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The Structure of Canine Distemper Virus
Res. ver. Sci., 1962. 3, 485
The Structure of Canine Distemper Virus J.
G.
CRUICKSHANK AND
A. P.
WATERSON
Department of Pathology, University of Cambridge A. D.
KANAREK
Wellcome Research Laboratories, Betkenham, England AND
D. M.
BERRY
Glaxo Laboratories, Middlesex, Englalld SUMMARY. The particles of canine distemper virus have been studied in the electron microscope. Their structure resembles that of thelarger myxoviruses. They have anouter coat or membrane bearing projections and an internal helical component ISO to 170 A in width. The particles vary from BOO to 5500 A in diameter. CANINE DISTEMPER has been recognised as due to a filterable agent since 1905 (Carre). Because of the difficulty of purifying the virus, studies on the particle have largely been confined to determinations of its size, work pioneered by Laidlaw and Dunkin (I926). In 1954 Bindrich and Gralheer, using gradocol membrane filtration techniques, estimated the particle diameter to be between 70 and lOS mu, The only electron microscope studies are those of Reagan and Brueckner (I95I), who examined extracts of infected tissues by the metal shadowing technique. They found spherical bodies with a mean diameter Or2IO A. This paper describes the electron-microscopic appearances of canine distemper virus particles prepared in sufficiently high titre and purity for examination by the negative staining technique of Brenner and Home (1959).
MATERIALS AND METHODS
Preparation of the Virus Concentrates The Onderstepoort egg-adapted strain of canine distemper virus was grown in chickembryo tissue cultures. The culture fluids were harvested after 5 or 6 days and were stored at - 20° C. before concentration. The titres of the fluids were between I0 5 . 2 and ro 5 ' 6 EID 5o/ I ' 0 ml. Two methods were used for concentrating the material. (I) One litre of harvested fluid was thawed and spun at 78,000 g for 60 minutes. The resulting pellets were resuspended in ro ml. of phosphate buffered saline, and the suspension was clarified at 13,000 g for IO minutes. The supernatant fluid was then spun at I05,000 g for 30 minutes, and the pellet was resuspended in o· 5 ml. of I per cent ammonium acetate for electron microscope studies. The titre of the concentrate was IoMEIDso/I'O ml. (2) The harvested fluid was thawed, triturated in an equal volume of stabilising medium, and spun at 500 g for 30 minutes at 4° C. The supernatant fluid was harvested and gelatin was added to give a final concentration of 0'06 per cent. The virus was then concentrated by
+
485
486
J. G. Cruickshank, A. P. Waterson, A. D. Kanarek and D. M. Berry
centrifuging at 85,000 g for 60 minutes (Baron, 1957). The pellet was resuspended in I per cent ammonium acetate. The final titre of this fluid was I06 • 6 EID so!I·0 ml.
Preparation for Electron Microscopy 0'1 ml. of virus concentrate was mixed with 0'2 ml. of 2 per cent phosphotungstic acid (adjusted to pH 7'0 with N KOH) and sprayed on to carbon-eoated grids with a Vaponefrin nebulizer. The preparations were examined in a Siemens Elmiskop electron microscope at an instrumental magnification of 40,000 times using double condenser illumination. RESULTS
The appearances of both concentrates were the same. A moderate number of both intact and disrupted particles were outlined in each phosphotungstate droplet. There was some variation in shape, but most particles were approximately spherical. They varied in size from rroo A to 5500 A at their greatest diameters, though most fell into the 1500 A to 3000 A range. Intact particles showed a fairly conspicuous outer membrane, about 50 A in thickness, enclosing the inner component. From this membrane arose projections of about 90 A length covering the whole surface. The inner component was not seen in intact particles (Fig. I). In partially-disrupted particles the phospho-tungstate had penetrated through the virus coat and outlined some internal structures. Figure 2 shows a particle from which a greater part of the outer layer had been stripped. In some places the material within can be seen to consist of rod-shaped structures. These have a helical pattern. This helical material is released from completely disrupted particles (Fig. 3). The helix has a width of ISO to 170 A, with a central hole of about 50 A diameter. The rod surface was serrated and had a periodicity of 50 to 60 A. DISCUSSION
The appearances described above indicate that canine distemper virus is structurally similar to the larger myxoviruses, e.g. Newcastle disease virus (Rott and Schafer, 1961; Home and Waterson, 1960). The same structure is found in the viruses of both measles (Waterson, Cruickshank, Laurence and Kanarek, 1961) and rinderpest (Plowright, Cruickshank and Waterson, 1962). Measles, rinderpest and distemper viruseshave long been grouped together, mainly for their serological relationships (Warren, 1960), and these findings serve to emphasize the close kinship between the three. Receivedfor publication February 23rd,
19<>2.
