JOURNAL
OF
Vol. 32, No. 1
VIROLOGY, Oct. 1979, p. 323-328
0022-538X/79/10-0323/06$02.00/0
NOTES Further Morphological Characterization and Structural Proteins of Infectious Bursal Disease Virus K. HIRAI,* N. KATO,t A. FUJIURA,: AND S. SHIMAKURA Department of Veterinary Microbiology, Faculty ofAgriculture, Gifu University, Kakamigahara-shi, Gifu 504, Japan Received for publication 4 May 1979
An outer layer surrounding the capsid of infectious bursal disease virus was evident from electron micrographs of intact virus particles having diameters of 62 to 63 nm. The capsid was found to be composed of large morphological units or capsomeres, measuring about 12 nm in diameter. The architecture of the capsid appears to be that of T = 3 symmetry, with a probable 32 morphological units by rotational enhancement of image detail. Structural proteins of infectious bursal disease virus consist of seven species, two major and five minor polypeptides. These are P1 to P7, with molecular weights of 133 x 103, 124 x 103, 98 x 103, 51 X 103, 33 x 103, 26 x 103, and 23 x 103, respectively.
Infectious bursal disease (IBD) is a naturally occurring virus-induced immune deficiency of chickens characterized by necrosis and depletion of lymphoid elements. A recent study has suggested that surface immunoglobulin M-bearing B lymphocytes are target cells for infection by the IBD virus (IBDV) (3). Electron microscopy has shown that IBDV is a nonenveloped icosahedron with diameters ranging from 55 to 65 nm (1, 2, 5, 13). In a previous study (5), we suggested that the capsid of the virion consisted of 32 capsomeres, each composed of five or six structural units, and that IBDV resembled bluetongue virus in morphology. However, it is known that there are biological and physicochemical differences between IBDV and bluetongue virus. Apart from the initial study by Nick et al. (10), little information is available on the structural proteins of IBDV, using polyacrylamide gel electrophoresis. The exact number of polypeptides and their locations within the virion are not yet clearly understood. This report is concerned with a reexamination of two criteria: the fine structure of virions viewed with high-resolution electron microscopy and rotational enhancement of image detail and the polypeptide structure of IBDV, using polyacrylamide gel electrophoresis. Samples of the GBF-1 strain of IBDV were t Present address: Department of Urology, School of Medicine, Gifu University, Tsukasa, Gifu 500, Japan. : Present address: Aichi Prefectural Institute of Public Health, Nagare 7-6: Kitaku, Nagoya, Japan.
obtained as previously described (4, 6). Briefly, susceptible chickens were inoculated with virus from passage 15 to 20, and affected bursae of Fabricius were harvested for virus extraction. The purification procedure was essentially the same as that described previously (5). Electron microscopy was performed as previously described (5). Rotational enhancement of image detail was accomplished with a double-wheel device modified from the pinwheel apparatus described by Markham et al. (8). These modifications eliminated the hole produced at the axis of rotation. Virus samples were solubilized by incubation for 2 h at 37°C with equal amounts of the 0.01 M phosphate buffer (pH 7.2) containing 2% sodium dodecyl sulfate, 6 to 8% 2-mercaptoethanol, and 50% glycerol, followed by heating for 2 min at 1000C before electrophoresis. The polypeptide composition of the virus was determined in 7.5% polyacrylamide gels as described by Spandidos and Graham (16). Electrophoresis was carried out at a constant current of 5 mA/gel for 8 h. The molecular weights of polypeptides determined on 7.5% gels by the procedure of Shapiro et al. (15) were used for the final results. After three equilibrium centrifugations in cesium chloride, the major band of IBDV occurred at a buoyant density of 1.34 g/ml, and three faint minor bands occurred at buoyant densities of 1.35, 1.36, and 1.37 g/ml, respectively. No cellular debris was observed in any band when examined by electron microscopy. Bands at den-
323
324
NOTES
