RU2560268C1 - FOOT-AND-MOUTH DISEASE VIRUS STRAIN Aphtae epizooticae TYPE A FOR PRODUCING AND CONTROLLING BIOLOGICAL PREPARATION FOR DIAGNOSTICS AND SPECIFIC PREVENTION OF TYPE A FOOT-AND-MOUTH DISEASE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. What is presented is the foot-and-mouth disease strain Aphtae epizooticae type A, Picornaviridae family, Aphtovirus genus deposited in the collection of FGBU VNIIZZh (Federal State Budgetary Institution Federal Animal Healthcare Centre), under the registration number the foot-and-mouth disease strain A No.2171/Kabardino-Balkar/2013. The strain is reproducible in a primary trypsinised monolayer swine kidney cell culture, grafted cell cultures PSGK-30, VNK-21 and IB-RS-2. For 18÷24 incubation hours, the viral infectious activity titre in the above cell culture achieves 6.0÷7.5 lg TCD₅₀/cm³.

EFFECT: presented viral strain can be used in producing and controlling the biological preparations for the diagnostics and specific prevention of the foot-and-mouth disease type A.

1 dwg, 6 tbl, 6 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 for the manufacture of biological products for the diagnosis 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 Museum of Strains of the Federal State Budget Scientific Institution VNIIZZh [1, 2, 3].

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, used throughout Russia and in the countries of the former Soviet Union [4].

In the 1990-2000s, two genetic lines of type A foot and mouth disease virus dominated in Central Asia and the Middle East: line A / Iran / 96 (the Russian production strain A / Armenia / 98 belongs to this genetic group) and A / Iran / 2005 . Genetic line A / Iran / 99 is not as widespread as the two mentioned above. The strains from group A / Iran / 99 and A / Iran / 96 are not currently included in the priority list of vaccine strains recommended by the OIE / FAO World Reference Laboratory for foot and mouth disease, while strain A / Iran / 2005 (A / Turkey / 06) is to high priority [5].

A known strain of foot and mouth disease virus Aphtae epizooticae A No. 1707 "Armenia-98" for the manufacture of diagnostic and vaccine preparations [6].

Isolation of type A FMD virus from cattle in the Amassi region of Armenia from cattle in 1998 and a study of its phylogenetic relationship showed that in a comparative study of the primary structure of the VP ₁ gene of FMD virus strain A No. 1707 “Armenia-98” with production strains and field isolates found that the isolates "Armenia - 98" are identical to each other and are related to strains of type A Turkey / 97, Turkey / 98 and Iran / 96. It is also found that the isolates examined have very different from all earlier isolates, including the subtype A virus ₂₂ (18% differences), which relates to the production and use of specific diagnostics and prevention means ₂₂ strain A №550.

Production strain A No. 1707 “Armenia-98” was used as part of FMD vaccines in the buffer zone of Transcaucasia.

A strain of A (Georgia) 1999 / No. 1721 [7] is known which belongs to the same line and was isolated in 1999 from sick cows in the private sector of the village of Ude in the Adigen region of the Republic of Georgia.

Since 2003, type A foot and mouth disease virus isolate has been isolated in Iran, significantly different from previously studied strains of this type. During 2005-2006 Type A foot and mouth disease is widespread in countries of 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 using strain A ₂₂ in Thrace in February-March 2006 prevented the spread of foot and mouth disease to neighboring Greece and Bulgaria, but fresh cases appeared after 3-4 months [8].

A known foot and mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007, isolated in the Kyrgyz Republic from a foot-and-mouth disease heifer in 2007, belongs to a genetic line called A Iran / 2005 [9].

The disadvantages of the above strains are that vaccines prepared on their basis provide effective protection of animals only against infection with a homologous virus.

The closest set of essential features to the present invention is strain A No. 2045 / Kyrgyzstan / 2007 (prototype).

An outbreak of foot and mouth disease in the Russian Federation was recorded in July 2013 in the yards of residents of the village of Nizhny Kurkuzhin, Baksan district, Kabardino-Balkaria, in the buffer zone, where cattle and small cattle were vaccinated against type 1, A, Asia-1 foot and mouth disease. Out of the population of the village, 2721 head of cattle of different ages and 941 head of cattle, 23 heads of cattle were ill.

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 the manufacture of sensitive and highly specific diagnosticum and immunogenic vaccine drugs homologous to the epizootic virus that appeared in Russia.

