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Hemolytic activity of rubella virus
VIROLOGY
89.610412
(1978)
Hemolytic
Activity
of Rubella
NOBUYOSHI Public
Health
institute
of
Yokohomn
City, Accepted
Virus
KOBAYASHI Takignshirn May
l-2-17,
Isogo,
Yokohama
235, Japan
19, 1978
Hemolytic activity of rubella virus was demonstrated by chelating Ca” with EDTA after adsorption of the virus to erythrocytes in the presence of the ion. The hemolysis was inhibited by rabbit serum immunized with the virus. Furthermore, the hemolytic activity co-sedimented in sucrose density gradient centrifugation with the hemagglutinating and plaque-forming activity of the virus, suggesting the association of the hemolytic activity with rubella virions.
Since the study by Stewart et al. (1) in 1967, hemagglutination (HA) by rubella virus has been extensively utilized for the study of the virus and for the diagnosis of the disease. Hemolytic activity of rubella virus, however, has not been demonstrated so far, although the HA activity of the virus and “fusion from within” (2) of the virusinfected cells has suggested that the virus can carry out hemolysis. Inasmuch as virus-induced hemolysis in general is inhibited by Ca2+ (3-5), hemolysis by rubella virus would also be inhibited by Ca” and the extent of the inhibition might be so high that any hemolysis could not be observed in the presence of trace Ca’+. Ca”, on the other hand, seems to be essential for HA by rubella virus, as it is stimulated by the ion and extremely reduced by EDTA (6). Therefore, complete removal of Ca”’ from a reaction mixture would inhibit the adsorption of the virus to erythrocytes; no hemolysis would then be observed. The present paper shows, however, that hemolytic activity of rubella virus can be demonstrated by separating the reaction between the virus and erythrocytes into two steps: adsorption of the virus to erythrocytes in the presence of Ca2+ and cell lysis by chelating the ion with EDTA. The media from Vero cell cultures infected with the M-33 strain of rubella virus, after removing cell debris by centrifugation, were treated by three cycles of freezing and thawing, and used as virus preparation for
the analysis of hemolysis. One milliliter of the virus, 64 HA units/ml, was mixed with 1 ml of 2% chick erythrocyte suspension in phosphate-buffered saline containing 1 mM CaCL, 1 mM MgCl2, 0.1% bovine serum albumin, and 0.001% gelatine, pH 6.4, and incubated at 4” for 90 min. Various concentratins of EDTA were then added to each reaction mixture which were further incubated at 37’ for 120 min. The extent of hemolysis was determined by measuring the absorbance at 540 nm of released hemoglobin in the supernatant obtained by centrifugating the reaction mixture. Significant development of hemolysis was observed when more than 1 mM EDTA was added, although little hemolysis was seen without or with 0.5 mM EDTA (Table 1). The highest hemolysis was obtained with 1.5 mM EDTA with which approximately 15% of the cells were hemolysed. In contrast, when an uninfected cell culture fluid was processed in a same manner, no hemolysis occurred at all concentrations of EDTA tested (Table 1). To determine whether the hemolytic activity thus demonstrated is associated with rubella virus, the effect of specific antiserum on the hemolysis was studied. The serum was obtained by immunizing a rabbit with rubella virus (strain M-33) (7); it showed a HA inhibition titer of 1:1+X4. The result showed that more than 90% reduction of hemolysis occurred at the dilution of 1:40 of the immune serum (Fig. 1). In 610
004%6822/78/0892-0610$02.00/O Copy-right 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
611
COMMUNICATIONS
contrast, only slight reduction was yielded with nonimmune serum at lower dilution. To study whether the hemolytic activity is revealed by infectious virus particles, rubella virus was pelleted from an infectious culture fluid and subjected to equilibrium sedimentation through a 5 to 50% sucrose density gradient (8). Fractions were assayed for hemolytic activity, HA activity, infectivity, and density. As shown in Fig. 2, the profile of hemolytic activity coincided with those of HA activity and of infectivity. TABLE
1
HEMOLYSISINIXICEDBYVARIOUSCONCENTRATIONS OF EDTA AFTERTHEADSORPTION OFRUBELLA V~RU~TOER~THROCYT& Absorbance EDTA concentrations (mJ4)
Infected culture
0
cell fluid
Uninfected cell culture fluid
0.01 0.04 0.17 0.23 0.21 0.19 0.18 0.11
0.5 1
1.5 2 3 4 5
at 540 nm
0.00 0.00 0.00 0.01 0.00 0.01 0.01 0.01
Fraction
number
FIG. 2. Association of hemolytic activity with rubella virions. Media from Vero cell culture infected with rubella virus, after removing cell debris, were treated with 30 mM EDTA for 1 hr at 4’ (8). Viruses were pelleted down at 40,OOOg for 2 hr at 4”, suspended in 1 ml phosphate-buffered saline. layered on a 5 to 50% sucrose gradient in 50 n&f Tris-HCl, 1 n* EDTA, pH 7.3, and centrifuged at 100,000 g for 2 hr at 4O in a Hitachi RPS40T-2 rotor. Hemolysis activity (w) was measured as described in the text with I.5 mM EDTA. Hemagglutination (n-.-n) was carried out as previously described (1). Infectivity (0- - -0) was determined by plaque formation on Vero cell monolayers (7). Densities (0-0) were measured using a Shimazu type 1 Abbe refractometer.
_,-,-z-T-7 . ,d 0’ Id’ ,’r /’P
” The procedure
is described
in the text
i’
/ L 40
Dilution-’
102d
640 of
serum
FIG. 1. Effects of immune serum on the hemolysis by rubella virus. Immune serum prepared by immunizing a rabbit with rubella virus (7). Rubella virus, 64 HA units in 0.5 ml, was treated with the same volume of diluted immune or normal sera at room temperature for 60 min. The treated virus was then assayed for hemolytic activity using 1.5 m&f EDTA as described in the text. u, immune serum; 0- - -0, normal serum.
The density of these peaks was 1.17 g/ml, being in agreement with a value previously obtained for the density of rubella virus (8). The present finding suggests that some of those viruses which contain HA activity but have not yet shown any hemolysis by conventional methods may reveal hemolytic activity by the procedure described here. Of particular interest is to test other members of the togavirus group for hemolytic activity by the procedure, since rubella virus has recently been placed in the togavirus group (9) and two other members of the group are also known to carry out hemolysis ( 10). ACKNOWLEDGMENTS
I am grateful to Drs. Y. Yogo and H. Shibuta for a critical reading of the manuscript and to Drs. Y. Saburi and H. Mori for helpful suggestions.
612
SHORT
COMMUNICATIONS
Gesamte.
REFERENCES
6. FURUKAWA, 1. STEWART, DOUGLAS,
G. L., PARKMAN, P. I)., HOPPS, H. E., D. D., HAMILTON, J. P., and MEYER,
H. M., JR., N. Engl. J. Med. 2. BRATT, M. A., and GALLAHER,
Acad. 3. BURNET, 4. MORGAN,
W.
(1967).
R., Proc.
Sci. USA, 64.536-543 (1969). F. M., Nature 164, 1008 (1949). H. R., Proc. Sot. Exp. Biol. Med.
276-278 5. NORRBY,
276,554-557
(1951). E. C. J., and
Nat.
and
L. G., Arch.
PROFETA, K., and
15(5),
39-46
14, 474-486 (1964). S. A., SEDWICK,
M. L., Proc.
Sot. Exp.
Biol.
W.
D.,
Med.
(1967). MATUMOTO,
397-406 (1971). A., BONSDORFF vow, T., AND VAXNANEN,
M.,
Jpn J. Microbial. C. H., VESIKARI, T., P., J. Gen. Virol. 5,
( 1969). F., Intervirology 7(1-2), 44-47 (1976). 20. KARABATOS, N., J. Zmmunol. 91, 76-82 (1963). 9. FENNER,
FALKSVEDEN,
T., PLOTKIN,
126,745-750 7. SODA, 8. VAHERI, Hovr,
77,
Vzrusforsh.
89.610412
(1978)
Hemolytic
Activity
of Rubella
NOBUYOSHI Public
Health
institute
of
Yokohomn
City, Accepted
Virus
KOBAYASHI Takignshirn May
l-2-17,
Isogo,
Yokohama
235, Japan
19, 1978
Hemolytic activity of rubella virus was demonstrated by chelating Ca” with EDTA after adsorption of the virus to erythrocytes in the presence of the ion. The hemolysis was inhibited by rabbit serum immunized with the virus. Furthermore, the hemolytic activity co-sedimented in sucrose density gradient centrifugation with the hemagglutinating and plaque-forming activity of the virus, suggesting the association of the hemolytic activity with rubella virions.
Since the study by Stewart et al. (1) in 1967, hemagglutination (HA) by rubella virus has been extensively utilized for the study of the virus and for the diagnosis of the disease. Hemolytic activity of rubella virus, however, has not been demonstrated so far, although the HA activity of the virus and “fusion from within” (2) of the virusinfected cells has suggested that the virus can carry out hemolysis. Inasmuch as virus-induced hemolysis in general is inhibited by Ca2+ (3-5), hemolysis by rubella virus would also be inhibited by Ca” and the extent of the inhibition might be so high that any hemolysis could not be observed in the presence of trace Ca’+. Ca”, on the other hand, seems to be essential for HA by rubella virus, as it is stimulated by the ion and extremely reduced by EDTA (6). Therefore, complete removal of Ca”’ from a reaction mixture would inhibit the adsorption of the virus to erythrocytes; no hemolysis would then be observed. The present paper shows, however, that hemolytic activity of rubella virus can be demonstrated by separating the reaction between the virus and erythrocytes into two steps: adsorption of the virus to erythrocytes in the presence of Ca2+ and cell lysis by chelating the ion with EDTA. The media from Vero cell cultures infected with the M-33 strain of rubella virus, after removing cell debris by centrifugation, were treated by three cycles of freezing and thawing, and used as virus preparation for
the analysis of hemolysis. One milliliter of the virus, 64 HA units/ml, was mixed with 1 ml of 2% chick erythrocyte suspension in phosphate-buffered saline containing 1 mM CaCL, 1 mM MgCl2, 0.1% bovine serum albumin, and 0.001% gelatine, pH 6.4, and incubated at 4” for 90 min. Various concentratins of EDTA were then added to each reaction mixture which were further incubated at 37’ for 120 min. The extent of hemolysis was determined by measuring the absorbance at 540 nm of released hemoglobin in the supernatant obtained by centrifugating the reaction mixture. Significant development of hemolysis was observed when more than 1 mM EDTA was added, although little hemolysis was seen without or with 0.5 mM EDTA (Table 1). The highest hemolysis was obtained with 1.5 mM EDTA with which approximately 15% of the cells were hemolysed. In contrast, when an uninfected cell culture fluid was processed in a same manner, no hemolysis occurred at all concentrations of EDTA tested (Table 1). To determine whether the hemolytic activity thus demonstrated is associated with rubella virus, the effect of specific antiserum on the hemolysis was studied. The serum was obtained by immunizing a rabbit with rubella virus (strain M-33) (7); it showed a HA inhibition titer of 1:1+X4. The result showed that more than 90% reduction of hemolysis occurred at the dilution of 1:40 of the immune serum (Fig. 1). In 610
004%6822/78/0892-0610$02.00/O Copy-right 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
611
COMMUNICATIONS
