GB1563284A - Production of mutant of aujeszky's virus - Google Patents

Description

(54) PRODUCTION OF MUTANT OF AUJESZKY'S VIRUS (71) We, DSO "PHARMACHIM", of 16, Iliensko Chaussee, Sofia, Bulgaria, a State Economic Corporation organised under the laws of Bulgaria, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a method for the production of an avirulent mutant of Aujeszky's virus, and to vaccines containing the same.

Viral genetics is a branch of modern virology which is becoming increasingly important. In preparing highly effective vaccines and solving problems associated with peculiarities of viral reproduction and the susceptibility of viruses towards different chemical agents, it is generally necessary to produce particular viruses having reproducable genetic characteristics.

However, viruses are known to be able to change their genetic characteristics.

Studies show that the alterations in the genetic characteristics of viruses may be connected with gene mutations, that is alterations in the structures of section of the molecules of the genetic material controlling the different functions of the viruses. Such alterations may also be attributable to mutations in chromosome structures. Bearing in mind the fact that the basic genetic material in viruses is constituted by a single molecule of nucleic acid, it is clear that their reproductability is largely determined by changes in genetic material according to the spotted (genic) mutation type. More particularly, it is clear that changes in viral form from generation to generation result from changes in specific sections of the viral nucleic acid.

According to the present invention, there is provided a method for the production of an avirulent mutant from Aujeszky's virus, which comprises subjecting Aujeszky's virus to the antimetabolite 5-iodo-2'-desoxyuridine until an apthogenic and immulogenic mutant of the virus is obtained as a result of replacement of thymidine units in the viral nucleus by 5-iodo-2'-desoxyuridine units.

Whilst it is known that many substances can inhibit viral reproduction, it has hitherto been found that a mutagenic action on the viruses only take place with some of them. Thus, it has hitherto been found that DNA-viruses alone are susceptible to the replacement of thymidine by a halogenated desoxyuridine. The present invention is based on the observation that if 5-iodo-2'desoxyuridine acts as antimetabolite on Aujeszky's virus in a controlled programme, a mutant is obtained which is apthogenic and immunogenic thus rendering it suitable for use as a live vaccine, as a result of replacement of thymidine units in the viral nucleus with 5-iodo-2'desoxyuridine units. In general, antimetabolites operate in such manner that other virus particles are not formed at all or so that irregular fitting of nucleic acid molecules to amino acids whereby proteins are formed occurs. In the present case, it is the latter action which occurs, this being, most probably, the result of false information leading to the synthesis of "defective" structural proteins or non-functional proteins regulating the fitting, in the parent virus, this false information being transferred to the new generation of virus particles.

When carrying out the method of the invention, the number of passages with the 5-iodo-2'-desoxyuridine to which the starting virus will be subjected will generally be 8. The highly immunogenic and apthogenic mutant of Aujeszky's virus obtained in this way yields vaccines which provide an immunity to Aujeszky's virus of long duration. Experiments which have been carried out using 5-iodo-2'-desoxyuridine antimetabolite for virulent virus cultivation in cell cultures obtained from chicken's embryo fibroblasts, show that an alteration in the genetic code of the virus has been attained which has lead to radical changes in the genetic characteristics of the biological properties of the virus. Not only does the 5-iodo-2'-desoxyuridine antimetabolite fit in the DNA chains in place of thymidine, one of the three nucleotide bases therein, thereby providing a number of changes in the DNA structure and loss of virulent and preservation of immunogeneity, but some of the other biological properties of the virus are changed too.

The viral mutant obtained by the method of this invention has been found to be apthogenic for sheep, sucking pigs, white mice, calves, rabbits and dogs. At the same time, it has been found to be highly immunogenic for these animals. Immunity provided thereby against Aujeszky's virus has lasted for one year. In lyophilised state, the virus can be stored for several years.

The following Example illustrates the invention.

EXAMPLE To prepare a mutant preparation, there were used 24 hour cell cultures from chicken's embro fibroblasts, these having been prepared in Petri dishes of 60 mm diameter. Culturing of the fibroblasts was effected in Hanks' solution to which had been added lactalbumin hydrolysate in an amount of 0.5% by weight, normal calf serum and the antibiotics penicillin (100 I.U. per ml) and streptomycin (100 mg per ml). Viral growth on the culture medium was effected by the plate technique, whereby plates of the virus identifiable by means of an indicator, are grown and the most successful plate is removed for use in a subsequent growth stage. Aujeszky's virus was added to the culture medium on the Petri dish and allowed to stand thereon for 1 hour at 37"C. The culture medium was then drained of liquid and added to a Difco (Registered Trade Mark) agar culture containing 1% by weight of agar and a culture medium having the aforesaid composition, but for the addition thereto of 15 ml of 5-iodo-2'-desoxyuridine solution. The Petri dish contents were then cultured for 2 days at 37"C. after which removal of liquid was effected and the same culture medium as was used in the initial 2 day cultivation, but for the omission of the 5-iodo-2'-desoxyuridine was added. At this stage, neutral red in a concentration of 1:10,000 by weight was added to the culture to indicate plate formation.

After a further cultivation for 24 hours at 37"C., the plate of largest diameter was isolated by means of a sterile pipette. This viral sample was used as starting material in the next passage.

The aforesaid procedure from the absorption of the virus onto the Difco agar culture containing, inter alia, 5-iodo-2'desoxyuridine, onwards was repeated a further seven times. Whilst in the first two passages the Difco agar culture employed in the first 48 hours of cultivation was added in an amount of 15 ml. in the final six stages or passages, the 5-iodo-2'desoxyuridine was employed in an amount of 35 ml.

The virus mutant obtained at the end of the eighth passage was apathogenic for sheep, sucking pigs, white mice, calves, rabbits and dogs, while being highly immunogenic for the animals.

WHAT WE CLAIM IS: - 1. A method for the production of an avirulent mutant from Aujeszky's virus, which comprises subjecting Aujeszky's virus to the antimetabolite 5-iodo-2'-desoxyuridine until an apthogenic and immunogenic mutant of the virus is obtained as a result of replacement of thymidine units in the viral nucleus by 4-iodo-2"-desoxyun.dine units.

2. A method as claimed in claim 1, wherein Aujeszky's virus is subjected to the action of the antimetabolite 5-iodo-2'-desoxyuridine for eight passages.

3. A method for the production of an avirulent mutant from Aujeszky's virus, substantially as described in the foregoing Example.

4. An avirulent mutant from Aujeszky's virus, whenever produced by the method claimed in any one of the preceding claims.

5. A vaccine comprising the avirulent mutant from Aujeszky's virus as claimed

Claims (1)

1. in claim 4.