) in vivo is rapid and peak viral titers are found at ten days after experimental infection of spleen, liver and lymph node, coincident with the development of anti-ADV antibody (19). Extremely high titers of ADV antibody were detected from serum of hypergammaglobulinemic mink (4, 14, 19). Circulating virusantibody complexes (18) are associated with immune complex arteritis (21) and glomerulonephritis (17, 19). Of particular interest, the role of host immune response in causing the disease condition has been shown by the prevention of lesions by immunosuppression (2) and by the enhancement of lesions by immunization with an inactivated virus vaccine followed by live virus challenge (20). Aleutian disease virions have been purified and visualized from infected mink tissues (3, 5, 11) and from cell cultures (23). Employing immunofluorescent techniques, Porter and co-workers (19) extensively examined ADV infected mink tissues. They reported finding viral antigen in the cytoplasm of macrophages in the spleen and lymph node and in Kupffer cells in the liver. These findings have been confirmed recently by others (8). In the present investigation, we examined ADV infected mink spleen, liver and mesenteric lymph node collected at ten to 13 days after infection by the electron microscope to identify the ADV in vivo. We also applied an immunoferritin technique to detect and localize the presence of ADV antigen in the cells from infected mink tissues and to determine the cell types the ADV replicates in vivo.
MATERIALS AND METHODS ANIMALS Mink (Mustela vison) with a violet coat colour which were homozygous for the autosomal recessive aleutian gene were purchased from a commercial mink ranch and maintained at the Animal Diseases Research Institute (Western). The mink were caged individually and fed a standard ranch diet. The mink were negative for ADV antibody
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as tested by counterimmunoelectrophoresis (4). The mink were vaccinated at the age of six months with live distemper vaccine, Clostridiium botulinurm type C toxoid and a formalin-inactivated mink viral enteritis vaccine. ALEUTIAN DISEASE VIRUS, INOCULUM AND COLLECTION OF TISSUES
Guelph strain of ADV (4, 10) was propagated in violet coat coloured mink. Virus was purified by fluorocarbon extraction and equilibrium density centrifugation in cesium chloride as described in an earlier publication (6). Aleutian disease virus, banded in a buoyant density of 1.405-1.416 g/ml, was inoculated intraperitoneally into eight mink. Each mink received 10" ID50 of the purified virus. Two mink were necropsied on each day from the tenth to the 13th day after infection and spleen, liver and mesenteric lymph node were collected. These organs were processed immediately for electron microscopy. The same tissues were collected from two uninfected mink and processed similarly. PROTEIN DETERMINATION
The protein concentration of the gammaglobulin fraction of mink serum was determined by the method of Lowry et al (13). CONJUGATION OF FERRITIN TO GAMMAGLOBULIN
Conjugation of ferritin to gammaglobulin was performed according to the method of Nicholson et al (15). Briefly, ferritin (E.M. Grade, Polyscience Inc., Rydal, Pa.) was recrystallized using cadmium sulfate precipitation and conjugated to gammaglobulin using toluene 2-4-diisocyanate. The crude conjugate was further purified by passing it through a column of agarose A-1.5 (Biorad 200-400 Mesh.). ELECTRON MICROSCOPY For morphological studies, tissues from three infected mink taken on the tenth, 12th and 13th days after infection and from one normal mink were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 at
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4°C for two hr. They were washed in 0.1 M phosphate buffer overnight, postfixed in osmium tetroxide and embedded in epon 812. For immunoelectron microscopy, tissues taken from the other five infected and one normal mink were fixed in freshly prepared 4% formaldehyde in 0.1 M phosphate buffer pH 7.2 at 4°C for 30 minutes. They were washed with the phosphate buffer (24 h, three changes of buffer, at 4°C) and embedded in water soluble glycol methacrylate (12). Thin sections were cut and treated with ferritin conjugated gammaglobulin as described previously (24). After final washing, sections were stained with uranyl acetate and examined in a Phillips EM 300 electron microscope.
