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Heterogeneity of Newcastle disease virus (NDV) particles with respect to their hemolytic activity
126
DISCUSSION
AND
PRELIMINARY
fective dye concentration at the time and site of viral maturation. The effective concentration at the time of eclipse might be considerably lower than at maturation because of a difference in sites, temporal changes, or both. Although it is likely that a photosensitive nucleic acid-dye complex occurs during eclipse, photooxidation of protein is not ruled out as a cause of the observed effect. Further experiments will be necessary to fully elucidate these problems.
REPORTS
The Blacksburg strain of NDV in its 26th and 27th egg passages was employed in these experiments. The virus from the infectious allantoic fluid was concentrated and partially purified by two cycles of differential centrifugation and treatment with ribonuclease and deoxyribonuclease. The final suspension of virus in Tris saline [0.02 M tris (hydroxymethyl) aminomethane, 0.13 M NaCl, pH 7.31 contained 4.9 x lo4 hemagglutinating (HA) units per milliliter. A DEAE-cellulose (Whatman, 1.0 meq. REFERENCES N per gram) column (1.4 x 16 cm) was Photobiol. 1, 1471. HACKETT, A. J., Photochem. loaded with 5 ml of this virus suspension 154 (1962). diluted with 10 ml of 0.02 M Tris buffer, 2. WILSON, J. N., and COOPER, P. D., Virology 17, pH 7.3. The virus was eluted at 25” with a 195-196 (1962). slightly concave gradient of NaCl at con3. MCBRIDE, W. D., Bactetiol. Proc. (Sot. Am. stant pH (7.3) and molarity of Tris buffer Bacteriologists), p. 130 (1962). (0.02). The undiluted effluent fractions were 18, 412-425 (1962). 4. SCHAFFER, F. L., Virology examined for absorbancy at 260 and 280 17, 28%294 (1962). 6. MANDEL, B., Virology mp. Their hemagglutination and hemolytic 6. MANDEL, B., Cold Spring Harbor Symp. Quant. Biol. 27,123-136 (1962). (after adjustment of the NaCl concentra7. DARNELL, J. E., JR., Cold Spring Harbor Symp. tion to 0.13 M) titers were determined as Quant. Biol. 27, 149-158 (1962). described previously (3). Proteins were deF. L. SCHAFFER termined as described by Lowry et al. (4). A. J. HACKETT The chromatographic pattern of viral heNaval Biological Laboratory magglutinins eluted at 0.125-0.250 M salt University of California was composed of several overlapping peaks Berkeley, California (Fig. 1). The virus eluted in this region Accepted May 28, 196s showed low EID&HA ( <104.0) (5) and high HA/% H (about 500; % H = percentage of hemolysis) ratios. The next two Heterogeneity of Newcastle Disease Virus peaks of hemagglutinins corresponded to (NDV) Particles with Respect to their fully infectious virus (EID,,/HA about 106.0), the maxima of hemagglutination acHemolytic Activity tivity coinciding with those of infectivity. The reason for the separation of fully infecIt has been shown that various preparatious virus into two distinct fractions is not tions of NDV-infected allantoic fluids differ in their hemolytic activities (1, 6), a quite clear. It is possible that the first of finding indicating heterogeneity with re- these fractions corresponded to a complex of virus with the normal component of spect to this property of the virus particles. allantoic fluid eluted from It was also shown that the hemolysin is noninfective DEAE-cellulose columns at 0.25 M NaCl. bound to the virus particle (a). However, This was indicated by control experiments no attempt was made to separate the virus with normal components of allantoic fluid particles into fractions exhibiting different concentrated by sedimentation, as well as hemolytic activities. Experiments described by the fact that in chromatogram of virus in the present communication show that treated with the receptor-destroying enchromatography on diethylaminoethyl zyme from Vibrio cholerae this first peak (DEAE) -cellulose columns, or density grawas not present. It is, however, evident dient centrifugation, can be successfully that in both peaks the maxima of hemolytic used for such fractionation.
