CA1069821A - Method for the preparation of avirulent viral mutant from the aujeszky's virus - Google Patents
Method for the preparation of avirulent viral mutant from the aujeszky's virusInfo
- Publication number
- CA1069821A CA1069821A CA281,836A CA281836A CA1069821A CA 1069821 A CA1069821 A CA 1069821A CA 281836 A CA281836 A CA 281836A CA 1069821 A CA1069821 A CA 1069821A
- Authority
- CA
- Canada
- Prior art keywords
- virus
- aujeszky
- preparation
- mutant
- desoxyuridin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16761—Methods of inactivation or attenuation
- C12N2710/16763—Methods of inactivation or attenuation by chemical treatment
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to the preparation of an avirulent viral mutant from the Aujeszky's virus which consists in reacting the nucleus of said virus, containing desoxyribonucleic acid, with the antimetabolite 5-iodine-2-desoxyuridin and after the eighth passage into a predetermined culture medium, there is obtained an apathogentic and immuno-genic mutant to be used as a live vaccine.
The present invention relates to the preparation of an avirulent viral mutant from the Aujeszky's virus which consists in reacting the nucleus of said virus, containing desoxyribonucleic acid, with the antimetabolite 5-iodine-2-desoxyuridin and after the eighth passage into a predetermined culture medium, there is obtained an apathogentic and immuno-genic mutant to be used as a live vaccine.
Description
31(~6~8~1 This invention relates to a method or the prepara-tion of an avirulent viral mutant ~rom -the Aujes~ky's virus.
It is well kno~n that viral genetics represen-t one of the most important trends in modern virusology. The obtainment of virus lines with standard genetic characteristics is an essential moment in the preparation of highly effective vaccines, for the solution of theoretical problems associated with the peculiarities of viral reproduction and of their susceptibility towards different chemical substances etc. It is also known that viruses are able to change their hereditary properties. The studies conducted show that the alterations in the hereditary material could be connected both wlth the genes mutation i.e. disorders in definite limited sections of the hereditary material controlling the different functions, and with the chromosome structure mutations. Taking into consideration that the genetic material in the viruses represents by itself only one molecule of nucleinic acid, it is obvious that in their heredity essential role is played by the changes in the genetic material after the spotted (genic) mutation type. It is evident, that as a basis of heredity alterations the mutation comes forth whose material expression consists in changes in definite sections of the viral nucleinic acid.
At present, many substances are known, which can inhibit the viral reproduction, but a mutagenic action with respect to the viruses is established only in some of them.
The DNA-viruses are exclusively susceptible to the replacement of Thymidine with a halogenized desoxyuridin. If the reproduc-tion of the Aujeszky's diseas~'virus takes place in -the presence of the 5-iodine-2-desoxyuridin antimetabolite, viral DNA and antigen accumulate, but virus particles are either not formed or an irregular fi.tting occurs. Most probably, that is ~o~z~
a result o~ a false information, leadin~ to a synthesi~ of "defective" structural proteins or o~ non-functional proteins regula-ting the fitting. This false information can be also transferred to the new generation of the virus particles.
The object of the present invention is to create a method for the preparation of avirulent viral mutan-t from the Aujeszky's virus with a view to the production of a highly immunogenic and apathogenic mutant with a long duration of the immunity obtained, which can be used as a qualitative effective vaccine. In the 5-iodine-2-desoxyuridin antimetabolite utilization in a definite program for the virulent virus cultivation in cell cultures from chicken's embryo fibroplasts, an alteration in the genetic code of the virus is attained, which leads to radical changes in the genetic chart of its biological properties. If the synthesis of the new virus particles takes place in the presence of the antimetabolite, the desoxyuridin fits ln the place of Thymidine, one of the triplets in one of the DNA chains, which leads to a number of changes in the DNA structure. As a consequence, the virulence of the virus is lost, but its immunogeneity is preserved.
Parallel with that, some of the other biological properties of the virus are changed too. A purposeful change therefore is present, based on the discovered regularities, that the genetic structure of the DNA-viruses can undergo lasting, purposeful, regulated and controlled changes under the effect ,i of a definite antimetabolite mutagen.
