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Inactivation of Newcastle disease virus by polyenes
VIROMQY
43, 536-538
(1971)
Inactivation
of Newcastle
Disease Virus by Polyenes
Polyene antibiotics are characterized by a large macrolide ring containing a series of conjugated double bonds (1). Compounds such as nystatin, amphotericin, and filipin bind to sterols in the membranes of susceptible organisms (e.g., fungi, protozoa, mycoplasma) with resulting leakage of vital cellular components (2). The binding of polyenes to sterols suggested experiments on the interaction of these compounds with enveloped viruses. The effect of polyene antibiotics on the properties of Newcastle disease virus (NDV) was studied in an attempt to gain information on the relationship between the viral envelope and other membranes. The OS and Roakin strains of NDV were propagated in the allantoic cavity of embryonated chick eggs. Ten-day eggs were inoculated with 200-500 hemadsorption focal units (HFU) of virus per egg and incubation continued at 37” for 4044 hours prior to collection of allantoic fluid. Infectivity assays were performed on HeLa cell monolayers as reported earlier (3). NDV was purified by precipitation with 6% polyethylene glycol (4) followed by banding in a preformed gradient of 245 % potassium tartrate (5). Virus collected from a sharp band (density 1.18 g cc-l) was dialyzed against several changes of phosphate-buffered saline (0.01 M, pH 7.2) prior to use. Filipin (Lot 5956) was a gift of the Upjohn Company. Nystatin was purchased from a local vendor. Polyene antibiotics were dissolved in a small volume of 95 % ethanol before suspending in serum-free minimal essential medium (Eagle) (6) for experiments. Controls in which the same ethanol concentration was employed (0.5 % or less) showed no virus inactivation. The absorption spectrum of the filipin was checked to verify the purity and authenticity of the compound under study. Characteristic peaks were obtained at 322, 338, 536
and 357 nm when filipin was dissolved in methanol (Fig. 1). The inactivation of NDV by various concentrations of filipin is presented in Fig. 2. Significant inactivation of the virus is evident at low concentrations of the polyene. The degree of inactivation is of the same order of magnitude for both strains. A tetraene, nystatin, was compared with filipin (a pentaene) for its effects on NDV. On a
3
2’
2
.l
3io F‘ra.1. ^__.
*
340
360 nm
-. Absorption
spectrum of filipin. Filipin was dissolved in methanol and the absorbancy determined in a Beckman DU-2 spectrophotometer.
molar basis, filipin was 5-7 times more active than nystatin in reducing virus infectivity. The kinetics of inactivation of NDV by filipin are shown in Fig. 3. Control virus preparations did not exhibit significant loss of infectivity at 37”. The decline of infectious virus in the presence of filipin appeared to be exponential and occurred rapidly at 37” (virus half-life, 5.6 minutes) ; inactivation was less marked at 22“ (virus half-life, 16 minutes). The decrease in virus
SHORT
pg FILlPIN
COMMUNICATIONS
per ml
FIG. 2. Inactivation of NDV by filipin. The Roakin (R) and OS (0) strains of NDV were mixed with various concentrations of filipin and assayed after incubation for 60 minutes at 37”. Control virus preparations were incubated under the same conditions without filipin.
I
45
15
60
M%“TES
FIG. 3. Kinetics of inactivation of NDV by illipin. Control virus preparations (Con.), incubated at 37” did not contain filipin. Incubation of virus suspensions with fdipin (100 pg/ml) was at 22 or 37”.
titer due to filipin was dependent upon temperature. Incubation of filipin-virus mixtures at 1, 23, 30, and 37” for 60 minutes yielded inactivation values of 18, SO,94, and 96.7%, respectively when the fllipin concentration was 125 pg/ml. The effect of filipin on the hemagglutinin (HA) of NDV was tested. The HA titers of both NDV strains were unaffected by filipin
537
sufficient to inactivate concentrations greater than 90 % of the virus infectivity. The decreasein virus titers observed with filipin could be due to clumping of virus particles. To exclude this possibility, the inactivation of filipin-treated NDV by ultraviolet light was examined. Virus inactivated to 4 % survival by filipin (100 pg/ml for 60 minutes at 37”) was irradiated with W light for various periods of time. Single-hit kinetics of inactivation of NDV were obtained when the surviving fraction was plotted versus W dose. This suggests that significant clumping of virus by filipin does not occur. Mengovirus, a picornavirus lacking an envelope, was incubated in the presence of filipin (200 pg/ml for 60 minutes at 37”) and assayed for infectivity on L cells (7). No effect on the infectivity of mengovirus was noted. This was expected since this virus possessesno membrane-like components. The present experiments have indicated a similarity between NDV and other organisms with respect to the effect of filipin. Bacterial cells or protoplasts which normally lack sterols are unaffected by polyenes, whereas fungi, mycoplasma, protozoa, and mammalian cells exhibit membrane damage in the presence of these compounds (2). This selectivity in polyene action has been attributed to the presence of sterols in this latter group of cells. The inactivation of NDV by filipin is similar to the effects on yeasts in two respects: (1) The inactivation process is temperature-dependent; it has been reported that binding of polyenes to yeast membranes does not occur at low temperatures (8). (2) Filipin is more active than nystatin, in accord with published data on yeast (8). It would appear that polyenes can interact with a wide variety of membranes and membrane-like structures containing sterols. Data obtained thus far indicate that studies on the inactivation of NDV infectivity by filipin may provide a means for examining the structural and functional arrangement of sterols in those membranes in which they are present. Studies on the alteration of the viral envelope by polyenes are in progress.