RESULTS Spleen, liver and mesenteric lymph nodes of mink infected with ADV were processed for electron microscopy. For comparison, tissues from uninfected mink were similarly prepared and examined. In cells from mink ten days after infection, granules and particles of 22 nm in diameter could be observed in the cytoplasm. These granules resembling virus particles were densely stained and could be differentiated from glycogen aggregates. Their presence was confined only to the cells from infected mink. The virus-like particles observed in tissue after epon embedding and positive staining were 1-2 nm smaller than the virus observed with negative staining of fluorocarbon purified virus (3, 5, 6, 23). The virus-like particles were mainly observed in macrophages of spleen and lymph node and Kupffer cells of liver but not in hepatocytes. There, they were usually surrounded by a membrane or membrane-delimited and thus appeared as cytoplasmic vacuoles of various sizes containing virus-like particles (Figs. 1 and 2). About 10% of the cells of infected spleen in these sections contained virus-like particles. Examination of a large number of sections from tissues of uninfected mink did not reveal the presence of such particles. The particles were always present in scattered form and no crystalline aggregation of them was observed in all of the tissues examined. Tissues of mink 12 and 13 days postinfection were also exam-
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ined and similar virus-particles were seen (Fig. 3) and the number of cells containing virus-like particles seemed to be similar to the number in those tissues collected at ten days postinfection. DEMONSTRATION OF ADV ANTIGEN IN CELLS FROM INFECTED MINK
Tissues of infected mink were fixed in formaldehyde and embedded in glycol methacrylate. Under these conditions the antigenicity of the embedded material has beeni shown to be preserved (24, 25). The disadvantage of this method of fixation and embedding was the lack of good preservation of the cellular fine structure. Membrane structures were not very distinct and mitochondria were not well preserved. In addition, in order to visualize ferritin molecules with a good contrast, thin sections were not stained with lead citrate. This also caused difficulties in making a clear distinction of the fine structure of cellular components. Treatment of thin sections of liver, spleen and lymph nodes from mink infected with ADV revealed the presence of ADV antigen in some cells as they were labelled with ferritin conjugated to ADV antibody. Viral antigen was present mainly in the cytoplasm and was usually found in membrane bound vacuoles (Fig. 4). Because of the lack of good preservation due to the fixation and embedding, no structures resembling virus-like particles could be seen inside these membrane bound vacuoles. The vacuoles containing the viral antigen varied in size and number and were always present in the cytoplasm of cells. About 12-15% of cells from the spleen and lymph nodes of infected animals contained ADV antigen. Viral antigen was also detected in the cytoplasm of some cells as being associated with some granular dark staining material (Fig. 5). Presence of ADV antigen was not confined to the cytoplasm of cells. In some cells from infected spleen, liver and lymph node, the nucleus was also labelled with ferritin (Fig. 6). Although no virus-like particles could be seen inside the nucleus of glycol methacrylate embedded cells, the presence of ferritin indicated the existence of ADV antigen in the nucleus. For control, thin sections of similarly infected tissues were treated either with ferritin conjugated to antibody against adenovirus, or with ferritin conjugated to normal mink gammaglo-
bulin. In both cases a large number of cells in these sections were examined and no ferritin labelled cell could be observed (Fig. 7). Tissues from uninfected mink were also processed similarly and sections were treated with ferritin conjugated to ADV gammaglobulin; no attachment of ferritin to the uninfected cells was seen.
DISCUSSION Although AD of mink is generally regarded as a "slow virus" disease, the initial replication of the virus in mink is rapid, anl peak viral titers of 108 to 109 per gram
f~~~~~~~~~Fig. 1. Thin section of a macrophage from mink spleen ten days after infection. Virus-like particles (arrow) are observed in a membrane bound compartment. Tissue was fixed in glutaraldehyde and osmium tetroxide, then embedded in epoxy resin. Bar=500 nm.
438
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of spleen and liver are found at ten days after experimental infection (19). These organs of the early infected mink were used for the production of ADV antigens (4, 14, 16) and the purification and visualization of ADV (3, 5, 16). By the use of immunofluorescent techniques, the only cell type in vivo which has been found to contain ADV antigen is the macrophage (19). In this study we examined thin sections of spleen, mesenteric lymph node and liver,
collected from ten to 13 days after ADV infection, to detect and visualize the ADV in vivo. We found 22 nm virus-like particles usually in the cytoplasm of the macrophages of these organs. These particles were usually found in membrane bound vacuoles. The present virus-like structures are believed to be different from those observed by Tsai and co-workers (27). These workers observed virus-like structures in the form of crystalline arrays within the cytoplasm of
Fig. 2. Thin section of a similar cell as above from infected lymph node. A large vacuole containing many viruslike particles is seen in the cytoplasm (arrow). Bar =500 nm.
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Fig. 3. Thin section of Kupffer cell from liver of a mink 13 days after infection. Virus-like particles are present in a small vacuole (arrow). Bar = 500 mn.
endothelial cells in the kidney of ADV infected mink. However, others (22) have failed to find similar particles in endothelial cells of kidney at any stage of the infection. In the present study, we were unable to observe such crystalline arrays of virus-like particles in cells from infected mink. They were always present in a scattered form, usually in cytoplasmic membrane bound vacuoles. Although 22 nm virus-like particles could
440
be detected only in the cells of infected mink, it could not be proven conclusively that these particles were ADV. Therefore, to determine the nature of these particles, we carried out the technique of staining thin sections of cells with ferritin labelled antibody (24). One of the main problems in staining intracellular antigens with ferritin labelled antibody is the inactivatioii of antigens during the process of fixation and embedding. Conventional fixatives such
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as glutaraldehyde and osmium teteroxide used for electron microscopy will destroy the antibody binding ability of antigens. In addition, embedding of tissue in epoxy resins will prevent the reaction between the embedded material and the specific antibody. For these reasons, the only available method which will protect the antigenicityof cellular material is to fix the cells in formaldehyde and embed them in glycol me-
thacrylate. In spite of the inadequate pre servation of tissue structure, the antigenicity of the embedded material processed by this technique is preserved and staining with ferritin conjugate has been proven to be specific (24, 25). Using ferritin conjugated to anti-ADV antibody, it was found that in some cells, areas in the cytoplasm were labelled with ferritin. These areas were bound by membrane and contained
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w ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~. . .
Fig. 4. Thin section of a cell from the spleen of ten days infected mink. Tissue was fixed in formaldehyde and embedded in glycol methacrylate. Sections were treated with ferritin conjugated to anti-ADV gammaglobulin. ADV antigen is present in a vacuole Inside the cytoplasm as it is labelled with ferritin. The section was stained lightly with uranyl acetate to visualize the ferritin mole cules. Bar = 500 nm.
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Fig. 6. A portion of the cytoplasm of a cell from infected spleen. The dark staining granular material is labelled with ferritin. Bar = 500 nm.