For the mutant MK preparation, 24-hour cell cultures from chicken,'s embryo fibroplasts have been used, prepared after the known method6 in Petri dishes with 60 mm in diameter. For the culture medium the ~lenks' solution was used with the addition of lactalbumin hydrolysate 0.5 percent, normal calf serum and antibiotics (Pen:Lcillin 100 I~ per ml . , ~ 2 -, z~
and Streptomycin 100 mg per ml). For -the virus ~btainment it is proceeded from a plate, and ~or that purpose th~ pl~te technique is used i.e. the virus iS selected With the aid of a plate technique. The virus is adsorbed at 37~C for one hour, whereupon the volume liquid is removed and khe infected with the vi~us cell cultures are spilled with a Difco agar culture containing 1 percent of this agar and the culture medium whose composition has been described above. Before the u'cilization of the agar culture , a 5-iodine-2-desoxyuridin is added to it, by 15 ml to the first two passages and by 35 ml to the following six. After a two-day cultivation of the Petri dishes at 37C they are spilled again with a second layer of the covering culture medium composed of the same components as the first one, but for the 5-iodine-2-desoxy-uridin. Neutral red in final concentration of 1:10000 is added to the culture as an indicator of the plate. After another 2~-hour cultivation at 37C, a plate having the biggest diameter is isolated with the aid of a special sterile pipette.
From this plate, the virus is obtained in cell cultures from chicken's embryo fibroplasts, with which the next passage is conducted ater the above described technique.
The virus mutant obtained is apathogenic for sheep, sucking piglets, white mice, calves, rabbits and dogs. ~t the same time it is highly immunogenic for the animals. The immunity created lasts one year. In lyophilized state the , virus can be stored for several years.
' The method provides the following advantages.
From the highly virulent virus of the Aujeszky's disease, an apathogenic and immunogenic viral mutant is obtained by the use of a very qu,ick and economic method, i.e.
by provoking changes in the structure of the viral DNA with ' the aid of antimetabolite S-iodine-2-desoxyuridin.
The mutant obtained is used for the production of - a vaccine.
It is well kno~n that viral genetics represen-t one of the most important trends in modern virusology. The obtainment of virus lines with standard genetic characteristics is an essential moment in the preparation of highly effective vaccines, for the solution of theoretical problems associated with the peculiarities of viral reproduction and of their susceptibility towards different chemical substances etc. It is also known that viruses are able to change their hereditary properties. The studies conducted show that the alterations in the hereditary material could be connected both wlth the genes mutation i.e. disorders in definite limited sections of the hereditary material controlling the different functions, and with the chromosome structure mutations. Taking into consideration that the genetic material in the viruses represents by itself only one molecule of nucleinic acid, it is obvious that in their heredity essential role is played by the changes in the genetic material after the spotted (genic) mutation type. It is evident, that as a basis of heredity alterations the mutation comes forth whose material expression consists in changes in definite sections of the viral nucleinic acid.
At present, many substances are known, which can inhibit the viral reproduction, but a mutagenic action with respect to the viruses is established only in some of them.
The DNA-viruses are exclusively susceptible to the replacement of Thymidine with a halogenized desoxyuridin. If the reproduc-tion of the Aujeszky's diseas~'virus takes place in -the presence of the 5-iodine-2-desoxyuridin antimetabolite, viral DNA and antigen accumulate, but virus particles are either not formed or an irregular fi.tting occurs. Most probably, that is ~o~z~
a result o~ a false information, leadin~ to a synthesi~ of "defective" structural proteins or o~ non-functional proteins regula-ting the fitting. This false information can be also transferred to the new generation of the virus particles.
The object of the present invention is to create a method for the preparation of avirulent viral mutan-t from the Aujeszky's virus with a view to the production of a highly immunogenic and apathogenic mutant with a long duration of the immunity obtained, which can be used as a qualitative effective vaccine. In the 5-iodine-2-desoxyuridin antimetabolite utilization in a definite program for the virulent virus cultivation in cell cultures from chicken's embryo fibroplasts, an alteration in the genetic code of the virus is attained, which leads to radical changes in the genetic chart of its biological properties. If the synthesis of the new virus particles takes place in the presence of the antimetabolite, the desoxyuridin fits ln the place of Thymidine, one of the triplets in one of the DNA chains, which leads to a number of changes in the DNA structure. As a consequence, the virulence of the virus is lost, but its immunogeneity is preserved.
Parallel with that, some of the other biological properties of the virus are changed too. A purposeful change therefore is present, based on the discovered regularities, that the genetic structure of the DNA-viruses can undergo lasting, purposeful, regulated and controlled changes under the effect ,i of a definite antimetabolite mutagen.