sities of 1.34 and 1.35 g/ml both contained numerous particles with hexagonal or roughly spherical profiles. Hexagonal virus particles predominated at the density of 1.34 g/ml, whereas the band at the density of 1.35 g/ml contained mostly spherical particles. Intact virions were generally hexagonal in profile, and surface capsomere arrangement of the morphological unit was not clearly resolved (Fig. la and b). These particles averaged from 62 to 63 nm in diameter and appeared to be covered by an outer layer composed of T-shaped structures (Fig. lb). The outer layer was continuous. Spherical particles without an apparent outer layer predominated at the density of 1.35 g/ml (Fig. lc). Their diameters were 54 to 55 nm, and morphological units were clearly observed. At a higher magnification, each morphological unit seemed to be composed of smaller subunits in hexagonal-pentagonal arrays around a central hole (Fig. 2a and d). Subunits appeared to be shared by neighboring structural units. In previous ultrastructural studies on the morphology of IBDV (5), we suggested that the capsid of the virion consisted of 32 capsomeres arranged in 5:3:2 symmetry. In an attempt to further elucidate the capsomere arrangement, rotational enhancement of image detail has been accomplished with a few IBDV particles. When viewed on a threefold vertex '(Fig. 2a), six capsomeres were clearly seen on an n = 6 rotation (Fig. 2b) but not on an n = 5 rotation (Fig. 2c). Peripheral capsomeres may not have been penetrated by stain and, thus, were not clearly seen. On a fivefold axis of symmetry (Fig. 2e), capsomeres were enhanced by an n = 5 rotation but not by an n = 6 rotation (Fig. 2f). Peripheral capsomeres were clearly enhanced by an n = 5 rotation. The diameters of the morphological unit and central hole were about 12 and 4 nm, respectively. Bands at densities of 1.36 and 1.37 g/ml both contained degraded virus particles and other smaller particles. However, a higher percentage of smaller particles was observed in the band at the density of 1.36 g/ml than in the band at the density of 1.37 g/ml. The latter band contained mostly stringlike structures. The smaller particles had a mean diameter of 15 nm and a range of 10 to 20 nm. These particles occurred singly, in large groups, and occasionally on the surface of degrading IBDV particles (Fig. ld). The stringlike structures observed in the 1.37-g/ml CsCl fraction (Fig. le) were 10 to 23 nm in width and appeared to be composed of smaller linked doughnut-shaped structures. Degraded virions were observed occasionally at a stage in which structural components were being released like an unwinding ball of string. The 5 and 10% gels used for electrophoresis
J. VIROL.
did not alter the IBDV polypeptide profiles obtained in 7.5% gels. Further heat dissociation of viral proteins before electrophoresis also did not alter the electrophoretic profiles. Electrophoresis of the major CsCl fraction having a buoyant density of 1.34 g/ml was done in more than 30 gels. A representative stained gel is shown in Fig. 3a. From these results, seven polypeptides representing viral structural proteins were evident and designated as P1, P2, P3, P4, P5, P6, and P7 (descending molecular weight). Two major bands, P3 and P4, were obvious, as indicated by their dense staining and high profiles. Five minor bands were also evident, of which two, P1 and P2, were seen as sharp but faint bands. The other minor bands, P5, P6, and P7, stained more diffusely. The molecular weights of viral polypeptides were determined by comparison with the relative mobilities of proteins of known molecular weights. An almost linear relationship was obtained between ratios of migration and molecular weight in the range of 12 x 103 to 160 X 103 for 7.5% gels (Fig. 4). The molecular weights of the seven structural components, representing averages of 15 separate determinations, were 133 x 103 (P1), 124 x 103 (P2), 98 x 103 (P3), 51 x 103 (P4), 33 x 103 (P5), 26.5 x 103 (P6), and 23 x 103 (P7). In Table 1, the molecular weights of the IBDV polypeptides are compared with those of rotavirus, bluetongue virus, and avian reovirus. The stained gel of the CsCl fraction at a density of 1.35 g/ml (Fig. 3b) did not differ from the gel of the major fraction shown in Fig. 3a. Electrophoresis of fractions at densities of 1.36 and 1.37 g/ml revealed that the polypeptide concentration of P4 was apparently less than that of P3, contrary to the standard profile of the virion. Other minor bands were not detected in these fractions (Fig. 3c). IBDV apparently has an outer layer, as indicated by the large size of intact particles with hexagonal outlines. Moreover, the capsomeric detail on the main capsid surface of IBDV often appeared partially obscured by such a layer. This outer layer was very thin (7 to 8 nm) and continuous, with T-shaped structures suggestive of the configuration reported for rotavirus but not as clearly defined (9, 12, 17). Although the outer layers of reovirus and orbivirus are also indistinct, they are featureless (7, 17). The outer layer of IBDV may exert some stability to the structure of the underlying major capsid layer. By rotational enhancement of image detail, the results of this study confirmed our previous report (5) that IBDV has an icosahedral symmetry of T = 3, with a probable 32 large capsomeres. This type of symmetry and the unique feature of subunit sharing are morphological characteristics reported for the Reoviridae family (11, 12).
