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Cryo-gamma radiation inactivation of bovine herpesvirus type-1
Radiation Physics and Chemistry 55 (1999) 469±471
Technical note
Cryo-gamma radiation inactivation of bovine herpesvirus type-1 C. FernaÂndez Degiorgi*, E.E. Smolko, J.H. Lombardo ComisioÂn Nacional de EnergõÂa AtoÂmica, Av. Libertador 8250, 1429, Buenos Aires, Argentina Received 12 December 1997; accepted 20 September 1998
Abstract The radioresistance of bovine herpesvirus-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), suspended in free serum Glasgow-MEM medium and frozen at ÿ788C was studied. The number of surviving virus at a given dose of gamma-radiation was determined by a plaque assay system. D10 values were calculated before and after removal of cell debris. The D10 values obtained were 4.72 kGy and 7.31 kGy before and after removal of cell debris, respectively. Our results indicate that the inactivated viral particles could be used for vaccine preparation or diagnostic reagents. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Infectious bovine rhinotracheitis virus; Bovine herpes virus-1; Cryo-gamma radiation; Viral inactivation
1. Introduction The action of ionizing radiation on biologically important molecules has been intensively studied for many years. However, radioinactivation of viruses has received sporadic attention during the past 30 years because the success of attenuated viral vaccines overshadowed the concept of killed vaccine preparations. Recently, renewed interest has been generated in the use of gamma radiation as a method for inactivation of viruses due to the fact that side eects or genetic alterations conducing to infective viruses has been observed associated to the use of some attenuated vaccines. Also, a number of viruses capable of infecting humans have been isolated from food and feed (Abinanti, 1961; Bagdasarlyan, 1964; Bendinelli and Ruschi, 1969; Ernek et al., 1968) and this has prompted extensive studies on viral inactivation by gamma radiation on these materials (Danes et al.,
* Corresponding author.
1966; Logan and Whitmore, 1964; Polley, 1962; Lombardo and Smolko, 1990). Bovine herpesvirus type-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), has been associated with respiratory, reproductive, enteric, ocular, central nervous system, neonatal and dermal infections of cattle (Schroeder and Moys, 1954; Kahrs, 1977; Moorthi, 1985). Also, infections in other species have been reported (Lupton et al, 1980; Joo et al., 1961). In this paper we report studies on the radioinactivation of BHV-1 viral particles in free serum GlasgowMEM medium before and after removal of cell debris at ÿ788C. 2. Materials and methods 2.1. Virus and cells Bovine herpesvirus type 1 (BHV-1) Los Angeles (LA) strain was used throughout this study. Viral particles were grown on Madin Darby bovine kidney cells
0969-806X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 9 9 ) 0 0 1 9 9 - 1
470
C. FernaÂndez Degiorgi et al. / Radiation Physics and Chemistry 55 (1999) 469±471
ing the inoculum. Infected cultures were incubated at 378C, until extensive cytopathic eect (generally at 48± 72 h). The agar overlay was then removed and 3 ml of 1% crystal violet in 10% ethanol plus 5% formalin in H2O were added. After 10 min, the bottles were rinsed with water, air dried and the plaques counted. 2.3. Radiation source and sample irradiation Viral suspensions in cryotubes were frozen at ÿ788C and irradiated with a gamma-ray 60Co source of 18.5 PBq at the ComisioÂn Nacional de EnergõÂ a AtoÂmica, Buenos Aires. The irradiations were carried out at doses ranging from 2.5 to 30 kGy. Dosimetry was performed using a modi®ed Fricke dosimeter (Swallow, 1960). After irradiation the samples were stored at ÿ208C until they were used in viral assays. Negative controls were introduced during this process. Triplicate samples from each tube were assayed for viral PFU. 3. Results Fig. 1. Gamma radiation inactivation of infectious bovine rhinotracheitis virus with q, and without cell debris r, at ÿ788C. Data points represent the average of at least three independent trials.