ADV antigen. These ferritin labelled vacuoles resembled the vacuoles containing virus-like particles in epon embedded tissue. The lack of appearance of the particles in; the vacuoles of ferritin stained sections is considered to be due to the lack of adequate fixation in formaldehyde and embedding in glycol methacrylate, since the cells processed by the above method were not well preserved. Mitochondria seemed to be extracted, nuclear chromatin appeared without contrast and granularity as compared to osmium fixed and epon embedded cells. Since the virus-like particles were not observed in tissues of uninfected mink and there were no cells of uninfected animal labelled with ferritin, we suggest that the particles observed in the membrane bound vacuoles in the tissues of infected mink
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(Figs. 1 to 3) were ADV particles. These areas, although without adequate contrast, reacted with ADV antibody conjugated to ferritin. Nuclei of some cells from spleen, lymph nodes and liver also contained some ADV antigen. It seems that during the replication cycle of the virus, viral antigen also appeared in the nucleus. Although the ADV replicated in the nucleus in a continuous line of feline renal cells in vitro (23), ADV antigen was principally detected in the cytoplasm of macrophages in vivo by immunofluorescent technique (19). However, it has not been determined if ADV replicates in these cells or is phagocytized as an immune complex. In the present study, the fact that ADV antigen was present in both nucleus and cytoplasm of macrophages (Fig. 6) would suggest that the ADV par-
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ticle is not phagocytized as an immune complex but it replicates in these cells. Although, using the Connecticut strain of ADV, Kenyon and co-workers (11) detected RNA from purified virus eluted from an immunoadsorbent column, and the purified virus has been shown to be labelled with 3H-uridine (30), it is generally believed that three other strains of ADV (Guelph, Pullman and Utah 1 strain) are parvoviruses (3, 6, 9, 23). Our recent analysis of ADV (26) showed that nucleic acid ex-
tracted from the virus is single stranded DNA. In addition, polyacrylamide gel electrophoresis of ADV revealed a marked difference between the polypeptides of ADV and picornaviruses. The present results agree with this parvovirus theory because it was found that ADV antigen was present in the nucleus of the cell. It is tempting to speculate that some stage of virus replication occurs in the nucleus, and the viral proteins, which are synthesized in the cytoplasm are transferred inside the nu-
Fig. 6. Thin section of a cell from infected spleen showing a portion of the nucleus and cytoplasm. The nucleus is labelled with ferritin (double arrows) indicating the presence of ADV antigen inside the nucleus. Viral antigen is also present in a vacuole inside the cytoplasm (single arrow). Bar = 500 nm.
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Fig. 7. Thin section of a cell from infected spleen show ing a large cytoplasmic vacuole. The section was treated with ferritin conjugated to gammaglobulin from uninfected mink. No ferritin is attached to the vacuole and cytoplasmic material. Bar = 500 nm.
cleus for virus assembly. The matured virus particles then accumulate inside the cytoplasmic membrane bound vacuoles; subsequently they are released, and, upon exposure to antibody, form virus-antibody complex. The in vivo persistence of ADV in the form of virus-antibody complex in the blood circulation, the development of hypergammaglobulinemia with extremely high titers of anti-ADV antibody, and the lack of neu444
tralizing ability of anti-ADV antibody could be explained by the suggestion that ADV replicates inside the macrophages. The present findings are in favour of Porter and co-workers' hypothesis (19) that ADV replicates in the macrophages, which they may also serve as an antigen processing function (7, 28), and act as the scavenger of antigen-antibody complexes (1, 29). The macrophages phagocytize the ADV-antibody complexes, reactivate the virus, and allow
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the virus to replicate in the macrophages. Thus the anti-ADV antibody might promote the viral persistence rather than terminate the infection.
ACKNOWLEDGMENTS This investigation was supported partly by the University of Alberta Medical Research Fund. REFERENCES 1. BENACERRAF, B., M. SEBESTYEN and N. S. COOPER. The clearance of antigen-antibody complexes from the blood by the reticuloendothelial system. J. Immunol. 82: 131-137. 1959. 2. CREEMA, A., J. B. HENSON and J. R. GORHAM. Aleutian disease of mink. Prevention of lesions by immunosuppression. Am. J. Path. 66: 548-552. 1972. 3. CHESEBRO, B., M. BLOOM, W. HADLOW and R. RACE. Purification and ultrastructure of Aleutian disease virus of mink. Nature, Lond. 254: 456457. 1975. 4. CHO, H. J. and D. G. INGRAM. Antigen and antibody In Aleutian disease in mink. I. Precipitation reaction by agar-gel electrophoresis. J. Immunol. 108: 556-557. 1972. 5. CHO, H. J. and D. G. INGRAM. Isolation, purification and the structure of Aleutian disease virus by immunological techniQue. Nature, New Biol. 243: 174-176. 1973. 6. CHO, H. J. Purification and structure of Aleutian disease virus. In Slow Virus Diseases of Animals and Man. R. H. Kimberlin, Editor. pp. 169-174. Amsterdam: Elsevier/North-Holland. 1976. 7. FISHMAN, M. and F. L. ADLER. Antibody formation initiated in vitro. II. Antibody synthesis in xirriated recipients of diffusion chambers containing nucleic acid derived from macrophages incubated with antigen. J. exp. Med. 117: 595-602. 1968. 8. HENSON, J. B., J. R. GORHAM, T. C. McGUIRE and T. B. CRAWFORD. Pathology and pathogenesis of Aleutian disease. In Slow Virus Diseases of Animals and Man. R. H. Kimberlin, Editor. pp. 175-205. Amsterdam: Elsevier/North-Holland. 1976. 9. JNGRAM, D. G. and H. J. CHO. Aleutian disease In mink: Virology, immunology and pathogenesis. J. Rheumatol. 1: 741-92. 1974. 10. KARSTAD, L. and T. J. PRIDHAM. Aleutian disease of mink I. Evidence of its viral etiology. Can. J. comp. Med. 26: 97-102. 1962. 11. KENYON, A. J., J. E. GANDER, C. LOPEZ and R. A. GOOD. Isolation of Aleutian mink diease virus by affinity chromatography. Science 179: 187-189. 1973.