REFERENCES S. (1957). Proc. Soc. exp.• Bioi. N.Y., 95, 760. PLOWRIGHT, W., CRUICKSHANK, ). G., and WATERSON, BINDRICH, H., and GRALHEER, H. (1954). Arch. expo Vet. A. P. (19<>2). Virology, 17, 1I8. Med•• 8,204. REAGAN, R. L., and BRUECKNER, A. L. (1951). Cornell BRENNER. S.• and HORNE, R. W. (1959). Biochim. biophys. Vet. 41, 14I. Acta., 34. 103. Ron, R., and SCHAFER, W. (19<>1). Virology, 14. 298. CARRE. M. C. (1905). C. R. Acad. Sci., 140. 689. WARREN, j., (1960). Advances in Virus Research, 7. 27. HORNE, R. W., and WATERSON, A. P. (1960).). mol. Biol., WATERSON, A. P., CRUICKSHANK,). G., LAURENCE, G. D., 2. 75. and KANAREK, A. D. (1961). Virology, IS. 379. l.AmLAW, P. P., and DUNKIN. G. W. (1926). ]. compo Path., 39. 222. BARON,
J.
G. Cmickslu1IIk, A. P. Watersoll, A. D. Kauarcl: alld D. M. Berry
FIG. I.
Two distemper virus particles, The membranes arc conspicuous. and short projections can be sccn in profile on thc surface. ,', 1\)0,000.
FH;. 2. A partly-disrupted particle, whose outer cO.n missin~. Helical rods can be seen within the particle.
is Llt~dy 135.000.
]. G. Cruicltshanle, A. P. WatersOIl, A. D. Kanarek and D. M. Berry
FIG. 3.
The helical inner component from a disrupted particle. ><
1')0,000.
The Structure of Canine Distemper Virus J.
G.
CRUICKSHANK AND
A. P.
WATERSON
Department of Pathology, University of Cambridge A. D.
KANAREK
Wellcome Research Laboratories, Betkenham, England AND
D. M.
BERRY
Glaxo Laboratories, Middlesex, Englalld SUMMARY. The particles of canine distemper virus have been studied in the electron microscope. Their structure resembles that of thelarger myxoviruses. They have anouter coat or membrane bearing projections and an internal helical component ISO to 170 A in width. The particles vary from BOO to 5500 A in diameter. CANINE DISTEMPER has been recognised as due to a filterable agent since 1905 (Carre). Because of the difficulty of purifying the virus, studies on the particle have largely been confined to determinations of its size, work pioneered by Laidlaw and Dunkin (I926). In 1954 Bindrich and Gralheer, using gradocol membrane filtration techniques, estimated the particle diameter to be between 70 and lOS mu, The only electron microscope studies are those of Reagan and Brueckner (I95I), who examined extracts of infected tissues by the metal shadowing technique. They found spherical bodies with a mean diameter Or2IO A. This paper describes the electron-microscopic appearances of canine distemper virus particles prepared in sufficiently high titre and purity for examination by the negative staining technique of Brenner and Home (1959).
MATERIALS AND METHODS
Preparation of the Virus Concentrates The Onderstepoort egg-adapted strain of canine distemper virus was grown in chickembryo tissue cultures. The culture fluids were harvested after 5 or 6 days and were stored at - 20° C. before concentration. The titres of the fluids were between I0 5 . 2 and ro 5 ' 6 EID 5o/ I ' 0 ml. Two methods were used for concentrating the material. (I) One litre of harvested fluid was thawed and spun at 78,000 g for 60 minutes. The resulting pellets were resuspended in ro ml. of phosphate buffered saline, and the suspension was clarified at 13,000 g for IO minutes. The supernatant fluid was then spun at I05,000 g for 30 minutes, and the pellet was resuspended in o· 5 ml. of I per cent ammonium acetate for electron microscope studies. The titre of the concentrate was IoMEIDso/I'O ml. (2) The harvested fluid was thawed, triturated in an equal volume of stabilising medium, and spun at 500 g for 30 minutes at 4° C. The supernatant fluid was harvested and gelatin was added to give a final concentration of 0'06 per cent. The virus was then concentrated by
+
485
486
J. G. Cruickshank, A. P. Waterson, A. D. Kanarek and D. M. Berry
centrifuging at 85,000 g for 60 minutes (Baron, 1957). The pellet was resuspended in I per cent ammonium acetate. The final titre of this fluid was I06 • 6 EID so!I·0 ml.