FIG. 1. Electron micrographs of negatively stained IBDV particles. (a) Intact virions of IBDV recovered from the 1.34-g/ml CsCl. Bar = 100 nm. x130,000. (b) Higher magnification of an intact virion, showing the outer layer. The surface of the particles appear to have T-shaped morphology which is continuous. Bar = 50 nm. x450,000. (c) IBDV particles recovered from the 1.35-g/ml CsCl fraction. Intact virions of IBDV (arrow) and particles with clear capsomeres showing the loss of the outer layer (double arrow). Bar = 100 nm. x190,000. (d) Smaller particles recovered from the 1.36-g/ml CsCl fraction. Smaller particles on the surface of a degraded particle. Each particle appears to be a structural unit of the capsid. Bar = 50 nm. x380,000. (e) Stringlike structures recovered from the 1.37-g/ml CsCl fraction. Bar = 100 nm. x33,000. 325
326
NOTES
J. VIROL.
FIG. 2. (a-c) Single IBDV particle viewed on a threefold axis of symmetry, representing n = 0, n = 6, and n = 5 rotations, respectively. Enhancement of capsomeres is evident in the n = 6 rotation. Bar = 50 nm. x390,000. (d-f) Single IBDV particle viewed on a fivefold axis of symmetry, representing n = 0, n = 5, and n = 6 rotations, respectively. Enhancement of capsomeres is evident in the n = 5 rotation. Bar = 50 nm.
x390,000.
NOTES
VOL. 32, 1979
327
10'
4-
:
.1-i
P1 P2
co 0 H-
mo
P3
le-0L
U
P4
0.2 0.4 0.6 0.8 1.0+ Relative mobility
FIG. 4. Semilogarithmic plot of molecular weight against relative distance of migration ofmarkerproteins (0) and IBDVproteins (0) separated by electrophoresis on 7.5%polyacrylamide gels.
P5 P6 P7
TABLE 1. Comparison of molecular weights of IBDVproteins with those of rotavirus, bluetongue virus, and avian reovirus Mol wt (X103) IBDV
a
b
c
FIG. 3. Polyacrylamide gel electrophoresis of IBDV proteins. Virus purification was by isopycnic banding in CsCl gradients. Stained acrylamide gels of viral polypeptides from fractions at densities of 1.34 (a), 1.35 (b), and 1.36 and 1.37 (c) g/ml. Migration is from top to bottom.
The results of this study demonstrated that IBDV is composed of seven polypeptides, two major and five minor proteins. The major polypeptides were associated with the smaller subunit particles and stringlike structures, both having the same electrophoretic profile. Therefore, these polypeptides may be the capsomeral proteins of IBDV. These results differed from those reported by Nick et al. (10) with regard to the number of polypeptides and their molecular weights. Only four polypeptides were found in the previous study, three of which did compare closely in molecular weight with our P4, P5, and P6 proteins. However, a polypeptide having a molecular weight of 11 x 103 was not detected in
133 124 98 51 33 26.5 23
Human rotavirus'
127 103 97 88 58 32 26 21
a Rodger et al. (14). b Verwoerd et al. (18). CSpandidos and Graham
Avian reoviBluetongue rus' virusb
140 110 101 82 61 42 29
140 125 115 85 72 40 36 32
(16).
our preparations. Their failure to detect no more than four structural polypeptides may have resulted from partial disruption of virus particles before sodium dodecyl sulfate-polyacrylamide gel analysis. Thus, the limited amount of purified IBDV obtained may not have provided sufficient material or detection of all structural polypeptides. As shown in Table 1, the molecular weights of the IBDV proteins compared more closely with those reported for the human rotavirus (14). However, the major polypeptide species (P3 and P4) of IBDV did not correspond with those major proteins of the human rotavi-
328
NOTES
did IBDV have an eighth polypeptide of molecular weight 88 x 103. Our results concerning the morphology of IBDV and the similarity of its protein composition to the rotavirus group suggest its tentative inclusion into the Reoviridae family.