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12. LEDUC, E. H. and W. BERNHARD. Recent modifications of the glycol methacrylate embedding procedure. J. Ultrastruct. Res. 19: 196-199. 1967. 13. LOWRY, 0. H., M. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL. Protein measurement with Folin phenol reagent. J. biol. Chem. 193: 265-275. 1951. 14. McGUIRE, T. C., T. B. CRAWFORD, J. B. HENSON and J. R. GORHAM. Aleutian disease of mink: Detection of large quantities of complement-fixing antibody to viral antigen. J. Immunol. 107: 14811482. 1971. 15. NICOLSON, G. L., V. T. MARCHESI and S. J. SINGER. The localization of spectrin on the inner surface of humaui red blood cell membranes by ferritin-conjugated antibody. J. cell Biol. 51: 265272. 1971. 16. NOTANI, G. W., E. C. HAHN, N. H. SARKAR and A. J. KENYON. Characterization of Aleutian disease antigen. Nature, Lond. 261: 56-58. 1976. 17. PAN, I. C., K. S. TSAI and L. KARSTAD. Glomerulo-nephritis in Aleutian disease of mink. Histological and immunofluorescence studies. J. Path. 101: 119-127. 1970. 18. PORTER, D. D. and A. E. LARSEN. Aleutian disease of mink: Infectious virus-antibody complexes in the serum. Proc. Soc. exp. Med. 126: 680-682. 1967. 19. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. I. In vivo replication and the host antibody response to viral antigen. J. exp. Med. 130: 575-589. 1969. 20. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. II. Enhancement of tissue lesions following the administration of a killed virus vaccine or passive antibody. J. Immunol. 109: 1-7. 1972. 21. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. III. Immune complex arteritis. Am. J. Path. 71: 331-344. 1973. 22. PORTER, D. D. and A. E. LARSEN. Aleutian disease of mink. Prog. med. Virol. 18: 32-47. 1974. 23. PORTER, D. D., A. E. LARSEN, N. A. COX, H. G. PORTER and S. C. SUFFIN. Isolation of Aleutian disease virus of mink in cell culture. Intervirol. 8: 129-144. 1977. 24. SHAHRABADI, M. S. and T. YAMAMOTO. A method for staining intracellular antigens in thin sections with ferritin-labelled antibody. J. cell Biol. 50: 246-250. 1971. 25. SHAHRABADI, M. S. and T. YAMAMOTO. Localization of canine adenovirus capsid antigens in a MDCK cell line by immunoferritin and immunofluorescent techniques. Can. J. Microbiol. 18: 12991305. 1972. 26. SHAHRABADI, M. S., H. J. CHO and R. G. MARUSYK. Characterization fo the protein and nucleic acid of aleutian disease virus. J. Virol. 23: 353-362. 1977. 27. TSAI, K. S., I. GRINYER, I. C. PAN and L. KARSTAD. Electron microscopic observation of crystalline arrays of virus-like particles in tissues of mink with Aleutian disease. Can. J. Microbiol. 15: 138-140. 1969. 28. UNAUE, E. R. and B. A. ASKONAS. The immune response of mice to antigen in macrophages. Immunology 15: 287-296. 1968. 29. WARD, P. A. Chemotaxis of mononuclear cells. J. exp. Med. 128: 1201-1221. 1968. 30. YOON, J. W., A. K. DUNKER and A. J. KENYON. Characterization of Aleutian mink disease virus. Virology 64: 575-580. 1975.
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On demontra la presence de particules d'aspect viral et d'un diametre d'environ 22 nm, dans Tissues from mink infected with aleutian des macrophages de la rate, des ganglions disease virus were examined by the electron lymphatiques mesenteriques et dans les cellumicroscope for the presence of virus par- les de Kupffer dans du foie, de dix i 13 jours ticles. Virus-like particles, measuring 22 nm apres l'infection. Ces particules occupaient in diameter, were observed in macrophages of ordinairement des vacuoles cytoplasmiques des spleen, mesenteric lymph node and in Kupffer macrophages et les cellules de Kupffer et on cells in liver of mink ten to 13 days after in- les retrouvait aussi de temps en temps a l'infection. The virus-like particles were usually terieur du noyau. Les cellules des visons tepresent in vacuoles inside the cytoplasm of moins ne recelaient pas de telles particules. macrophages and Kupffer cells and, occa- Afin d'etablir une relation entre la presence de sionally, similar particles were observed in- ces particules et celle du virus de la maladie des side the nucleus. Cells from uninfected mink Iles Aleoutiennes, on examina les tissus infecdid not contain such patricles. To correlate tes a l'aide de la technique 'a l'immunoferritine. the existence of these virus-like particles with On demontra ainsi la presence de ce virus dans the presence of aleutian disease virus antigen les vacuoles cytoplasmiques des macrophages in infected cells, tissues were processed for de la rate, des ganglions lymphatiques mesenimmunoferritin technique. It was found that teriques et du foie des visons infectes; on reaaleutian disease virus antigen was present lisa aussi que la localisation du virus corresin vacuoles inside the cytoplasm of cells from pondait it celle des particules qui ressemblaient the infected spleen, lymph node and liver, and it un virus et dont le diametre atteignait 22 that the location was similar to that of the 22 nm. On decela aussi de l'antigene viral, sous nm virus-like particles. In addition, some viral la forme de materiel cytoplasmique granuleux. antigen was also detected as cytoplasmic Le noyau de certaines cellules infectees congranular material. The nuclei of some cells tenait egalement le virus de la maladie des also contained aleutian disease virus antigen. Iles Ale'outiennes. La distribution de ce virus The pattern of aleutian disease virus antigen i l'interieur des cellules infectees corresponwas similar to the distribution of virus-like dait 'a celle des particules ressemblant 'a un particles in cells of infected tissue. It is sug- virus. II semble par consequent que le virus gested that virus replication occurs inside the se multiplie 'a l'interieur du noyau et s'accunucleus with subsequent accumulation of mule ensuite dans les vacuoles cytoplasmivirus in the vacuoles of the cytoplasm. ques.
RESUME
Cette experience visait particules virales, 'a l'aide electronique, dans certains atteints de la maladie des
'a rechercher des de la microscopie organes de visons Iles Aleoutiennes.
*Department
of Medical Bacteriology, University of Alberta, Edmonton, Alberta T6G 2E1 (Shahrabadi) and Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta TlJ 3Z4 (Cho). Slbmitted November 19, 1976.
Volume 41
October, 1977
INTRODUCTION Aleutian disease (AD) is a common persistent viral infection of mink which causes serious economic loss to commercial mink ranches throughout the world. The disease is characterized by generalized plasmacytosis, hypergammaglobulinemia and immunologically-mediated lesions (8, 9, 22). The
435
progress of the disease is "slow", although the initial replication of the aleutian disease virus (ADV) in vivo is rapid and peak viral titers are found at ten days after experimental infection of spleen, liver and lymph node, coincident with the development of anti-ADV antibody (19). Extremely high titers of ADV antibody were detected from serum of hypergammaglobulinemic mink (4, 14, 19). Circulating virusantibody complexes (18) are associated with immune complex arteritis (21) and glomerulonephritis (17, 19). Of particular interest, the role of host immune response in causing the disease condition has been shown by the prevention of lesions by immunosuppression (2) and by the enhancement of lesions by immunization with an inactivated virus vaccine followed by live virus challenge (20). Aleutian disease virions have been purified and visualized from infected mink tissues (3, 5, 11) and from cell cultures (23). Employing immunofluorescent techniques, Porter and co-workers (19) extensively examined ADV infected mink tissues. They reported finding viral antigen in the cytoplasm of macrophages in the spleen and lymph node and in Kupffer cells in the liver. These findings have been confirmed recently by others (8). In the present investigation, we examined ADV infected mink spleen, liver and mesenteric lymph node collected at ten to 13 days after infection by the electron microscope to identify the ADV in vivo. We also applied an immunoferritin technique to detect and localize the presence of ADV antigen in the cells from infected mink tissues and to determine the cell types the ADV replicates in vivo.
MATERIALS AND METHODS ANIMALS Mink (Mustela vison) with a violet coat colour which were homozygous for the autosomal recessive aleutian gene were purchased from a commercial mink ranch and maintained at the Animal Diseases Research Institute (Western). The mink were caged individually and fed a standard ranch diet. The mink were negative for ADV antibody
436
as tested by counterimmunoelectrophoresis (4). The mink were vaccinated at the age of six months with live distemper vaccine, Clostridiium botulinurm type C toxoid and a formalin-inactivated mink viral enteritis vaccine. ALEUTIAN DISEASE VIRUS, INOCULUM AND COLLECTION OF TISSUES
Guelph strain of ADV (4, 10) was propagated in violet coat coloured mink. Virus was purified by fluorocarbon extraction and equilibrium density centrifugation in cesium chloride as described in an earlier publication (6). Aleutian disease virus, banded in a buoyant density of 1.405-1.416 g/ml, was inoculated intraperitoneally into eight mink. Each mink received 10" ID50 of the purified virus. Two mink were necropsied on each day from the tenth to the 13th day after infection and spleen, liver and mesenteric lymph node were collected. These organs were processed immediately for electron microscopy. The same tissues were collected from two uninfected mink and processed similarly. PROTEIN DETERMINATION
The protein concentration of the gammaglobulin fraction of mink serum was determined by the method of Lowry et al (13). CONJUGATION OF FERRITIN TO GAMMAGLOBULIN
Conjugation of ferritin to gammaglobulin was performed according to the method of Nicholson et al (15). Briefly, ferritin (E.M. Grade, Polyscience Inc., Rydal, Pa.) was recrystallized using cadmium sulfate precipitation and conjugated to gammaglobulin using toluene 2-4-diisocyanate. The crude conjugate was further purified by passing it through a column of agarose A-1.5 (Biorad 200-400 Mesh.). ELECTRON MICROSCOPY For morphological studies, tissues from three infected mink taken on the tenth, 12th and 13th days after infection and from one normal mink were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 at
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4°C for two hr. They were washed in 0.1 M phosphate buffer overnight, postfixed in osmium tetroxide and embedded in epon 812. For immunoelectron microscopy, tissues taken from the other five infected and one normal mink were fixed in freshly prepared 4% formaldehyde in 0.1 M phosphate buffer pH 7.2 at 4°C for 30 minutes. They were washed with the phosphate buffer (24 h, three changes of buffer, at 4°C) and embedded in water soluble glycol methacrylate (12). Thin sections were cut and treated with ferritin conjugated gammaglobulin as described previously (24). After final washing, sections were stained with uranyl acetate and examined in a Phillips EM 300 electron microscope.