For the mutant MK preparation, 24-hour cell cultures from chicken,'s embryo fibroplasts have been used, prepared after the known method6 in Petri dishes with 60 mm in diameter. For the culture medium the ~lenks' solution was used with the addition of lactalbumin hydrolysate 0.5 percent, normal calf serum and antibiotics (Pen:Lcillin 100 I~ per ml . , ~ 2 -, z~
and Streptomycin 100 mg per ml). For -the virus ~btainment it is proceeded from a plate, and ~or that purpose th~ pl~te technique is used i.e. the virus iS selected With the aid of a plate technique. The virus is adsorbed at 37~C for one hour, whereupon the volume liquid is removed and khe infected with the vi~us cell cultures are spilled with a Difco agar culture containing 1 percent of this agar and the culture medium whose composition has been described above. Before the u'cilization of the agar culture , a 5-iodine-2-desoxyuridin is added to it, by 15 ml to the first two passages and by 35 ml to the following six. After a two-day cultivation of the Petri dishes at 37C they are spilled again with a second layer of the covering culture medium composed of the same components as the first one, but for the 5-iodine-2-desoxy-uridin. Neutral red in final concentration of 1:10000 is added to the culture as an indicator of the plate. After another 2~-hour cultivation at 37C, a plate having the biggest diameter is isolated with the aid of a special sterile pipette.
From this plate, the virus is obtained in cell cultures from chicken's embryo fibroplasts, with which the next passage is conducted ater the above described technique.
The virus mutant obtained is apathogenic for sheep, sucking piglets, white mice, calves, rabbits and dogs. ~t the same time it is highly immunogenic for the animals. The immunity created lasts one year. In lyophilized state the , virus can be stored for several years.
' The method provides the following advantages.
From the highly virulent virus of the Aujeszky's disease, an apathogenic and immunogenic viral mutant is obtained by the use of a very qu,ick and economic method, i.e.
by provoking changes in the structure of the viral DNA with ' the aid of antimetabolite S-iodine-2-desoxyuridin.
The mutant obtained is used for the production of - a vaccine.
Claims
The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:
A method for the preparation of avirulent viral mutant from the Aujeszky's virus wherein on the nucleous of the virus, containing desoxyribonucleinic acid is acted on with the antimetabolite 5-iodine-2-desoxyuridin and after the eighth passage an apathogenic and immunogenic mutant is obtained, which is used as a live vaccine.
A method for the preparation of avirulent viral mutant from the Aujeszky's virus wherein on the nucleous of the virus, containing desoxyribonucleinic acid is acted on with the antimetabolite 5-iodine-2-desoxyuridin and after the eighth passage an apathogenic and immunogenic mutant is obtained, which is used as a live vaccine.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BG3365276 | 1976-07-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1069821A true CA1069821A (en) | 1980-01-15 |
Family
ID=3902412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA281,836A Expired CA1069821A (en) | 1976-07-02 | 1977-06-30 | Method for the preparation of avirulent viral mutant from the aujeszky's virus |
Country Status (7)
| Country | Link |
|---|---|
| AR (1) | AR216081A1 (en) |
| BE (1) | BE856351A (en) |
| CA (1) | CA1069821A (en) |
| ES (1) | ES460321A1 (en) |
| FR (1) | FR2356726A1 (en) |
| GB (1) | GB1563284A (en) |
| NL (1) | NL7707341A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3122669A1 (en) * | 1980-06-12 | 1982-02-11 | Asta-Werke Ag, Chemische Fabrik, 4800 Bielefeld | "METHOD FOR PRODUCING NEW MUTANTS OF HERPES SIMPLEX VIRUS TYPE 1 AND TYPE 2" |
| HU187421B (en) * | 1982-10-27 | 1986-01-28 | Phylaxia Oltoanyagtermeloe Vallalat,Hu | Process for producing inactivated, adjuvated serum against aujeszky's illness |
| US4514497B1 (en) * | 1983-12-30 | 1998-02-24 | Novagene Inc | Modified live pseudorabies viruses |
-
1977
- 1977-06-29 AR AR268231A patent/AR216081A1/en active
- 1977-06-30 GB GB27522/77A patent/GB1563284A/en not_active Expired
- 1977-06-30 CA CA281,836A patent/CA1069821A/en not_active Expired
- 1977-07-01 BE BE2056044A patent/BE856351A/en not_active IP Right Cessation
- 1977-07-01 NL NL7707341A patent/NL7707341A/en not_active Application Discontinuation
- 1977-07-01 ES ES460321A patent/ES460321A1/en not_active Expired
- 1977-07-01 FR FR7720399A patent/FR2356726A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| FR2356726A1 (en) | 1978-01-27 |
| ES460321A1 (en) | 1978-11-01 |
| FR2356726B1 (en) | 1981-01-16 |
| NL7707341A (en) | 1978-01-04 |
| AR216081A1 (en) | 1979-11-30 |
| BE856351A (en) | 1977-10-31 |
| GB1563284A (en) | 1980-03-26 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |