RU2316346C2 - Inactivated emulsion vaccine against swine's reproductive-respiratory syndrome - Google Patents

Abstract

FIELD: veterinary virology, biotechnology.

SUBSTANCE: the suggested vaccine contains an active substance and a target additive. As the active substance it contains the suspension of avirulent and purified antigenic material out of "KPR-96" strain of the virus of swine's reproductive-respiratory syndrome (SRRS), Arteriviridae type, Arterivirus genus, FGU collection "VGNKI" "KPR-96"-DEP in efficient quantity. The vaccine in question induces high level of antibodies against SRRS, provides reliable passive maternal immunity in youngsters up to the age of 25-30 d that enables to protect the youngsters against field virulent SRRS virus in earlier period of life.

EFFECT: higher efficiency.

7 cl, 3 dwg, 7 ex, 16 tbl

Description

The invention relates to veterinary virology and biotechnology and can be used in the development and manufacture of means for the specific prevention of pig reproductive and respiratory syndrome (PRRS).

PRRS is a contagious viral disease characterized by abortion at the end of gestation, the birth of a dead and weak offspring that dies in the first 2-3 weeks of life, and respiratory damage in piglets. The death of piglets can reach 80-90%.

The disease was first recorded and described in 1987 in the United States and Canada. The first outbreak with similar symptoms was noted in Germany in 1990, whence the infection spread to most European countries.

In Russia, the disease was first noted in 1991 in the farms of the Kursk region. The disease spread rapidly and today PRRS is diagnosed in many regions and regions of Russia.

Currently, PRRS is widespread in many countries of the world with developed pig husbandry and often proceeds in enzootic (chronic) form (1-8).

The causative agent of the disease is an RNA-containing virus belonging to the genus Arterivirus, a family of Arteriviridae of the order Nidovirales.

For the first time, PRRS virus was isolated by Dutch scientists at the Central Veterinary Institute in 1991 in a culture of pig alveolar macrophage cells (9).

PRRSV strains isolated in various countries of Europe, North America and Asia differ in virulence, antigenic structure, and nucleotide sequence in genomic RNA.

At least two PRRS virus genotypes are known - European and American. The homology between them at the nucleotide level is only 55-70%, and within the genotypes 97-99%.

The differences relate not only to variations in the nucleotide and amino acid structure, but also to the size of the viral proteins. So the size of the nucleocapsid protein of American strains of the PRRS virus is 123 amino acids, and the size of the nucleocapsid protein of European strains is 128 amino acids (10-12).

When conducting molecular characterization of domestic PRRS virus isolates, it was found that they belong to the European group, however, at the amino acid sequence level, differences of up to 16% are found, and the size of the nucleocapsid protein differs from that of both American (123 amino acids) and European strains (128 amino acids) and is 125 amino acids (13).

These structural features of domestic PRRS virus isolates concerning nucleocapsid as one of the most conserved viral proteins indicate their antigenic differences compared with the corresponding characteristics of West European and American virus isolates.

When comparing isolates of the PRRSV virus isolated on the territory of Russia, their extremely genetic and antigenic variability is found.

This circumstance forces us to constantly search for new PRRS virus isolates suitable for the manufacture of diagnostic and vaccine preparations.

Specific PRRS prophylaxis is carried out using live and inactivated vaccines.

Although the advantage of live vaccines due to manufacturability and ease of use is not in doubt, the immune response in pigs is not always stable when administered and in some cases causes complications after vaccination.

At present, emulsion inactivated vaccines prepared from various strains of the virus grown in sensitive biological systems are widely used in the global industrial pig breeding for specific prophylaxis of PRRSs (9, 14).

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from strain CNCM No. I-1102 (Lelystad) of PRRS virus obtained in sensitive hetero- or homologous cell cultures, and the target additive in the form of an oil adjuvant in an effective relation (15).

A known vaccine against PRRS inactivated emulsion containing an active substance in the form of a suspension of avirulent and purified antigenic material from strain CNCM No. I-1140 of PRRS virus obtained in sensitive hetero- or homologous cell cultures and a target additive in the form of an oil adjuvant in an effective ratio (16 )

There is a known vaccine against PRRS inactivated emulsion containing an active substance in the form of a suspension of avirulent and purified antigenic material from strain CNCM No. I-1153 of PRRS virus obtained in sensitive hetero- or homologous cell cultures, and a target additive in the form of an oil adjuvant in an effective ratio (17 )

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from ECACC strain No. V-93070108 of PRRS virus obtained in sensitive hetero- or homologous cell cultures, and the target additive in the form of an oil adjuvant in an effective ratio (18 )

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from strain CNCM No. I-1387 or strain CNCM No. I-1388 of PRRS virus obtained in sensitive hetero- or homologous cell cultures, and the target additive in the form oil adjuvant in an effective ratio (19).

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from the ATCC strain No. VR-2402 of PRRS virus obtained in sensitive hetero- or homologous cell cultures, and the target additive in the form of an oil adjuvant in an effective ratio (20 )

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from the ATCC strain No. VR-2509 of PRRS virus obtained in sensitive hetero- or homologous cell cultures and the target additive in the form of an oil adjuvant in an effective ratio (21 )

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from the ATCC strain No. VR-2525 of PRRS virus obtained in sensitive hetero- or homologous cell cultures and the target additive in the form of an oil adjuvant in an effective ratio (22 )

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from strain JK-100 (CCTSS V 20005) of the PRRS virus obtained in sensitive hetero- or homologous cell cultures, and the target additive in the form of an oil adjuvant in an effective relation (23).

A known vaccine against PRRS inactivated emulsion containing the active substance in the form of a suspension of avirulent and purified antigenic material from the strain "DB" of the PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and the target additive in the form of an oil adjuvant in the ratio, wt.%:

Antigenic material 33.3-50.0 Oil adjuvant up to 100.0 (24)

A common drawback of the known PRRS vaccines is their low antiepizootic efficacy for the Russian Federation, due to the fact that the PRRS strains used for their manufacture have antigenic and immunobiological differences compared with similar characteristics of epizootic isolates of the PRRS virus isolated in Russia at different time intervals .