RESULTS Spleen, liver and mesenteric lymph nodes of mink infected with ADV were processed for electron microscopy. For comparison, tissues from uninfected mink were similarly prepared and examined. In cells from mink ten days after infection, granules and particles of 22 nm in diameter could be observed in the cytoplasm. These granules resembling virus particles were densely stained and could be differentiated from glycogen aggregates. Their presence was confined only to the cells from infected mink. The virus-like particles observed in tissue after epon embedding and positive staining were 1-2 nm smaller than the virus observed with negative staining of fluorocarbon purified virus (3, 5, 6, 23). The virus-like particles were mainly observed in macrophages of spleen and lymph node and Kupffer cells of liver but not in hepatocytes. There, they were usually surrounded by a membrane or membrane-delimited and thus appeared as cytoplasmic vacuoles of various sizes containing virus-like particles (Figs. 1 and 2). About 10% of the cells of infected spleen in these sections contained virus-like particles. Examination of a large number of sections from tissues of uninfected mink did not reveal the presence of such particles. The particles were always present in scattered form and no crystalline aggregation of them was observed in all of the tissues examined. Tissues of mink 12 and 13 days postinfection were also exam-
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ined and similar virus-particles were seen (Fig. 3) and the number of cells containing virus-like particles seemed to be similar to the number in those tissues collected at ten days postinfection. DEMONSTRATION OF ADV ANTIGEN IN CELLS FROM INFECTED MINK
Tissues of infected mink were fixed in formaldehyde and embedded in glycol methacrylate. Under these conditions the antigenicity of the embedded material has beeni shown to be preserved (24, 25). The disadvantage of this method of fixation and embedding was the lack of good preservation of the cellular fine structure. Membrane structures were not very distinct and mitochondria were not well preserved. In addition, in order to visualize ferritin molecules with a good contrast, thin sections were not stained with lead citrate. This also caused difficulties in making a clear distinction of the fine structure of cellular components. Treatment of thin sections of liver, spleen and lymph nodes from mink infected with ADV revealed the presence of ADV antigen in some cells as they were labelled with ferritin conjugated to ADV antibody. Viral antigen was present mainly in the cytoplasm and was usually found in membrane bound vacuoles (Fig. 4). Because of the lack of good preservation due to the fixation and embedding, no structures resembling virus-like particles could be seen inside these membrane bound vacuoles. The vacuoles containing the viral antigen varied in size and number and were always present in the cytoplasm of cells. About 12-15% of cells from the spleen and lymph nodes of infected animals contained ADV antigen. Viral antigen was also detected in the cytoplasm of some cells as being associated with some granular dark staining material (Fig. 5). Presence of ADV antigen was not confined to the cytoplasm of cells. In some cells from infected spleen, liver and lymph node, the nucleus was also labelled with ferritin (Fig. 6). Although no virus-like particles could be seen inside the nucleus of glycol methacrylate embedded cells, the presence of ferritin indicated the existence of ADV antigen in the nucleus. For control, thin sections of similarly infected tissues were treated either with ferritin conjugated to antibody against adenovirus, or with ferritin conjugated to normal mink gammaglo-
bulin. In both cases a large number of cells in these sections were examined and no ferritin labelled cell could be observed (Fig. 7). Tissues from uninfected mink were also processed similarly and sections were treated with ferritin conjugated to ADV gammaglobulin; no attachment of ferritin to the uninfected cells was seen.
DISCUSSION Although AD of mink is generally regarded as a "slow virus" disease, the initial replication of the virus in mink is rapid, anl peak viral titers of 108 to 109 per gram
f~~~~~~~~~Fig. 1. Thin section of a macrophage from mink spleen ten days after infection. Virus-like particles (arrow) are observed in a membrane bound compartment. Tissue was fixed in glutaraldehyde and osmium tetroxide, then embedded in epoxy resin. Bar=500 nm.
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of spleen and liver are found at ten days after experimental infection (19). These organs of the early infected mink were used for the production of ADV antigens (4, 14, 16) and the purification and visualization of ADV (3, 5, 16). By the use of immunofluorescent techniques, the only cell type in vivo which has been found to contain ADV antigen is the macrophage (19). In this study we examined thin sections of spleen, mesenteric lymph node and liver,
collected from ten to 13 days after ADV infection, to detect and visualize the ADV in vivo. We found 22 nm virus-like particles usually in the cytoplasm of the macrophages of these organs. These particles were usually found in membrane bound vacuoles. The present virus-like structures are believed to be different from those observed by Tsai and co-workers (27). These workers observed virus-like structures in the form of crystalline arrays within the cytoplasm of
Fig. 2. Thin section of a similar cell as above from infected lymph node. A large vacuole containing many viruslike particles is seen in the cytoplasm (arrow). Bar =500 nm.
