CS211190B1 - Aujeszky distemper virus strain avirulent for pigs and method of its selection - Google Patents

Abstract

Obtaining the avirulent Aujeszky tribe virus harmless to everyone's piss age, with very good immunizations properties. Weaving method the strain consists of a serial passage selected Aujeszky's disease virus strain on smokers embryonic cells at 37 ° C using media defined composition and targeted choice multiplicity of infection, derivation of pure line by plaque technique, subsequent adapted and passaged. selected lines at low temperatures, their adaptation and propagation on primary fetal cultures bovine kidneys in the first subassembly after defined conditions of infection and multiplication and ensuring the homogeneity of the viral population selected line by plaque purification with multiple repetitions.

Description

The invention relates to the Aujeszky's disease strain (Au). b./virulent for pigs capable of industrial multi-placement on in vitro cultured cell cultures for the preparation of a live vaccine, p.p. ih $ izQlác-le. and .p, Q, unitÍA · pra. immunizáci.u Šípaných.

Ouch. ch. has become one of the most significant swine infections of a viral nature in the last decade. This is because so far we have not managed to deal with Au. ch, not one of the more infested states, and vice versa, appearing in states in which it was recently unknown, and in some sporadic states, its last epizootic spread has occurred / Herrick J. B: Highlights of the Pseudorabies Fact-Finding Conference, Vet.-Med. Smáli Anim. Clin. , 1 360-1 362, 1 977, Torna B., Goret P .: La Maladie d'Aujeszky en France, Can. Med. Vet., 43, 225-236 (1974). Failure of applied anti-infection measures and high economic damage which Au. ch. under the conditions of large-scale farming causes, they required the introduction of specific prophylaxis.

Until recently, unsuccessful results of all attempts to prepare an effective inactivated vaccine / Wittman G .: Die Schüchzimpfen gegen Aujeszkysche Krenkheit (AK) have inactivierten Vakzinen Dtsch. Tierärztl. Wschr. 6, 316-322, 1979] have shown that the main focus was on the height and use of live vaccines. Live vaccines prepared from animal attenuated strains / Toneva V .: Variability du virus de la maladie d 'Aujeszky. Vaccin contre la maladie d ^- Aujeszky. Vaccin contre la maladie d ^- Aujeszky obtenu de souche du virus Aujeszky attenué a l'aide de passages par der pigeons, Vet.-Med. Nauki, I. 35--42, 1964] were too virulent and therefore unsuitable for the preparation of live vaccines. Not even live vaccines prepared from strains attenuated by serial passages on smoking embryos / Bran L., Suhaci I., Ursache R .: Imminizing the value of the egg-adapted pseudorabies virus strain Bucarest after various number of chick embryo passages, Archiva Teter., 5 1968, 75-81, Bazylev P. Μ. , Prochorova E, M ,: Suchaja virusvakcina protiv bolezni Aujeszki iz stamma GNKI, Veterinarija, č. 2, 44-45, 1970, did not meet the scientifically submitted requirements for the preparation of live vaccines.

Modern live vaccines are intended to represent homologous populations of virus grown from genetically stable clones derived from virus strains by plaque technique. Bartha / Bartha A .: Experimental reduction of virulence of Aujeszkys disease virus, Magi. Állatorv. Lap.

16, 42-45, 1961 / cleaved from the virulent strain by serial passaging the cultures of porcine clouded maloplakový clone "C, which is used for the preparation of a live vaccine in mCr / Bartha A.ί Le vaccin contre la maladie hongroic d ^- Aujeszky, Cah. Med. Vet., 43, 338-340, 1974].

Žuffa isolated four strains from a Bucharest strain serially passaged on embryonic cell culture cultures showing different degrees of residual virulence for laboratory and livestock animals. The Buk-TK-300 / 9.2 line is used to prepare NORVAK 1 / Žuffa A .: Immunization against Aujeszky's disease - X-isolation of an avirulent clone for rabbits and guinea pigs and evaluation of its immunogenicity, Vet.-Med. / Prague / 16, 525-538. 1971, Žuffa A .: Salaj J .: Immunization against Aujeszky's disease. Immunogenicity of the avirulent clone Buk-TK-900 / 6.K for the preparation of the live vaccine NORVAK 2 / Žuffa A .: Salaj I .: Immunization against Aujeszky's disease immunogenicity of the highly attenuated strain Buk-TK-900 / IV, and for pigs, Vet.-Med . (Prague, 16, 221-223, 1971), which are used to immunize fur animals, sheep and cattle.

For the preparation of live vaccines SUIVAK 1 and SUIVAK 2 intended for the vaccination of pigs serve the lines Buk-TK-900 / IV, and / Žuffa A., Salaj J .: Immunization of Aujeszky's disease sweats - immunogenicity of the highly attenuated Buk-TK-900 / IV strain, and over pigs. Vet.-Med. / Prague / 1 6, 221-233, 1971 / respectively. Buk-TK-200 / 3.PPM / Žuffa A .: Immunisierung gegen die Aujeszkysche Krankheit, I. Gewinnung des Modifizierten Virus der Aujeszkyschen Krankheit, Arch. exp. J. Med., 17, 1325-1344, 1963]. From the Bucharest strain, Skoda et al. line / Skoda R., Brauner I., Sadecky E.,

Maxer V .: Iramunization against Aujeszky's disease with live vaccine I. Attenuation of virus and some properties of attenuated strains. Acta virol., Et al., 1-9, 1964 / at 336, and 621.

passages on smoked fibroblasts and used to prepare experimental live vaccines: for an undesirable degree of residual virulence, which they have retained even after over 1,000 tissue pasa211190 women / Jamrichová 0., Skoda R .: Multiplicatio and dstribution of attenuated pseudorabies virus in the organism of vaccinater pigs. | Acta virol., 13, 42-51, 19697, however, were not used for commercial production.

The bovine avirulent virus line was isolated by Svobodova et al. (Svobodová J., Sabo A., BlaŠkovič D .: Immunogenicity of the attenuated pseudorabies virus in cattle, Acta virol., 1, 4, 263, 1970) from the attenuated or Buk-621 strain via long-term persistent infection of cultures from calf kidneys whose immunogenicity was checked only on rabbits and cattle. Analogous to Žuffa in the selection of the Buk-TK-300 / 9.2 line, Torna / Torna B, a study of a temperature sensitive strain of Aujeszky virus, Proc. 5th vorls 9.P.V.S. Congresa, Zagreb, 1978 /, isolated the thermosensitive strain Alfort from virus long-term passaged on embryonic cell culture and used it to prepare a live vaccine. Finally Tatarov / Tatarov G .: Apathogener Mutant des Aujeszky-Virus, induced by 5-Iodo-2-Deoxyuridine / JUDR7, Zbl. Vet.-Met., B, 15, 847-853, 1968] isolated the avirulent mutant MK from virus propagated on cultured from smoking germs by selecting plaques resistant to 5-iodo-deoxyuridine,

In the study of the innocuousness and immunization efficacy of individual avirulent and attenuated Au virus strains. ch. used for the preparation of live vaccines were found to be significant differences between them. The strain Buk-TK-900 / IV, and is avirulent for all ages of pigs at any route of administration, but at the same time unable to reproduce in the organism of vaccinated animals and therefore elicits the desired immune response in pigs only upon administration of a large dose of virus; the presence of colostral flood antibodies interferes with the development of active immunity when vaccinating mammalian pigs and weaning. Vaccination of sows stimulates antibody production only to low to medium titers, and therefore the antibody content in the colony does not provide reliable protection to the pigs / Zuffa A., Grunert Z .: Les · vaccines utilisés en Técoslovaquie contre la maladie d'Aujeszky, Csh «Méd. Vet., 43, 334-337, 1974]. The interfering effect of coletral antibodies with the development of active immunity in vaccinating pigs with this virus requires a double-phase vaccination in order to achieve reliable protection, the latter only taking place after the maternal antibodies have been excluded.

Strain K ”is virulent for porcine fetuses throughout intrauterine development / Sokol A., Gallo T .: Distribution of Bartha ^- with Vaccine of Aujeszky's Disease in Pig Foetuses after Intraperitoneal Application, Folia microbiol., 16, 544, 1971 / a therefore, its use for immunizing pregnant sows is associated with some risk. In addition, the irrational response ⁽ after a single vaccination course is inadequate, and therefore a two-phase vaccination of piglets is required at intervals of 3 to 4 weeks and sows always in the last trimester of pregnancy / Bartha A .:

Le vaccin hongrois contre la maladie d ^- Aujeszky, Cah. Med. Vet., 43, 338-340 (1974).