The closest to the invention according to the set of essential features is an inactivated emulsion PRRS vaccine containing the active substance in the form of a suspension of avirulent and purified antigenic material from strain CNCM No. I-1102 (Lelystad) of the PRRS virus obtained in a sensitive biological system and the target additive in the form oil adjuvant in an effective ratio (15).

The disadvantage of the prototype vaccine also lies in its low antiepizootic efficacy for the Russian Federation, due to the fact that the strain CNCM No. I-1102 (Lelystad) used for its production has antigenic and immunobiological differences compared with similar characteristics of the PRRS virus epizootic isolates isolated on the territory Russia at various time intervals.

In addition, the strain CNCM No. I-1102 (Lelystad) is virulent, which always creates problems when meeting the requirements of veterinary and sanitary safety.

In this regard, the problem of creating new vaccines against PRRS emulsion inactivated on the basis of new strains of PRRS virus with high antigenic and immunogenic activity remains relevant.

The task of creating the present invention was the development of a vaccine against PRRS inactivated emulsion, which creates a tense and prolonged immunity in vaccinated animals against the epizootic PRRS virus circulating in Russia.

The technical result from the use of the present invention is to expand the arsenal of vaccines against PRRS inactivated emulsion, which have high antigenic and immunogenic activity and harmlessness and create intense and long-lasting immunity in vaccinated animals against the epidemic virus PRRS circulating in Russia.

The specified technical result is achieved by creating a vaccine against PRRS inactivated emulsion, characterized by the following set of features.

The proposed invention contains the active substance in the form of a suspension of avirulent and purified antigenic material from strain "CRC-96" (copyright) of PRRS virus obtained in a mammalian cell culture, and a target additive in the form of an oil adjuvant in an effective ratio.

The original virus for obtaining the KPR-96 strain was isolated in 1996 from the blood serum of aborted sows with clinical signs of PRRS from the Victory Agricultural Union of the Leninsk-Kuznetsk District of the Kemerovo Region and adapted by serial passages to the kidney cell culture of the African green monkey Marc-145. The infectious activity of strain KPR-96 of the PRRS 6 virus of passage 6 averaged 5.17 lg TCD ₅₀ / ml, and passage 17 - 5.09 lg TCD ₅₀ / ml. In its native form, the strain "CRC-96" is weakly virulent for pigs.

The strain "KPR-96" was deposited in the All-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, Federal State Institution "All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" (FGU "VGNKI") October 28, 2004 under registration number (link) "KPR-96" -DEP.

To obtain antigenic material from strain "CRC-96" of PRRS virus, a Marc-145 cell culture is used as a sensitive biological system.

The resulting virus-containing material is purified from ballast impurities by centrifugation or any other known method.

To inactivate the virus, aminoethylethyleneimine (AEEI) is used, which is added to the purified suspension to a concentration of 0.05-0.08 wt.%. Inactivation of the virus is carried out at 37 ° C for 20-22 hours with periodic stirring. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to the suspension.

In accordance with the invention, the vaccine contains a suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml, in an amount of 33.0 wt. %

As a target additive, the vaccine contains an oil adjuvant in an amount of 67.0 wt.%.

Of the oil adjuvants, the vaccine contains an oil adjuvant of the brand Montanide ISA-70 manufactured by Seppic (France).

The present invention includes the following set of essential features that provide a technical result in all cases for which legal protection is sought:

1. Inactivated emulsion vaccine against PRRS.

2. The active substance in the form of a suspension of avirulent and purified antigenic material from strain "CRC-96" of the PRRS virus in an effective amount.

3. Targeted supplement.

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. Inactivated emulsion vaccine against PRRS.

2. The active substance.

3. Targeted supplement.

Compared with the prototype vaccine, an essential distinguishing feature of the present invention is that as an active substance it contains a suspension of avirulent and purified antigenic material from strain KPR-96 of the PRRS virus in an effective amount.

The present invention is also characterized by other distinctive features expressing specific forms of execution or special conditions for its use:

1. Suspension of avirulent and purified antigenic material from strain "CRC-96" PRRS virus obtained in a sensitive biological system.

2. Suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml.

3. Suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml, in an amount of 33.0 wt.%.

4. Targeted supplement in the form of an oil adjuvant.

5. Of the oil adjuvants, the Montanide ISA-70 brand oil adjuvant.

6. Oil adjuvant of the brand Montanide ISA-70 in an amount of 67.0 wt.%.

7. Suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml, and an oil adjuvant in the ratio, wt.%:

Antigenic material 33.0 Oil adjuvant to 100.0

The present invention extends the arsenal of vaccines against PRRS emulsion inactivated, with high antigenic and immunogenic activity and harmlessness and creating a tense and prolonged immunity in vaccinated animals against the epizootic PRRS virus circulating in Russia.

The achievement of the technical result from the use of the invention is explained by the fact that it contains a suspension of avirulent and purified antigenic material from strain "CRC-96" of the PRRS virus, which retains its native immunobiological properties after inactivation and has high antigenic and immunogenic activity and harmlessness.

The possibility of its use for the manufacture of a vaccine against PRRS inactivated emulsion inactivated was experimentally confirmed.

The strain "CRC-96" of the PRRS virus provides an effective inactivated vaccine against PRRS, which protects susceptible animals from the causative agent of the disease circulating in Russia.

The invention is illustrated in the graphic image, which presents the results of comparing the amino acid sequences of the GP5 protein of strain "PRC-96" and strain CNCM No. I-1102 (Lelystad) of the PRRS virus, as well as in the sequence listing, in which:

SEQ ID NO: 1 represents the nucleotide sequence of the OPC5 gene of strain "CRC-96" PRRS virus;

SEQ ID NO: 2 — GP5 Protein Amino Acid Sequence

strain "CRC-96" virus PRRS.

The strain "CRC-96" PRRS virus is characterized by the following features and properties.

Morphological properties

The strain KPR-96 of the PRRS virus belongs to the family Arferiviridae, the genus Arterivirus, is an RNA genomic, has morphological characteristics characteristic of the PRRS virus: the shape of the virion is spherical. The size of the virion is 45–75 nm, with a nucleus occupying 3/4 of the virion with a diameter of 25–35 nm, surrounded by a lipid membrane.

Antigenic properties

The PRRS virus of the KPR-96 strain is stably neutralized by homologous antiserum.