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Fig. 3. Thin section of Kupffer cell from liver of a mink 13 days after infection. Virus-like particles are present in a small vacuole (arrow). Bar = 500 mn.
endothelial cells in the kidney of ADV infected mink. However, others (22) have failed to find similar particles in endothelial cells of kidney at any stage of the infection. In the present study, we were unable to observe such crystalline arrays of virus-like particles in cells from infected mink. They were always present in a scattered form, usually in cytoplasmic membrane bound vacuoles. Although 22 nm virus-like particles could
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be detected only in the cells of infected mink, it could not be proven conclusively that these particles were ADV. Therefore, to determine the nature of these particles, we carried out the technique of staining thin sections of cells with ferritin labelled antibody (24). One of the main problems in staining intracellular antigens with ferritin labelled antibody is the inactivatioii of antigens during the process of fixation and embedding. Conventional fixatives such
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as glutaraldehyde and osmium teteroxide used for electron microscopy will destroy the antibody binding ability of antigens. In addition, embedding of tissue in epoxy resins will prevent the reaction between the embedded material and the specific antibody. For these reasons, the only available method which will protect the antigenicityof cellular material is to fix the cells in formaldehyde and embed them in glycol me-
thacrylate. In spite of the inadequate pre servation of tissue structure, the antigenicity of the embedded material processed by this technique is preserved and staining with ferritin conjugate has been proven to be specific (24, 25). Using ferritin conjugated to anti-ADV antibody, it was found that in some cells, areas in the cytoplasm were labelled with ferritin. These areas were bound by membrane and contained
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Fig. 4. Thin section of a cell from the spleen of ten days infected mink. Tissue was fixed in formaldehyde and embedded in glycol methacrylate. Sections were treated with ferritin conjugated to anti-ADV gammaglobulin. ADV antigen is present in a vacuole Inside the cytoplasm as it is labelled with ferritin. The section was stained lightly with uranyl acetate to visualize the ferritin mole cules. Bar = 500 nm.
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Fig. 6. A portion of the cytoplasm of a cell from infected spleen. The dark staining granular material is labelled with ferritin. Bar = 500 nm.
ADV antigen. These ferritin labelled vacuoles resembled the vacuoles containing virus-like particles in epon embedded tissue. The lack of appearance of the particles in; the vacuoles of ferritin stained sections is considered to be due to the lack of adequate fixation in formaldehyde and embedding in glycol methacrylate, since the cells processed by the above method were not well preserved. Mitochondria seemed to be extracted, nuclear chromatin appeared without contrast and granularity as compared to osmium fixed and epon embedded cells. Since the virus-like particles were not observed in tissues of uninfected mink and there were no cells of uninfected animal labelled with ferritin, we suggest that the particles observed in the membrane bound vacuoles in the tissues of infected mink
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(Figs. 1 to 3) were ADV particles. These areas, although without adequate contrast, reacted with ADV antibody conjugated to ferritin. Nuclei of some cells from spleen, lymph nodes and liver also contained some ADV antigen. It seems that during the replication cycle of the virus, viral antigen also appeared in the nucleus. Although the ADV replicated in the nucleus in a continuous line of feline renal cells in vitro (23), ADV antigen was principally detected in the cytoplasm of macrophages in vivo by immunofluorescent technique (19). However, it has not been determined if ADV replicates in these cells or is phagocytized as an immune complex. In the present study, the fact that ADV antigen was present in both nucleus and cytoplasm of macrophages (Fig. 6) would suggest that the ADV par-
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ticle is not phagocytized as an immune complex but it replicates in these cells. Although, using the Connecticut strain of ADV, Kenyon and co-workers (11) detected RNA from purified virus eluted from an immunoadsorbent column, and the purified virus has been shown to be labelled with 3H-uridine (30), it is generally believed that three other strains of ADV (Guelph, Pullman and Utah 1 strain) are parvoviruses (3, 6, 9, 23). Our recent analysis of ADV (26) showed that nucleic acid ex-
tracted from the virus is single stranded DNA. In addition, polyacrylamide gel electrophoresis of ADV revealed a marked difference between the polypeptides of ADV and picornaviruses. The present results agree with this parvovirus theory because it was found that ADV antigen was present in the nucleus of the cell. It is tempting to speculate that some stage of virus replication occurs in the nucleus, and the viral proteins, which are synthesized in the cytoplasm are transferred inside the nu-
Fig. 6. Thin section of a cell from infected spleen showing a portion of the nucleus and cytoplasm. The nucleus is labelled with ferritin (double arrows) indicating the presence of ADV antigen inside the nucleus. Viral antigen is also present in a vacuole inside the cytoplasm (single arrow). Bar = 500 nm.
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Fig. 7. Thin section of a cell from infected spleen show ing a large cytoplasmic vacuole. The section was treated with ferritin conjugated to gammaglobulin from uninfected mink. No ferritin is attached to the vacuole and cytoplasmic material. Bar = 500 nm.
cleus for virus assembly. The matured virus particles then accumulate inside the cytoplasmic membrane bound vacuoles; subsequently they are released, and, upon exposure to antibody, form virus-antibody complex. The in vivo persistence of ADV in the form of virus-antibody complex in the blood circulation, the development of hypergammaglobulinemia with extremely high titers of anti-ADV antibody, and the lack of neu444
tralizing ability of anti-ADV antibody could be explained by the suggestion that ADV replicates inside the macrophages. The present findings are in favour of Porter and co-workers' hypothesis (19) that ADV replicates in the macrophages, which they may also serve as an antigen processing function (7, 28), and act as the scavenger of antigen-antibody complexes (1, 29). The macrophages phagocytize the ADV-antibody complexes, reactivate the virus, and allow
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the virus to replicate in the macrophages. Thus the anti-ADV antibody might promote the viral persistence rather than terminate the infection.