538
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author thanks Dr. R. E. Barnhart of Vantress Farms for his advice and assistance. This study was supported by a grant from the McCandless Fund. This is publication No. 981, Department of Microbiology, Division of Basic Health Sciences, Emory University.
1. KINSKY,
REFERENCES S. C., LUSE, S. A., and VAN DEENGN,
L.L.M., Fed. Amer. 1503-1510 (1966). 2. KINSKY,
S. C.,
Ann.
142 (1970). 3. ASH, R. J., and BUBEL, 211,891~892 (1966).
Sot. Rev.
Exp.
Pharmacol.
Biol.
25,
10, 119-
H. C., Nature London
4. MCSHARRY,
J., and BENZINGER, R., Virology 40, 745-746 (1970). 6. MCCREA, J. F., EPSTEIN, R. S., and BARRY, W. H., Nature London 189, 229-221 (1961). 6. EAGLE, H., Science 130,432437 (1959). 7. BUBEL, H. C., Proc. See. Ezp. Biol. Med. 125, 783-786 (1967). 8. KINSKY, S. C. In “Antibiotics. I. Mechanism of Action,” pp. 122-141. Springer-Verlag, New York (1967). RONALD J. ASH Department of Microbiology Emory University Atlanta, Georgia, SO%8 Accepted
December
lb,
1970
43, 536-538
(1971)
Inactivation
of Newcastle
Disease Virus by Polyenes
Polyene antibiotics are characterized by a large macrolide ring containing a series of conjugated double bonds (1). Compounds such as nystatin, amphotericin, and filipin bind to sterols in the membranes of susceptible organisms (e.g., fungi, protozoa, mycoplasma) with resulting leakage of vital cellular components (2). The binding of polyenes to sterols suggested experiments on the interaction of these compounds with enveloped viruses. The effect of polyene antibiotics on the properties of Newcastle disease virus (NDV) was studied in an attempt to gain information on the relationship between the viral envelope and other membranes. The OS and Roakin strains of NDV were propagated in the allantoic cavity of embryonated chick eggs. Ten-day eggs were inoculated with 200-500 hemadsorption focal units (HFU) of virus per egg and incubation continued at 37” for 4044 hours prior to collection of allantoic fluid. Infectivity assays were performed on HeLa cell monolayers as reported earlier (3). NDV was purified by precipitation with 6% polyethylene glycol (4) followed by banding in a preformed gradient of 245 % potassium tartrate (5). Virus collected from a sharp band (density 1.18 g cc-l) was dialyzed against several changes of phosphate-buffered saline (0.01 M, pH 7.2) prior to use. Filipin (Lot 5956) was a gift of the Upjohn Company. Nystatin was purchased from a local vendor. Polyene antibiotics were dissolved in a small volume of 95 % ethanol before suspending in serum-free minimal essential medium (Eagle) (6) for experiments. Controls in which the same ethanol concentration was employed (0.5 % or less) showed no virus inactivation. The absorption spectrum of the filipin was checked to verify the purity and authenticity of the compound under study. Characteristic peaks were obtained at 322, 338, 536
and 357 nm when filipin was dissolved in methanol (Fig. 1). The inactivation of NDV by various concentrations of filipin is presented in Fig. 2. Significant inactivation of the virus is evident at low concentrations of the polyene. The degree of inactivation is of the same order of magnitude for both strains. A tetraene, nystatin, was compared with filipin (a pentaene) for its effects on NDV. On a
3
2’
2
.l
3io F‘ra.1. ^__.