The virus does not have hemagglutinating properties. When vaccinated, the antigen from the strain "CRC-96" induces the formation of specific antibodies detected in the enzyme-linked immunosorbent assay (ELISA).

The antigenic activity and specificity of the PRRSV strain KPR-96 strain was determined by detecting specific antibodies in the serum of pigs obtained 28 days after their immunization with an inactivated emulsion vaccine made from the PRRSV strain KPR-96 strain.

Prior to immunization in animals' blood sera, antibodies to PRRS virus were not detected, and 28 days after vaccination in all samples specific antibodies ranging from 1.79 to 2.05 were detected in ELISA.

Biotechnological characteristics

The strain "KPR-96" exhibits high biological activity.

The biological activity of the strain was determined by titration on a Marc-145 cell culture.

Virus 6 and 17 passages obtained in a Marc-145 cell culture were used for typing after double freezing-thawing and low speed centrifugation to precipitate cell detritus. The results of typing on a Marc-145 cell culture showed that the biological activity of the CRC-96 strain of the PRRS virus of passage 6 averaged 5.17 lg TCD ₅₀ / ml, and passage 17 - 5.09 lg TCD ₅₀ / ml.

The difference in the titers of the virus 6 and 17 passages is not significant, i.e. as a result of 11 passages, the biological activity of the strain KPR-96 of the PRRS virus remained unchanged.

Chemo- and genotaxonomic characterization

The genome of the PRRS virus strain "CRC-96" consists of a single molecule of linear positive single-stranded RNA about 15,000 nucleotides in size. The 5'-end of RNA is capped, and the 3'-end is polyadenylated. The capsid protein gene is located on the 3'-terminal part of the genome. The genome of the strain KPR-96, like other strains and isolates of the PRRS virus, contains eight open reading frames (ORFs) that encode replicase genes (ORS 1a and 1b), envelope proteins (ORS 2-6), and nucleocapsid protein (ORS) 7). Six structural proteins of PRRSV virus and their corresponding genes were identified: a non-glycosylated nucleocapsid protein N (molecular weight 15 kDa) encoded by OPC 7, a non-glycosylated transmembrane protein M (molecular mass 18 kDa) encoded by OPC 6, and four N-glycosylated proteins with a pier. weight 25; 31-35; 45-50 and 29-30 kDa, encoded by OPC 5, OPC 4, OPC 3 and OPC 2, respectively. Spherical virions have a diameter of 45-75 nm, include a core with a diameter of 25-35 nm. The virion membrane contains lipids and carbohydrates as part of glycoproteins.

The identification and specificity of the PRRS virus was determined by polymerase chain reaction (PCR) and nucleotide sequencing.

As a result of the studies, only the PRRS virus genome was detected. The nucleotide sequencing of the OPC 5 gene confirmed that the strain "CRC-96" belongs to the European PRRS virus genotype. Comparison of the GP5 protein amino acid sequences derived from the OPC nucleotide sequences of the 5 amino acid sequences showed that the KPR-96 strain differs from the CNCM strain No. I-1102 (Lelystad) in two amino acid residues (amino acid residues). Twentieth A.O. KPR-96 is represented by phenylalanine, and CNCM strain No. I-1102 (Lelystad) is represented by leucine; Asparagine and aspartic acid are in the 37th position of the GP5 protein from KPR-96 and CNCM No. I-1102 (Lelystad), respectively.

Physical properties

The virion mass is from 49 × 10 ³ Da to 55 × 10 Da. The floating density in the gradient of cesium chloride is 1.18-1.2 g / ml.

Resistance to external factors

The strain "KPR-96" is unstable to ether, chloroform and detergents, sensitive to formaldehyde, ultraviolet radiation, gamma radiation and drying.

Additional features and properties

Immunogenic activity - 14 days after the immunization of pigs it causes them to form specific antibodies.

Reactogenicity is absent.

It is slightly virulent for pigs.

Oncogenicity is absent.

It is contagious in contact (the joint keeping of infected and healthy pigs).

Based on the data obtained, it can be argued that the strain “CRC-96" of the PRRS virus according to antigenic and immunological spectra is an original, taxonomically new, previously unknown strain of the PRRS virus.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, allowed to establish that the applicant did not find sources characterized by signs that are identical (identical) to all the essential features of the invention.

The definition from the list of identified analogues of the prototype as the closest in the aggregate essential in relation to perceived by the applicant to the technical result of the distinguishing features of the invention, set forth in the independent claim.

Therefore, the claimed solution meets the condition of patentability "novelty."

To verify the conformity of the proposed solution to the patentability condition “inventive step”, an additional search for known solutions was carried out to identify features included in the distinctive part of the independent claim. The search results showed that the proposed solution does not follow explicitly for the specialist from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the present invention have been identified), and the effect of the transformations provided for by the essential features of the proposed invention has not been identified to achieve a technical result.

Therefore, the proposed solution meets the condition of patentability "inventive step".

The essence of the invention is illustrated by examples of its implementation and use, which do not limit its scope.

Example 1

In 1996, during a virological study of pig blood serum with clinical signs of PRRS obtained from the Victory agricultural complex of the Leninsk-Kuznetsk district of the Kemerovo region, a PRRS virus isolate was isolated on a Marc-145 cell culture. To isolate the PRRS virus from the field material, plastic culture mattresses with a monolayer of Marc-145 cell culture were used. A small volume (1 cm ³ ) of the total sample from a suspension of pathological material (lungs and transudate from stillborn piglets) and blood serum of sows with clinical signs of PRRS infected the Marc-145 cell culture and it was placed in a CO ₂ incubator for one hour at 37 ° C . After that, Needle medium with 10% content of fetal cattle of cattle and antibiotics was added to infected cells. A pronounced cytopathic effect (CPD) was manifested after 6 passages on a Marc-145 cell culture. Identification and specificity of the virus was determined in ELISA (IDEXX, USA) with specificity control. The virus can also be isolated from the lungs, lymph nodes, spleen and other internal organs of infected piglets.

Isolated virus was used to infect a 3-day culture of Marc-145 cells. Before the virus was introduced, the cell monolayer was washed once with phosphate-buffered saline (PBS). The culture was infected with a virus at a dose of 0.01-0.1 TCD ₅₀ per cell. Virus adsorption was carried out at 37 ° C for 1 hour. After that, Needle medium was introduced into the material in a volume of 150-200 cm ³ with the addition of 5% fetal cattle serum, 50 μl / ml gentamicin and 0.3 mg / ml glutamine.

