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Nuclear Instruments and Methods in Physics Research B 236 (2005) 249-253 www.elsevier.com/locate/nimb

Virus inactivation studies using ion beams, electron and gamma irradiation
Eduardo E. Smolko
a

a,* ,

Jorge H. Lombardo

b

Ä Laboratorio de Polimeros, Grupo Aplicaciones Industriales, Unidad de Aplicaciones Tecnologicas y Agropecuarias, Ä Ä Ä Ä Centro Atomico Ezeiza, Comision Nacional de Energia Atomica, Pbro. Juan Gonzalez y Aragon 15, C.P. B1802AYA Ezeiza, Buenos Aires, Argentina b Biotech S.A., C.P. 1754 Buenos Aires, Argentina Available online 24 May 2005

Abstract Known methods of virus inactivation are based on the chemical action of some substances such as acetylethylenimine, betapropiolactone, glycidalaldehyde, formaldehyde, etc. In such a process, the viral suspension should be kept at room or higher temperatures for 24-48 h. Under these conditions, physical and chemical agents act to degrade the virus antigenic proteins. On the contrary with ionizing radiations at low temperatures, the treatment does not cause such degradation allowing the study of different viral functions. In this work, particle (a, d and ï) and c irradiations were used for partial and total inactivation of Foot and Mouth Disease Virus (FMDV), Rauscher Leukemia Virus (RLV) and Herpes Simplex Virus (HSV). Obtention of the D37 dose from survival curves and the application of the target theory, permitted the determination of molecular weight of the nucleic acid genomes, EBR values and useful information for vaccine preparation. For RLV virus, a two target model of the RNA genome was deduced in accordance with biological information while from data from the literature and our own work on the structure of the scrapie prion, considering the molecular weight obtained by application of the theory, a new model for prion replication is presented, based on a trimer molecule. ã 2005 Elsevier B.V. All rights reserved.
PACS: 87.50.a; 87.50.Gi Keywords: Virus inactivation; Gamma; Electron; Alpha; Deuteron; Irradiation

1. Introduction
Corresponding author. Tel.: +54 11 6779 8571; fax: +54 11 6779 8322. E-mail address: smolko@cae.cnea.gov.ar (E.E. Smolko).
*

The different processes for preparing vaccines against viral diseases may be classified as follows:

0168-583X/$ - see front matter ã 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2005.04.055


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virus production, virus inactivation and vaccine formulation. Virus production is the first step in the preparation of antigenic particles for vaccine formulation. It is normally carried out by infecting the cells which are particularly sensitive to the virus, and removing the viral material present in the cell culture. Nowadays for that purposes, BHK cells cultures in suspension are commonly used. The next step is the virus inactivation. Known methods are based on the chemical action of some chemicals which disrupts the three-dimensional structure of the virus, attacking fundamentally the nucleic acid of the virus and rendering it incapable of proliferation. In such stringent processes the viral suspension should be kept at room and possible higher temperatures for one or more days. Inactivation of viruses by ionizing radiations was studied by many authors: Pollard [1], Ginoza et al. [2], Dertinger and Jung [3], among others. Virus inactivated by ionizing radiations were reported to retain most of their antigenicity by Polley [4], Jordan and Kempe [5]. Particularly inactivation of FMDV with ionizing radiations was studied by Johnson [6], Massa [7], Baldelli [8], Polatnick and Bachrach [8] and Lombardo and Smolko [9]. In this work, we present data of partial and total inactivation of various virus by using different ionizing radiations, calculating some of the parameters using the target theory.

logy Institute of the German Cancer Investigation Center (DKFZ) in Heidelberg and Herpes Simplex Virus was obtained from the Medical Virology Institute of Heidelberg University, corresponding to ``Thea'' and ``Muller'' strains. ? 2.2. Irradiation facilities FMDV was irradiated with gamma rays from a Cobalt 60 source at the Semi-industrial Irradiation Plant of the Ezeiza Atomic Center and with a 27 MeV deuterons beam from the Synchrocyclotron of Buenos Aires. RLV and HSV, were irradiated with gamma rays from a Co-60 AECL Gammacell 220 source at the Institut fur ? Strahlenbiologie and with a 10 MeV linear electron accelerator (Varian V-7703) at the Bundesforschungsanstalt fur Enahrung, both instalations ? ? at Karlsruhe. RLV was irradiated also with a 52 MeV energy deuteron and with 104 MeV alpha particle beams from the isochronous cyclotron at the Institute fur Angewandte Kernphysics also in ? Karlsruhe, Germany. 2.3. Virus titration The virus titre was determined by unit mass of the particular tissue or organ selected for the study. FMDV and RLV titrations were made by calculating the percentage of mortality of experimentally infected animals according to the method of Reed and Muench, by taking eight animals per dilution and seven dilution of each sample, progressively diluted by steps of 0.1. FMDV titration was made in newborn Pirbright mice, seven days old, i.m. inoculated with 0.05 ml of the viral suspension. RLV titration was made in BALB/c mice, 4-8 weeks old, with intraperitoneally injection of 0.2 ml and was also titrated by weighing the murine spleen. HSV was titrated by the method of plate forming units (PFU/ml) in embryo fibroblast cell cultures.

