THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO. I • JULY 1978 © 1978 by The University of Chicago. 0022-1899/78/3801-0013$00.75
Inactivation of Cytomegalovirus and Semliki Forest Virus by Butylated Hydroxytoluene From the New York State Institute for Basic Research in Mental Retardation, Staten Island, New York
K. S. Kim, H. M. Moon, V. Sapienza, R. I. Carp, and R. Pullarkat
Cytomegalovirus (CMV) is recognized as an important human pathogen that produces a broad clinical spectrum varying from classic "cytomegalic inclusion disease" to intrauterine death, prematurity, congenital defects, mental retardation, infectious mononucleosis, postperfusion syndromes, and interstitial pneumonia in organ transplant patients. Recurrence of excretions of human CMV during pregnancy, reactivation of CMV replication in immunosuppressed patients, and the presence of the infectious CMV in samples of fresh blood from donors strongly indicate the existence of latency of human CMV, as found with other viruses of the herpesvirus group [I-
castle disease virus (NDV) were reduced greatly when the viruses were exposed to BHT [9, 10]. More significant was the finding that chickens fed on diets containing BHT (100-200 ppm) did not die when exposed to virulent NDV. Also, chickens fed on a BHT-containing diet did not show any appreciable humoral immune response when exposed to avirulent NDV [9, 10]. These findings prompted us to study the effect of BHT on CMV in vitro and to evaluate its possible usefulness as a chemotherapeutic agent against CMV in vivo. This paper presents results of exposure of BHT to human and murine CMV, Semliki Forest virus (SFV), and vaccinia virus.
5]. At present there are no effective, safe chemotherapeutic agents or vaccines available for treatment or prevention of CMV diseases. Butylated hydroxytoluene (BHT) is one of the several antioxidants that have been widely used in human and animal foods to maintain freshness and prevent spoilage by delaying degradation of lipid components of food [6]. Usually, 50-200 parts per million (ppm) by weight of BHT is added to foods to maintain their freshness. However, in higher concentrations (2,000-10,000 ppm), BHT is known to exhibit various biological effects [7, 8]. Recently, the infectivity titers of the lipid-containing viruses herpes simplex virus, ep6, and New-
Materials and Methods
Viruses. Murine CMV (Smith strain) was obtained from the American Type Culture Collection, Rockville, Md., and propagated for 52 passages in secondary mouse embryo fibroblasts (MEF). The Towne strain of human CMV, obtained from Dr. Stanley Plotkin of Wi star Institute, Philadelphia, Pa., was propagated in human fetal tonsil cells. SFV (strain A774, MG4519), obtained from Dr. Robert E. Shope, Yale Arbovirus Research Unit of Yale University Medical School, New Haven, Conn., was passaged once in mice and once in secondary MEF cells before use. Poliovirus type 2, provided by Dr. R. Bablanian, Department of Microbiology, Downstate Medical Center, Brooklyn, N.Y., was passaged once in Detroit-f continuous cell line. Vaccinia virus (WR [mouse neurotropic] strain) was passaged in MEF cells.
Received for publication December 27,1977. We thank Ms. Peggy Clark for her assistance in the preparation of the manuscript. Please address requests for reprints to Dr. K. S. Kim, New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 10314.
91
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
Butylated hydroxy toluene (BHT) is an antioxidant that is widely used in foods because it prevents spoilage by delaying degradation of lipid components. This hydrophobic compound inactivated human and murine cytomegalovirus (CMV) and Semliki Forest virus (SFV). Both human and murine CMV were inactivated more than 90% by 40 JAg of BHT Iml after incubation for I hr at 37 C. Under the same conditions, SFV was inactivated about 75%, whereas poliovirus, which does not contain lipid membrane as a part of its structure, was not inactivated at all. Vaccinia virus was less sensitive to BHT than was CMV or SFV.
92
Kim et al,
Chemicals. BHT (2.6-di-tert-butyl-p-cresol) was obtained from Sigma Chemical Co., St. Louis, Mo. Experimental procedures. Each solution of stock virus (2 ml) was mixed with 20 ILl of various concentrations of BHT dissolved in absolute ethanol. As a control, virus was mixed with 20 ILl of absolute ethanol alone. The BHT-virus mixtures and the controls were incubated in a water bath for either 30 or 60 min at 37 C with agitation at 5-min intervals. The mixtures were then chilled in ice, diluted, and assayed for plaque formation in appropriate cells by the methods described above. Results
Effect of BHT on infectivity of human and murine CMV. At 30 and 60 min after mixtures of virus and various concentrations of BHT were placed in a water bath at 37 C, 0.2 ml was removed immediately from each mixture, mixed with 1.8 ml of complete medium, and diluted serially by Ill-fold steps. Plaque titers were determined in appropriate monolayers. As shown in table 1, both human and murine CMV were inactivated rapidly by BHT. At the end of incubation for 1 hr, >99% of the human CMV was inactivated in the presence of 40 ILg of BHT jmI, whereas >90% of the murine CMV was inactivated at this concentration of BHT. More than 99% of the murine CMV was inactivated at a concentration of 320 ILg of BHTjml. Effect of BHT on infectivity of SFV, vaccinia virus, and poliovirus. Two additional lipidcontaining viruses, SFV and vaccinia virus, and
Table 1. Inactivation of human and murine cytomegalovirus (CMV) incubated at 37 C for 30 and 60 min with various concentrations of butylated hydroxytoluene (BHT). Concentration ofBHT (J.lg/ml) 0 10 20 40 80 160 320
6o-min incubation
3O-min incubation Murine CMV 4.17 3.54 2.30 2.00 -2.17)
HumanCMV
Murine CMV
HumanCMV
8.00 (0.00) 7.86 (-0.14) 7.93 (-0.07) 7.29 (-0.71) 7.29 (-0.71) 6.98 (-1.02) 6.98 (-1.02)
4.10 3.23 2.30 -2.10)
NOTE. Data are loglO pfu/ml before incubation (changes in loglo pfujml after incubation).
(0.00) (-0.23) (-0.27) (-0.83) (-1.26) (-1.47) (-2.05)
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
Plaque assay methods. Human and murine CMV were assayed for plaque formation by methods described by Kim and Carp [11] and Wentworth and French [12]. For plaque assay of poliovirus, the method described for murine CMV was used except that monolayers of Detroit-f cells were infected and plaques were counted on the third day. SFV was assayed for plaque formation by the method described for murine CMV except that the plaques were counted on the second or third day. Vaccinia virus was assayed for plaque formation by the method described by Sharp and Kim [13] except that MEF cells were used. Medium. Eagle's minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) with 10% fetal calf serum containing penicillin (100 unitsjml) and streptomycin (50 ILgjml) was used for propagation of cells and for maintenance of infected cells. Preparation of virus pools. Fluid from virusinfected cultures was centrifuged at 500 g for 10 min (model GLC-l centrifuge; Sorvall Inc., Newtown, Conn.) for elimination of large pieces of cellular debris. The supernatants of human and murine CMV and vaccinia virus culture fluids were centrifuged at 110,000 g for 1 hr with use of an International ultracentrifuge rotor (A147; International Equipment Co., Needham Heights, Mass.). The supernatants obtained after lowspeed centrifugation of SFV and poliovirus preparations were centrifuged at 200,000 g for 3 hr in an International ultracentrifuge rotor (A321). Pellets of each virus were resuspended in Earle's balanced salt solution, pH 7.2, without NaHCO a and containing 10% tryptose phosphate broth (Difco, Detroit, Mich.).
93
Inactivation of CMV and SFV
Table 2. Effects of butylated hydroxytoluene (BHT) on the titers of Semliki Forest virus (SFV), poliovirus type 2, and vaccinia virus during incubation for 60 min at 37 C. Concentration of BHT (J.lg/ml) 0 10 20 40 80 160 320
SFV 8.07 8.11 7.47 7.13 6.95 7.04 7.04
(0.00) (+0.04) (-0.60) (-0.94) (-1.12) (-1.03) (-1.03)
Poliovirus 7.77 7.60 8.04 8.04 7.77 7.30 7.69
(0.00) (-0.17) (+0.27) (+0.27) (0.00) (-0.4 7) (-0.08)
Vaccinia virus 4.11 (0.00) 4.27 (+0.16) 4.00(-0.11) 3.77 (-0.34) 3.84 (-0.27) 3.95 (-0.16) 4.04 (-0.07)
NOTE. Data are logiO pfu/ml before incubation (changes in logiO pfu/ml after incubation).
Discussion
Although the reason(s) for the effectiveness of BHT as an antiviral agent against CMV and SFV is not known, interaction of the drug with either virus effectively inactivated infectivity without any apparent adverse effect on the host cells. Perhaps the interaction of BHT with lipid-containing viral envelopes somehow disturbs the proper function of the viral envelope during adsorption of viral particles to host cell membrane [14]. Although vaccinia virus is also one of the lipid-containing viruses, for an unknown reason(s) BHT had a slight inactivating effect. BHT is one of the antioxidants classified as a safe food additive by the Food and Drug Administration [6]. Of prime importance is the fact that this compound effectively inactivated CMV and SFV by >90% in 1 hr at 37 C at concentrations that are known to have no biologically adverse effect when BHT is fed to animals and humans as a food additive. A concentration of BHT of 160 j.Lgjml was more than sufficient to inactivate >95% of the CMV and SFV. BHT concentrations of up to 200 ppm are known to have no side effects and are routinely added to foods [6]. In vivo the effectiveness of BHT against infection with enveloped virus has already been re-
ported. Brugh [l0] reported that chickens maintained on feed containing 200-2,000 ppm did not show a normal humoral immunological response when challenged with an avirulent strain of NDV. Each of these findings is due to inactivation of the infecting virus by BHT. Another significant point is that BHT is effective against either RNA or DNA viruses that have a lipidcontaining membrane. Since BHT has no adverse effect on the host and selectively inactivates the enveloped viruses tested thus far, it has great potential as a broad-spectrum antiviral agent. Further experiments are now in progress to determine whether BHT is an effective antiviral agent in the mouse-murine CMV model.
References
J. Cytomegalovirus infections in organ transplantation and post transfusion. An hypothesis. Arch Gesamte Virusforsch. 37:365-377, 1972. 2. Weller, T. H. The cytomegaloviruses: the ubiquitous agents with protean clinical manifestations. N. Engl. 1. Lang, D.
J. ~ed.285:203-214,1971. 3. Montgomery, R., Youngblood, L., Medearis, D. N., Jr. Recovery of cytomegalovirus from the cervix in pregnancy. Pediatrics 49:524-531, 1972. 4. Diosi, P., Moldovan, E., Tomescu, N. Latent cytomegalovirus infection in blood donors. Br. Med. J. 4:660-662, 1969. 5. Kaariaiuen, L., Pa1oheimo, J., Klemo1a, E., ~ake1a, T., Koivuniemi, A. Cytomegalovirus-mononucleosis: isolation of the virus and demonstration of subclinical infections after fresh blood transfusion in connection with open·heart surgery. Annales Medicine Experimentalis et Biologiae Fenniae 44:297-301, 1966. 6. Stuckey, B. N. Butylated hydroxytoluene is classified "generally recognized as safe" by the Food and Drug Administration. In T. E. Furia [ed.]. Handbook of
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
non-lipid-containing poliovirus were exposed to BHT under identical experimental conditions. As shown in table 2, >90% of the SFV was inactivated in the presence of 80 j.Lg of BHT jml, whereas poliovirus was not inactivated by BHT at all concentrations tested. Although vaccinia virus was also inactivated, the extent of inactivation was far less than that of either SFV or CMV.
94
7.
8.
9.
10.
11.
Kim et al.
food additives. Chemical Rubber Company, Cleveland, Ohio, 1972, p. 185. Branen, A. L. Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. J. Am. Oil Chern. Soc. 52:59-63,1975. March, B. E., Coates, V., Biely, J. Reticulocytosis in response to dietary antioxidants. Science 64:13981400,1969. Snipes, W., Person, S., Keith, A., Cupp, J. Butylated hydroxytoluene inactivates lipid-containing viruses. Science 188:64-66, 1975. Brugh, M., Jr. Butylated hydroxytoluene protects chickens exposed to Newcastle disease virus. Science 197:1291-1292,1977. Kim, K. S., Carp, R. I. Abortive infection of human
diploid cells by murine cytomegalovirus. Infec. Immun. 6:793-797,1972. 12. Wentworth, B. B., French, L. Plaque assay of cytomegalovirus strains of human origin. Proc. Soc. Exp. Bioi. Med. 135:253-258, 1970. 13. Sharp, D. G., Kim, K. S. Multiplicity reactivation and radiation survival of aggregated vaccinia virus. Calculation of plaque titer based on MR and particle aggregation seen in the electron microscope. Virology 29:359-366, 1966. 14. Eletr, S., Williams, M. A., Watkins, T., Keith, A. D. Perturbat ions of the dynamics of lipid alkyl chains in membrane systems: effect on the activity of membrane-bound enzymes. Biochim. Biophys. Acta 339: 190-201,1974. Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
Inactivation of Cytomegalovirus and Semliki Forest Virus by Butylated Hydroxytoluene From the New York State Institute for Basic Research in Mental Retardation, Staten Island, New York
K. S. Kim, H. M. Moon, V. Sapienza, R. I. Carp, and R. Pullarkat
Cytomegalovirus (CMV) is recognized as an important human pathogen that produces a broad clinical spectrum varying from classic "cytomegalic inclusion disease" to intrauterine death, prematurity, congenital defects, mental retardation, infectious mononucleosis, postperfusion syndromes, and interstitial pneumonia in organ transplant patients. Recurrence of excretions of human CMV during pregnancy, reactivation of CMV replication in immunosuppressed patients, and the presence of the infectious CMV in samples of fresh blood from donors strongly indicate the existence of latency of human CMV, as found with other viruses of the herpesvirus group [I-
castle disease virus (NDV) were reduced greatly when the viruses were exposed to BHT [9, 10]. More significant was the finding that chickens fed on diets containing BHT (100-200 ppm) did not die when exposed to virulent NDV. Also, chickens fed on a BHT-containing diet did not show any appreciable humoral immune response when exposed to avirulent NDV [9, 10]. These findings prompted us to study the effect of BHT on CMV in vitro and to evaluate its possible usefulness as a chemotherapeutic agent against CMV in vivo. This paper presents results of exposure of BHT to human and murine CMV, Semliki Forest virus (SFV), and vaccinia virus.
5]. At present there are no effective, safe chemotherapeutic agents or vaccines available for treatment or prevention of CMV diseases. Butylated hydroxytoluene (BHT) is one of the several antioxidants that have been widely used in human and animal foods to maintain freshness and prevent spoilage by delaying degradation of lipid components of food [6]. Usually, 50-200 parts per million (ppm) by weight of BHT is added to foods to maintain their freshness. However, in higher concentrations (2,000-10,000 ppm), BHT is known to exhibit various biological effects [7, 8]. Recently, the infectivity titers of the lipid-containing viruses herpes simplex virus, ep6, and New-
Materials and Methods
Viruses. Murine CMV (Smith strain) was obtained from the American Type Culture Collection, Rockville, Md., and propagated for 52 passages in secondary mouse embryo fibroblasts (MEF). The Towne strain of human CMV, obtained from Dr. Stanley Plotkin of Wi star Institute, Philadelphia, Pa., was propagated in human fetal tonsil cells. SFV (strain A774, MG4519), obtained from Dr. Robert E. Shope, Yale Arbovirus Research Unit of Yale University Medical School, New Haven, Conn., was passaged once in mice and once in secondary MEF cells before use. Poliovirus type 2, provided by Dr. R. Bablanian, Department of Microbiology, Downstate Medical Center, Brooklyn, N.Y., was passaged once in Detroit-f continuous cell line. Vaccinia virus (WR [mouse neurotropic] strain) was passaged in MEF cells.
Received for publication December 27,1977. We thank Ms. Peggy Clark for her assistance in the preparation of the manuscript. Please address requests for reprints to Dr. K. S. Kim, New York State Institute for Basic Research in Mental Retardation, Staten Island, New York 10314.
91
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
Butylated hydroxy toluene (BHT) is an antioxidant that is widely used in foods because it prevents spoilage by delaying degradation of lipid components. This hydrophobic compound inactivated human and murine cytomegalovirus (CMV) and Semliki Forest virus (SFV). Both human and murine CMV were inactivated more than 90% by 40 JAg of BHT Iml after incubation for I hr at 37 C. Under the same conditions, SFV was inactivated about 75%, whereas poliovirus, which does not contain lipid membrane as a part of its structure, was not inactivated at all. Vaccinia virus was less sensitive to BHT than was CMV or SFV.
92
Kim et al,
Chemicals. BHT (2.6-di-tert-butyl-p-cresol) was obtained from Sigma Chemical Co., St. Louis, Mo. Experimental procedures. Each solution of stock virus (2 ml) was mixed with 20 ILl of various concentrations of BHT dissolved in absolute ethanol. As a control, virus was mixed with 20 ILl of absolute ethanol alone. The BHT-virus mixtures and the controls were incubated in a water bath for either 30 or 60 min at 37 C with agitation at 5-min intervals. The mixtures were then chilled in ice, diluted, and assayed for plaque formation in appropriate cells by the methods described above. Results
Effect of BHT on infectivity of human and murine CMV. At 30 and 60 min after mixtures of virus and various concentrations of BHT were placed in a water bath at 37 C, 0.2 ml was removed immediately from each mixture, mixed with 1.8 ml of complete medium, and diluted serially by Ill-fold steps. Plaque titers were determined in appropriate monolayers. As shown in table 1, both human and murine CMV were inactivated rapidly by BHT. At the end of incubation for 1 hr, >99% of the human CMV was inactivated in the presence of 40 ILg of BHT jmI, whereas >90% of the murine CMV was inactivated at this concentration of BHT. More than 99% of the murine CMV was inactivated at a concentration of 320 ILg of BHTjml. Effect of BHT on infectivity of SFV, vaccinia virus, and poliovirus. Two additional lipidcontaining viruses, SFV and vaccinia virus, and
Table 1. Inactivation of human and murine cytomegalovirus (CMV) incubated at 37 C for 30 and 60 min with various concentrations of butylated hydroxytoluene (BHT). Concentration ofBHT (J.lg/ml) 0 10 20 40 80 160 320
6o-min incubation
3O-min incubation Murine CMV 4.17 3.54 2.30 2.00 -2.17)
HumanCMV
Murine CMV
HumanCMV
8.00 (0.00) 7.86 (-0.14) 7.93 (-0.07) 7.29 (-0.71) 7.29 (-0.71) 6.98 (-1.02) 6.98 (-1.02)
4.10 3.23 2.30 -2.10)
NOTE. Data are loglO pfu/ml before incubation (changes in loglo pfujml after incubation).
(0.00) (-0.23) (-0.27) (-0.83) (-1.26) (-1.47) (-2.05)
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
Plaque assay methods. Human and murine CMV were assayed for plaque formation by methods described by Kim and Carp [11] and Wentworth and French [12]. For plaque assay of poliovirus, the method described for murine CMV was used except that monolayers of Detroit-f cells were infected and plaques were counted on the third day. SFV was assayed for plaque formation by the method described for murine CMV except that the plaques were counted on the second or third day. Vaccinia virus was assayed for plaque formation by the method described by Sharp and Kim [13] except that MEF cells were used. Medium. Eagle's minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) with 10% fetal calf serum containing penicillin (100 unitsjml) and streptomycin (50 ILgjml) was used for propagation of cells and for maintenance of infected cells. Preparation of virus pools. Fluid from virusinfected cultures was centrifuged at 500 g for 10 min (model GLC-l centrifuge; Sorvall Inc., Newtown, Conn.) for elimination of large pieces of cellular debris. The supernatants of human and murine CMV and vaccinia virus culture fluids were centrifuged at 110,000 g for 1 hr with use of an International ultracentrifuge rotor (A147; International Equipment Co., Needham Heights, Mass.). The supernatants obtained after lowspeed centrifugation of SFV and poliovirus preparations were centrifuged at 200,000 g for 3 hr in an International ultracentrifuge rotor (A321). Pellets of each virus were resuspended in Earle's balanced salt solution, pH 7.2, without NaHCO a and containing 10% tryptose phosphate broth (Difco, Detroit, Mich.).
93
Inactivation of CMV and SFV
Table 2. Effects of butylated hydroxytoluene (BHT) on the titers of Semliki Forest virus (SFV), poliovirus type 2, and vaccinia virus during incubation for 60 min at 37 C. Concentration of BHT (J.lg/ml) 0 10 20 40 80 160 320
SFV 8.07 8.11 7.47 7.13 6.95 7.04 7.04
(0.00) (+0.04) (-0.60) (-0.94) (-1.12) (-1.03) (-1.03)
Poliovirus 7.77 7.60 8.04 8.04 7.77 7.30 7.69
(0.00) (-0.17) (+0.27) (+0.27) (0.00) (-0.4 7) (-0.08)
Vaccinia virus 4.11 (0.00) 4.27 (+0.16) 4.00(-0.11) 3.77 (-0.34) 3.84 (-0.27) 3.95 (-0.16) 4.04 (-0.07)
NOTE. Data are logiO pfu/ml before incubation (changes in logiO pfu/ml after incubation).