DISCUSSION
0
100
AND PRELIMINARY
200 Volume in ml.
REPORTS
127
300
400
FIG. 1. Fractionation of NDV particles by chromatography on DEAE-cellulose column. Six-milliliter fractions were collected at a rate of 1.2 ml per minute. Proteins were determined in 0.5-ml amounts of the fractions made up to 3.25 ml with the reagents. The recovery of hemagglutinins (HA), hemolysin (H), and proteins was 100, 63, and 52%, respectively.
activity did not, coincide with those of hemagglutinating activity, the virus particles with relatively high hemolytic activity being eluted at markedly higher salt concentrations than the bulk of the virus. Virus particles exhibiting the lowest HA/% H ratio (about 35) were eluted at 0.325 and 0.450-0.625111 NaCI, respectively, whereas those eluted at the maxima of the two peaks had HA/% H ratios of about 200. Two virus preparations obtained by the procedure described and containing 2.4 x lo4 and 6.6 x lo4 HA units of virus per milliliter, respectively, were used in sucrose density gradient centrifugation experiments. Tubes were filled by layering successively 0.8 ml each of 60, 50, 40, 20, 10, and 5% (w/v) sucrose in Tris saline and kept overnight at 4”. Then 0.25 ml of virus suspension was layered over the sucrose solution and the tubes were centrifuged at, room temperature in the Spinco swinging-bucket rotor SW 39 model L at 20,000 rpm for 15 minutes. Fractions of 5 drops each were collected by puncturing the bottom of the tube. These were diluted by the addition of 0.75 ml of 0.1 M Tris pH 7.8, and their hemagglutination titers were estimated. For esti-
mation of hemolytic activity, the samples were further diluted 4 times, and after hemolysis the optical densities of the solutions read against appropriate controls. The presence of excess sucrose did not, cause nonspecific hemolysis. The results are presented in Fig. 2. Three hemagglutinating components are discernible in the sedimentation pattern, the bottom component being poorly separated from the middle one. The top component is probably identical with the hemagglutinin fraction eluted from the DEAE-cellulose column at 0.125&0).250 M NaCl, as it is only slightly hemolytic (HA/% H ratio about 470) and more than 100 times less infective than the fully infectious viruses in the middle and bottom components. These particles are similar to or identical with the noninfective, nonhemolytic hemagglutinins derived from NDV-infected tissue or allantoic fluid (6). The virus particles in the middle component have a HA/% H ratio of about 200. The virus
particles
with
the highest
exhibit (HA/% studies
the
H
in the
bottom
highest
constant
hemolytic
19.2 in fraction
by Neurath
component
sedimentation
activity
8).
Recent
(7) also have revealed
128
DISCUSSION
AND
PRELIMINART
30
20 x 2 O
10
0
4
8
12
16
20
number FIG. 2. Fractionation of NDV particles by sucrose density gradient centrifugation. The arrow points to the bottom fraction. Hemolysin and hemagglutinins were quantitatively recovered after centrifugation. Ordinate on the right: HA units per fraction. Fraction
20
2
REPORTS
density gradient centrifugation of untreated NDV and of virus subjected to 5 cycles of freezing and thawing showed (Fig. 3) that after freezing and thawing a new peak of hemolysins appeared aproximately in the middle component. The hemolytic activities of the virus particles in the bottom and top components were csscntially unaffected by this treatment. It must be added, however, that in treated sample the sedimentation rate of hcmagglutinins from the middle component was decreased (8). It appears that only virus particles from the middle component contain masked hemolysins which can be liberated by repeated freezing and thawing. The results of chromatographic and of density gradient centrifugation experiments described here were confirmed in repeated experiments. Investigations with plaquepurified fractions of NDV are now in progress to determine whether the variation in hemolytic activity is of heritable nature. REFERENCES
1. BURNET,
F. M., and LIND, P. E., Australian J. Biol. Med. Sci. 28, 129-150 (1950). a. SAGIK, B. P., and LEVIKE, S., Virology 3, 401-416 (1957). 3. NEKJRATH, A. R., and SOKOL, F., Acta Virol. (Prague) 6,6676 (1962). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RAYDALL, R. J., J. Rid. Chem. 193, 265 275 (1951). 5. WILSOK, D. E., J. Hacte?iol. 84, 295-301 (1962). 6. GRANOFF, A., and HENLE, W., J. Zmmz~nol. 72, 329-339 (1954). Y. NEURATH, A. R., Acta Viral. (Prague) 7, in press (1963). 8. BANG, F. B., J. Exptl. Med. N&251-266 (1948). FRANTIBEK SOKOL Exptl.