2. Material and methods 2.1. Virus Virus was obtained from stocks in Argentina as well as from Germany. FMDV was obtained from large stocks of strains A, C and O existing in South America. It was produced in BHK cell cultures and further concentrated in PEG-6000. The precipitated material was resuspended to 1/10 of the original volume in Hanksåsolution, pH = 7.6. Samples were obtained by fractionation of this solution and were kept frozen before and during the irradiation. After the irradiation and until titration the samples were kept at Ð40 ÀC. Rauscher Leukemia Virus was obtained from the Experimental Patho-

3. Results Radiobiological data of the molecular weight of the target sensitive to virus replication are given


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for the three virus studied. Inactivation curves showed to depend not only on virus external factors such as dose, radiation type, temperature and nature of the virus substrate during the irradiation process, but also on the morphological characteristics of the virus per se, such as its chemical composition and the nature of the molecular structure. In Fig. 1 the survival response for different viruses and different radiations to the irradiation dose is shown. Dose-response curves for FMDV and HSV were interpreted under the assumption of a response of a single hit, single target exponential curve. In the figure, the survival curves for FMDV are represented as FMDV (d) and FMDV(c) for d and c irradiations respectively and HSV-i(c) and HSV-ii(c) for irradiations with

gamma rays of the Thea and Muller strains of ? the HSV virus. The interpretation of the dose-response curve for the inactivation of RLV was under the assumption of a single hit, two target response and is represented in the figure as curve RLV(c). In Table 1 the inactivation dose D37 in kGy and the molecular weight of the target of virus is shown, as obtained from the application of the target theory. The effectiveness for different kinds of radiation measured as the ratio between dose for the same response under identical conditions: RBE Ì D37 ?cî=D37 ?particleî is bigger for single chain targets like those of FMDV and RLV virus. The effectiveness of the 10 MeV energy electrons is in general of about 50%, but the dose rate of any of the particle beams used is much higher than the gamma irradiation taken as reference. The particular example of the response to irradiation of RLV virus is noticeable because it is the first case in which it was possible to separate

Table 1 Inactivation dose and molecular weight of different virus Virus FMDV HSV (a) (b) RLV (i) Radiation c d c b c b c b d a c b d a D37 (kGy) 2.7 3.7 1.12 2.92 1.34 2.92 0.57 1.14 1.18 0.96 2.08 3.52 4.33 5.00 MW (kDa) 2,200 5,100 4,300 10,100

(ii) Fig. 1. Radiation inactivation curves of several virus FMDV(d) and FMDV(c) correspond to the response of foot and mouth disease virus to d and c irradiation, respectively. HSV-i(c) and HSV-ii(c) are the dose-inactivation curves corresponding to Herpes Simplex Virus irradiated with c rays, of HSV strains Thea and Muller respectively. RLV(c), is the dose-inactivation ? curve of Rauscher Leukemia Virus subjected to c irradiation.

2,800

FMDV, foot and mouth disease virus. HSV, Herpes Simplex Virus (a): Thea strain, (b): Muller strain. ? RLV, Rauscher Leukemia Virus (i): Target 1, (ii): Target 2. D37 (kGy): Inactivation radiation dose in kilogray. MW (kDa): Target molecular weight in kilodalton.


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Fig. 2. The figure shows the dose-inactivation response of a wide range of biological objects subjected to different types of ionizing radiations. Most of the present results for target molecular weights between 2.2 and 10.1 kDa are represented in a thick line near the origin. The curve of mouse brain Scrapie is from [11] and [12]. The shadowed area corresponds to date from [10] on radiation target sizes of Scrapie prions from hamster brain. Interferon response has been taken from [15].

the response curve for different radiations in two parts, according to a model of two single hit target of different volumes V1 and V2: N =N 0 Ì a Ñ exp?ÐV 1Ó Dî??1 Ð aîÓ exp?ÐV 2Ó Dî and these two targets have been recognized as a particular type of virus with two different functions called RMLV and SFFV, the competent virus and the defective one, respectively. In Fig. 2, on a logarithmic scale, the extensive range of dose necessary for obtaining the target molecular weight of most of the biological objects generally in study is given. The range of dose for most of the virus are represented by a thick line near the origin, for target molecular weights between 2 and 10 MDa. These doses can be obtained in around 5 h irradiation time with the most powerful Co-60 facility. For scrapie proteins and interferon studies, with molecular weights of around 20 kDa the necessary dose in reasonable times is

mainly obtained by irradiation with particle beams. In the same figure the molecular weight of scrapie proteins by different authors [10,11] is represented obtained by application of the target theory. It is important to remember that all these ample results for doses of the order of various MGy could be arisen from differences in dose rates, energies and in the preparation of different specimens for the irradiation. As a good result of the application of the target theory, it was possible to obtain inactivated virus, which was used successfully as antigen in the the preparation of a vaccine against FMDV. The first commercial lot of this antiviral radiovaccine was of the order of one million doses which effectively protected the inoculated cattle against the disease [13]. The application of ionizing radiation to enravel the size of the molecular weight of the scrapie prion, has given up to now confusing results. The target molecular weight of 55 kDa obtained as an average of inactivation doses of scrapie samples prepared in different ways [10]: homogenates from mouse or hamster brains, sonicated rods, purified rods, liposomes, microsomes, etc., were given place, at present abandoned - Prusiner mortal dimer model - of the infective disease. More recently chemical and structural information obtained on scrapie prions plus a deeper analysis of its radiation inactivation response, has permitted the incorporation of new hypothesis concerning to the multiplication of the scrapie proteins [14].

References
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[13] J.H. Lombardo, private communication, BIOSUR S.A. Moreno, Prov. Bs. As. Ä [14] E.E. Smolko, D. Adrogue and J.H. Lombardo, EnfermedÄ ades por Priones: un desafio para la Sanidad Humana y Ä Animal, Catalogos S.R.L., Buenos Aires, 2004, p. 9. [15] S. Pestka, B. Kelder, P.C. Familletti, J.A. Moschera, R. Crowl, E.S. Kempner, J. Biol. Chem. 258 (1983) 9706.