contrast, only slight reduction was yielded with nonimmune serum at lower dilution. To study whether the hemolytic activity is revealed by infectious virus particles, rubella virus was pelleted from an infectious culture fluid and subjected to equilibrium sedimentation through a 5 to 50% sucrose density gradient (8). Fractions were assayed for hemolytic activity, HA activity, infectivity, and density. As shown in Fig. 2, the profile of hemolytic activity coincided with those of HA activity and of infectivity. TABLE
1
HEMOLYSISINIXICEDBYVARIOUSCONCENTRATIONS OF EDTA AFTERTHEADSORPTION OFRUBELLA V~RU~TOER~THROCYT& Absorbance EDTA concentrations (mJ4)
Infected culture
0
cell fluid
Uninfected cell culture fluid
0.01 0.04 0.17 0.23 0.21 0.19 0.18 0.11
0.5 1
1.5 2 3 4 5
at 540 nm
0.00 0.00 0.00 0.01 0.00 0.01 0.01 0.01
Fraction
number
FIG. 2. Association of hemolytic activity with rubella virions. Media from Vero cell culture infected with rubella virus, after removing cell debris, were treated with 30 mM EDTA for 1 hr at 4’ (8). Viruses were pelleted down at 40,OOOg for 2 hr at 4”, suspended in 1 ml phosphate-buffered saline. layered on a 5 to 50% sucrose gradient in 50 n&f Tris-HCl, 1 n* EDTA, pH 7.3, and centrifuged at 100,000 g for 2 hr at 4O in a Hitachi RPS40T-2 rotor. Hemolysis activity (w) was measured as described in the text with I.5 mM EDTA. Hemagglutination (n-.-n) was carried out as previously described (1). Infectivity (0- - -0) was determined by plaque formation on Vero cell monolayers (7). Densities (0-0) were measured using a Shimazu type 1 Abbe refractometer.
_,-,-z-T-7 . ,d 0’ Id’ ,’r /’P
” The procedure
is described
in the text
i’
/ L 40
Dilution-’
102d
640 of
serum
FIG. 1. Effects of immune serum on the hemolysis by rubella virus. Immune serum prepared by immunizing a rabbit with rubella virus (7). Rubella virus, 64 HA units in 0.5 ml, was treated with the same volume of diluted immune or normal sera at room temperature for 60 min. The treated virus was then assayed for hemolytic activity using 1.5 m&f EDTA as described in the text. u, immune serum; 0- - -0, normal serum.
The density of these peaks was 1.17 g/ml, being in agreement with a value previously obtained for the density of rubella virus (8). The present finding suggests that some of those viruses which contain HA activity but have not yet shown any hemolysis by conventional methods may reveal hemolytic activity by the procedure described here. Of particular interest is to test other members of the togavirus group for hemolytic activity by the procedure, since rubella virus has recently been placed in the togavirus group (9) and two other members of the group are also known to carry out hemolysis ( 10). ACKNOWLEDGMENTS
I am grateful to Drs. Y. Yogo and H. Shibuta for a critical reading of the manuscript and to Drs. Y. Saburi and H. Mori for helpful suggestions.
612
SHORT
COMMUNICATIONS
Gesamte.
REFERENCES
6. FURUKAWA, 1. STEWART, DOUGLAS,
G. L., PARKMAN, P. I)., HOPPS, H. E., D. D., HAMILTON, J. P., and MEYER,
H. M., JR., N. Engl. J. Med. 2. BRATT, M. A., and GALLAHER,
Acad. 3. BURNET, 4. MORGAN,
W.
(1967).
R., Proc.
Sci. USA, 64.536-543 (1969). F. M., Nature 164, 1008 (1949). H. R., Proc. Sot. Exp. Biol. Med.
276-278 5. NORRBY,
276,554-557
(1951). E. C. J., and
Nat.
and
L. G., Arch.
PROFETA, K., and
15(5),
39-46
14, 474-486 (1964). S. A., SEDWICK,
M. L., Proc.
Sot. Exp.
Biol.
W.
D.,
Med.
(1967). MATUMOTO,
397-406 (1971). A., BONSDORFF vow, T., AND VAXNANEN,
M.,
Jpn J. Microbial. C. H., VESIKARI, T., P., J. Gen. Virol. 5,
( 1969). F., Intervirology 7(1-2), 44-47 (1976). 20. KARABATOS, N., J. Zmmunol. 91, 76-82 (1963). 9. FENNER,
FALKSVEDEN,
T., PLOTKIN,
126,745-750 7. SODA, 8. VAHERI, Hovr,
77,
Vzrusforsh.