0.5
0 5 t0 15 Fraction number
20
FIG. 3. Sucrose density gradient centrifugation of untreated and 5 times frozen and thawed NDV. The samples of untreated (bottom figure) and treated virus (top figure) were centrifuged simultaneously. Hemolysin and hemagglutinins were quantitatively recovered in fractions of untreated virus. The recovery rate of hemolysin in fractions of treated virus was 175%.
Irstitute
that Sendai virus particles sedimenting with the highest velocity exhibit the highest hemolytic activity. On the other hand, it was not possible after chromatographic separation or density gradient centrifugation to obtain fractions of viral hemagglutinins completely devoid of hemolytic activity. Freezing and thawing increases the hemolytic activity of NDV (6). It was therefore attempted to find out which fractions of t.he virus particles are involved in this enhancement of hemolytic activity. Sucrose
of Virology,
Czechoslovak
Academy
of
Sciences Rratislava, Czechoslovakia
Accepted
Mechanism
May 25,196S
of the Cytopathic Virus
Effect
of Rabies
in Tissue Culture’
Even though propagation of rabies virus in several tissue culture systems has been i This investigation was supported (in part) by USPHS Research Grant AI-02954 from the National Institute of Allergy and Infectious Diseases.
DISCUSSION
AND
PRELIMINARY
fective dye concentration at the time and site of viral maturation. The effective concentration at the time of eclipse might be considerably lower than at maturation because of a difference in sites, temporal changes, or both. Although it is likely that a photosensitive nucleic acid-dye complex occurs during eclipse, photooxidation of protein is not ruled out as a cause of the observed effect. Further experiments will be necessary to fully elucidate these problems.
REPORTS
The Blacksburg strain of NDV in its 26th and 27th egg passages was employed in these experiments. The virus from the infectious allantoic fluid was concentrated and partially purified by two cycles of differential centrifugation and treatment with ribonuclease and deoxyribonuclease. The final suspension of virus in Tris saline [0.02 M tris (hydroxymethyl) aminomethane, 0.13 M NaCl, pH 7.31 contained 4.9 x lo4 hemagglutinating (HA) units per milliliter. A DEAE-cellulose (Whatman, 1.0 meq. REFERENCES N per gram) column (1.4 x 16 cm) was Photobiol. 1, 1471. HACKETT, A. J., Photochem. loaded with 5 ml of this virus suspension 154 (1962). diluted with 10 ml of 0.02 M Tris buffer, 2. WILSON, J. N., and COOPER, P. D., Virology 17, pH 7.3. The virus was eluted at 25” with a 195-196 (1962). slightly concave gradient of NaCl at con3. MCBRIDE, W. D., Bactetiol. Proc. (Sot. Am. stant pH (7.3) and molarity of Tris buffer Bacteriologists), p. 130 (1962). (0.02). The undiluted effluent fractions were 18, 412-425 (1962). 4. SCHAFFER, F. L., Virology examined for absorbancy at 260 and 280 17, 28%294 (1962). 6. MANDEL, B., Virology mp. Their hemagglutination and hemolytic 6. MANDEL, B., Cold Spring Harbor Symp. Quant. Biol. 27,123-136 (1962). (after adjustment of the NaCl concentra7. DARNELL, J. E., JR., Cold Spring Harbor Symp. tion to 0.13 M) titers were determined as Quant. Biol. 27, 149-158 (1962). described previously (3). Proteins were deF. L. SCHAFFER termined as described by Lowry et al. (4). A. J. HACKETT The chromatographic pattern of viral heNaval Biological Laboratory magglutinins eluted at 0.125-0.250 M salt University of California was composed of several overlapping peaks Berkeley, California (Fig. 1). The virus eluted in this region Accepted May 28, 196s showed low EID&HA ( <104.0) (5) and high HA/% H (about 500; % H = percentage of hemolysis) ratios. The next two Heterogeneity of Newcastle Disease Virus peaks of hemagglutinins corresponded to (NDV) Particles with Respect to their fully infectious virus (EID,,/HA about 106.0), the maxima of hemagglutination acHemolytic Activity tivity coinciding with those of infectivity. The