Discussion
Although the reason(s) for the effectiveness of BHT as an antiviral agent against CMV and SFV is not known, interaction of the drug with either virus effectively inactivated infectivity without any apparent adverse effect on the host cells. Perhaps the interaction of BHT with lipid-containing viral envelopes somehow disturbs the proper function of the viral envelope during adsorption of viral particles to host cell membrane [14]. Although vaccinia virus is also one of the lipid-containing viruses, for an unknown reason(s) BHT had a slight inactivating effect. BHT is one of the antioxidants classified as a safe food additive by the Food and Drug Administration [6]. Of prime importance is the fact that this compound effectively inactivated CMV and SFV by >90% in 1 hr at 37 C at concentrations that are known to have no biologically adverse effect when BHT is fed to animals and humans as a food additive. A concentration of BHT of 160 j.Lgjml was more than sufficient to inactivate >95% of the CMV and SFV. BHT concentrations of up to 200 ppm are known to have no side effects and are routinely added to foods [6]. In vivo the effectiveness of BHT against infection with enveloped virus has already been re-
ported. Brugh [l0] reported that chickens maintained on feed containing 200-2,000 ppm did not show a normal humoral immunological response when challenged with an avirulent strain of NDV. Each of these findings is due to inactivation of the infecting virus by BHT. Another significant point is that BHT is effective against either RNA or DNA viruses that have a lipidcontaining membrane. Since BHT has no adverse effect on the host and selectively inactivates the enveloped viruses tested thus far, it has great potential as a broad-spectrum antiviral agent. Further experiments are now in progress to determine whether BHT is an effective antiviral agent in the mouse-murine CMV model.
References
J. Cytomegalovirus infections in organ transplantation and post transfusion. An hypothesis. Arch Gesamte Virusforsch. 37:365-377, 1972. 2. Weller, T. H. The cytomegaloviruses: the ubiquitous agents with protean clinical manifestations. N. Engl. 1. Lang, D.
J. ~ed.285:203-214,1971. 3. Montgomery, R., Youngblood, L., Medearis, D. N., Jr. Recovery of cytomegalovirus from the cervix in pregnancy. Pediatrics 49:524-531, 1972. 4. Diosi, P., Moldovan, E., Tomescu, N. Latent cytomegalovirus infection in blood donors. Br. Med. J. 4:660-662, 1969. 5. Kaariaiuen, L., Pa1oheimo, J., Klemo1a, E., ~ake1a, T., Koivuniemi, A. Cytomegalovirus-mononucleosis: isolation of the virus and demonstration of subclinical infections after fresh blood transfusion in connection with open·heart surgery. Annales Medicine Experimentalis et Biologiae Fenniae 44:297-301, 1966. 6. Stuckey, B. N. Butylated hydroxytoluene is classified "generally recognized as safe" by the Food and Drug Administration. In T. E. Furia [ed.]. Handbook of
Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
non-lipid-containing poliovirus were exposed to BHT under identical experimental conditions. As shown in table 2, >90% of the SFV was inactivated in the presence of 80 j.Lg of BHT jml, whereas poliovirus was not inactivated by BHT at all concentrations tested. Although vaccinia virus was also inactivated, the extent of inactivation was far less than that of either SFV or CMV.
94
7.
8.
9.
10.
11.
Kim et al.
food additives. Chemical Rubber Company, Cleveland, Ohio, 1972, p. 185. Branen, A. L. Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. J. Am. Oil Chern. Soc. 52:59-63,1975. March, B. E., Coates, V., Biely, J. Reticulocytosis in response to dietary antioxidants. Science 64:13981400,1969. Snipes, W., Person, S., Keith, A., Cupp, J. Butylated hydroxytoluene inactivates lipid-containing viruses. Science 188:64-66, 1975. Brugh, M., Jr. Butylated hydroxytoluene protects chickens exposed to Newcastle disease virus. Science 197:1291-1292,1977. Kim, K. S., Carp, R. I. Abortive infection of human
diploid cells by murine cytomegalovirus. Infec. Immun. 6:793-797,1972. 12. Wentworth, B. B., French, L. Plaque assay of cytomegalovirus strains of human origin. Proc. Soc. Exp. Bioi. Med. 135:253-258, 1970. 13. Sharp, D. G., Kim, K. S. Multiplicity reactivation and radiation survival of aggregated vaccinia virus. Calculation of plaque titer based on MR and particle aggregation seen in the electron microscope. Virology 29:359-366, 1966. 14. Eletr, S., Williams, M. A., Watkins, T., Keith, A. D. Perturbat ions of the dynamics of lipid alkyl chains in membrane systems: effect on the activity of membrane-bound enzymes. Biochim. Biophys. Acta 339: 190-201,1974. Downloaded from http://jid.oxfordjournals.org/ at Emory University on August 24, 2015
