RU2451745C2 - A 2045/KYRGYZSTAN/ 2007 STRAIN OF FOOT-AND-MOUTH DISEASE VIRUS Aphtae epizooticae TYPE A FOR ANTIGENIC AND IMMUNOGENIC PERFORMANCE CONTROL OF FOOT-AND-MOUTH DISEASE VACCINES AND FOR PRODUCTION OF BIOPREPARATIONS FOR DIAGNOSIS AND SPECIFIC PREVENTION OF FOOT-AND-MOUTH DISEASE TYPE A - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to veterinary virology and biotechnology, as well as the foot-and-mouth disease virus Aphtae epizooticae type A Picornaviridae, genus Aphtovirus, which is deposited in the Russian State Centre for Quality and Standardisation of Veterinary Drugs and Feed under registration number A No.2045/Kyrgyzstan/2007 - DEP. The disclosed strain is reproduced in a monolayer swine kidney cell culture, passaged Siberian mountain goat kidney cell cultures (PSGK-30), VHK-21 and IB-RS-2. After 18-24 hours of incubation, virus yield in said cell cultures reaches 6.0-7.5 Ig TCD₅₀/cm³. For multiplicity of infection (1-10 TCD/cell), the strain causes CPD after 5 hours, while preserving the initial characteristics when passaging in cell cutures for 10 passages.

EFFECT: disclosed strain can be used to control antigenic and immunogenic performance of foot-and-mouth disease vaccines and to produce biopreparations for diagnosis and specific prevention of foot-and-mouth disease type A.

6 cl, 1 dwg, 10 tbl, 12 ex

Description

Strain A No. 2045 / Kyrgyzstan / 2007, foot and mouth disease virus Aphtae epizooticae type A for controlling 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 A.

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 A.

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, 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, revenues in dairy and beef cattle breeding are reduced by (%): 30–40.

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.

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 foot and mouth disease virus is caused by amino acid substitutions in polypeptide fragments (antigenic epitopes) exposed on the surface of capsid proteins.

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.

Type A foot and mouth disease virus strains have been used as production strains in the USSR and the Russian Federation for the past 50 years.

These include the following strains: A ₇ No. 103, isolated in 1962 in the Kuibyshev region; A ₇ No. 2, allocated in 1965 in the Tajik SSR; And No. 717/73, allocated in 1973 in the Stavropol Territory.

These strains were used to obtain diagnosticums and FMD vaccines used in various regions of the country.

However, after the elimination of foot-and-mouth disease caused by strains of the virus that are closely related to them in antigenicity, they were discontinued and are currently only supported in the collection of microorganism strains of the Federal State Institution “ARRIAH” (1–7).

The known strain No. 550 of foot-and-mouth disease virus A ₂₂ , isolated in 1964 in Azerbaijan and used in the Russian Federation as an industrial one in the manufacture of specific prophylaxis and diagnostics, is used throughout Russia and in the countries of the former Soviet Union (8, 9).

The disadvantages of this strain are its lack of antigenic and immunogenic activity against epizootic isolates of FMD virus type A, currently circulating in the countries of the Caucasus, Central Asia and the Middle East (Turkey, Iran, etc.) due to the significant antigenic differences.

The known strain No. 1707 "Armenia-98" of foot and mouth disease virus Aphtae epizooticae type A for the manufacture of diagnostic and vaccine preparations (10).

Also known is strain A (Georgia) 1999 / No. 1721 of the FMD virus Aphtae epizooticae type A for the manufacture of diagnostic and vaccine preparations (11).

In recent years, two genetic lines of type A foot and mouth disease virus have dominated in Central Asia and the Middle East: line A / Iran / 96 (this genetic group includes the Russian production strain No. 1707 “Armenia-98”) and A / Iran / 2005. The genetic line A / Iran / 99 is not as widespread as the two mentioned, and perhaps therefore the strains from group A / Iran / 99 are not included in the priority list of the recommended OIE / FAO vaccine strains.

The isolation of FMD virus type A from the cattle of the Amassi region of the Republic of Armenia in 1998 from the cattle from the cattle and study of its phylogenetic relationship showed that in a comparative study of the primary structure of the VP ₁ gene _of strain No. 1707 “Armenia-98”, FMD virus of type A with production strains and field isolates found that the isolates Armenia / 98 are identical to each other and are related to type A strains Turkey / 97, Turkey / 98 and Iran / 96. It was also found that the studied isolates significantly differ from all previously isolated isolates, including the A ₂₂ subtype virus (18% of the differences), which includes strain A ₂₂ No. 550 used for the production of diagnostic tools and specific prophylaxis. Strain A (Georgia) 1999 / No. 1721 of foot and mouth disease virus type A. Strain A No. 1707 “Armenia-98” and A (Georgia), isolated in 1999 from the sick cows in the private sector of the village of Ude of the Adigen region of the Republic of Georgia, belongs to the same line 1999 / No.1721 was deposited on November 12, 1998 and August 19, 2003, respectively, into the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, FGU VGNKI.

Production strain A No. 1707 “Armenia-98” of type A foot and mouth disease virus was used as a part of FMD vaccines in the buffer zone of Transcaucasia.

The closest to the invention according to the set of essential features is strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus for the manufacture of diagnostic and vaccine preparations (11).

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

In 2003, a new type A FMD virus isolate was isolated in Iran, significantly different from the previously studied strains of this type. During 2005 ÷ 2006 Type A foot and mouth disease has become widespread in West Asia - Iran, Turkey, Saudi Arabia and Pakistan. In February 2006, Type A foot and mouth disease of the Iran / 05 line came to Thrace, the European part of Turkey, which borders Bulgaria and Greece. The 100% vaccination of all ruminants with the use of strain A ₂₂ in Thrace in February-March 2006 prevented the spread of foot and mouth disease to neighboring Greece and Bulgaria, however, fresh cases of the disease appeared 3–4 months later (12).

In this regard, it became necessary to obtain a new production strain from the epizootic virus of foot and mouth disease serotype A 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 A, 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 FMD vaccines and for the manufacture of sensitive and highly specific diagnostics and highly immunogenic vaccine preparations homologous to the epizootic virus, appeared on the territory of the Republic of Kyrgyzstan.

This problem was solved by obtaining strain A No. 2045 / Kyrgyzstan / 2007 (author's name) of type A foot and mouth disease virus used to control antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for the diagnosis and specific prevention of type A foot and mouth disease.

The viral isolate, which served as the source for strain A No. 2045 / Kyrgyzstan / 2007, was isolated from samples of aphthous material from a sick heifer, received at the Federal State Research Institute of Health Protection in December 2007 from the Republic of Kyrgyzstan (examination No. 2045 "Kyrgyzstan / 07"). Production strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus was obtained by successive passages on sensitive hetero- and homologous cell cultures.

Strain A No. 2045 / Kyrgyzstan / 2007, type A foot-and-mouth disease virus was deposited on July 7, 2009 at the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, Federal State Institution “All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" (FGU " VGNKI ”) under the registration number (link): production strain A No. 2045 / Kyrgyzstan / 2007-DEPT of FMD virus type A.

Compared with the prototype strain, strain A No. 2045 / Kyrgyzstan / 2007 has a higher infectious, antigenic and immunogenic activity in its native form and after inactivation.

The experimentally confirmed the possibility of using strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus to control 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 A.

The invention is illustrated in a graphical image showing a dendrogram reflecting the phylogenetic relationships of strain A No. 2045 / Kyrgyzstan / 2007 of foot and mouth disease virus serotype A with other epizootic and vaccine strains of foot and mouth disease virus serotype A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₁ gene.

The invention is illustrated in the list of sequences in

which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP1 protein gene of strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus.

Strain A No. 2045 / Kyrgyzstan / 2007 of the FMD virus type A is characterized by the following features and properties.

Morphological features

Strain A No. 2045 / Kyrgyzstan / 2007, type A foot and mouth disease virus belongs to the family Picornaviridae, genus Aphtovirus, serotype A 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 A No. 2045 / Kyrgyzstan / 2007 of the FMD virus belongs to serotype A. 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, RDP and RMN.

During hyperimmunization of guinea pigs, the concentrated antigen from inactivated foot and mouth disease type A virus of strain A No. 2045 / Kyrgyzstan / 2007 induces the formation of virus-specific and virus-neutralizing antibodies detected in CSC in dilutions of 1: 80 ÷ 1: 160 and in ELISA in dilutions of 1: 10000 ÷ 1: 12,000.

The method of nucleotide sequencing was determined the primary structure of VP ₁ gene of FMD virus type A strain A №2045 / Kyrgyzstan / 2007 and the deduced primary structure of the protein VP _1. A comparative analysis of the nucleotide sequences showed that type A strain A No. 2045 / Kyrgyzstan / 2007 of foot and mouth disease virus has a close phylogenetic relationship with strains A / Iran / 05 and A / Turkey / 06 (the percentage of nucleotide differences 3.82 ÷ 4.3 and 4, 46 ÷ 5.57, respectively) and is significantly different from vaccine strain A ₂₂ No. 550. In addition, it differs from strains of the virus of this type isolated in 1998 in Armenia and Turkey, as well as in Georgia and Iran in 1999 (see dendrogram). The degree of nucleotide differences in the sequences of strain A No. 2045 / Kyrgyzstan / 2007 of FMD virus type A with strains of FMD virus of serological type A amounted to: with strain A ₂₂ No. 550 - 18.15%, with strain A No. 1707 “Armenia-98” - 17, 68%, with strain A / Turkey / 98 - 17.2%, with strain A (Georgia) 1999 / No.1721 - 18.79%, with strain A / Iran / 99 - 17.68%.

Thus, phylogenetic analysis showed that strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus belongs to the new genetic line of foot and mouth disease virus of type A.

The antigenic affinity of strain A No. 2045 / Kyrgyzstan / 2007 of type A foot-and-mouth disease virus with the production of foot-and-mouth disease virus strain A ₂₂ No. 550 and strains previously isolated on the Asian continent was studied in CSC, ELISA and RMN.

The results of studies in CSCs are presented in table 1. Antigenic affinity (R) of strain A No. 2045 / Kyrgyzstan / 2007 with other FMD viruses of serological type A amounted to 8% for A / Iran /, A ₂₂ / Iraq 24/64 - 28%, A / Iran / 05 - 68%, A / Turkey / 06 - 66%, A ₂₂ No. 550 - 14%.

The results of the studies in the RMN are presented in table 2. Antigenic compliance (r ₁ ) was for A / Iran / 97 - 0.125, A ₂₂ No. 550 - 0.17, A ₂₂ / Iraq 24/64 - 0.6, A / Turkey / 06 - 0.5. When r ₁ ≥0.3, the field isolate and 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 from an epizootic virus (13).

The results of studies in ELISA are presented in table 3. Antigenic compliance (r ₁ ) amounted to A ₂₂ No. 550 - 0.24. A ₂₂ / Iraq 24/64 - 0.24, A / Iran / 97 - 0.19, A / Turkey / 06 - 0.5. When the value of r ₁ equal to 0.4 ÷ 1.0, the field isolate and production strain are in close antigenic relationship; if 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 (14).

Biotechnological characteristics

Strain A No. 2045 / Kyrgyzstan / 2007 is reproduced in a monolayer culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), VNK-21 and IB-RS-2 and during 18-24 hours of incubation the crop virus in these cell cultures reaches values from 6.0 to 7.5 Ig 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).

Genotaxonomic characteristic

Strain A No. 2045 / Kyrgyzstan / 2007, type A foot and mouth disease virus 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 (VP _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 A No. 2045 / Kyrgyzstan / 2007 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 A No. 2045 / Kyrgyzstan / 2007, was isolated at VNIIZZH from samples of aphthous material obtained in December 2007 from a cattle foot and mouth disease suspect from the Republic of Kyrgyzstan (examination No. 2045 “Kyrgyzstan / 07” "). Samples of aphthous material arrived at the Federal State Institution "ARRIAH" on December 28, 2007.

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 (15).

Biological and virological methods included:

virus isolation on a culture of primary trypsinized SP cells, followed by adaptation on cultures of transplantable cell lines PSGK-30, IB-RS-2, BHK-21 and cattle (2 passage). To set up bioassays on primary and transplanted cell cultures, they were grown on appropriate nutrient media under stationary conditions in 50 ÷ 100 cm ³ vials, 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 ° С, 5–10 cm ^{3 of the} supporting medium were introduced into the vials and incubated at 37 ° С 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 the CSC for the presence of a viral antigen, and a set of commercial type-specific sera stored in the museum of strains of the Federal State Institution VNIIZZh was used. The virus was considered adapted to cell cultures if, within 18–24 hours (%): 90–100 CPP in a monolayer appeared.

Adaptation of epizootic isolate No. 2045 “Kyrgyzstan / 07” to various cell lines occurred at the level of 3–5 passage. 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 the experimental vaccine series, as well as to obtain the antigen for hyperimmunization of guinea pigs.

The virus adapted to the IB-RS-2 cell culture was used to produce antigen for CSC and ELISA.

The results of the adaptation of the virus to various cell cultures are presented in table 4.

The data shown in table 4 indicate a good adaptive ability of strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus to cell cultures used.

Isolated using the above methods, the viral preparation was studied in RSK and ELISA to identify its type affiliation (tables 5 and 6). 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 5 and 6 indicate that in the aphthous test material No. 2045 “Kyrgyzstan / 07” an FMD virus antigen of type A was detected at a dilution of 1: 4 in RSK and 1:32 in ELISA. No contamination with bacterial and fungal microflora, mycoplasmas and extraneous viruses was found.

The resulting strain of FMD virus type A is assigned the copyright name: strain A No. 2045 / Kyrgyzstan / 2007.

Strain A No. 2045 / Kyrgyzstan / 2007, type A foot-and-mouth disease virus was deposited on July 7, 2009 at the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, Federal State Institution “All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" (FGU " VGNKI ”) under the registration number (link): production strain A No. 2045 / Kyrgyzstan / 2007-DEPT of FMD virus type A.

Example 2

For hyperimmunization of guinea pigs, the antigen from strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus reproduced in a monolayer PSGK-30 cell culture is used. The virus-containing suspension is cleaned of ballast impurities by the addition of 0.05% polyhexamethylene guanidine (PHMG). The purified virus is concentrated 100 times by adding 10% polyethylene glycol (PEG) m.m. 6000 and inactivate aminoethylethyleneimine (AEEI) at a concentration of 0.025 ÷ 0.05% at a pH value 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 methodological recommendations for the identification and identification of FMD virus strains (15).

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 ampoules 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 7.

The data shown in table 7 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 immunochemical reactions, FMD virus of type A strain A No. 2045 / Kyrgyzstan / 2007, adapted to the cell culture of the transplantable lines BHK-21, PSGK-30 and IB-RS-2, is used. For adaptation, virus-containing material is used in the form of a 10% suspension of cattle aft. 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 diagnostic components for ELISA.

In this way, 3 series of diagnostic antigen were prepared, the characteristics of which are shown in table 8.

The research results, shown in table 8, 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 a vaccine inactivated sorbed foot and mouth disease virus type A strain A No. 2045 / Kyrgyzstan / 2007 reproduced in suspension culture cells VNK-21. As a supporting medium, an Earle solution without serum is used with the addition of FGMS, GBKS and antibiotics at a pH of 7.2–7.4. The cell culture is infected with a virus at the rate of 0.001 ÷ 0.05 TCD 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 is continued for another 2 ÷ 3 hours. Upon reaching the number of dead cells (%): 90 ÷ 95, the cultivation is stopped and the virus-containing suspension is monitored for sterility and the content of 146S and 75S 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% solution of AEEI, 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 (%): 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 (%): 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 that forms at the bottom of the vial during storage and easily breaks up into a homogeneous suspension with shaking.

Example 5

Tests for cattle of FMD vaccine inactivated adsorbed from strain A No. 2045 / Kyrgyzstan / 2007, manufactured as described in example 4, were performed.

Cattle weighing 200 ÷ 250 kg were immunized subcutaneously, administering 2 cm ^{3 of a} whole vaccine and in dilutions of 1: 4 and 1:16. The vaccine in its entirety already on the 10th day after vaccination (DPV) induced a sufficient level of neutralizing antibodies (4.50 ± 0.52 log ₂ ), and by 20 DPV the number of neutralizing antibodies (VNA) doubled to 5.55 ± 0. 20 log ₂ . The protective (protective) dose of the vaccine for 50% of the animals (PD ₅₀ ) was 0.25 cm ³ , i.e. in a vaccine dose of 2 cm ³ contained 8 PD ₅₀ , indicating a high immunogenic activity of the sorbed vaccine.

Example 6

Comparative tests on guinea pigs of the immunogenic activity of the vaccine inactivated adsorbed against foot and mouth disease type A from strain A No. 2045 / Kyrgyzstan / 2007 and the vaccine inactivated adsorbed against foot and mouth disease from strain A ₂₂ No. 550, made as described in example 4.

To determine the immunogenic activity of each drug, 128 guinea pigs were used, and in addition to control 4 groups of guinea pigs, 6 animals each. Vaccines were administered intact and diluted with phosphate buffer in a ratio of 1: 3, 1: 9 and 1:27. Eight guinea pigs were taken for each vaccine dilution. Each group of 8 pigs was kept in a separate enclosure, on which a label was fixed indicating the vaccine series, its breeding, the date of the start of testing and the number of animals vaccinated with this breeding.

Each vaccine was administered to 6 groups of guinea pigs intramuscularly in a volume of 2 cm ³ in the region of the right and left thigh 1 cm ³ . Unvaccinated guinea pig groups were used to test the activity of control serotypes of foot and mouth disease virus.

Vaccinated guinea pigs were monitored for 21 days. After that, all vaccinated and control pigs were infected with foot-and-mouth Guinea pig-adapted virus of strains A ₂₂ No. 550 and A No. 2045 / Kyrgyzstan / 2007. The viral suspension was injected intradermally into the plantar surface of one of the hind legs at a dose of 10 ⁴ HD / 0.1 cm ³ . The results of infection were taken into account after 3, 5, and 7 days and were assessed by generalization of the foot-and-mouth disease process on the front and one of the hind legs. The generalization was the formation of secondary aphthae on at least one paw into which the virus was not introduced. Vaccine control was considered valid if out of 6 unvaccinated pigs, 6 animals fell ill with a generalized form of foot and mouth disease. Table 9 presents the results of a study on guinea pigs of the immunogenic activity of monovalent FMD vaccines inactivated adsorbed from strains A No. 2045 / Kyrgyzstan / 2007 and A ₂₂ No. 550 with a control infection with homologous and heterologous FMD virus strains No. 2045 / Kyrgyzstan / 2007 and A ₂₂ No. 550. The vaccine prepared from strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus, when tested in guinea pigs, turned out to be a highly immunogenic preparation against homologous virus (ImD ₅₀ is 0.050) and less immunogenic against FMD virus of strain A ₂₂ No. 550 (Im ₅₀ = 0 , 25).

Example 7

For the manufacture of an inactivated emulsion type A vaccine against foot and mouth disease vaccine, strain A virus No. 2045 / Kyrgyzstan / 2007 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 use 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 colloid mills. Of the oil adjuvants, the VNIIZH oil adjuvant can be used (16), as well as the Montanide ISA-70 or Montanide ISA-206 oil adjuvant manufactured by Seppic (France, standard ISO 9001).

The result is an inactivated emulsion vaccine against foot and mouth disease virus type A, which is a milk-like liquid insoluble in water. The vaccine has a liquid consistency, easily resolves at the injection site, does not cause abscesses, a general reaction in the form of a rise in temperature, and has a pronounced immunogenicity for pigs and cattle at a dose of 2 cm ³ through 28 DPV. The vaccine is administered intramuscularly to pigs, and cattle subcutaneously. Inoculated volume should contain at least 4 μg of 146S and 75S components.

Example 8

The immunogenic activity of the FMD vaccine inactivated emulsion from strain A No. 2045 / Kyrgyzstan / 2007, made as described in Example 7, was tested. The immunogenic activity of this vaccine was tested on gilts weighing 40–50 kg. The research results are presented in table 10. ImD ₅₀ vaccines amounted to 0.05 cm ³ , i.e. in the inoculated volume of 2 cm ³ contained 40 PD ₅₀ for pigs. Gilts vaccinated with whole vaccine at 21 WPV had a BHA titer of 6.50 ± 0.20 log ₂ . After infection, 2 control gilts and 1 gilts vaccinated with a 1:25 dilution vaccine became ill with a generalization of the process.

The tested emulsion vaccine had a high immunogenic activity in pigs (40 PD ₅₀ / 2.0 cm ³ ).

Example 9

The FMD virus type A strain A No. 2045 / Kyrgyzstan / 2007, designed to control the immunogenic activity of FMD vaccines in cattle, is prepared as follows. The resulting virus is preliminarily 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. A virus suspension containing 10,000 ÷ 10,000,000 ID ₅₀ / cm ³ with antibiotics is administered at 0.2 ÷ 0.3 cm ³ in 20 ÷ 40 channels intradermolingually over the entire surface of the tongue. Infected animals are not fed until the virus is removed. After 20–30 hours after infection, the 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 as described above, followed by the addition of antibiotics and glycerol. The resulting suspension of the virus is evaluated in CSC and by RT-PCR followed by nucleotide sequencing for type and strain specificity. Virus activity in CSCs should be at least 1: 4.

Infectious activity of the virus is determined on cattle. The control virus must have an infectivity titer of at least 10 ^4.0 ID ₅₀ / 0.2 cm ³ . Vials with a suspension of the virus in the presence of glycerol are stored in the refrigerator at minus (40 ° C) ÷ (70 ° C).

Example 10

The FMD virus type A strain A No. 2045 / Kyrgyzstan / 2007, 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-free zones of the country, where livestock was not 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 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 9. Titration of the infectious activity of the foot and mouth disease virus is carried out on pigs and in the culture of SP cells.

Example 11

Monitoring the immunogenic and antigenic activity of the vaccine inactivated adsorbed from FMD virus type A strain A No. 2045 / Kyrgyzstan / 2007, made as described in example 4, was carried out as follows. To test the immunogenic activity of the drug, 17 heads of cattle weighing 200 ÷ 250 kg were used, delivered from the country's FMD zones. The first group of animals from 5 heads of cattle was administered the vaccine subcutaneously in their entirety. The second group of animals from 5 goals of cattle was injected subcutaneously with a dilution of the vaccine in phosphate-buffered saline in a ratio of 1: 4 and the third group of 5 animals at a dilution of 1:16.

The vaccine dose volume was 2.0 cm ³ . At 20 DPA at 15 and 2 vaccinated control head of cattle blood samples taken and held challenged animal by transmucosal administration language suspensions of FMD virus type A strain A №2045 / Kyrgyzstan / 2007 the third passage to the RNC in a dose of 10 ^4.0 ID _50/0 , 2 cm ³ .

The antigenic activity of the vaccine was monitored by the level of BHA in serum in the pH on the monolayer of the primary trypsinized culture of SP cells. At 20 DPA, the VNA level in cattle vaccinated with the whole vaccine was 5.50 ± 0.20 log ₂ p <0.001. 8 days after infection, a pathological examination of cattle was performed. 2 control animals, 1 animal vaccinated with a dilution of 1: 4 vaccine, and 4 animals vaccinated with a 1:16 vaccine dilution were ill. PD _{50 of the} vaccine was 0.25 cm ³ . The graft dose of the vaccine contained 8 PD ₅₀ , which indicated the effectiveness of the drug. Such a vaccine is considered immunogenic and suitable for practical use.

Example 12

The control of the immunogenic and antigenic activity of the inactivated emulsion vaccine from foot and mouth disease virus type A strain A No. 2045 / Kyrgyzstan / 2007, made as described in example 7, was carried out as follows.

To test the immunogenic activity of the drug, 17 pigs weighing 30–50 kg were used. Pigs were divided into 3 groups of 5 animals each. Two animals were used as controls. The first group of animals was immunized intramuscularly with a whole dose of 2.0 cm ³ vaccine containing 6.0 μg of the immunogenic components of foot and mouth disease virus type A strain A No. 2045 / Kyrgyzstan / 2007. The second group of animals was immunized with a dilution of the vaccine 1: 5, and the third - a dilution of 1:25. At 21 DPV, 15 vaccinated and 2 control pigs received a suspension of aphthous foot-and-mouth disease virus A of strain A No. 2045 / Kyrgyzstan / 2007 adapted to pigs under the tongue mucosa at a dose of 10 ⁴ ID ₅₀ / 0.2 cm ³ . The amount of VNA in pigs vaccinated with a whole dose of the vaccine was 6.50 ± 0.20 log ₂ p <0.001. On the 8th day after infection, a pathological study of the animals was performed. 2 control and 1 animals, vaccinated with 1:25 vaccine dilution, got sick with a generalized form of foot and mouth disease. ImD _{50 of the} vaccine was 0.05 cm ³ . The graft dose of the vaccine contained 40 PD ₅₀ for pigs.

Thus, the vaccine is considered immunogenic and suitable for practical use.

Table 1 Antigenic spectrum of strain A No. 2045 / Kyrgyzstan / 2007, foot and mouth disease virus of serological type A according to DSC Compare strains Indicators r ₁ r ₂ R% A / Iran / 97 0.05 0.11 8 A ₂₂ / Iraq 24/64 0.08 1,0 28 A / Iran / 05 0.46 1,0 68 A / Turkey / 06 0.59 0.76 66 A ₂₂ No. 550/64 0.02 1,0 fourteen Note: R≥40% - FMD virus strains belong to the same subtype R = (%): 25 ÷ 40 - to different subtypes.

table 2 Antigenic compliance (r ₁ ) in RMN of isolate A No. 2045 / Kyrgyzstan / 2007 with type A foot and mouth disease virus strains Strains Exponent r ₁ A / Iran / 97 0.125 A ₂₂ No. 550 0.17 A ₂₂ Iraq 24/64 0.6 A Turkey / 06 0.5

Table 3 Antigenic compliance (r ₁ ) in the ELISA of strain A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus to type A foot and mouth disease virus Strains Exponent r ₁ A ₂₂ No. 550 0.24 A ₂₂ Iraq 24/64 0.24 A Iran / 97 0.19 A Turkey / 06 0.5

Table 4 Biological properties of the virus of epizootic strain A No. 2045 / Kyrgyzstan / 2007, type A foot and mouth disease virus Virus reproduction system The time of manifestation of biological activity (hours) Number of adaptation passages Adapted material characteristics Activity
in RSK The titer of infectious activity (lgTCD ₅₀ / cm ³ ) Joint venture twenty 3 1: 2 6.5 PSGK-30 twenty 3 1: 6 7 IB-RS-2 24 four 1: 4 6.33 VNK-21 24 5 1:12 7.5 ÷ 8.25

Table 5 Determination of the type of virus in a 33% suspension of aphthous material in the CSC (examination No. 2045 "Kyrgyzstan / 07") g / and serum in working dilution Examination No. 2045 The control target
ny 1: 2 1: 4 1: 8 to / a A _/ Turkey / 06 O / Seaside / 2000 Asia-1 Amur / 2005 Sat-1 Sat-2 Sat-3 b / a A / Turkey / 06 four four 3 - four - - - - - - O / Seaside / 2000 - - - - - four - - - - - Asia-1 Amur / 2005 - - - - - - four - - - - Sat-1 - - - - - - - four - - - Sat-2 - - - - - - - - four - - Sat-3 - - - - - - - - - four - b / s - - - - - - - - - - - b / c four four four four four four four four four four four Note: 4-100% delayed hemolysis; "-" - complete hemolysis b / s - without serum b / a - without antigen b / c - no complement

Table 6 Determination of the type of virus in a 10% suspension of aphthous material in ELISA (examination No. 2045 "Kyrgyzstan / 07") Antigen Activity in the diagnostic system A ₂₂ O ₁ Asia 1 A Kyrgyzstan / 07 1:32 - - Control A ₂₂ 1: 128 - - Control O ₁ - 1: 128 - Control Asia-1 - - 1: 256

Table 7 The characteristic of the diagnostic serum obtained for the antigen from strain A No. 2045 / Kyrgyzstan / 2007, foot and mouth disease virus serological type A Named
preparation Series Volume (cm ³ ) Activity in RSK with homologous diagnosticum with heterologous diagnostics A A ₂₂ No. 550 A Turkey / 06 A ₂₂ Iraq 24/64 A Iran / 97 Hyperimmune Serum one 200 1:80 <1: 4.9 1: 152 1:20 1:14

Table 8 Characterization of diagnostic antigens from strain A No. 2045 / Kyrgyzstan / 2007 of the FMD virus of serological type A grown on various cell cultures The name of the drug Series Volume (cm ³ ) Activity in RSK with homologous diagnosticum A No. 2045 / Kyrgyzstan / 2007 with heterologous diagnosticum A ₂₂ No. 550 PSGK-30 culture antigen one 40 1:16 <1: 2 Cultural Antigen IB-RS-2 one 60 1:12 <1: 2 Culture antigen VNK-21 one one hundred 1:16 <1: 2

Table 9 Cross-testing in Guinea Pigs of the immunogenic activity of vaccines from strain A ₂₂ No. 550 of foot and mouth disease virus and strain A No. 2045 / Kyrgyzstan / 2007 of foot and mouth disease FMD strains for infection control The vaccine from strain A ₂₂ No. 550 of the virus of foot and mouth disease type A The vaccine from the strain of the virus of foot and mouth disease A No. 2045 / Kyrgyzstan / 2007 ImD ₅₀ MS PD ₅₀ MS ImD ₅₀ MS PD ₅₀ MS A ₂₂ No. 550 0,07 28.5 0.25 8.0 A No. 2045 / Kyrgyzstan / 2007 0.21 9.5 0.05 40,0 Note: ImD ₅₀ - 50% immunizing dose; PD ₅₀ - 50% protective dose in vaccination volume

Table 10 The results of the test vaccine inactivated emulsion against foot and mouth disease type A from strain A No. 2045 / Kyrgyzstan / 2007 in pigs Vaccinating
naya dose, cm ³ Vaccine dilutions The number of 146S + 75S components in the vaccination dose (mcg) The amount of VNA (log ₂ ) in serum at 21 DPA The number of gilts protected from infection control PD ₅₀ (cm ³ ) 2 Ts 6.0 6.50 ± 0.20 5/5 40 p <0.001 2 1: 5 1,2 3.67 ± 0.45 5/5 p <0.001 2 1:25 0.24 2.43 ± 0.61 4/5 p <0,025 The control 0/2

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention “Strain A No. 2045 / Kyrgyzstan / 2007, foot and mouth disease virus Aphtae epizooticae type A for monitoring the antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for diagnosis and specific prevention foot and mouth disease type A ".

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Claims (6)

1. The strain of FMD virus Aphtae epizooticae type A sem. Picornaviridae, of the genus Aphtovirus, deposited in the collection of the Federal State Institution VGNKI under registration number A No. 2045 / Kyrgyzstan / 2007 - DEPT, for the control of antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for the diagnosis and specific prophylaxis of foot and mouth disease type A, characterized in that it was obtained during sequential passage in cultures of cells of homologous and heterologous origin, has high biological activity in its native form and retains high antigenic and immunogenic activity s after inactivation. 2. The strain according to claim 1, characterized in that it is obtained for 3 consecutive passages in a culture of pig kidney cells (SP) with a titer of infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction ( RSK) 1: 2. 3. The strain according to claim 1, characterized in that it was obtained for 3 consecutive passages in a culture of kidney cells of the Siberian mountain ibex (PSGC-30) with a titer of infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ and antigenic activity in RSK 1: 4. 4. The strain according to claim 1, characterized in that it was obtained for 4 consecutive passages in cell culture, IB-RS-2 with a titer of infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and antigenic activity in CSC 1: 12. 5. The strain according to claim 1, characterized in that it was obtained for 5 consecutive passages in cell culture, BHK-21 with a titer of infectious activity of at least 7.0 Ig TCD ₅₀ / cm ³ and antigenic activity in RAC 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 and virus-neutralizing antibodies in the body of guinea pigs detected in CSC in dilutions of 1: 80-1: 160 and in an enzyme-linked immunosorbent assay (ELISA) in dilutions 1: 10000-1: 12000.

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