ACKNOWLEDGMENTS This investigation was supported partly by the University of Alberta Medical Research Fund. REFERENCES 1. BENACERRAF, B., M. SEBESTYEN and N. S. COOPER. The clearance of antigen-antibody complexes from the blood by the reticuloendothelial system. J. Immunol. 82: 131-137. 1959. 2. CREEMA, A., J. B. HENSON and J. R. GORHAM. Aleutian disease of mink. Prevention of lesions by immunosuppression. Am. J. Path. 66: 548-552. 1972. 3. CHESEBRO, B., M. BLOOM, W. HADLOW and R. RACE. Purification and ultrastructure of Aleutian disease virus of mink. Nature, Lond. 254: 456457. 1975. 4. CHO, H. J. and D. G. INGRAM. Antigen and antibody In Aleutian disease in mink. I. Precipitation reaction by agar-gel electrophoresis. J. Immunol. 108: 556-557. 1972. 5. CHO, H. J. and D. G. INGRAM. Isolation, purification and the structure of Aleutian disease virus by immunological techniQue. Nature, New Biol. 243: 174-176. 1973. 6. CHO, H. J. Purification and structure of Aleutian disease virus. In Slow Virus Diseases of Animals and Man. R. H. Kimberlin, Editor. pp. 169-174. Amsterdam: Elsevier/North-Holland. 1976. 7. FISHMAN, M. and F. L. ADLER. Antibody formation initiated in vitro. II. Antibody synthesis in xirriated recipients of diffusion chambers containing nucleic acid derived from macrophages incubated with antigen. J. exp. Med. 117: 595-602. 1968. 8. HENSON, J. B., J. R. GORHAM, T. C. McGUIRE and T. B. CRAWFORD. Pathology and pathogenesis of Aleutian disease. In Slow Virus Diseases of Animals and Man. R. H. Kimberlin, Editor. pp. 175-205. Amsterdam: Elsevier/North-Holland. 1976. 9. JNGRAM, D. G. and H. J. CHO. Aleutian disease In mink: Virology, immunology and pathogenesis. J. Rheumatol. 1: 741-92. 1974. 10. KARSTAD, L. and T. J. PRIDHAM. Aleutian disease of mink I. Evidence of its viral etiology. Can. J. comp. Med. 26: 97-102. 1962. 11. KENYON, A. J., J. E. GANDER, C. LOPEZ and R. A. GOOD. Isolation of Aleutian mink diease virus by affinity chromatography. Science 179: 187-189. 1973.
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12. LEDUC, E. H. and W. BERNHARD. Recent modifications of the glycol methacrylate embedding procedure. J. Ultrastruct. Res. 19: 196-199. 1967. 13. LOWRY, 0. H., M. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL. Protein measurement with Folin phenol reagent. J. biol. Chem. 193: 265-275. 1951. 14. McGUIRE, T. C., T. B. CRAWFORD, J. B. HENSON and J. R. GORHAM. Aleutian disease of mink: Detection of large quantities of complement-fixing antibody to viral antigen. J. Immunol. 107: 14811482. 1971. 15. NICOLSON, G. L., V. T. MARCHESI and S. J. SINGER. The localization of spectrin on the inner surface of humaui red blood cell membranes by ferritin-conjugated antibody. J. cell Biol. 51: 265272. 1971. 16. NOTANI, G. W., E. C. HAHN, N. H. SARKAR and A. J. KENYON. Characterization of Aleutian disease antigen. Nature, Lond. 261: 56-58. 1976. 17. PAN, I. C., K. S. TSAI and L. KARSTAD. Glomerulo-nephritis in Aleutian disease of mink. Histological and immunofluorescence studies. J. Path. 101: 119-127. 1970. 18. PORTER, D. D. and A. E. LARSEN. Aleutian disease of mink: Infectious virus-antibody complexes in the serum. Proc. Soc. exp. Med. 126: 680-682. 1967. 19. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. I. In vivo replication and the host antibody response to viral antigen. J. exp. Med. 130: 575-589. 1969. 20. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. II. Enhancement of tissue lesions following the administration of a killed virus vaccine or passive antibody. J. Immunol. 109: 1-7. 1972. 21. PORTER, D. D., A. E. LARSEN and H. G. PORTER. The pathogenesis of Aleutian disease of mink. III. Immune complex arteritis. Am. J. Path. 71: 331-344. 1973. 22. PORTER, D. D. and A. E. LARSEN. Aleutian disease of mink. Prog. med. Virol. 18: 32-47. 1974. 23. PORTER, D. D., A. E. LARSEN, N. A. COX, H. G. PORTER and S. C. SUFFIN. Isolation of Aleutian disease virus of mink in cell culture. Intervirol. 8: 129-144. 1977. 24. SHAHRABADI, M. S. and T. YAMAMOTO. A method for staining intracellular antigens in thin sections with ferritin-labelled antibody. J. cell Biol. 50: 246-250. 1971. 25. SHAHRABADI, M. S. and T. YAMAMOTO. Localization of canine adenovirus capsid antigens in a MDCK cell line by immunoferritin and immunofluorescent techniques. Can. J. Microbiol. 18: 12991305. 1972. 26. SHAHRABADI, M. S., H. J. CHO and R. G. MARUSYK. Characterization fo the protein and nucleic acid of aleutian disease virus. J. Virol. 23: 353-362. 1977. 27. TSAI, K. S., I. GRINYER, I. C. PAN and L. KARSTAD. Electron microscopic observation of crystalline arrays of virus-like particles in tissues of mink with Aleutian disease. Can. J. Microbiol. 15: 138-140. 1969. 28. UNAUE, E. R. and B. A. ASKONAS. The immune response of mice to antigen in macrophages. Immunology 15: 287-296. 1968. 29. WARD, P. A. Chemotaxis of mononuclear cells. J. exp. Med. 128: 1201-1221. 1968. 30. YOON, J. W., A. K. DUNKER and A. J. KENYON. Characterization of Aleutian mink disease virus. Virology 64: 575-580. 1975.
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