The same procedure is followed for the irrigation of pigs with live vaccines prepared from MK / Tatarov strains G., Boyadjiew St., Wesselinova A., Gergow P .: Untersuchung der Pathogenese der Aujeszkyschen Krankheit, Tagung zur ”Probleme und Prophylaxe der Industriemassigen Schweinepze , Bulgarion, 14, 1975) and the thermoresistant strain Alfort / Torna B., Brun A., Chappuis G., Terre J .: Prophesives of Biological and Thermosensible Sets (Alfort · 26) by the maladia virus of Aujeszky, Res. Med. Vet., 1555, 245-252, 1979]. Strains of Buk-TK-200/3. PPM / Zuffa A .: Immunisierung gegen die Aujeszkyschen Krankheit. I. Gewinnung des Modifizierten Virus der Aujeszkyschen Krankheit, Arch. exp. Vet.-Med., 17, 1 325-1 344 (1963) and Buk-621 / Skoda R., Brauner I., Sadecky E., Mayer V .: Immunization against Aujeszky's disease with live vaccine I. Attenuation of virus and Acta virol., 8, 1-9, 1964] are immunogenic to pigs even at low doses, but after injection, wounds are not only injected into the tissues of the injection site and adjacent lymph nodes, but occasionally also penetrate the blood, central nerves, tonsils and mucous membranes of the upper respiratory tract, which is associated with the risk of secretion of the virus by secretions into the environment and transmission to co-secreted susceptible pigs. Immunisierung gegen die Aujeszkysche Krankheit. I. Gewinnung des Modifizierten Virus der Aujeszkyschen Krankheit, Arch. exp. Vet.-Med., 17, 325-134, 1963]. Because of the lack of attenuation of virulence, these strains are from an epidemiological point of view for the preparation of live Au vaccines. ch. inappropriate.

Although effective inactivated vaccines have been developed in recent years

Ouch. ch. / Wíttmann G .: Die Schutzimpfung gegen Aujeszkysche Krankheit / AK / mit inaktivierten

Vakzinen, Dtsch. Tierarztl. Wschr., 86, 316-322, 1979] immunoproxylaxis of this disease in areas with enzootic or epizootic occurrence by the method of choice using live vaccines remains / Žuffa A., Salaj J .: Resistance of pigs Immunized with live or inactivated Aujeszkys disease vaccines against experimental infection , IPVS Congress, Copenhagen, Denmark, 1980.

Although the inactivated vaccine attributed eyewitness epizooto1ogické advantages are even more effective when zničovaní economic losses, even in eliminating the virulent virus from infected flocks / Skoda R .: The control of Aujeszky ^- a disease in swine Karge units by use of live attenuated vaccines. IPVS Congress, Ames Iowa, USA, 1976, G6 /.

Taking into account the technical demands, the cost of vaccination but also the development of local and overall responses associated with the use of inactivated vaccines / Wittnamnn G .:

Die Schutzimpfung gegen Aujeszkysche Krankheit /&.YJ mit Inaktivierten Vakzinen, Dtsch. Tierärztl. Wschr., 8, 6, 31, 6322, 1979, in endemic infestation of Aujeszky virus swine breeds, it is preferable to perform immunoprophylaxis with harmless, effective live vaccines.

These shortcomings are associated with the quality of live Au vaccines. ch. are solved according to the invention, which is based on the breeding of an avirulent immunogenic virus clone via long-term passage of the Bucharest strain on primary cultures from smoking germs at a temperature of 36 ° C to 38 ° C - 150 to 250 serial passages followed by line selection, by dilution method or plaque technique with a six-fold repeat which is further passaged on the same tissue type, gradually decreasing the culture temperature by 1 ° C to 30 ° C to 33 ° C and passage at that temperature 20 to 10 times, adapting the obtained virus to growth to fetal bovine kidney cultures; plaque selection of viral clone on six-fold embryonic cells in embryonic cells.

The susceptible vaccine strain exhibits advantageous growth properties on beech cultures prepared in a known manner from smoking germs or duck germs, containing 10? up to 10% virus / ml in infectious fluids from said tissues. It is harmless to pigs of all ages at parenteral, i. us. or by inhalation, does not harm developing fetuses in vaccinating sows at any stage of pregnancy, induces an immune response in mammalian pigs even in the presence of low and medium titers of maternal antibodies, induces a high degree of disease protection in natural and experimental infections after single administration. virus, and vaccinating sows in infested holdings will reliably protect mammalian pigs against clinical disease with antibodies received by colostrum; following parenteral administration, the virus is not eliminated from vaccinated pigs and does not spread by contact with co-proprietary susceptible pigs.

The immunobiological properties of the bred strain allow the preparation of a live vaccine thereof, by application of which, in endemically infested breeds, will protect the entire pig population against a clinical disease using the simplest immunization schedule so far in the immunoprophylaxis of Au. ch. used.

The strain Buk-TK / 650 deposited in the collection of microorganisms at the Research Institute of Veterinary Medicine in Brno Medlánky, Hudcova 60 under number CAMP V-265 was isolated by the method according to the invention from the virulent strain Bucharest from the collection of the Pasteur Institute in Bucharest. Bran L., Suthaci I., Ursache R.: Immunizing the value of the egg-adapted pseudorabies virus strain Bucarest after varicus number of chick embryo passages, Archiva Teter., 5, 75-81 (1968) 200 times on primary cultures of smoking germs. The virus in the 200th tissue passage was a gradual destruction of the culture. temperature and thereby selection of virus particles with good multiplication properties at low temperatures, adapted to grow at a temperature of 30 to 33 ° C, preferably at 32 ° C. At this stage of the selection process, a line that was tested for is isolated at the 30th serial passage level at 32 ° C by a six-fold repeat method on tube cultures from specific embryonic germ-free embryo (SPF) tube cultures. four week old pigs free of antibodies against the Aujeszky virus with regard to residual degree of virulence and immunogenic capabilities.

The residual virulence of this line was totally attenuated by passage on fetal kidney cell cultures. The cultures of fetal bovine kidneys in the first sub-passage were infected 24 hours later. from loading, i. j. before forming a continuous monolayer after the growth medium was mixed with a dose of 0.2 ml of undiluted virus, the virus allowed to adsorb 1 point. at 37 ° C and cultures were quenched with 1 ml of Earl's medium with 2% fetal bovine serum. Infected cultures were incubated at 37 ° C for 3-4 days during a period of almost complete virus-specific tissue breakdown. The same procedure was followed for the other passages of the virus to. fetal bovine kidney cultures incubated for 3 to 5 days, with the development of cytopathic changes gradually decreasing only in the 5th direct passage on said tissue type, the presence of viral infection showed only minor tissue damage characterized by ring-cell degeneration of isolated cells. At this stage, the virus has multiplied on cultures of smoking embryonic cells on which it induced complete lysis of tissue with a high content of infectious virus in tissue fluids.

The virus thus propagated was used for passage on bovine fetal kidney cultures in the same manner. The virus from the 5th straight passage was abbreviated on cultures of smoking germs, and further passed exclusively on fetal bovine kidney cultures. Influence of virus growth on this tissue type with the development of significant cytopathic changes was the infection of cultures in the sub-passage after 24 hours. growth before forming a continuous monolayer. By this procedure, the breeding strain crossed 10 straight passages on fetal bovine kidney cultures. From this virus, clones representing homologous viral populations were selected by plaque technique with six-fold repeats in smokers of embrionic cells. The homogeneity of the viral population was confirmed by maintaining unchanged virulence for susceptible mammalian pigs after 10-fold passage in rabbits. The virus clone thus obtained shows good growth properties on cultures prepared from smokers and duck germs.

This procedure yields a strain designated CAMP V-265 Buk-TK / 650 which has the characteristics of a virus suitable for the preparation of a live Au vaccine. ch., as it is characterized by a loss of virulence for pigs of all ages at parenteral, i. us. and by inhalation, it is not detected in blood, central nervous system tissues, almonds or mucous membranes of the upper respiratory tract after parenteral administration, is not excreted by secretions or excretions into the environment and is not transmitted by contact to co-diagnosed susceptible pigs. If all the requirements for harmlessness are met, the bred strain is suitable for the production of a live Au vaccine. ch. especially for pigs in that it induces an irrational response even with a small dose of virus (10 ΤΚΙϋ ^ θ and more), induces protection against clinical disease in both natural and experimental infections even after single administration, protection induced by single vaccination lasts at least one year, biphasic Vaccination with a 10 ^ TKXD ^ qa virus will induce medium and high titer antibody development with a developer of both clinical and partial biological immunity; once a year, vaccinating sows gives the mammalian pigs colostrum a degree of passive immunity that protects them against disease for up to 10 weeks. age, will elicit a positive immune response in pigs with low to moderate maternal antibody titers.

The use of a live vaccine prepared from a bred strain of these immunobiological properties provides protection for the entire pig population against Au. ch. applying the simplest immunization schedule that has been used in immunoprophylaxis to date; the immunization procedure consists in weaning pigs for single-time vaccinations of piglets for fattening, for breeding pigs for single-sowing of sows and boars aged 10 to 12 weeks and re-vaccinating before mating and one-year vaccination of production pigs and sows without fattening . When compared to a live vaccine from a susceptible Buk-TK / 650 strain with other live vaccines of both domestic and foreign origin, it was shown to be the best in terms of harmlessness, immunogenicity, protection of vaccinated pigs and simplicity of the immunization schedule.

The novel strain of the present invention is distinguishable from the virulent Rucarest strain according to the growth characteristics and nature of cytopathic changes found on beech cultures, animals, and animal immunological properties.

Depending on the growth characteristics of beech cultures, the newly selected strain is distinguished from the virulent starting strain by faster propagation and higher virus yield when grown on cultures from chicken embryos, round-cell degeneration as well as lower infectious titers and lower fluorescence PK fluorescence cell culture localization of fluorescent antigens almost exclusively in the cytoplasm of cells of the stable TL-72 line. X-intranasal application of the initial virulent strain to susceptible pigs induces intensive multiplication of the virus on the mucosa of the upper respiratory tract containing up to 10 ^ TKID / ml, the virus persists on the mucosa of the oropharynx until 14 to 21 days, regularly penetrates into the central nerve and induces clinical disease characterized respiratory inflammation and CNS disorders, with about 50 deaths from infected pigs t.

Selected selected strain is on the mucous membranes of the upper respiratory tract after i. us. application does not reproduce and proves only to 24 hours. and the health of the animals remains completely intact. Animals infected with the virulent parent strain will produce neutralizing antibodies in blood serum at titers of 1:64 to 1: 1024, whereas animals treated with the bred strain at titers of 1: 1 to 1:16 only. Application of both the parent and the bred strain to rabbits causes death, but in the first case, the predominant symptom of the disease is intense pruritus, whereas the death of rabbits treated with the bred strain is free of itching.

Execution example

Virulent strain of Au virus. ch. Bucharest is serum passaged on primary cultures of chicken embryos using routine laboratory methods. The presence of virus in tissue fluids is controlled at different levels of passage by the development of viral-specific cytopathic changes. The process of attenuating the strain of virulence of the strain is controlled by applying 0.5 ml of log 10 virus dilutions i. cer, and 2-4 ml of undiluted virus i. us. Creation of weekly pigs, detection of virus content in nasal secretions, development of local changes, thermal and general clinical reactions. Upon reaching a degree of attenuation of the virulence of the passaged strain at which it does not induce i. cer. application of 10 ^. ^ θ virus and in. us. administration of 10 ΤΚΙϋ virus of clinically observable disruption to the health of treated pigs, with a raaxal increase in body temperature of 1 ° C within 14 days of administration, the virus is purified by borderline dilution or plaque techniques and adapted to growth at subnormal temperatures.

In this process, the infected cultures are incubated at 35 ° C at the earliest and the virus passage on the tube cultures is continued until the onset and development of cytopathic changes until complete tissue disintegration occurs no later than 72-96 hours. incubation. Thereafter, the temperature at which the infected tube cultures are incubated by 1 ° C according to the same criteria as above was lowered. Subsequently, direct passages are performed at 32 ° C and residual virulence of the virus is tested in 2-4 week old pigs. They are administered 1 ml of undiluted infectious tissue fluid to the pre-knee algae and checked for the presence of virus on the mucous membranes of the nose and trachea, tonsils and CNS by always examining at least two pigs on days 3, 5 and 7 after vaccination. Virus passage at 32 ° C is terminated when isolation experiments on cell cultures or rabbits prove that said tissues are free of infectious virus.

This virus is serum-passaged on fetal bovine kidney tube cultures, with cultures showing only the presence of infection by morphological changes only in single cells, multiplication of the virus on smokers of embryonic cultures, and the passage of the virus on bovine kidney cells with the same. After adapting the virus to this type of tissue, 10 to 20 straight serial passages are made. The condition for success is that fetal bovine kidney cultures in the subpassage after one hour are used to passage the virus. growth before forming a continuous monolayer. From the 20th passage virus on bovine kidney cells, a line is repeated on plasmid embryonic cells using a six-fold plaque technique, which is subjected to identity, attenuation and immunogenicity screening assays.