J. VIROL.
rus, nor
We are grateful to T. R. Meyers of Cornell University for the critical reading of and suggestions for the manuscript. This work was supported by a grant from The Ministry of Education, Science and Culture, Japan.
1.
2.
3.
4.
5. 6.
7.
LITERATURE CITED Almeida, J. D., and R. Morris. 1973. Antigenically-related virus associated with infectious bursal disease. J. Gen. Virol. 20:369-375. Harkness, J. W., D. J. Alexander, M. Pattison, and A. C. Scott. 1975. Infectious bursal disease agent: morphology by negative stain electron microscopy. Arch. Virol. 48:63-73. Hirai, K., and B. W. Calnek. 1979. In vitro replication of infectious bursal disease virus in established lymphoid cell lines and chicken B lymphocytes. Infect. Immun. 25:964-970. Hirai, K., E. Kawamoto, and S. Shimakura. 1974. Some properties of precipitating antigens associated with infectious bursal disease virus. Infect. Immun. 10: 1235-1240. Hirai, K., and S. Shimakura. 1974. Structure of infectious bursal disease virus. J. Virol. 14:957-964. Hirai, K., S. Shimakura, E. Kawamoto, N. Taguchi, S. T. Kim, C. N. Chang, Y. Adachi, Y. Suzuki, C. Itakura, F. Funakoshi, Y. Tsushio, and M. Hirose. 1973. Isolation of infectious bursal disease virus and distribution of precipitating antibodies in chicken sera. Jpn. J. Vet. Sci. 35:61-70. Luftig, R. B., S. S. Kilham, A. J. Hay, H. J. Zweerink,
8.
9.
10. 11.
12.
13.
14.
15.
16. 17.
18.
and W. K. Joklik. 1972. An ultrastructural study of virions and cores of reovirus type 3. Virology 48:171181. Markham, R., S. Frey, and G. J. Hills. 1963. Method for enhancement of image detail and accentuation of structure in electron microscopy. Virology 20:88-102. Martin, M. L., E. L. Palmer, and P. J. Middleton. 1975. Ultrastructure of infantile gastroenteritis virus. Virology 68:146-153. Nick, H., D. Cursiefen, and H. Becht. 1976. Structural and growth characteristics of infectious bursal disease virus. J. Virol. 18:227-234. Palmer, E. L., and M. L. Martin. 1977. The fine structure of the capsid of reovirus type 3. Virology 76:109-113. Palmer, E. L., M. L. Martin, and F. A. Murphy. 1977. Morphology and stability of infantile gastroenteritis virus: comparison with reovirus and bluetongue virus. J. Gen. Virol. 35:403-414. Pattison, M., D. J. Alexander, and J. W. Harkness. 1975. Purification and preliminary characterization of a pathogenic strain of infectious bursal disease virus. Avian Pathol. 4:175-187. Rodger, S. M., R. D. Schnagl, and L. N. Holmes. 1977. Further biochemical characterization, including the detection of surface glycoproteins of human, calf, and simian rotaviruses. J. Virol. 24:91-98. Shapiro, A. L., E. Virnela, and J. V. Maizel. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. Spandidos, D. A., and A. F. Graham. 1976. Physical and chemical characterization of an avian reovirus. J. Virol. 19:968-976. Stannard, L. M., and B. D. Schoub. 1977. Observations on the morphology of two rotaviruses. J. Gen. Virol. 37: 435-439. Verwoerd, D. W., H. J. Els, E.-M. de Villiers, and H. Huismans. 1972. Structure of the bluetongue virus capsid. J. Virol. 10:783-794.
OF
Vol. 32, No. 1
VIROLOGY, Oct. 1979, p. 323-328
0022-538X/79/10-0323/06$02.00/0
NOTES Further Morphological Characterization and Structural Proteins of Infectious Bursal Disease Virus K. HIRAI,* N. KATO,t A. FUJIURA,: AND S. SHIMAKURA Department of Veterinary Microbiology, Faculty ofAgriculture, Gifu University, Kakamigahara-shi, Gifu 504, Japan Received for publication 4 May 1979
An outer layer surrounding the capsid of infectious bursal disease virus was evident from electron micrographs of intact virus particles having diameters of 62 to 63 nm. The capsid was found to be composed of large morphological units or capsomeres, measuring about 12 nm in diameter. The architecture of the capsid appears to be that of T = 3 symmetry, with a probable 32 morphological units by rotational enhancement of image detail. Structural proteins of infectious bursal disease virus consist of seven species, two major and five minor polypeptides. These are P1 to P7, with molecular weights of 133 x 103, 124 x 103, 98 x 103, 51 X 103, 33 x 103, 26 x 103, and 23 x 103, respectively.
Infectious bursal disease (IBD) is a naturally occurring virus-induced immune deficiency of chickens characterized by necrosis and depletion of lymphoid elements. A recent study has suggested that surface immunoglobulin M-bearing B lymphocytes are target cells for infection by the IBD virus (IBDV) (3). Electron microscopy has shown that IBDV is a nonenveloped icosahedron with diameters ranging from 55 to 65 nm (1, 2, 5, 13). In a previous study (5), we suggested that the capsid of the virion consisted of 32 capsomeres, each composed of five or six structural units, and that IBDV resembled bluetongue virus in morphology. However, it is known that there are biological and physicochemical differences between IBDV and bluetongue virus. Apart from the initial study by Nick et al. (10), little information is available on the structural proteins of IBDV, using polyacrylamide gel electrophoresis. The exact number of polypeptides and their locations within the virion are not yet clearly understood. This report is concerned with a reexamination of two criteria: the fine structure of virions viewed with high-resolution electron microscopy and rotational enhancement of image detail and the polypeptide structure of IBDV, using polyacrylamide gel electrophoresis. Samples of the GBF-1 strain of IBDV were t Present address: Department of Urology, School of Medicine, Gifu University, Tsukasa, Gifu 500, Japan. : Present address: Aichi Prefectural Institute of Public Health, Nagare 7-6: Kitaku, Nagoya, Japan.
obtained as previously described (4, 6). Briefly, susceptible chickens were inoculated with virus from passage 15 to 20, and affected bursae of Fabricius were harvested for virus extraction. The purification procedure was essentially the same as that described previously (5). Electron microscopy was performed as previously described (5). Rotational enhancement of image detail was accomplished with a double-wheel device modified from the pinwheel apparatus described by Markham et al. (8). These modifications eliminated the hole produced at the axis of rotation. Virus samples were solubilized by incubation for 2 h at 37°C with equal amounts of the 0.01 M phosphate buffer (pH 7.2) containing 2% sodium dodecyl sulfate, 6 to 8% 2-mercaptoethanol, and 50% glycerol, followed by heating for 2 min at 1000C before electrophoresis. The polypeptide composition of the virus was determined in 7.5% polyacrylamide gels as described by Spandidos and Graham (16). Electrophoresis was carried out at a constant current of 5 mA/gel for 8 h. The molecular weights of polypeptides determined on 7.5% gels by the procedure of Shapiro et al. (15) were used for the final results. After three equilibrium centrifugations in cesium chloride, the major band of IBDV occurred at a buoyant density of 1.34 g/ml, and three faint minor bands occurred at buoyant densities of 1.35, 1.36, and 1.37 g/ml, respectively. No cellular debris was observed in any band when examined by electron microscopy. Bands at den-
323
324
NOTES
sities of 1.34 and 1.35 g/ml both contained numerous particles with hexagonal or roughly spherical profiles. Hexagonal virus particles predominated at the density of 1.34 g/ml, whereas the band at the density of 1.35 g/ml contained mostly spherical particles. Intact virions were generally hexagonal in profile, and surface capsomere arrangement of the morphological unit was not clearly resolved (Fig. la and b). These particles averaged from 62 to 63 nm in diameter and appeared to be covered by an outer layer composed of T-shaped structures (Fig. lb). The outer layer was continuous. Spherical particles without an apparent outer layer predominated at the density of 1.35 g/ml (Fig. lc). Their diameters were 54 to 55 nm, and morphological units were clearly observed. At a higher magnification, each morphological unit seemed to be composed of smaller subunits in hexagonal-pentagonal arrays around a central hole (Fig. 2a and d). Subunits appeared to be shared by neighboring structural units. In previous ultrastructural studies on the morphology of IBDV (5), we suggested that the capsid of the virion consisted of 32 capsomeres arranged in 5:3:2 symmetry. In an attempt to further elucidate the capsomere arrangement, rotational enhancement of image detail has been accomplished with a few IBDV particles. When viewed on a threefold vertex '(Fig. 2a), six capsomeres were clearly seen on an n = 6 rotation (Fig. 2b) but not on an n = 5 rotation (Fig. 2c). Peripheral capsomeres may not have been penetrated by stain and, thus, were not clearly seen. On a fivefold axis of symmetry (Fig. 2e), capsomeres were enhanced by an n = 5 rotation but not by an n = 6 rotation (Fig. 2f). Peripheral capsomeres were clearly enhanced by an n = 5 rotation. The diameters of the morphological unit and central hole were about 12 and 4 nm, respectively. Bands at densities of 1.36 and 1.37 g/ml both contained degraded virus particles and other smaller particles. However, a higher percentage of smaller particles was observed in the band at the density of 1.36 g/ml than in the band at the density of 1.37 g/ml. The latter band contained mostly stringlike structures. The smaller particles had a mean diameter of 15 nm and a range of 10 to 20 nm. These particles occurred singly, in large groups, and occasionally on the surface of degrading IBDV particles (Fig. ld). The stringlike structures observed in the 1.37-g/ml CsCl fraction (Fig. le) were 10 to 23 nm in width and appeared to be composed of smaller linked doughnut-shaped structures. Degraded virions were observed occasionally at a stage in which structural components were being released like an unwinding ball of string. The 5 and 10% gels used for electrophoresis
J. VIROL.
did not alter the IBDV polypeptide profiles obtained in 7.5% gels. Further heat dissociation of viral proteins before electrophoresis also did not alter the electrophoretic profiles. Electrophoresis of the major CsCl fraction having a buoyant density of 1.34 g/ml was done in more than 30 gels. A representative stained gel is shown in Fig. 3a. From these results, seven polypeptides representing viral structural proteins were evident and designated as P1, P2, P3, P4, P5, P6, and P7 (descending molecular weight). Two major bands, P3 and P4, were obvious, as indicated by their dense staining and high profiles. Five minor bands were also evident, of which two, P1 and P2, were seen as sharp but faint bands. The other minor bands, P5, P6, and P7, stained more diffusely. The molecular weights of viral polypeptides were determined by comparison with the relative mobilities of proteins of known molecular weights. An almost linear relationship was obtained between ratios of migration and molecular weight in the range of 12 x 103 to 160 X 103 for 7.5% gels (Fig. 4). The molecular weights of the seven structural components, representing averages of 15 separate determinations, were 133 x 103 (P1), 124 x 103 (P2), 98 x 103 (P3), 51 x 103 (P4), 33 x 103 (P5), 26.5 x 103 (P6), and 23 x 103 (P7). In Table 1, the molecular weights of the IBDV polypeptides are compared with those of rotavirus, bluetongue virus, and avian reovirus. The stained gel of the CsCl fraction at a density of 1.35 g/ml (Fig. 3b) did not differ from the gel of the major fraction shown in Fig. 3a. Electrophoresis of fractions at densities of 1.36 and 1.37 g/ml revealed that the polypeptide concentration of P4 was apparently less than that of P3, contrary to the standard profile of the virion. Other minor bands were not detected in these fractions (Fig. 3c). IBDV apparently has an outer layer, as indicated by the large size of intact particles with hexagonal outlines. Moreover, the capsomeric detail on the main capsid surface of IBDV often appeared partially obscured by such a layer. This outer layer was very thin (7 to 8 nm) and continuous, with T-shaped structures suggestive of the configuration reported for rotavirus but not as clearly defined (9, 12, 17). Although the outer layers of reovirus and orbivirus are also indistinct, they are featureless (7, 17). The outer layer of IBDV may exert some stability to the structure of the underlying major capsid layer. By rotational enhancement of image detail, the results of this study confirmed our previous report (5) that IBDV has an icosahedral symmetry of T = 3, with a probable 32 large capsomeres. This type of symmetry and the unique feature of subunit sharing are morphological characteristics reported for the Reoviridae family (11, 12).
FIG. 1. Electron micrographs of negatively stained IBDV particles. (a) Intact virions of IBDV recovered from the 1.34-g/ml CsCl. Bar = 100 nm. x130,000. (b) Higher magnification of an intact virion, showing the outer layer. The surface of the particles appear to have T-shaped morphology which is continuous. Bar = 50 nm. x450,000. (c) IBDV particles recovered from the 1.35-g/ml CsCl fraction. Intact virions of IBDV (arrow) and particles with clear capsomeres showing the loss of the outer layer (double arrow). Bar = 100 nm. x190,000. (d) Smaller particles recovered from the 1.36-g/ml CsCl fraction. Smaller particles on the surface of a degraded particle. Each particle appears to be a structural unit of the capsid. Bar = 50 nm. x380,000. (e) Stringlike structures recovered from the 1.37-g/ml CsCl fraction. Bar = 100 nm. x33,000. 325
326
NOTES
J. VIROL.
FIG. 2. (a-c) Single IBDV particle viewed on a threefold axis of symmetry, representing n = 0, n = 6, and n = 5 rotations, respectively. Enhancement of capsomeres is evident in the n = 6 rotation. Bar = 50 nm. x390,000. (d-f) Single IBDV particle viewed on a fivefold axis of symmetry, representing n = 0, n = 5, and n = 6 rotations, respectively. Enhancement of capsomeres is evident in the n = 5 rotation. Bar = 50 nm.
x390,000.
NOTES
VOL. 32, 1979
327
10'
4-
:
.1-i
P1 P2
co 0 H-
mo
P3
le-0L
U
P4
0.2 0.4 0.6 0.8 1.0+ Relative mobility
FIG. 4. Semilogarithmic plot of molecular weight against relative distance of migration ofmarkerproteins (0) and IBDVproteins (0) separated by electrophoresis on 7.5%polyacrylamide gels.
P5 P6 P7
TABLE 1. Comparison of molecular weights of IBDVproteins with those of rotavirus, bluetongue virus, and avian reovirus Mol wt (X103) IBDV
a
b
c
FIG. 3. Polyacrylamide gel electrophoresis of IBDV proteins. Virus purification was by isopycnic banding in CsCl gradients. Stained acrylamide gels of viral polypeptides from fractions at densities of 1.34 (a), 1.35 (b), and 1.36 and 1.37 (c) g/ml. Migration is from top to bottom.
The results of this study demonstrated that IBDV is composed of seven polypeptides, two major and five minor proteins. The major polypeptides were associated with the smaller subunit particles and stringlike structures, both having the same electrophoretic profile. Therefore, these polypeptides may be the capsomeral proteins of IBDV. These results differed from those reported by Nick et al. (10) with regard to the number of polypeptides and their molecular weights. Only four polypeptides were found in the previous study, three of which did compare closely in molecular weight with our P4, P5, and P6 proteins. However, a polypeptide having a molecular weight of 11 x 103 was not detected in
133 124 98 51 33 26.5 23
Human rotavirus'
127 103 97 88 58 32 26 21
a Rodger et al. (14). b Verwoerd et al. (18). CSpandidos and Graham
Avian reoviBluetongue rus' virusb
140 110 101 82 61 42 29
140 125 115 85 72 40 36 32
(16).
our preparations. Their failure to detect no more than four structural polypeptides may have resulted from partial disruption of virus particles before sodium dodecyl sulfate-polyacrylamide gel analysis. Thus, the limited amount of purified IBDV obtained may not have provided sufficient material or detection of all structural polypeptides. As shown in Table 1, the molecular weights of the IBDV proteins compared more closely with those reported for the human rotavirus (14). However, the major polypeptide species (P3 and P4) of IBDV did not correspond with those major proteins of the human rotavi-
328
NOTES
did IBDV have an eighth polypeptide of molecular weight 88 x 103. Our results concerning the morphology of IBDV and the similarity of its protein composition to the rotavirus group suggest its tentative inclusion into the Reoviridae family.
J. VIROL.
rus, nor
We are grateful to T. R. Meyers of Cornell University for the critical reading of and suggestions for the manuscript. This work was supported by a grant from The Ministry of Education, Science and Culture, Japan.
1.
2.
3.
4.
5. 6.
7.
LITERATURE CITED Almeida, J. D., and R. Morris. 1973. Antigenically-related virus associated with infectious bursal disease. J. Gen. Virol. 20:369-375. Harkness, J. W., D. J. Alexander, M. Pattison, and A. C. Scott. 1975. Infectious bursal disease agent: morphology by negative stain electron microscopy. Arch. Virol. 48:63-73. Hirai, K., and B. W. Calnek. 1979. In vitro replication of infectious bursal disease virus in established lymphoid cell lines and chicken B lymphocytes. Infect. Immun. 25:964-970. Hirai, K., E. Kawamoto, and S. Shimakura. 1974. Some properties of precipitating antigens associated with infectious bursal disease virus. Infect. Immun. 10: 1235-1240. Hirai, K., and S. Shimakura. 1974. Structure of infectious bursal disease virus. J. Virol. 14:957-964. Hirai, K., S. Shimakura, E. Kawamoto, N. Taguchi, S. T. Kim, C. N. Chang, Y. Adachi, Y. Suzuki, C. Itakura, F. Funakoshi, Y. Tsushio, and M. Hirose. 1973. Isolation of infectious bursal disease virus and distribution of precipitating antibodies in chicken sera. Jpn. J. Vet. Sci. 35:61-70. Luftig, R. B., S. S. Kilham, A. J. Hay, H. J. Zweerink,
8.
9.
10. 11.
12.
13.
14.
15.
16. 17.
18.
and W. K. Joklik. 1972. An ultrastructural study of virions and cores of reovirus type 3. Virology 48:171181. Markham, R., S. Frey, and G. J. Hills. 1963. Method for enhancement of image detail and accentuation of structure in electron microscopy. Virology 20:88-102. Martin, M. L., E. L. Palmer, and P. J. Middleton. 1975. Ultrastructure of infantile gastroenteritis virus. Virology 68:146-153. Nick, H., D. Cursiefen, and H. Becht. 1976. Structural and growth characteristics of infectious bursal disease virus. J. Virol. 18:227-234. Palmer, E. L., and M. L. Martin. 1977. The fine structure of the capsid of reovirus type 3. Virology 76:109-113. Palmer, E. L., M. L. Martin, and F. A. Murphy. 1977. Morphology and stability of infantile gastroenteritis virus: comparison with reovirus and bluetongue virus. J. Gen. Virol. 35:403-414. Pattison, M., D. J. Alexander, and J. W. Harkness. 1975. Purification and preliminary characterization of a pathogenic strain of infectious bursal disease virus. Avian Pathol. 4:175-187. Rodger, S. M., R. D. Schnagl, and L. N. Holmes. 1977. Further biochemical characterization, including the detection of surface glycoproteins of human, calf, and simian rotaviruses. J. Virol. 24:91-98. Shapiro, A. L., E. Virnela, and J. V. Maizel. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. Spandidos, D. A., and A. F. Graham. 1976. Physical and chemical characterization of an avian reovirus. J. Virol. 19:968-976. Stannard, L. M., and B. D. Schoub. 1977. Observations on the morphology of two rotaviruses. J. Gen. Virol. 37: 435-439. Verwoerd, D. W., H. J. Els, E.-M. de Villiers, and H. Huismans. 1972. Structure of the bluetongue virus capsid. J. Virol. 10:783-794.
