RU2563522C1 - STRAIN O №2102/Zabaikalsky/2010 FMD VIRUS Aphtae epizooticae OF TYPE O FOR CONTROL OF ANTIGENIC AND IMMUNOGENIC ACTIVITY OF FMD VACCINES AND FOR PRODUCTION OF BIOLOGICAL PRODUCTS FOR DIAGNOSTICS AND SPECIFIC PROPHYLAXIS OF FMD OF TYPE O - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed strain is reproduced in a monolayer culture of pig kidney cells (PK), passaged cultures PSGK-30, BHK-21 and IBRS-2, for 18-24 hours of incubation the virus yield in these cell cultures reaches the values of 6.63-7.5 lg TCD 50/cm³. At high multiplicity of infection (1-10 TCD/cell) it causes TCD after 5 hours, while maintaining the initial characteristics in passaging in cell cultures for 10 passages.

EFFECT: strain may be used to control the antigenic and immunogenic activity of FMD vaccines and for production of biological products for diagnostics and specific prevention of FMD of type O.

6 cl, 1 dwg, 8 tbl, 9 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular to a new strain of foot and mouth disease virus Aphtae epizooticae, and can be used to control antigenic and immunogenic activity of FMD vaccines, as well as in the development and manufacture of diagnostic tools and specific prevention of foot and mouth disease type O.

Foot and mouth disease is an acute, contagious viral disease of artiodactyl animals, manifested by fever, vesicular (aphthous) lesions of the mucous membrane of the oral cavity, hairless areas of the scalp, udder, corolla, and inter-hoof gap and accompanied by impaired movement. This pathogen is characterized by a tendency to widespread and epizootic course. The disease is accompanied by large losses of milk, meat and other types of livestock products, complicates commercial operations and economic activity. Years of experience show that with endemic foot and mouth disease, income in dairy and beef cattle breeding is reduced by (%): 30–40 [1].

The foot and mouth disease virus belongs to the family Picornaviridae, the genus Aphtovirus. It has 7 antigenic types, a large number of subtypes and many strains [2].

The causative agent of foot and mouth disease has significant antigenic variability of strains within the same serotype, which is detected at different time intervals and in different territories and depends on the species composition of the susceptible population, its immune status and many other various factors. The antigenic variability of the foot and mouth disease virus is caused by amino acid substitutions in polypeptide fragments (antigenic epitopes) exposed on the surface of capsid proteins [2, 3].

The shifts of the antigenic spectrum corresponding to the renewal of the structure of a new field strain can vary from insignificant ones captured by monoclonal antibodies to significant ones recorded using traditional polyclonal immunoglobulins. Significant changes in the antigenic characteristics of a natural strain are very likely to weaken specific immunity induced by a non-homologous antigen. They also cause strain-specific diagnosis difficulties.

As a result, it becomes necessary to create new diagnostic tools and specific immunoprophylaxis.

Known strains of foot and mouth disease virus type O, used as production in Russia over the past 50 years. These include the following strains: O ₁ No. 1618 Chechen-Ingush, isolated in 1966; O ₁ No. 194, allocated in 1957 in the Volgograd region. These strains were used to obtain diagnosticum and FMD vaccines used in various regions of the country, and are currently supported in the collection of microorganism strains of the Federal State Budget Scientific Institution VNIIZZH.

A known type O strain No. 1736 / Abkhazia / 2000, isolated from cattle in the Gali district of Abkhazia, for the manufacture of diagnostic and vaccine preparations.

A known type O strain No. 1964 / Mongolia / 2004, isolated from cattle in February 2004 in the East Gobian aimak of Mongolia, for the manufacture of diagnostic and vaccine preparations [4].

A known strain of type O No. 1734 "Primorsky-2000", isolated 13.04.2000 from pigs in the farm "Stepnoe" with. The elite of the Ussuri district of Primorsky Krai [5, 6].

A comparative analysis of the nucleotide sequences showed that the strain of type O No. 1734 "Primorsky-2000" belongs to the Pan-Asian genetic line of the FMD virus type O. The pan-Asian virus caused a pandemic of foot and mouth disease in 1999-2000 in most countries in Asia, including Vietnam, Japan, Korea, Mongolia, as well as Armenia and Georgia. The devastating epidemic of foot and mouth disease in the UK in 2001 was also caused by a pan-Asian virus. The studied virus has the highest homology level (98.74%) with isolates O Vietnam / 99 and O Taiwan / 99. It was found that isolate O No. 1734 "Primorsky-2000" differs from all previously isolated isolates, including strains O ₁ No. 194 and O ₁ No. 1618 used for the manufacture of diagnostic tools and specific prophylaxis.

Production strain No. 1734 "Primorsky-2000" is used in the Russian Federation as an industrial strain in the manufacture of specific prophylaxis and diagnostics that are used throughout Russia and in the CIS countries.

In 2010, in Japan, South Korea, Hong Kong, and mainland China, type O foot and mouth disease outbreaks were caused by the Southeast Asia topotype virus belonging to the Mua-98 genetic line [7].

The closest to the invention according to the set of essential features is the strain No. 1734 "Primorsky-2000" of the FMD virus type O for the manufacture of diagnostic and vaccine preparations.

The disadvantages of the known strains, including the prototype strain, are antigenic differences from type O foot and mouth disease virus isolates isolated at different time intervals, and vaccines prepared on their basis provide effective protection of animals only against infection with a homologous virus.

An outbreak of foot and mouth disease in the Russian Federation was recorded in July 2010 in the yards of residents of the village of Abagaytu, Zabaykalsky District, 12 km from the border with China in the buffer zone, where cattle and small cattle were vaccinated against foot and mouth disease types O, A, Asia-1. Of the 2,256 cattle of various ages that were available to residents, 112 were ill (5%), of 50 pigs, 4.

In this regard, it became necessary to obtain a new production strain from the epizootic virus of foot and mouth disease serotype O to ensure the safety of the territory of Russia and neighboring states from this pathogen.

The problem to which the present invention is directed, is to expand the arsenal of production strains of foot and mouth disease virus serotype O, which have high infectious, antigenic and immunogenic activity in their native form and retain antigenic and immunogenic activity after inactivation, suitable for controlling the antigenic and immunogenic activity of vaccines, manufacturing sensitive and highly specific diagnosticums and highly immunogenic vaccine preparations homologous to the epizootic virus that appeared on ter Ithor Russia.

This problem was solved by obtaining strain O No. 2102 / Zabaykalsky / 2010 (copyright) of foot and mouth disease virus to control the antigenic and immunogenic activity of vaccines and the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type O.

The viral isolate, which served as the source for strain O No. 2102 / Zabaykalsky / 2010, was isolated in July 2010 from a sick pig in the village of Abagaytuy, Zabaykalsky District, Zabaykalsky Krai (examination No. 2102). Production strain O No. 2102 / Zabaykalsky / 2010 of type O FMD virus was obtained by successive passages on sensitive hetero- and homologous cell cultures.

Strain O No. 2102 / Zabaykalsky / 2010 of the FMD virus of type O was deposited on March 01, 2012 to the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”) under registration number (link): foot and mouth disease virus strain O No. 2102 / Zabaykalsky / 2010 (production).

Compared with the strain of the prototype strain 0№2102 / Zabaykalsky / 2010 has a higher infectious, antigenic and immunogenic activity in its native form and after inactivation.

The possibility of using strain O No. 2102 / Zabaykalsky / 2010 of type O foot and mouth disease virus for controlling the antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type O has been experimentally confirmed.

The invention is illustrated in the graphic image. A dendrogram is presented that reflects the phylogenetic relationships of strain O No. 2102 / Zabaykalsky / 2010 of foot and mouth disease virus with epizootic and vaccine strains of foot and mouth disease virus of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

The invention is illustrated by the following list of sequences:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain O No. 2102 / Transbaikal / 2010 of type O foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP1 protein gene of strain O No. 2102 / Transbaikal / 2010 of type O foot and mouth disease virus.

Strain O No. 2102 / Transbaikal / 2010 of the foot and mouth disease virus is characterized by the following features and properties.

Morphological features

Strain O No. 2102 / Zabaykalsky / 2010, type O foot and mouth disease virus belongs to the family Picomaviridae, genus Aphtovirus, serotype O and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the form of the virion is icosahedral, size 23-25 nm. Virion consists of an RNA molecule enclosed in a protein coat. The protein shell consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, strain O No. 2102 / Zabaykalsky / 2010 of the foot and mouth disease virus belongs to the O serotype. The virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity (GA activity). In sick animals, antibodies are formed in the blood serum that are detected in an enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN). When cattle are immunized with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA and RMN.

During hyperimmunization of guinea pigs, a concentrated antigen from an inactivated type O strain No. 2102 / Transbaikal / 2010 of foot and mouth disease virus induces the formation of virus-specific antibodies detected in CSC at a dilution of 1:30 and an ELISA at a dilution of 1: 10000.

Using the method of nucleotide sequencing, the primary structure of the VP1 gene of strain O No. 2102 / Transbaikal / 2010 of foot and mouth disease virus was determined and the primary structure of the VP1 protein was derived. A comparative analysis of the nucleotide sequences showed that strain O No. 2102 / Transbaikal / 2010 of type O foot and mouth disease virus has a close phylogenetic relationship with isolates isolated in Hong Kong, South Korea, Japan and mainland China in 2010 and is significantly different from O ₁ Manisa strains. About No. 1734 "Primorsky-2000" of the PanAsia subline, as well as from the Middle East - South Asia topotype virus (ME-SA) of the PanAsia2 subline. The homology of isolates О No. 2102 / Zabaykalsky / 2010 and О / Hong Kong / 20/2010 is 97%.

Thus, phylogenetic analysis showed that strain O No. 2102 / Zabaykalsky / 2010 belongs to the topotype Southeast Asia (SEA) of the genetic line Mua-98 of the FMD virus of serological type O.

Antigenic affinity of strain O No. 2102 / Zabaykalsky / 2010 with production strains of foot-and-mouth disease virus O ₁ Manisa, O No. 1734 / Primorsky / 2000 and O PanAsia2 was studied in ELISA and RMN.

The results of studies at the RMN are presented in Table 1. Antigenic compliance (r ₁ ) was 0.31 for O ₁ Manisa, O Primorsky 2000, No. 1734, 0.36, and 0.3 O for PanAsia2. When r ₁ > 0.3, the field isolate and the production strain are closely related, and the vaccine from the production strain will protect against epizootic virus, when r ₁ <0.3 the field isolate is different from the production strain, and the vaccine from this strain does not protect against epizootic virus [8].

The results of studies in ELISA are presented in table 2. Antigenic compliance (r ₁ ) was for O ₁ No. 1618 - 0.22, About No. 1734 / Primorsky / 2000 - 0.35, About PanAsia2 - 0.23. When the value of r ₁ - 0.4-1.0 production strain is in close antigenic relationship; when r ₁ is 0.2-0.39 - the field isolate is antigenically related to the production strain, the vaccine from the production strain can be used if a more related strain is not found and provided that the animals are immunized more than 1 time; when r ₁ <0.2, the field isolate differs from the production strain, the vaccine from which is not acceptable for protection against infection with the field virus.

Biotechnological characteristics

Strain O No. 2102 / Zabaykalsky / 2010 is reproduced in a monolayer culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), Syrian hamster kidney (VNK-21) and pig kidney (IB-RS-2) . Within 18-24 hours of incubation, the virus harvest in these cell cultures reaches values from 6.0 to 7.5 lg TCD ₅₀ / cm ³ . With a high multiplicity of infection (1-10 TCD / cell), it causes CPD after 5 hours. It retains its original characteristics when passaged in cell cultures for 10 passages (observation period).

Geno and chemotaxonomic characteristics

Strain O No. 2102 / Zabaykalsky / 2010 of the FMD virus type O is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ D.

Nucleic acid is represented by a single-chain linear molecule with a molecular weight of 2.8 × 10 ⁶ D. The virion has a protein shell consisting of four basic proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The lipoprotein membrane is absent.

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Virionic RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VR _66a ) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

Physical properties

The virion mass is 8.4 × 10 ⁻¹⁸ g. A sedimentation coefficient of 146S in the sucrose gradient. The floating density of 1.45 g / cm ³ .

Resistance to external factors

Strain O No. 2102 / Zabaykalsky / 2010 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.2-7.6. PH shifts to both the acidic and alkaline sides lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-radiation, high temperatures.

Additional features and properties

Immunogenic activity - immunogen in the composition of an inactivated vaccine.

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals, newborn mice, guinea pigs.

Virulence - virulence for naturally susceptible animals with contact, aerosol and parenteral infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 10 passages (observation period).

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

Example 1

The viral isolate, which served as the source for strain O No. 2102 / Zabaykalsky / 2010, was isolated from the VNIIZZh Federal State Budget Scientific Institution from samples of aphthous material obtained in July 2010 from pigs suspected of foot and mouth disease from the village of Abagaytuy, Zabaykalsky Krai, Transbaikal Territory (examination No. 2102 / 2010). Samples of aphthous material were received at the Federal State Budget Scientific Institution VNIIZZH July 18, 2010.

When isolating a virus in order to obtain a homogeneous population with optimal biotechnological properties, a complex of biological, virological, and biochemical methods was used, provided for by guidelines for identifying and identifying FMD virus strains [9].

Biological and virological methods included the isolation of the virus and its adaptation.

Virus isolation on a culture of primary trypsinized SP cells, transplanted PSGK-30, IB-RS-2, BHK-21 cell lines and pigs with subsequent adaptation (3 passages). For bioassays on primary and transplanted cell cultures, they were grown on appropriate nutrient media, under stationary conditions in bottles with a surface area of 25 cm ² , washed from the growth medium and infected with a 10% suspension of aphthous material (the multiplicity of infection was 1-10 TCD ₅₀ per cell ) prepared in a Hanks solution with 0.5% lactalbumin hydrolyzate (GLA) and antibiotics according to a standard formulation. To remove microflora and ballast cell components, the suspension was treated with 10% chloroform. After 30 minutes of contact at 37 ° C, 5 cm ^{3 of the} support medium was added to the vials and incubated at 37 ° C until the appearance of the virus CPD. In the presence of CPD (rounding of cells, increasing their optical density, degeneration and separation of cells from glass), the bottles were subjected to freezing, thawing, purification of the cell suspension with chloroform and centrifugation at 3000 g for 15 min. The obtained virus-containing material was used for subsequent passages and studies in RSK and ELISA for the presence of viral antigen, using commercial type-specific sera stored in the museum of strains of FSBI "ARRIAH" and "Kit for detecting antigen of foot and mouth disease in IFA", manufactured by FSBI "ARRIAH" . The virus was considered adapted to cell cultures if, within 18-24 hours, (%) 90-100 CPDs appeared in the monolayer.

The adaptation of the epizootic isolate About No. 2102 / Zabaykalsky / 2010 to various cell lines occurred at the level of 3 passages. The virus adapted to the PSGK-30 cell culture was used to infect the VNK-21 cell suspension in order to obtain antigen for the manufacture of an experimental vaccine series, as well as for hyperimmunization of guinea pigs.

Virus adapted to IB-RS-2 cell cultures? used to obtain antigen for RSK and ELISA.

The results of the adaptation of the virus to various cell cultures are presented in table 3.

The data shown in table 3 indicate a high adaptive ability of strain O No. 2102 / Transbaikal / 2010 of the FMD virus type O to the used cell cultures.

Isolated using the above methods, the viral preparation was studied in the reactions: CSC and ELISA to identify its type (Tables 4 and 5). The strain was tested for sterility, the absence of contamination with bacterial and fungal microflora and mycoplasmas, as well as extraneous viruses in PCR and RT-PCR.

The results shown in tables 4 and 5 indicate that in the aphthous test material No. 2102 / Zabaykalsky / 2010 an FMD virus antigen of type O was detected at a dilution of 1: 2 in RSK and 1:16 in ELISA.

No contamination with bacterial and fungal microflora, mycoplasmas and extraneous viruses was found.

Example 2

For hyperimmunization of guinea pigs, the antigen from strain O No. 2102 / Zabaykalsky / 2010 of type O foot and mouth disease virus reproduced in a monolayer culture of PSGK-30 cells is used. The virus-containing suspension is concentrated 100 times by adding 8-10% polyethylene glycol (PEG) m.m. 6000, cleaned from ballast impurities by adding 10% chloroform. The purified virus is inactivated by aminoethylethyleneimine (AEEI) at a concentration of 0.025-0.05% at a pH of 8.0-8.3.

The inactivated antigen in the concentrate was mixed with an equal volume type oil adjuvant incomplete Freund's adjuvant are administered to guinea pigs in a volume of 1.0 cm ³ intramuscularly. After 21 and 7 days, the immunization is repeated and 10 days after the last administration of the antigen, the animals are bled. Individual blood serum samples are checked for type specificity and activity in CSCs in accordance with the guidelines for the identification and identification of foot and mouth disease virus strains [9].

After that, a series is prepared by mixing type-specific individual serum samples of the same activity. The strain specificity of a serial preparation is determined in one- and two-sided reactions with homo- and heterologous antigens.

After preservation with sodium azide (1: 5000) and holding at 4 ° C for 30 days, the resulting serum is Packed in bottles of 0.5-1.0 cm ³ and dried by freeze-drying under vacuum.

By the method described in example 2, was prepared 1 series of hyperimmune serum, the characteristics of which are presented in table 6.

The data shown in table 6 indicate that a diagnostic serum has been obtained that, according to its specific activity, meets the requirements of GOST 25384-82.

Example 3

To obtain the antigen for serological and immunochemical reactions, strain O No. 2102 / Zabaykalsky / 2010 of foot and mouth disease virus Aphtae epizooticae adapted to the cell culture of the transplantable lines VPK-21, PSGK-30 and IB-RS-2 is used. For adaptation, virus-containing material is used in the form of a 10% aphthous suspension. Passaging is carried out for 3-5 consecutive passages. The resulting virus is used to produce viral raw materials. Infection of cell cultures and the collection of viral material is carried out according to standard methods.

The resulting virus-containing suspension is concentrated 100 times by adding (%) 8-10 PEG. The resulting concentrate is inactivated by AEEI, Packed in bottles and dried by sublimation under vacuum.

The antigen obtained on the cell culture of BHK-21, is used to obtain immunospecific components for ELISA.

In this way, 3 series of diagnostic antigen were prepared, the characteristics of which are given in table 7.

The research results, shown in table 7, indicate that diagnostic antigens have been obtained, the specific activity of which meets the requirements of GOST 25384-82.

Example 4

For the manufacture of an inactivated sorbed type O vaccine of strain O No. 2102 / Zabaykalsky / 2010, foot and mouth disease virus is reproduced in a suspension culture of BHK-21 cells. Serum without serum with the addition of FGMS, GBKS and antibiotics at a pH of 7.2-7.4 is used as a supporting medium. The cell culture is infected with a virus at the rate of 0.001-0.05 TCC ₅₀ per cell. The cultivation of the virus is carried out at a temperature of (36 ° C) - (37 ° C). After 8-10 hours of cultivation, live and dead cells are counted by trypan blue staining. If the number of living cells is (%) 15-20, then cultivation continues for another 2-3 hours. When the number of dead cells (%) 90-95 is reached, cultivation is stopped and the virus-containing suspension is checked for sterility and the content of 146S and 75 S components.

The amount of 146S and 75S components in the suspension should be at least 0.5 μg / cm ³ . Immediately after the end of the virus reproduction cycle, without stopping thermostating, a 15–20% AEEI solution, acidified with glacial acetic acid to pH 8.0–8.5, is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to (%) 0.025-0.05. Inactivation of the infectiousness of the virus is carried out for 12-24 hours at (36 ° C) - (37 ° C) and a pH of 7.2-7.6 with stirring after 5-6 hours for 3-5 minutes. At the end of inactivation, the antigen suspension is cooled to (4 ° C) - (8 ° C). A 10% solution of PHMG is added to the cooled suspension to a concentration of 0.005-0.007% for flocculation of ballast impurities and inactivation of possible contaminants. Flocculated ballast impurities are subjected to sedimentation followed by decantation. The resulting antigen is monitored for avirulence, virus-specific protein content, 146S and 75S virus components, and sterility. The required concentration of 146S and 75S components in a graft dose of an adsorbed vaccine is obtained by concentrating the antigen with gel of aluminum oxide hydrate (GOA).

The calculated volume of the GOA gel of 3% concentration is added to the cooled antigen suspension with the stirrer operating. Stirring is carried out for 30 minutes. After sedimentation of the GOA gel, the calculated volume of the remaining suspension is drained. The final concentration of GOA should be in the range of 1.62 ± 0.488 mg / cm ³ P <0.01 n = 10, and the concentration of 146S and 75S components of the FMD virus is not less than 3.0 μg / cm ³ . Then an additional 10% saponin solution is added to the suspension to a final concentration of at least 0.075%.

The resulting vaccine is packaged in glass bottles and sterility control is performed according to GOST 28085-89.

The virulence and harmlessness of the vaccine is checked on 5 heads of cattle, introducing the vaccine first under the mucous membrane of the tongue at a dose of 2 cm ³ , then subcutaneously at a dose of 10 cm ³ . Observation of the clinical condition of the animals is carried out for 10 days. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

The resulting vaccine is a light yellow liquid with a loose white precipitate of sorbent, which is formed at the bottom of the vial during storage and is easily broken into a homogeneous suspension with shaking.

Example 5

Tests for cattle of FMD vaccine inactivated sorbed from strain O No. 2102 / Zabaykalsky / 2010, manufactured as described in example 4, were performed.

Cattle weighing 200-250 kg were immunized subcutaneously by administering 2 cm ^{3 of a} whole vaccine. The vaccine in its entirety already on the 7th day after vaccination induced a sufficient level of neutralizing antibodies (1.78 ± 0.11 lg), and by the 35th day after vaccination the number of neutralizing antibodies (VNA) increased to 2.11 ± 0.26 lg (table 8). After infection, 2 control heads of cattle and 1 vaccinated head of cattle fell ill with generalization of the process.

Example 6

For the manufacture of an inactivated emulsion type O vaccine against foot and mouth disease, type O virus strain No. 2102 / Zabaykalsky / 2010 is reproduced in a suspension culture of BHK-21 cells, inactivated, purified from ballast impurities, the resulting antigen is checked for avirulence, the content of virus-specific protein, 146S and 75S components of the virus and sterility as described in example 4.

The required concentration of 146S and 75S components in the vaccine dose of the emulsion vaccine is obtained by concentration of the antigen by flow ultrafiltration. For this, BTU 0.5-2 ultrafilters are used. The resulting antigen concentrate is stored at 4-6 ° C until used in the vaccine.

An emulsion vaccine is obtained by dispersing antigen concentrate and oil adjuvant in a ratio of 3: 7-1: 1, respectively, in colloidal mills. From oil adjuvants that are used in FSBI "ARRIAH" (GOA, saponin), as well as oil adjuvant brand Montanide ISA-70 or Montanide ISA-206 manufactured by Seppic (France, standard ISO 9001).

The result is an inactivated emulsion vaccine against foot and mouth disease virus type O, which is a milk-like liquid insoluble in water. The vaccine has a liquid consistency, easily dissolves at the injection site, does not cause the formation of abscesses, a general reaction in the form of a rise in temperature, and has a pronounced immunogenicity for cattle at a dose of 2 cm ³ 35 days after vaccination. The vaccine is administered cattle subcutaneously. Inoculated volume should contain at least 4 μg of 146S and 75S components.

Example 7

Tests of the immunogenic activity of FMD vaccine inactivated emulsion from strain O No. 2102 / Zabaykalsky / 2010, manufactured as described in example 6. The immunogenic activity of this vaccine was tested for cattle weighing 200-250 kg The results of the studies are presented in table 8. Cattle grafted with a whole vaccine based on the Montanide ISA-70 brand adjuvant, on day 35 after vaccination had a BHA titer of 2.63 ± 0.16 lg, and based on the Montanide ISA-206 brand adjuvant 2.57 ± 0.16 log. After infection control, only 2 control heads of cattle fell ill with generalization of the process.

Example 8

Strain O No. 2102 / Zabaykalsky / 2010 of foot and mouth disease virus Aphtae epizooticae type O, designed to control the immunogenic activity of FMD vaccines in cattle, is prepared as follows. The resulting virus is pre-refreshed on cattle, infecting 1-2 animals weighing 250-300 kg, delivered from the FMD-free zones of the country, where animals have not been vaccinated against FMD over the past 2 years. The number of passages of the virus should not exceed 20, starting from the original virus. The virus suspension containing 10000-10000000 ID _50/1 cm ³ with antibiotics administered by 0.2-0.3 cm ³ 20-40 intradermalingvalno channels across the tongue surface. Infected animals are not fed until the virus is removed. After 20-30 hours after infection, aphthae are removed as they mature. Lymph is collected by syringe. Based on the collected aphthous material, a 20% suspension of the virus is prepared, which is purified from ballast impurities, followed by the addition of antibiotics and an equal volume of glycerin. The resulting 10% virus suspension was evaluated in CSC for type-specificity and by RT-PCR followed by nucleotide sequencing to match the original virus according to the primary structure of the VP1 gene. The activity of the virus in CSC should not be lower than 1: 4.

Infectious activity of the virus is determined on cattle and in the primary culture of SP cells. The control virus should have a titer of infectious activity of at least 10 ^4.0 ID ₅₀ / 0.1 cm ³ . Vials with a suspension of the virus in the presence of glycerol are stored in the refrigerator at a temperature of minus (70 ° C - 40 ° C).

Example 9

Strain O No. 2102 / Zabaykalsky / 2010 of the FMD virus type O, designed to control the immunogenic activity of FMD vaccines in pigs, is prepared as follows. The resulting virus is pre-refreshed in pigs, infecting 1-2 animals weighing 30-50 kg, delivered from the FMD-safe areas of the country where cattle against foot and mouth disease have not been vaccinated for 2 years. The virus-containing suspension is injected under the mucous membrane of the tongue or into the skin of the corolla of the extremities. Mature aphthae are removed 24-72 hours after infection. The preparation of a suspension of the virus and the control of its activity is carried out as described in example 8. Titration of the infectious activity of the virus of foot and mouth disease is carried out in pigs and in cell culture SP. The control virus should have a titer of infectious activity of at least 10 ^4.0 ID ₅₀ / 0.1 cm ³ .

Information sources

1. Rörer X. Foot and mouth disease. Per. with him. G.A. Surkova. / Ed. and with the foreword. Cand. vet. sciences. P.V. Malyarets. - M .: Kolos, 1971, 432 p.

2. Foot and mouth disease / A.N. Burdov, A.I. Dudnikov, P.V. Malyarets [et al.] - M., Agropromizdat, 1990, 320 pp.

3. Viral diseases of animals / Syurin VP, Samuilenko A.Ya., Soloviev B.V. [and others] - M., VNIITIBP, 1998, S.532-548.

4. Immunobiological properties of the epizootic strain of foot and mouth disease virus type O No. 1964 / Mongolia / 2004 / T.A. Fomina, V.K. Spirin, A.I. Egorova [et al.] // Proceedings of the Federal Center for Animal Health. - Vladimir, 2005. - T. 3. - S. 94-104.

5. Immunobiological properties of the epizootic strain of foot and mouth disease virus type O No. 1734 "Primorsky-2000" / V.K. Spirin, A.I. Egorova, S.R. Kremenchug [et al.] // Proceedings of the Federal Center for Animal Health. - Vladimir, 2007.- T. 5. - S. 52-57.

6. Pat. RF №2204599, C12N 7/00, А61К 39/135, 05.20.2003

7. Southeast Asian Foot-and-Mouth Disease Vimses in Eastern Asia / N.J. Knowles, J.J. He, Y. Shang [et al] // Emerg Infect Dis. - 2012 .-- V. 18 (3): P. 499-501.

8. OIE. Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and beers). - 7th Edition, Paris, 2008. - Vol. 1. - P. 203-207

9. Guidelines for the identification and identification of FMD virus strains / Gusev AA, Zakharov VM, Shazhko Zh.A. [and etc.]. Vladimir, 2002, 31 p.

Claims (6)

1. The strain of FMD virus Aphtae epizooticae type O, fam. Picornaviridae, genus Aphtovirus, deposited in the collection of microorganism strains of the Federal State Budget Scientific Institution VNIIZZH under registration number foot and mouth disease virus strain No. 2102 / 3abaykalsky / 2010 (production) for the control of antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type O characterized by the fact that it was obtained during sequential passage in cell cultures of homologous and heterologous origin, has a high biological activity in native form and maintains a high antigenic and immunogenic activity after inactivation. 2. The strain according to claim 1, characterized in that it was obtained for 3 consecutive passages in a primary trypsinized cell culture of the kidneys of the Siberian mountain ibex (PSGK-30) with an infectious activity titer of at least 6.63 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (CSC) 1: 4. 3. The strain according to claim 1, characterized in that it is obtained for 3 consecutive passages in a transplanted cell culture of porcine kidney (SP) with a titer of infectious activity of at least 7.0 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (RSK) 1: 8. 4. The strain according to claim 1, characterized in that it was obtained for 3 consecutive passages in an inoculated culture of pig kidney cells (IB-RS-2) with a titer of infectious activity of at least 6.75 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (CSC) 1: 6. 5. The strain according to claim 1, characterized in that it was obtained for several consecutive passages in an inoculated culture of Syrian hamster kidney cells (BHK-21) with an infectious activity titer of at least 7.5 lg TCD ₅₀ / cm ³ and antigenic activity in complement fixation reaction (CSC) 1:12. 6. The strain according to any one of claims 1 to 5, characterized in that after inactivation it induces the formation of virus-specific antibodies in the guinea pigs organism, detected in CSC at dilutions of 1:30 and in ELISA at a dilution of 1: 10000.

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