reason for the separation of fully infecIt has been shown that various preparatious virus into two distinct fractions is not tions of NDV-infected allantoic fluids differ in their hemolytic activities (1, 6), a quite clear. It is possible that the first of finding indicating heterogeneity with re- these fractions corresponded to a complex of virus with the normal component of spect to this property of the virus particles. allantoic fluid eluted from It was also shown that the hemolysin is noninfective DEAE-cellulose columns at 0.25 M NaCl. bound to the virus particle (a). However, This was indicated by control experiments no attempt was made to separate the virus with normal components of allantoic fluid particles into fractions exhibiting different concentrated by sedimentation, as well as hemolytic activities. Experiments described by the fact that in chromatogram of virus in the present communication show that treated with the receptor-destroying enchromatography on diethylaminoethyl zyme from Vibrio cholerae this first peak (DEAE) -cellulose columns, or density grawas not present. It is, however, evident dient centrifugation, can be successfully that in both peaks the maxima of hemolytic used for such fractionation.
DISCUSSION
0
100
AND PRELIMINARY
200 Volume in ml.
REPORTS
127
300
400
FIG. 1. Fractionation of NDV particles by chromatography on DEAE-cellulose column. Six-milliliter fractions were collected at a rate of 1.2 ml per minute. Proteins were determined in 0.5-ml amounts of the fractions made up to 3.25 ml with the reagents. The recovery of hemagglutinins (HA), hemolysin (H), and proteins was 100, 63, and 52%, respectively.
activity did not, coincide with those of hemagglutinating activity, the virus particles with relatively high hemolytic activity being eluted at markedly higher salt concentrations than the bulk of the virus. Virus particles exhibiting the lowest HA/% H ratio (about 35) were eluted at 0.325 and 0.450-0.625111 NaCI, respectively, whereas those eluted at the maxima of the two peaks had HA/% H ratios of about 200. Two virus preparations obtained by the procedure described and containing 2.4 x lo4 and 6.6 x lo4 HA units of virus per milliliter, respectively, were used in sucrose density gradient centrifugation experiments. Tubes were filled by layering successively 0.8 ml each of 60, 50, 40, 20, 10, and 5% (w/v) sucrose in Tris saline and kept overnight at 4”. Then 0.25 ml of virus suspension was layered over the sucrose solution and the tubes were centrifuged at, room temperature in the Spinco swinging-bucket rotor SW 39 model L at 20,000 rpm for 15 minutes. Fractions of 5 drops each were collected by puncturing the bottom of the tube. These were diluted by the addition of 0.75 ml of 0.1 M Tris pH 7.8, and their hemagglutination titers were estimated. For esti-
mation of hemolytic activity, the samples were further diluted 4 times, and after hemolysis the optical densities of the solutions read against appropriate controls. The presence of excess sucrose did not, cause nonspecific hemolysis. The results are presented in Fig. 2. Three hemagglutinating components are discernible in the sedimentation pattern, the bottom component being poorly separated from the middle one. The top component is probably identical with the hemagglutinin fraction eluted from the DEAE-cellulose column at 0.125&0).250 M NaCl, as it is only slightly hemolytic (HA/% H ratio about 470) and more than 100 times less infective than the fully infectious viruses in the middle and bottom components. These particles are similar to or identical with the noninfective, nonhemolytic hemagglutinins derived from NDV-infected tissue or allantoic fluid (6). The virus particles in the middle component have a HA/% H ratio of about 200. The virus
particles
with
the highest
exhibit (HA/% studies
the
H
in the
bottom
highest
constant
hemolytic
19.2 in fraction
by Neurath
component
sedimentation
activity
8).