The virus identity is confirmed by: a. by neutralizing infectivity by a specific antiserum prepared in rabbits immunized with one of the inactivated Aujeszky virus collection strains containing thyroid adjuvants; b. by iraunofluorescence by staining cultures infected with the strain with a specific conjugated AV-immunoglobulin, c. by demonstrating the resistance of the vaccinated pigs to i. us. infection with a known virulent strain.

The degree of attenuation of virulence is checked in 2-4 week susceptible pigs. The virus is checked for pigs vaccinated with a dose of about 10% of the virus on the mucous membranes of the nose, tonsils, tracheal mucosa and in the CNS by inoculating organ suspensions for cell cultures and rabbits.

The virus immunogenicity is checked by inoculating susceptible logs. _n dilutions in a dose of 2 6 to 2 ml containing 10 to 10 TKID virus in inoculate animals are screened for antibody content and infect i. us. virulent virus. The infection must be survived by pigs in which the inoculum contained 10 ΤΚΙΏ of virus and above, while at least 75% not vaccinated in the same way with infected control pigs must have died. Two-phased administration of the virus at a dose of 10 ⁵ µm or more must induce antibody production in titers of 1: 16 to 1: 128, with an average titer of at least 1:40.

The newly bred strain according to the invention Buk-TK / 650 with good growth properties on duck embryo smoker's cultures is harmless to pigs of all ages at any application, induces protection against disease after a single administration at a small dose lasting at least one year and generates neutralizing antibodies in medium up to high titers after biphasic administration at a dose of 10 µm or more. Said characteristics of the strain of the invention allow the production of a safe and effective live Au vaccine. ch. using which achieves reliable clinical protection of the entire pig population against disease by applying the simplest immunization schedule as in immunoprophylaxis of Au. ch. so far in pigs.

Claims (2)

211190 6 24 h growth before formation of a continuous monolayer. Line 20 was isolated from the 20 passage virus on bovine kidney cells by repeating the plaque technique on a smoking membrane cell and subjected to identity, attenuation and immunogenicity control assays. The identity of the virus is confirmed by: a. Neutralizing the infectivity of a specific antiserum prepared on rabbits immunized with any of the Aujeszky virus collection strains in an inactivated donor adjuvanted state, b. vaccinated pigs against i. infection with a known virulent strain. The degree of virulence attenuation is checked on 2-4 week old susceptible pigs. The presence of virus in pigs vaccinated at about 10 µl of nose nasal, almond, tracheal mucosa and CNS vaccination with organ suspensions is checked! cell cultures and rabbits. Virus virus immunogenicity is checked by seeding susceptible wicks with log.n dilutions at dose 2 of 6 to 2 ml containing 10 to 10 TKID virus in inoculated animals are examined for antibody content infecting i. virulent virus. The infection must survive the pigs, which contained 3 inoculum of 10 ΤΚΙΏ θ virus and more, while at least 75% of the unvaccinated in the same way infected control pigs must die. The bipedal injection of the virus at a dose of 105 ΤΚΙϋ ^ θ and, in particular, must produce antibodies in titers of 1: 16 to 1: 128, with an average of at least 1: 40. The New-Breed Strain of Buk-TK / 650 with Good Growth Properties of Duck Embryo Smokers is harmless to pigs of any age in voluntary administration, induces protection against a single-dose disease at a small dose of at least one year, and the generation of neutralizing antibodies in medium to high titers after two-fold administration at a dose of 10 µC or more. Said properties of the strain of the invention allow the production of a safe and effective Au vaccine. ch. by which reliable clinical protection of the entire population of pigs against the disease can be achieved by applying the simplest immunization scheme for the immunoprophylaxis of Au. ch. she has used so far in pigs. SUBJECT OF THE INVENTION 1. Aujeszky's disease virus breeding method characterized in that the Aujeszky's disease virus strain Bucharest is first passaged 150 to 250 times in primary cultures of smoking germs at 36 ° C to 38 ° C, selected from the virus by the borderline dilution method or by a six-fold plaque technique which is further passaged through the same tissue type by gradually lowering the culture temperature by 1 ° C to 30 ° C to 33 ° C and passages 20 to 40 times at this temperature, this virus adapts to fetal bovine growth of renal culture, and from which it selects embryonic cells with a six-fold repetition of a hoihologically genetically stable line. 2. Aujeszky virus virus strain avirulent for pigs Buk-TK / 650 CAMP V-265 with enhanced reproductive capacity of its own viral substance in cell cultures, obtained as described under 1. Severography, n.