Preparation for Electron Microscopy 0'1 ml. of virus concentrate was mixed with 0'2 ml. of 2 per cent phosphotungstic acid (adjusted to pH 7'0 with N KOH) and sprayed on to carbon-eoated grids with a Vaponefrin nebulizer. The preparations were examined in a Siemens Elmiskop electron microscope at an instrumental magnification of 40,000 times using double condenser illumination. RESULTS
The appearances of both concentrates were the same. A moderate number of both intact and disrupted particles were outlined in each phosphotungstate droplet. There was some variation in shape, but most particles were approximately spherical. They varied in size from rroo A to 5500 A at their greatest diameters, though most fell into the 1500 A to 3000 A range. Intact particles showed a fairly conspicuous outer membrane, about 50 A in thickness, enclosing the inner component. From this membrane arose projections of about 90 A length covering the whole surface. The inner component was not seen in intact particles (Fig. I). In partially-disrupted particles the phospho-tungstate had penetrated through the virus coat and outlined some internal structures. Figure 2 shows a particle from which a greater part of the outer layer had been stripped. In some places the material within can be seen to consist of rod-shaped structures. These have a helical pattern. This helical material is released from completely disrupted particles (Fig. 3). The helix has a width of ISO to 170 A, with a central hole of about 50 A diameter. The rod surface was serrated and had a periodicity of 50 to 60 A. DISCUSSION
The appearances described above indicate that canine distemper virus is structurally similar to the larger myxoviruses, e.g. Newcastle disease virus (Rott and Schafer, 1961; Home and Waterson, 1960). The same structure is found in the viruses of both measles (Waterson, Cruickshank, Laurence and Kanarek, 1961) and rinderpest (Plowright, Cruickshank and Waterson, 1962). Measles, rinderpest and distemper viruseshave long been grouped together, mainly for their serological relationships (Warren, 1960), and these findings serve to emphasize the close kinship between the three. Receivedfor publication February 23rd,
19<>2.
REFERENCES S. (1957). Proc. Soc. exp.• Bioi. N.Y., 95, 760. PLOWRIGHT, W., CRUICKSHANK, ). G., and WATERSON, BINDRICH, H., and GRALHEER, H. (1954). Arch. expo Vet. A. P. (19<>2). Virology, 17, 1I8. Med•• 8,204. REAGAN, R. L., and BRUECKNER, A. L. (1951). Cornell BRENNER. S.• and HORNE, R. W. (1959). Biochim. biophys. Vet. 41, 14I. Acta., 34. 103. Ron, R., and SCHAFER, W. (19<>1). Virology, 14. 298. CARRE. M. C. (1905). C. R. Acad. Sci., 140. 689. WARREN, j., (1960). Advances in Virus Research, 7. 27. HORNE, R. W., and WATERSON, A. P. (1960).). mol. Biol., WATERSON, A. P., CRUICKSHANK,). G., LAURENCE, G. D., 2. 75. and KANAREK, A. D. (1961). Virology, IS. 379. l.AmLAW, P. P., and DUNKIN. G. W. (1926). ]. compo Path., 39. 222. BARON,
J.
G. Cmickslu1IIk, A. P. Watersoll, A. D. Kauarcl: alld D. M. Berry
FIG. I.
Two distemper virus particles, The membranes arc conspicuous. and short projections can be sccn in profile on thc surface. ,', 1\)0,000.
FH;. 2. A partly-disrupted particle, whose outer cO.n missin~. Helical rods can be seen within the particle.
is Llt~dy 135.000.
]. G. Cruicltshanle, A. P. WatersOIl, A. D. Kanarek and D. M. Berry
FIG. 3.
The helical inner component from a disrupted particle. ><
1')0,000.