Cultivation was carried out at 36-37 ° C for 96-144 hours. As a control, 3 mattresses were left uninfected, in which the environment was changed.

Virus propagation in a Marc-145 cell culture was determined by the characteristic CPD with the formation of a cluster of spherical cells towering above the monolayer. There should not be any destructive cell changes in the control mattresses. Mattresses in which CPD was observed with a lesion of 60% of the cell monolayer (usually after 48-96 hours) were selected and frozen at -20 ° C. The virus was obtained by freezing and thawing the infected cells twice, followed by removal of cell debris by centrifugation at 2500 rpm for 20 minutes. During 120 hours of incubation, passage 6 virus accumulated to a titer of 5.17 lg TCD ₅₀ / ml. The resulting strain of PRRSV virus (copyright name “KPR-96”) was deposited in the All-Russian State Collection of Microorganisms Used in Veterinary and Animal Husbandry, the Federal State Institution “The All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed” (FGU VGNKI) 28 October 2004, under the registration number (link) "KPR-96" -DEP.

Example 2

The biological properties of the vaccine strain "CRC-96" (passage 6) were checked for the following indicators:

- lack of bacterial and fungal contamination;

- specificity and lack of viral contamination (PCR method);

- antigenic activity and specificity (ELISA);

- biological activity (titration on a Marc-145 cell culture);

- virulence for pigs.

1. Determination of the absence of bacterial and fungal contamination of the strain "KPR-96".

For this, Saburo media, meat-peptone broth (MPB), meat-peptone agar (MPA) and Kitta-Tarozzi medium were used. All bacterial media used were tested for growth properties according to GOST 28085-89. For testing of 3 vials (separately from each) with the culture virus, 1 cm ^{3 of} virus-containing material was introduced into 4 test tubes with thioglycolic medium. The tubes were incubated at different temperature conditions.

Two tubes with contents from each vial were kept in a thermostat at a temperature of 37 ± 0.5 ° C, one at 30 ± 0.5 ° C, and one at 22 ± 0.5 ° C. After 7 days, from 3 tubes incubated at 37 ± 0.5 ° C, reseeding was carried out on the following bacterial media: agar and Saburo liquid medium, MPB and MPA, sugar broth and Kitta-Tarozzi medium. On Wednesday Kitta-Tarozzi was introduced in 1 cm ³ , and in the rest of the medium - 0.5 cm ^{3 of the} test material and kept for 7 days at a temperature of 37 ± 0.5 ° C, with Saburo medium - at a temperature of 22 ± 0, 5 ° C.

All tubes were subjected to daily visual inspection for 14 days.

The test results are presented in table 1. Tests showed that the strain “CRC-96" of the PRRS virus (passage 6) is not contaminated with bacterial and fungal microflora.

On all media with seeding and reseeding growth of bacterial and fungal microflora was not observed.

2. Testing the strain “CRC-96" of the PRRS virus for specificity and the absence of viral contamination by PCR.

Determination of specificity and the absence of viral contamination was carried out by examining a sample of a culture suspension of the PRRS virus of the KPR-96 strain by PCR for the presence of the genomes of the PRRS viruses, CSF, Aujeszky's disease, PVIS, coronaviruses, circoviruses and pathogens of pig mycoplasmoses (M. hyopneumoniae, M.hyorhinisis , M. hyosynoviae).

All studies by PCR were carried out according to the "Guidelines for the indication of the genome of the PRRS virus by polymerase chain reaction", approved by the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation February 21, 1997.

As a result of the studies, only the PRRS virus genome was detected in samples of the culture suspension. Genomes of CoES viruses, Aujeszky’s disease, PVIS, corona viruses, circoviruses and pathogens of pig mycoplasmoses (M.hyopneumoniae, M.hyorhinis, M.hyosynoviae) were not found.

Comparison of the complete nucleotide sequences of the nucleocapsid protein gene (OPC 7) of the KPR-96 strain and other known strains of the PRRS virus showed a high degree of homology with the CNCM strain No. I-1102 (Lelystad) of the PRRS virus.

The level of nucleotide homology according to OPC 7 between strains "CRC-96" and "DB" is only 60%.

Significant differences between the KPR-96 and BD strains were also confirmed in ELISA (kit Ingenasa, Spain) by cross-sectional studies against the American and European types of PRRS virus. The research results are presented in table 2. The data shown in table 2 indicate that the strain "CRC-96" belongs to the European gene group of the PRRS virus.

3. Determination of the specificity and lack of contamination of the strain "CRC-96" of PRRS virus in gilts.

For the test used 3 gilts weighing 25-30 kg. All animals were seronegative for PRRS, PVIS, Aujeszky’s disease, CoES, porcine transmissible gastroenteritis (THES), influenza and M.hyopneumoniae.

All animals were injected intramuscularly with 5 ml of a culture suspension of the PRRS virus of the KPR-96 strain with a titer of 10 ^5.0 TCD ₅₀ / ml. Before and 28 days after infection, blood samples were taken from animals and serum was examined for the presence of antibodies against the following pathogens:

- PRRS virus in ELISA using a kit from IDEXX (USA), s / p value <0.4 - no specific antibodies, s / p≥0.4 - specific antibodies;

- PVIS virus in RTGA using the “Set of drugs for serodiagnosis of PVIS in RTGA” manufactured by FSI “ARRIAH”, value ≥1: 256 - the presence of specific antibodies;

- Aujeszky virus using a Chekit kit, s / p value <50 - no specific antibodies, s / p> 100 - specific antibodies;

- CoES virus using a Chekit kit, s / p value <40 - no specific antibodies, s / p> 50 - specific antibodies;

- THES virus in the microneutralization reaction, value ≥3.0 log ₂ - the presence of specific antibodies;

- M.hyopneumoniae using a Chekit kit, s / p value <20 - no specific antibodies; s / p> 30 - the presence of specific antibodies;

- swine flu virus using an IDEXX kit (USA), s / p <0.4 - no specific antibodies, s / p≥0.4 - the presence of specific antibodies.

The results of studies of blood serum for the presence of antibodies to the above pathogens are presented in table 3.

The data presented in table 3 show that in the tested samples antibodies to the viruses of PVIS, Aujeszky's disease, CoES, THES, influenza and M.hyopneumoniae were not detected. This indicates the absence of contamination of the strain "CRC-96" by the above pathogens.

4. Determination of antigenic activity and specificity of the strain "CRC-96" of the PRRS virus by ELISA.

These studies were carried out by ELISA by detecting specific antibodies in the serum of pigs obtained 28 days after immunization with an inactivated emulsion vaccine made from PRRS-96 strain KPR-96.

For this, 4 seronegative gilts weighing 25-30 kg were used. A virus with an infectivity titer of 5.0 lg TCD ₅₀ / ml was inactivated with aminoethylethyleneimine (AEEI) and emulsified with Montanide ISA-70 oil adjuvant in a 1: 3 ratio. After checking for sterility, the drug was administered to animals intramuscularly at a dose of 2 ml per head.

Blood samples were taken from all animals before and 28 days after vaccination. Blood serum was examined in a commercial ELISA kit from IDEXX (USA). The results of the study of the antigenic activity of the strain "CRC-96" of the PRRS virus by ELISA are presented in table 4.

The data in table 4 indicate that prior to immunization in serum gilts of antibodies to the PRRS virus were not detected, and after 28 days after vaccination in all 4 samples revealed specific antibodies ranging from 1.79 to 2.05 lg TCD ₅₀ / cm ³ .

Studies have also been conducted to study the antigenic activity of strains "DB", CNCM No. I-1102 (Lelystad) and "CRC-96" of PRRS virus in pigs. The research results are presented in table 5.

From the data of table 5 it is clearly seen that the strain "CRC-96" has a pronounced antigenic activity.

After its introduction in blood serum of 2-3-month-old piglets, nonspecific antibodies to PRRS virus are detected at a sufficiently high level, both with intranasal and intramuscular methods of introducing virus-containing material. In addition, the level of antibodies to PRRS virus 21 days after immunization with the strain "CRC-96" was higher than in piglets immunized with the strain "DB".

5. Determination of the biological activity of the strain "CRC-96" PRRS virus.

The biological activity of the strain "CRC-96" was determined by titration on a culture of Marc-145 cells according to the standard method. Virus 6 and 17 passages grown in a Marc-145 cell culture were used for titration after freezing-thawing twice and low speed centrifugation to precipitate cell detritus.

For titration of PRRS virus, a 2-3-day monolayer of Marc-145 cell culture grown in test tubes was used. Initially, 10-fold dilutions (from 10 ^-1 to 10 ^-9 ) of virus-containing material were prepared on Eagle's medium without serum (pH 7.0-7.4). Each virus dilution was added to 4 cell culture tubes. Previously, growth medium was poured from the tubes, 0.1 cm ^{3 of the} diluted virus was added.

After 1 hour of contact, 0.9 cm ^{3 of} culture medium was added. On the tubes indicated the number of the test material (numerator) and dilution (denominator). The tubes were left in racks in an inclined position and placed in an incubator for incubation at a temperature of + 37 ° C.

At the same time, control was set with an uninfected cell culture. Every day for 6 days, titration results were recorded by viewing each tube under a small magnification of the microscope. After viewing, the viral CPD (conditionally in the crosses) was noted, the final results were recorded 144 hours after infection of the cell culture. The titration results were considered reliable while maintaining the monolayer in the control.

The titer was calculated according to the Kerber method. Each passage of the test virus was titrated in 3 replicates. The results of determining the biological activity of the strain KPR-96 of the PRRS virus are presented in table 6. The titration results on the Marc-145 cell culture showed that the biological activity of the strain KPR-96 strain of the PRRS 6 virus is 5.17 lg TCD ₅₀ / ml, and passage 17 - 5.09 lg TCD ₅₀ / ml. The difference in the titers of the virus 6 and 17 passages is not significant, i.e. as a result of 11 passages, the biological activity of the strain KPR-96 of the PRRS virus remained unchanged.

Studies were also carried out of the cultural properties of the strains "DB", CNCM No. I-1102 (Lelystad), NVSL, No. 2156 and "KPR-96." The results of these studies are presented in table 7. From table 7 it is seen that the CPD of the strain “CRC-96” in the Marc-145 cell culture appears from 3 days after infection and reaches its maximum manifestation on 5 days, while the virus titer is 5.1 ± 0.07 lg TCD ₅₀ / ml.

6. Determination of the degree of virulence of the strain "CRC-96" PRRS virus in pigs.

The degree of virulence of the strain "CRC-96" for pigs was studied on pigs of 2-4 months of age with a live weight of 25-30 kg. The results of these experiments are presented in table 8.

From table 8 it is seen that a check of the strain “CRC-96” on pigs of 2-4 months of age at passage 17 showed that it is weakly virulence, since in some infected gilts only a slight increase in body temperature was observed (up to 40.7 ° C) within 1-4 days.

The resulting strain "CRC-96" was also tested on pregnant sows. Two seronegative pregnant pigs were intranasally infected 4 weeks before farrowing. Both sows were farrowed on day 114 after insemination. From them received 23 pigs. The characteristics of the offspring of sows are presented in table 9.

According to the data in table 9, the sows brought 18 living healthy piglets (78.3%) and one non-viable piglet (8.7%) and 3 with eye pathology (13%) were born in one sow. Two non-viable piglets died on the first day after birth. During the suction period, the remaining piglets, including those born with eye pathology, did not observe any clinical signs of PRRS.

In the blood serum of piglets before receiving colostrum, antibodies to the PRRS virus were not detected. Examination of samples of the placenta of sows and internal organs of two forcedly killed piglets before receiving colostrum for the presence of the PRRS virus genome gave a negative result. At the same time, antibodies to the PRRS virus at a high level of s / p of 1.76 and 2.24 (in ELISA, IDEXX) were detected in the blood serum of sows taken on the day of farrowing.

A comparative study of the virulent properties of strains CNCM No. I-1102 (Lelystad), "DB" and "CRC-96" of PRRS virus in pigs was carried out. The research results are presented in table 10.

According to table 10, the clinical manifestation of PRRS in piglets infected with strains of "KPR-96", "DB" and CNCM No. I-1102 (Lelystad) is significantly different, which indicates the different virulence of these strains. After infection of animals with strains of “KPR-96” and “DB”, no clinical signs of the disease were detected, with the exception of hyperthermia, which is typical for weakly virulent and avirulent strains of PRRS virus.

Thus, the test results showed that the strain "CRC-96" PRRS virus is weakly virulent.

Example 3

An inactivated emulsion vaccine against PRRS is prepared from the mother virus of the KPR-96 strain grown in a 3-day old Marc-145 cell culture with a concentration of more than 100,000 cells / ml. A mother virus is considered suitable for producing viral raw materials if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Marc-145 cells is at least 5.0 lg TCD ₅₀ / ml, pH 7.2-7.4 and in the absence of contamination.

The virus is grown in mattresses with a capacity of 1.5 DM ³ with cell culture.

After washing off the growth medium and washing once with the FBI or medium, 5 ml of the virus-containing material of the matrovy series are introduced into the mattresses. Virus adsorption is carried out at 37 ° C for an hour. After that, Needle medium in the amount of 150-200 ml is added to the mattresses with the addition of 5% fetal serum with an antibiotic (gentamicin at a concentration of 50 μg / ml or its analogues). Cultivation is carried out at 37 ° C for 72-120 hours.

For control, 3 mattresses are left uninfected, in which the medium is replaced. Virus propagation in a Marc-145 cell culture is determined by the characteristic CPD with the formation of a cluster of spherical cells towering above the monolayer. There should not be any destructive cell changes in the control mattresses. Mattresses in which cytopathic changes are observed with damage to up to 60% of the cell monolayer (usually after 72-120 hours) are selected and frozen at -20 ° C.

The virus for the manufacture of the vaccine is obtained by freezing and thawing the infected cells twice, followed by draining the contents of the mattresses into one container, observing sterile conditions. The resulting virus is freed from cell detritus by centrifugation at 1000 rpm for 20 minutes. The suspension purified from detritus should look like a clear pinkish-cherry liquid. The resulting virus is monitored for infectious activity.

A production series of the PRRS virus of the KPR-96 strain is considered suitable for the manufacture of vaccine preparations if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Marc-145 cells is at least 5.0 lg TCD ₅₀ / ml, pH 7.2-7 , 4 and in the absence of contamination. The purified virus-containing suspension is subjected to inactivation. Inactivation of the PRRS virus is carried out using a 1-2% aqueous solution of AEI, which is introduced into the virus-containing suspension to a final concentration of 0.05-0.08 wt.%. For this, an AEEI solution is added to the suspension heated to 26-28 ° C with constant stirring and the pH is adjusted to 7.2-7.4 by adding a 5% succinic acid solution to the suspension. Inactivation of the virus is carried out in a thermostat at 37 ± 2 ° C for 24 hours with periodic stirring. At the end of inactivation, the antigenic material is cooled to 4-6 ° C, its pH value is set in the range of 7.2-7.4, and antigen samples are taken to test for sterility and avirulence using methods known to those skilled in the art.

An emulsion vaccine is prepared by dispersing a mixture of antigenic material and an oil adjuvant containing mineral oil with an emulsifier. As an oil adjuvant, a preparation of the company “Seppic” (France) under the brand name Montanide ISA-70 is used.

The ratio of antigenic material from strain "CRC-96" of the PRRS virus and the Montanide ISA-70 oil adjuvant is 33% and 67%, respectively. Each vaccine dose of a 2 ml vaccine should contain 0.7 ml of PRRS virus antigen with a titer of at least 5.0 lg TCD ₅₀ / ml (before inactivation) and 1.3 ml of Montanide ISA-70 oil adjuvant.

The vaccine is an emulsion of a white or pink-white with a slightly viscous consistency. During storage, slight exfoliation of mineral oil on the surface of the emulsion is allowed. When shaken, the vaccine acquires a homogeneous homogeneous structure.

The resulting vaccine has an optimal component composition, wt.%:

Suspension of Avirulent and Purified antigenic material from the strain "CRC-96" PRRS virus obtained in transplantable cell culture Marc-145 with infectious activity of at least 5.0 lg TCD ₅₀ / ml 33.0 Oil adjuvant up to 100.0

The antigen content in the vaccine in the above value is its effective amount in the drug, ensuring the achievement of a technical result.

The resulting vaccine is Packed in glass bottles and controlled in accordance with the technical conditions for sterility, safety and antigenic activity.

The vaccine was tested for sterility by plating on the following nutrient media: MPB, MPA, MPPB, Saburo medium and thioglycol medium. In all cases, microflora growth was not observed.

The safety of the vaccine was tested on six white mice and two gilts, which were injected subcutaneously and intramuscularly with 0.5 ml and 5.0 ml of the vaccine, respectively. The animals were monitored for 10 and 15 days, respectively, then they were euthanized and an autopsy was performed. During the observation period (10 days in mice and 15 days in gilts), all animals remained healthy. Slight swelling was observed at the injection site, and at the autopsy during examination of the injection site, single, small, encapsulated foci were observed with a diameter of 1-3 mm in mice and 5-7 mm in gilts, when the vaccine contents were separated.

Determination of the antigenic activity of the vaccine was carried out on six pigs 2.5-3 months old, seronegative with respect to PRSS, with a live weight of 25-30 kg. The vaccine was administered at a dose of 2 ml intramuscularly in the middle third of the neck (behind the ear) twice with an interval of 28 days. The animals were monitored for 45 days and blood samples were periodically taken. Blood serum was tested in ELISA using a commercial kit company IDEXX (USA) for the presence of specific antibodies to the PRRS virus. The results of the study of blood serum of piglets before and at various times after vaccination are presented in table 11.

According to table 11, specific antibodies to PRRS virus at s / p≥0.4 (positive values) in blood serum were detected by 21 days after vaccination in 5 animals out of 6 immunized.

28 days after vaccination, the average antibody level in the group significantly increased and amounted to s / p 0.95 ± 0.24, however, one animal remained seronegative.

A week after revaccination, a significant increase in antibodies to the PRRS virus was observed, including that of a previously seronegative gilt, and its s / p index increased from 0.2 to 1.13. 14 days after revaccination, the average antibody level in the group was s / p 2.45 ± 0.49. After revaccination, one of the piglets experienced a decrease in the level of antibodies from 0.5 to 0.35, which is probably due to the peculiarities of the immune system in this animal. The results of clinical observation of vaccinated gilts showed that after immunization in all animals, an increase in body temperature was not recorded at the injection site. The general condition of the animals remained within the physiological norm.

Thus, the resulting inactivated emulsion vaccine from the strain “KPR-96” is a sterile, harmless preparation and has sufficient antigenic activity. The vaccine is used for prophylactic immunization of pigs in farms threatened and dysfunctional by PRRS. In PRRS threatened farms, sows and repair pigs are vaccinated twice with an interval of 20-30 days three weeks before mating (insemination). Subsequently, previously immunized sows are inoculated once three to four weeks before mating (insemination). In PRRS dysfunctional farms, sows and repair pigs are vaccinated for the first time twice with an interval of 20-30 days three weeks before mating (insemination), then they are additionally immunized for 60-70 days of gestation. Subsequently, previously immunized sows are inoculated for the first time once three to four weeks before mating (insemination), the second time for 60-70 days of gestation. Boars-producers are immunized twice with an interval of 20-30 days three weeks before mating (insemination), subsequently vaccinated every 6 months. Piglets born from vaccinated sows are inoculated twice at 30-45 days of age with an interval of 20-30 days. Piglets obtained from unvaccinated sows against PRRS and not having colostral antibodies against the PRRS virus may be vaccinated from the first days of life. The vaccine is administered to pigs intramuscularly behind the ear in a dose of 2 cm ³ regardless of the age of the animals. Only clinically healthy animals are vaccinated. Immunity in vaccinated animals occurs 21-30 days after vaccination and lasts at least 6 months. The vaccine induces a high level of antibodies against PRRS and provides reliable passive maternal immunity in young animals up to 25-30 days of age, which helps to protect it from the field virulent virus in early life.

Example 4

Tests of the effectiveness of the vaccine against PRRS inactivated emulsion, manufactured as described in example 3, and containing, wt.%:

Suspension of Avirulent and Purified antigenic material from the strain "CRC-96" PRRS virus obtained in Marc-145 cell culture with infectious activity of 5.25 lg TCD ₅₀ / ml 33.0 Oil adjuvant up to 100.0

The effectiveness of the drug was tested at pig farm No. 1, numbering 57,000 pigs.

Here, abortions were observed in sows, accompanied by stillbirth, as well as hollows and a high mortality rate in suckling pigs.

The reproductive livestock of the farm began to be inoculated with the associated emulsion inactivated vaccine against PRRS and PVIS produced by FGI “VNIIZZH”, while piglets of the growing group were not vaccinated. Vaccination of the reproductive population has significantly reduced the number of abortions, the level of stillbirth, free-flowing, as well as to increase the number and safety of offspring.

However, after this, the manifestation of respiratory syndrome in piglets of 45-50 days of age was noted.

Multiple studies of material from sick animals conducted at the Federal State Institution “ARRIAH”, using PCR, isolated the PRRS virus gene, as well as mycoplasmas (M.hyopneumoniae, M.hyorhinis), Haemophilus parasuis, Pasteurella multocida, Actino-bacillus pleuropneumoniae and 2 circuses.

To test the inactivated emulsion vaccine against PRRS from the strain "CRC-96" experimental and control groups of animals were formed, each of which consisted of 3 sectors of 1000 animals, selected according to the type of analogues.

Pigs of the experimental group were vaccinated at 25 days of age with revaccination after 3-4 weeks at a dose of 2 ml per head, the control sectors were not vaccinated. All piglets were obtained from sows inoculated with the associated inactivated emulsion vaccine against PRRS and PVIS.

The animals of both groups were monitored throughout the growing period. After vaccination in the experimental group, thermometry was performed for 20 animals for 5 days, while the body temperature of the animals was within the normal range of 38.4-40.1 ° C. At the injection site, a small swelling was felt up to 1.0 cm in diameter.

The test results of the emulsion inactivated vaccine against PRRS from strain "CRC-96" on weaned piglets in pig complex No. 1 are presented in table 12.

The data in table 12 indicate that in the experimental group (vaccinated sectors) during the growing period, the number of sick animals forced to be killed and handed over for sanitary marriage was less than in the control group. At the same time, the average daily gain was higher by an average of 7.3 grams.

The death in both groups was almost the same, but the overall safety of the animals during the rearing period in the experimental group was higher.

An analysis of the incidence showed that in the experimental group, the majority of cases were animals with impaired gastrointestinal tract functions and injuries, and in the control group with respiratory disorders.

The results of the studies of blood serum of piglets in ELISA (FGU "ARRIAH") vaccinated with an inactivated emulsion inactivated vaccine against PRRS from strain "CRC-96" in pig complex No. 1, are presented in table 13.

According to table 13, it follows that prior to vaccination, the percentage of seropositive animals for PRRS animals was 52.5%. This is due to the fact that at this time (24-28 days of life) colostral antibodies were detected in the blood serum of animals. This is due to the fact that pigs were obtained from sows who had been ill and vaccinated against PRRS and PVIS. 26 days after vaccination, the number of seropositive animals decreased to 35%, which is associated with a decrease in the level of colostral and insufficient formation of post-vaccination antibodies. After revaccination, the percentage of immune animals increased to 94.7%. At the same time, the average antibody level increased from 66.1 ± 4.8 before vaccination to 82.2 ± 8.1 50 days after vaccination.

Example 5

Tests of the effectiveness of the vaccine against PRRS inactivated emulsion, manufactured as described in example 3, and containing, wt.%:

Suspension of Avirulent and Purified antigenic material from the strain "CRC-96" PRRS virus obtained in Marc-145 cell culture with infectious activity 5.17 lg TCD ₅₀ / ml 33.0 Oil adjuvant to 100.0

The effectiveness of the drug was tested at pig farm No. 2.

In this farm, the disease manifested itself mainly in respiratory form on piglets of the rearing group at 45-55 days of age. The overall level of death and forced slaughter averaged 40-50%.

For research, the experimental and control groups (sectors) of piglets of 25-28 days of age were formed. Animals were vaccinated at the age of 25-28 days of life with revaccination after 3-4 weeks. At 35 days of age, these piglets were transferred to the rearing group, and weak and lagging animals were rejected. The test results of the vaccine from strain "CRC-96" on weaned piglets in pig complex No. 2 are presented in table 14.

From the data presented in table 14, it follows that in the experimental group, the incidence and mortality were lower than in unvaccinated animals. The average daily gain was also higher in the group of vaccinated animals (190 g versus 167 g in the control).

Safety over the growing period was also higher in vaccinated animals (68.1% versus 54.3% in the control).

The results of the studies of blood serum of piglets in ELISA (FGU "ARRIAH"), obtained during the test vaccines from strain "KPR-96" in pig complex No. 2, are presented in table 15.

The data in table 15 indicate that before vaccination the percentage of animals seronegative in relation to PRRS was 52.5%.

This is due to the fact that at this time (24-28 days of life) colostral antibodies of a rather high level were detected in the blood serum of animals due to the fact that piglets were obtained from sows who had been ill and vaccinated against PRRS and PVIS. 26 days after vaccination, the number of seropositive animals decreased to 35%, which is associated with a decrease in the level of colostral and insufficient formation of post-vaccination antibodies. After revaccination, the percentage of immune animals increased to 94.7%. At the same time, the average antibody level increased from 56.7 ± 8.0 before vaccination to 102.6 ± 11.8 50 days after vaccination.

Example 6

Tests of the effectiveness of the vaccine against PRRS inactivated emulsion, manufactured as described in example 3, and containing, wt.%:

Suspension of Avirulent and Purified antigenic material from the strain "CRC-96" PRRS virus obtained in Marc-145 cell culture with infectious activity of 5.9 lg TCD ₅₀ / ml 33.0 Oil adjuvant up to 100.0

The effectiveness of the drug was tested at pig farm No. 2 for vaccination of the reproductive population.

Sows and maintenance pigs were divided into two groups of 60 animals each. The animals of the experimental group were vaccinated with the test vaccine according to the temporary instructions for its use: the first time twice with an interval of 20-30 days three weeks before mating (insemination), then the animals were re-immunized for 60-70 days of gestation. In the control group, sows against PRRS were not vaccinated.

After immunization with the test vaccine in vaccinated animals, the total body temperature and at the injection site remained within the physiological norm; no clinical signs of the disease and deviations were noted.

The results of the test "KPR-96" on sows in pig farm No. 2 are presented in table 16.

From the data presented in table 16 it follows that in the experimental group, the yield of piglets is 1 sow higher than in the control (10.4 pigs versus 8.4). In vaccinated sows, the average weight of the newborn piglet was also higher compared to the control group (1.24 versus 1.21 kg).

During subsequent insemination (in the first hunt after the transfer of piglets for rearing) among sows previously vaccinated with emulsion inactivated vaccine from the KPR-96 strain, the sowing rate was lower than in the control group (6.6% versus 12% in control animals).

Example 7

Tests of the effectiveness of the vaccine against PRRS inactivated emulsion, manufactured as described in example 3, and containing, wt.%:

Suspension of Avirulent and Purified antigenic material from strain "CRC-96" PRRS virus obtained in culture Marc-145 cells with infectious activity of 5.25 lg TCD ₅₀ / ml 33.0 Oil adjuvant up to 100.0

The effectiveness of the drug was tested at pig farm No. 3 for vaccination of the reproductive population. Sows and maintenance pigs were divided into two groups of 60 animals each. The animals of the experimental group were vaccinated with the test vaccine according to the temporary instructions for its use: the first time twice with an interval of 20-30 days three weeks before mating (insemination), then the animals were re-immunized for 60-70 days of gestation. In the control group, sows against PRRS were not vaccinated.

After immunization with the test vaccine in vaccinated animals, the total body temperature and at the injection site remained within the physiological norm; no clinical signs of the disease and deviations were noted.

As a result of vaccination, the productivity of sows in the experimental group became higher than in the control (11 pigs per 1 sow in the experimental group versus 10 pigs in the control).

In the experimental group, 31 pigs fell during the suction period, and in the control group 35 pigs, i.e. the difference between the groups was 17.8%. The safety of sucking piglets in the experimental group was 96%, and in the control - 94.1%.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:

- vaccine against PRRS inactivated emulsion, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form as described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed;

- vaccine against PRRS inactivated emulsion, made on the basis of the strain "KPR-96" in accordance with the invention, expands the arsenal of vaccines against PRRS inactivated emulsion, having high immunogenic activity and harmlessness and creating intense and prolonged immunity in vaccinated animals against circulating in Russia epizootic PRRS virus.

Therefore, the present invention meets the condition of patentability "industrial applicability".

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Claims (8)

1. The vaccine against pig reproductive and respiratory syndrome (PRRS) inactivated emulsion, containing the active substance and the target additive, characterized in that as an active substance it contains a suspension of avirulent and purified antigenic material from strain "CRC-96" of the PRRS virus, family Arteriviridae Arteriviridae , genus Arterivirus, collection of the Federal State Institution “VGNKI” “KPR-96” - DEPT, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains a suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a sensitive biological system. 3. The vaccine according to claim 2, characterized in that it contains a suspension of avirulent and purified antigenic material from the KPR-96 strain of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml . 4. The vaccine according to claim 3, characterized in that it contains a suspension of avirulent and purified antigenic material from the KPR-96 strain of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml in an amount of 33.0 wt.%. 5. The vaccine according to claim 1, characterized in that it contains an oil adjuvant as a target additive. 6. The vaccine according to claim 5, characterized in that it contains an oil adjuvant of the brand Montanide ISA-70 from oil adjuvants. 7. The vaccine according to claim 6, characterized in that it contains an oil adjuvant of the brand Montanide ISA-70 in an amount of 67.0 wt.%. 8. The vaccine according to any one of claims 1 to 7, characterized in that it contains a suspension of avirulent and purified antigenic material from strain "CRC-96" of PRRS virus obtained in a Marc-145 cell culture with an infectious activity of at least 5.0 lg TCD ₅₀ / ml, and oil adjuvant of the brand Montanide ISA-70 in the ratio, wt.%: Antigenic material 33.0 Oil adjuvant up to 100.0