(MDBK ATCC CLL 22) previously cultivated in Glasgow-MEM (Gibco) supplemented with 10% fetal bovine serum (Gibco, Burlington, Ontario), 0.2% tryptose phosphate broth (Gibco), 2 mM glutamine (Sigma), 0.021% NaHCO3 7%, 200 U/ml penicillin and 100 mg/ml streptomycin (Richet). 2% bactoagar (Difco) was used as agar overlay. Infected cells were cultivated in serum free medium. Cell monolayers showing cytopathic eects were frozen and thawed three times and the virus was harvested. The viral suspension was fractionated in two aliquots; one was clari®ed by centrifugation for 15 min at 1060 g at 48C and the other was used without clari®cation. Viral titers were determined and after this the viral preparation was dispensed into polycarbonate cryotubes and stored at ÿ208C until irradiation. 2.2. Virus assay To obtain viral titers, a plaque-forming unit (PFU) assay system was used. For infection, con¯uent MDBK monolayers were washed once with serum free medium. After this, tenfold serial dilution viral suspensions were prepared using serum free medium, pH 7.2± 7.4, as diluent. 0.2 ml of these dilutions were added to the cell monolayer and allowed to adsorb for 1 h at 378C. Then, 3 ml of agar overlay plus serum free Glasgow-MEM (2) (1:1) were added without remov-
Fig. 1 shows the summary of the gamma radiation inactivation curves of BHV-1 in free serum GlasgowMEM before and after removal of cellular debris at ÿ788C. Data from the experiments were analysed with the linear regression model and the D10 values (radiation dose that reduces the viral population by 90%) were computed. The D37 values (the dose that reduces the surviving virus to e ÿ1 or 37% of its original) were also calculated (D37=0.43 D10). The virus before removal of cell debris resulted in more radiosensitivity (D10=4.72 kGy and D37=2.03 kGy) than the virus after removal of cell debris (D10=7.31 kGy and D37=3.14 kGy) at the temperature investigated (ÿ788C). 4. Discussion Gamma radiation inactivation rate kinetics on BHV1 suspended in free serum Glasgow-MEM showed a ®rst-order curve con®guration. The high D10 value observed for the virus in the frozen material could be due to the inhibition of free-radical formation or to impeding the free-radical travel in the frozen material (Sullivan et al., 1973). Viruses in the frozen state have been reported to require more ionizing energy for inactivation and had higher D10 values (Jordan and Kempe, 1956). The viruses irradiated with cellular debris were more sensitive than the cleared virus. This result may be attributed to the presence in the medium of substances that induce the secondary eects of radiation (Ginoza, 1968).
C. FernaÂndez Degiorgi et al. / Radiation Physics and Chemistry 55 (1999) 469±471
There are dierent ways in which the radiation damage to a biological target can be modi®ed, resulting in sensitization or protection. In addition to primary radicals from the irradiated water, secondary radicals induced directly or indirectly in organic material must be considered as possible agents which might attach to DNA when irradiated in solution or in the frozen state (slower process) (Hotz 1978). Variation in D10 (or D37) according to various authors working with several viruses showed a wide range of dose-modi®cation factor (DMF), described by Jagger (1967) as the relationship between D10 values in the presence and absence of the modi®er. When Coxsackievirus B-2 was irradiated at temperatures between ÿ308C and ÿ608C, DMF = D10 at ÿ308C/ D10 at ÿ608C = 1.2 was obtained (Sullivan et al., 1973). In our case we observed a DMF value of 1.54, where DMF = D10 virus clear/D10 virus crude, which indicates a value similar to those caused by temperature variation. Another possibility is that of radicals trapped in debris that are unevenly distributed in the virus suspension, with a slow recombination process at low temperatures, but enhanced at melting temperatures. In practice, the D10 value for each virus in each medium and at a de®ned temperature must be measured. Results on irradiation of sera using ionizing radiation at very low temperatures have demonstrated that this is an eective way of viral inactivation for DNA or RNA virus. Furthermore, this treatment does not aect proteins or activity of neutralizing antibodies present in the media (Lombardo et al., 1992; Nonomiya et al., 1992; Lombardo et al., 1995). For the IBR virus, this methodology for inactivation has been used in vaccine preparation, as described by Smolko et al. (1992). The results obtained in the present work demonstrate that it could also be used for BHV-1 viral particles that could lead to vaccine or diagnostic reagent preparations. Acknowledgements We thank Dr J. Zorzopulos for critical reading of the manuscript. References Abinanti, F.R., 1961. Respiratory and enteric viruses in man and animals. Publ. Health Rep 76, 897. Bagdasarlyan, G.A., 1964. Survival of viruses of the enterovirus group (poliomyelitis, ECHO, coxsackie) in soil and on vegetables. J. Hyg. Epidemiol. Microbiol. Immunol 8, 497.
471
Bendinelli, M., Ruschi, A., 1969. Isolation of human enterovirus from mussels. Appl. Microbiol 18, 531. Danes, L., Hrukova, J., Blazek, K., 1966. Poxvirus ocinale inactivated by gamma radiation: immunogenicity of the antigen. J. Hyg. Epidemiol. Microbiol. Immunol 10, 210. Ernek, E., Kozuch, O., Nosek, J., 1968. Isolation of tickborne encephalitis virus from blood and milk of goats grazin in the Tribec focus zone. J. Hyg. Epid. Microbiol. Immunol 12, 32. Ginoza, W., 1968, Inactivation of viruses by ionizing radiation and by heat. In: Methods in Virology, Academic Press, New York, 139±179. Hotz, G., 1978. Modi®cation of radiation damage. In: Eects of Ionizing Radiation on DNA. Springer-Verlag, BerlinHeidelberg, pp. 304±311. Jagger, J., 1967. Introduction to research in ultraviolet photobiology. Prentice-Hall, Inc., Englewood Clis, New Jersey. Joo, H.B., Dee, S.A., Molitor, T.W., Thacker, B.J., 1961. In utero infection of swine fetuses with infectious bovine rhinotracheitis virus (bovine herpesvirus-1). Am. J. Vet. Med. Assoc 139, 236. Jordan, R.T., Kempe, L.L., 1956. Inactivation of some animal viruses with gamma radiation from 60Cobalt. Proc. Soc. Exp. Biol. Med 91, 212. Kahrs, R.F., 1977. Infectious bovine rhinotracheitis. A review and update. J. Am. Vet. Med. Assoc 171, 1055. Logan, D.M., Whitmore, G.F., 1964. Radiation eects on polyoma virus. Virology 25, 495. Lombardo, J.H., Smolko, E.E., 1990. A biotechnological project with a gamma radiation source of 100,000 Ci. Radiat. Phys. Chem 35, 585. Lombardo, J.H., FernaÂndez, Degiorgi C., Quattrini, D., Michelin, S., Smolko, E.E. 1992 Sera radiosterilization process. 8th International Meeting on Radiation Processing, Beijing, p. 200. Lombardo, J.H., FernaÂndez, Degiorgi C., Quattrini, D., Michelin, S., Smolko, E.E., 1995. Sera radiosterilization: studies and applications. Radiat. Phys. Chem 45, 275. Lupton, H.W., Barnes, H.J., Reed, D.E., 1980. Evaluation of the rabbit as a laboratory model for infectious bovine rhinotracheitis virus infection. Cornell Vet 70, 77. Moorthi, A.R.S., 1985. Outbreak of balanoposthitis in breeding bulls. Vet. Rec 116, 98. Nonomiya, T., Ito, M., Morimoto, A., Yamashiro, T., Ishigaki, T., Iwatsuki, K., Tsutsumi, T., 1992. Inactivation of RNA viruses by gamma irradiation. Fd Irradiat. Jap 27, 19. Polley, J.R., 1962. The use of gamma radiation for the preparation of virus vaccines. Can. J. Microbiol 8, 455. Schroeder, R.J., Moys, M.D., 1954. An acute upper respiratory infection of dairy cattle. J. Am. Vet. Med. Assoc 125, 471. Smolko, E.E., Lombardo, J.H., Rivenson, S., 1992. First antiviral vaccine commercially produced by gamma-ray processing technology (Abst.). 8th Int. Mtg on Radiat. Processing, Beijing, 347. Sullivan, R., Scarpino, P.V., Fassolitis, A.C., Larkin, E.P., Peeler, J.T., 1973. Gamma radiation inactivation of Coxsackievirus B-2. Appl. Microbiol 26, 14. Swallow, A.J., 1960. In: Radiation Chemistry of Organic Compounds. Pergamon Press, Oxford, p. 42.