Recent
(7) also have revealed
128
DISCUSSION
AND
PRELIMINART
30
20 x 2 O
10
0
4
8
12
16
20
number FIG. 2. Fractionation of NDV particles by sucrose density gradient centrifugation. The arrow points to the bottom fraction. Hemolysin and hemagglutinins were quantitatively recovered after centrifugation. Ordinate on the right: HA units per fraction. Fraction
20
2
REPORTS
density gradient centrifugation of untreated NDV and of virus subjected to 5 cycles of freezing and thawing showed (Fig. 3) that after freezing and thawing a new peak of hemolysins appeared aproximately in the middle component. The hemolytic activities of the virus particles in the bottom and top components were csscntially unaffected by this treatment. It must be added, however, that in treated sample the sedimentation rate of hcmagglutinins from the middle component was decreased (8). It appears that only virus particles from the middle component contain masked hemolysins which can be liberated by repeated freezing and thawing. The results of chromatographic and of density gradient centrifugation experiments described here were confirmed in repeated experiments. Investigations with plaquepurified fractions of NDV are now in progress to determine whether the variation in hemolytic activity is of heritable nature. REFERENCES
1. BURNET,
F. M., and LIND, P. E., Australian J. Biol. Med. Sci. 28, 129-150 (1950). a. SAGIK, B. P., and LEVIKE, S., Virology 3, 401-416 (1957). 3. NEKJRATH, A. R., and SOKOL, F., Acta Virol. (Prague) 6,6676 (1962). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RAYDALL, R. J., J. Rid. Chem. 193, 265 275 (1951). 5. WILSOK, D. E., J. Hacte?iol. 84, 295-301 (1962). 6. GRANOFF, A., and HENLE, W., J. Zmmz~nol. 72, 329-339 (1954). Y. NEURATH, A. R., Acta Viral. (Prague) 7, in press (1963). 8. BANG, F. B., J. Exptl. Med. N&251-266 (1948). FRANTIBEK SOKOL Exptl.
0.5
0 5 t0 15 Fraction number
20
FIG. 3. Sucrose density gradient centrifugation of untreated and 5 times frozen and thawed NDV. The samples of untreated (bottom figure) and treated virus (top figure) were centrifuged simultaneously. Hemolysin and hemagglutinins were quantitatively recovered in fractions of untreated virus. The recovery rate of hemolysin in fractions of treated virus was 175%.
Irstitute
that Sendai virus particles sedimenting with the highest velocity exhibit the highest hemolytic activity. On the other hand, it was not possible after chromatographic separation or density gradient centrifugation to obtain fractions of viral hemagglutinins completely devoid of hemolytic activity. Freezing and thawing increases the hemolytic activity of NDV (6). It was therefore attempted to find out which fractions of t.he virus particles are involved in this enhancement of hemolytic activity. Sucrose
of Virology,
Czechoslovak
Academy
of
Sciences Rratislava, Czechoslovakia
Accepted
Mechanism
May 25,196S
of the Cytopathic Virus
Effect
of Rabies
in Tissue Culture’
Even though propagation of rabies virus in several tissue culture systems has been i This investigation was supported (in part) by USPHS Research Grant AI-02954 from the National Institute of Allergy and Infectious Diseases.