*
340
360 nm
-. Absorption
spectrum of filipin. Filipin was dissolved in methanol and the absorbancy determined in a Beckman DU-2 spectrophotometer.
molar basis, filipin was 5-7 times more active than nystatin in reducing virus infectivity. The kinetics of inactivation of NDV by filipin are shown in Fig. 3. Control virus preparations did not exhibit significant loss of infectivity at 37”. The decline of infectious virus in the presence of filipin appeared to be exponential and occurred rapidly at 37” (virus half-life, 5.6 minutes) ; inactivation was less marked at 22“ (virus half-life, 16 minutes). The decrease in virus
SHORT
pg FILlPIN
COMMUNICATIONS
per ml
FIG. 2. Inactivation of NDV by filipin. The Roakin (R) and OS (0) strains of NDV were mixed with various concentrations of filipin and assayed after incubation for 60 minutes at 37”. Control virus preparations were incubated under the same conditions without filipin.
I
45
15
60
M%“TES
FIG. 3. Kinetics of inactivation of NDV by illipin. Control virus preparations (Con.), incubated at 37” did not contain filipin. Incubation of virus suspensions with fdipin (100 pg/ml) was at 22 or 37”.
titer due to filipin was dependent upon temperature. Incubation of filipin-virus mixtures at 1, 23, 30, and 37” for 60 minutes yielded inactivation values of 18, SO,94, and 96.7%, respectively when the fllipin concentration was 125 pg/ml. The effect of filipin on the hemagglutinin (HA) of NDV was tested. The HA titers of both NDV strains were unaffected by filipin
537
sufficient to inactivate concentrations greater than 90 % of the virus infectivity. The decreasein virus titers observed with filipin could be due to clumping of virus particles. To exclude this possibility, the inactivation of filipin-treated NDV by ultraviolet light was examined. Virus inactivated to 4 % survival by filipin (100 pg/ml for 60 minutes at 37”) was irradiated with W light for various periods of time. Single-hit kinetics of inactivation of NDV were obtained when the surviving fraction was plotted versus W dose. This suggests that significant clumping of virus by filipin does not occur. Mengovirus, a picornavirus lacking an envelope, was incubated in the presence of filipin (200 pg/ml for 60 minutes at 37”) and assayed for infectivity on L cells (7). No effect on the infectivity of mengovirus was noted. This was expected since this virus possessesno membrane-like components. The present experiments have indicated a similarity between NDV and other organisms with respect to the effect of filipin. Bacterial cells or protoplasts which normally lack sterols are unaffected by polyenes, whereas fungi, mycoplasma, protozoa, and mammalian cells exhibit membrane damage in the presence of these compounds (2). This selectivity in polyene action has been attributed to the presence of sterols in this latter group of cells. The inactivation of NDV by filipin is similar to the effects on yeasts in two respects: (1) The inactivation process is temperature-dependent; it has been reported that binding of polyenes to yeast membranes does not occur at low temperatures (8). (2) Filipin is more active than nystatin, in accord with published data on yeast (8). It would appear that polyenes can interact with a wide variety of membranes and membrane-like structures containing sterols. Data obtained thus far indicate that studies on the inactivation of NDV infectivity by filipin may provide a means for examining the structural and functional arrangement of sterols in those membranes in which they are present. Studies on the alteration of the viral envelope by polyenes are in progress.
538
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author thanks Dr. R. E. Barnhart of Vantress Farms for his advice and assistance. This study was supported by a grant from the McCandless Fund. This is publication No. 981, Department of Microbiology, Division of Basic Health Sciences, Emory University.
1. KINSKY,
REFERENCES S. C., LUSE, S. A., and VAN DEENGN,
L.L.M., Fed. Amer. 1503-1510 (1966). 2. KINSKY,
S. C.,
Ann.
142 (1970). 3. ASH, R. J., and BUBEL, 211,891~892 (1966).
Sot. Rev.
Exp.
Pharmacol.
Biol.
25,
10, 119-
H. C., Nature London
4. MCSHARRY,
J., and BENZINGER, R., Virology 40, 745-746 (1970). 6. MCCREA, J. F., EPSTEIN, R. S., and BARRY, W. H., Nature London 189, 229-221 (1961). 6. EAGLE, H., Science 130,432437 (1959). 7. BUBEL, H. C., Proc. See. Ezp. Biol. Med. 125, 783-786 (1967). 8. KINSKY, S. C. In “Antibiotics. I. Mechanism of Action,” pp. 122-141. Springer-Verlag, New York (1967). RONALD J. ASH Department of Microbiology Emory University Atlanta, Georgia, SO%8 Accepted
December
lb,
1970