Technical note
Cryo-gamma radiation inactivation of bovine herpesvirus type-1 C. FernaÂndez Degiorgi*, E.E. Smolko, J.H. Lombardo ComisioÂn Nacional de EnergõÂa AtoÂmica, Av. Libertador 8250, 1429, Buenos Aires, Argentina Received 12 December 1997; accepted 20 September 1998
Abstract The radioresistance of bovine herpesvirus-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), suspended in free serum Glasgow-MEM medium and frozen at ÿ788C was studied. The number of surviving virus at a given dose of gamma-radiation was determined by a plaque assay system. D10 values were calculated before and after removal of cell debris. The D10 values obtained were 4.72 kGy and 7.31 kGy before and after removal of cell debris, respectively. Our results indicate that the inactivated viral particles could be used for vaccine preparation or diagnostic reagents. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Infectious bovine rhinotracheitis virus; Bovine herpes virus-1; Cryo-gamma radiation; Viral inactivation
1. Introduction The action of ionizing radiation on biologically important molecules has been intensively studied for many years. However, radioinactivation of viruses has received sporadic attention during the past 30 years because the success of attenuated viral vaccines overshadowed the concept of killed vaccine preparations. Recently, renewed interest has been generated in the use of gamma radiation as a method for inactivation of viruses due to the fact that side eects or genetic alterations conducing to infective viruses has been observed associated to the use of some attenuated vaccines. Also, a number of viruses capable of infecting humans have been isolated from food and feed (Abinanti, 1961; Bagdasarlyan, 1964; Bendinelli and Ruschi, 1969; Ernek et al., 1968) and this has prompted extensive studies on viral inactivation by gamma radiation on these materials (Danes et al.,
* Corresponding author.
1966; Logan and Whitmore, 1964; Polley, 1962; Lombardo and Smolko, 1990). Bovine herpesvirus type-1 (BHV-1), commonly known as infectious bovine rhinotracheitis virus (IBRV), has been associated with respiratory, reproductive, enteric, ocular, central nervous system, neonatal and dermal infections of cattle (Schroeder and Moys, 1954; Kahrs, 1977; Moorthi, 1985). Also, infections in other species have been reported (Lupton et al, 1980; Joo et al., 1961). In this paper we report studies on the radioinactivation of BHV-1 viral particles in free serum GlasgowMEM medium before and after removal of cell debris at ÿ788C. 2. Materials and methods 2.1. Virus and cells Bovine herpesvirus type 1 (BHV-1) Los Angeles (LA) strain was used throughout this study. Viral particles were grown on Madin Darby bovine kidney cells
0969-806X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 9 9 ) 0 0 1 9 9 - 1
470
C. FernaÂndez Degiorgi et al. / Radiation Physics and Chemistry 55 (1999) 469±471
ing the inoculum. Infected cultures were incubated at 378C, until extensive cytopathic eect (generally at 48± 72 h). The agar overlay was then removed and 3 ml of 1% crystal violet in 10% ethanol plus 5% formalin in H2O were added. After 10 min, the bottles were rinsed with water, air dried and the plaques counted. 2.3. Radiation source and sample irradiation Viral suspensions in cryotubes were frozen at ÿ788C and irradiated with a gamma-ray 60Co source of 18.5 PBq at the ComisioÂn Nacional de EnergõÂ a AtoÂmica, Buenos Aires. The irradiations were carried out at doses ranging from 2.5 to 30 kGy. Dosimetry was performed using a modi®ed Fricke dosimeter (Swallow, 1960). After irradiation the samples were stored at ÿ208C until they were used in viral assays. Negative controls were introduced during this process. Triplicate samples from each tube were assayed for viral PFU. 3. Results Fig. 1. Gamma radiation inactivation of infectious bovine rhinotracheitis virus with q, and without cell debris r, at ÿ788C. Data points represent the average of at least three independent trials.
(MDBK ATCC CLL 22) previously cultivated in Glasgow-MEM (Gibco) supplemented with 10% fetal bovine serum (Gibco, Burlington, Ontario), 0.2% tryptose phosphate broth (Gibco), 2 mM glutamine (Sigma), 0.021% NaHCO3 7%, 200 U/ml penicillin and 100 mg/ml streptomycin (Richet). 2% bactoagar (Difco) was used as agar overlay. Infected cells were cultivated in serum free medium. Cell monolayers showing cytopathic eects were frozen and thawed three times and the virus was harvested. The viral suspension was fractionated in two aliquots; one was clari®ed by centrifugation for 15 min at 1060 g at 48C and the other was used without clari®cation. Viral titers were determined and after this the viral preparation was dispensed into polycarbonate cryotubes and stored at ÿ208C until irradiation. 2.2. Virus assay To obtain viral titers, a plaque-forming unit (PFU) assay system was used. For infection, con¯uent MDBK monolayers were washed once with serum free medium. After this, tenfold serial dilution viral suspensions were prepared using serum free medium, pH 7.2± 7.4, as diluent. 0.2 ml of these dilutions were added to the cell monolayer and allowed to adsorb for 1 h at 378C. Then, 3 ml of agar overlay plus serum free Glasgow-MEM (2) (1:1) were added without remov-
Fig. 1 shows the summary of the gamma radiation inactivation curves of BHV-1 in free serum GlasgowMEM before and after removal of cellular debris at ÿ788C. Data from the experiments were analysed with the linear regression model and the D10 values (radiation dose that reduces the viral population by 90%) were computed. The D37 values (the dose that reduces the surviving virus to e ÿ1 or 37% of its original) were also calculated (D37=0.43 D10). The virus before removal of cell debris resulted in more radiosensitivity (D10=4.72 kGy and D37=2.03 kGy) than the virus after removal of cell debris (D10=7.31 kGy and D37=3.14 kGy) at the temperature investigated (ÿ788C). 4. Discussion Gamma radiation inactivation rate kinetics on BHV1 suspended in free serum Glasgow-MEM showed a ®rst-order curve con®guration. The high D10 value observed for the virus in the frozen material could be due to the inhibition of free-radical formation or to impeding the free-radical travel in the frozen material (Sullivan et al., 1973). Viruses in the frozen state have been reported to require more ionizing energy for inactivation and had higher D10 values (Jordan and Kempe, 1956). The viruses irradiated with cellular debris were more sensitive than the cleared virus. This result may be attributed to the presence in the medium of substances that induce the secondary eects of radiation (Ginoza, 1968).
C. FernaÂndez Degiorgi et al. / Radiation Physics and Chemistry 55 (1999) 469±471
There are dierent ways in which the radiation damage to a biological target can be modi®ed, resulting in sensitization or protection. In addition to primary radicals from the irradiated water, secondary radicals induced directly or indirectly in organic material must be considered as possible agents which might attach to DNA when irradiated in solution or in the frozen state (slower process) (Hotz 1978). Variation in D10 (or D37) according to various authors working with several viruses showed a wide range of dose-modi®cation factor (DMF), described by Jagger (1967) as the relationship between D10 values in the presence and absence of the modi®er. When Coxsackievirus B-2 was irradiated at temperatures between ÿ308C and ÿ608C, DMF = D10 at ÿ308C/ D10 at ÿ608C = 1.2 was obtained (Sullivan et al., 1973). In our case we observed a DMF value of 1.54, where DMF = D10 virus clear/D10 virus crude, which indicates a value similar to those caused by temperature variation. Another possibility is that of radicals trapped in debris that are unevenly distributed in the virus suspension, with a slow recombination process at low temperatures, but enhanced at melting temperatures. In practice, the D10 value for each virus in each medium and at a de®ned temperature must be measured. Results on irradiation of sera using ionizing radiation at very low temperatures have demonstrated that this is an eective way of viral inactivation for DNA or RNA virus. Furthermore, this treatment does not aect proteins or activity of neutralizing antibodies present in the media (Lombardo et al., 1992; Nonomiya et al., 1992; Lombardo et al., 1995). For the IBR virus, this methodology for inactivation has been used in vaccine preparation, as described by Smolko et al. (1992). The results obtained in the present work demonstrate that it could also be used for BHV-1 viral particles that could lead to vaccine or diagnostic reagent preparations. Acknowledgements We thank Dr J. Zorzopulos for critical reading of the manuscript. References Abinanti, F.R., 1961. Respiratory and enteric viruses in man and animals. Publ. Health Rep 76, 897. Bagdasarlyan, G.A., 1964. Survival of viruses of the enterovirus group (poliomyelitis, ECHO, coxsackie) in soil and on vegetables. J. Hyg. Epidemiol. Microbiol. Immunol 8, 497.
471
Bendinelli, M., Ruschi, A., 1969. Isolation of human enterovirus from mussels. Appl. Microbiol 18, 531. Danes, L., Hrukova, J., Blazek, K., 1966. Poxvirus ocinale inactivated by gamma radiation: immunogenicity of the antigen. J. Hyg. Epidemiol. Microbiol. Immunol 10, 210. Ernek, E., Kozuch, O., Nosek, J., 1968. Isolation of tickborne encephalitis virus from blood and milk of goats grazin in the Tribec focus zone. J. Hyg. Epid. Microbiol. Immunol 12, 32. Ginoza, W., 1968, Inactivation of viruses by ionizing radiation and by heat. In: Methods in Virology, Academic Press, New York, 139±179. Hotz, G., 1978. Modi®cation of radiation damage. In: Eects of Ionizing Radiation on DNA. Springer-Verlag, BerlinHeidelberg, pp. 304±311. Jagger, J., 1967. Introduction to research in ultraviolet photobiology. Prentice-Hall, Inc., Englewood Clis, New Jersey. Joo, H.B., Dee, S.A., Molitor, T.W., Thacker, B.J., 1961. In utero infection of swine fetuses with infectious bovine rhinotracheitis virus (bovine herpesvirus-1). Am. J. Vet. Med. Assoc 139, 236. Jordan, R.T., Kempe, L.L., 1956. Inactivation of some animal viruses with gamma radiation from 60Cobalt. Proc. Soc. Exp. Biol. Med 91, 212. Kahrs, R.F., 1977. Infectious bovine rhinotracheitis. A review and update. J. Am. Vet. Med. Assoc 171, 1055. Logan, D.M., Whitmore, G.F., 1964. Radiation eects on polyoma virus. Virology 25, 495. Lombardo, J.H., Smolko, E.E., 1990. A biotechnological project with a gamma radiation source of 100,000 Ci. Radiat. Phys. Chem 35, 585. Lombardo, J.H., FernaÂndez, Degiorgi C., Quattrini, D., Michelin, S., Smolko, E.E. 1992 Sera radiosterilization process. 8th International Meeting on Radiation Processing, Beijing, p. 200. Lombardo, J.H., FernaÂndez, Degiorgi C., Quattrini, D., Michelin, S., Smolko, E.E., 1995. Sera radiosterilization: studies and applications. Radiat. Phys. Chem 45, 275. Lupton, H.W., Barnes, H.J., Reed, D.E., 1980. Evaluation of the rabbit as a laboratory model for infectious bovine rhinotracheitis virus infection. Cornell Vet 70, 77. Moorthi, A.R.S., 1985. Outbreak of balanoposthitis in breeding bulls. Vet. Rec 116, 98. Nonomiya, T., Ito, M., Morimoto, A., Yamashiro, T., Ishigaki, T., Iwatsuki, K., Tsutsumi, T., 1992. Inactivation of RNA viruses by gamma irradiation. Fd Irradiat. Jap 27, 19. Polley, J.R., 1962. The use of gamma radiation for the preparation of virus vaccines. Can. J. Microbiol 8, 455. Schroeder, R.J., Moys, M.D., 1954. An acute upper respiratory infection of dairy cattle. J. Am. Vet. Med. Assoc 125, 471. Smolko, E.E., Lombardo, J.H., Rivenson, S., 1992. First antiviral vaccine commercially produced by gamma-ray processing technology (Abst.). 8th Int. Mtg on Radiat. Processing, Beijing, 347. Sullivan, R., Scarpino, P.V., Fassolitis, A.C., Larkin, E.P., Peeler, J.T., 1973. Gamma radiation inactivation of Coxsackievirus B-2. Appl. Microbiol 26, 14. Swallow, A.J., 1960. In: Radiation Chemistry of Organic Compounds. Pergamon Press, Oxford, p. 42.