This problem was solved by obtaining strain A No. 2171 / Kabardino-Balkarian / 2013 (copyright) of type A foot and mouth disease virus for controlling antigenic and immunogenic activity 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. 2171 / Kabardino-Balkarsky / 2013, was isolated in July 2013 from sick animals in the village of Nizhny Kurkuzhin, Baksan district, Kabardino-Balkarian Republic (examination No. 2171). Production strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus was obtained by successive passages on sensitive hetero- and homologous cell cultures.

Strain A No. 2171 / Kabardino-Balkarian / 2013, type A foot-and-mouth disease virus was deposited on January 28, 2014 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under the registration number (link): virus strain foot and mouth disease A No. 2171 / Kabardino-Balkarian / 2013 (production).

Compared with the prototype strain, strain A No. 2171 / Kabardino-Balkarian / 2013 has a higher infectious and antigenic activity.

The possibility of using foot-and-mouth disease virus A No. 2171 / Kabardino-Balkarsky / 2013 for the manufacture of diagnostic tools and specific prophylaxis of foot and mouth disease type A was experimentally confirmed.

The invention is illustrated in a graphical image in which a dendrogram reflecting phylogenetic relationships of a strain of foot and mouth disease virus A No. 2171 / Kabardino-Balkarian / 2013 with epizootic and vaccine strains of foot and mouth disease virus of serological type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

The invention is explained in the list of sequences in which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain A No. 2171 / Kabardino-Balkarian / 2013 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. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus.

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus is characterized by the following features and properties.

Morphological features

Strain A No. 2171 / Kabardino-Balkarian / 2013 of 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 icosahedral form of the virion is 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. 2171 / Kabardino-Balkarian / 2013 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).

During hyperimmunization of guinea pigs, the concentrated antigen from inactivated foot and mouth disease virus type A No. 2171 / Kabardino-Balkarsky / 2013 induces the formation of virus-specific antibodies detected in CSC at a dilution of 1:30.

Using the method of nucleotide sequencing, the primary structure of the type 1 FMD virus of the FMD virus No. 2171 / Kabardino-Balkarsky / 2013 was determined and the primary structure of the VP1 protein was derived. A comparative analysis of the nucleotide sequences showed that the type A foot-and-mouth disease virus strain No. 2171 / Kabardino-Balkarsky / 2013 is significantly different from the production strains of type A. The degree of nucleotide differences in the sequences of strain A No. 2171 / Kabardino-Balkarian / 2013 sequences with foot-and-mouth disease virus strains of serological type A was : A ₂₂ No. 550 - 22.65%, A ₂₂ / Iraq / 64 - 21.66%, A / Iran / 96 - 21.43%, A / Armenia / 98 - 21.98%, A / Turkey / 06 - 8.23%, A / Iran / 05 - 8.75%.

Thus, phylogenetic analysis showed that strain A No. 2171 / Kabardino-Balkarian / 2013 belongs to the Iran 2005 genetic line of the Asia topotype.

The antigenic affinity of strain VYA A No. 2171 / Kabardino-Balkarsky / 2013 with existing production strains of VYA A was investigated by the serological method in the cross microneutralization reaction (RMN). A strain №2171 / Kabardino-Balkaria / 2013 antigenically different from the production strains # 550 A _22, A ₂₂ Iraq / 64, A / Iran / 97, A / Turkey / 2006 and A / Kyrgyzstan / 07. Strain VIA A No. 2171 / Kabardino-Balkarian / 2013 is not closely related to them.

The results of the studies in the RMN are presented in table 1. Antigenic compliance (r ₁ ) was for A ₂₂ No. 550 - 0.013, A ₂₂ Iraq / 64 - 0.02, A / Iran / 97 - 0.03, A / Turkey / Ob - 0.19 and A / Kyrgyzstan / 2007 - 0.03. With a value of 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, with a value of 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 [10, 11].

Biotechnological characteristics

Strain A No. 2171 / Kabardino-Balkarsky / 2013 is reproduced in monolayer cell cultures: a primary trypsinized cell culture of pig kidneys (SP), transplantable cultures of kidney cells of the Siberian mountain ibex (PSGK-30), BHK-21 and 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), the virus causes CPD after 5 hours. The virus retains its original characteristics when passaged in cell cultures for 5 passages (observation period).

Genotaxonomic characteristic

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus is an RNA-containing virus with a molecular weight of 7x10 ⁶ D.

Nucleic acid is 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. 2171 / Kabardino-Balkarsky / 2013 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.

Antigenic activity - administration of an inactivated antigen to guinea pigs induces the formation of virus-specific antibodies.

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 5 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. 2171 / Kabardino-Balkarsky / 2013, was isolated at the All-Russian Scientific Research Institute of Health and Agriculture from samples of aphthous material obtained in July 2013 from cattle foot and mouth disease suspected of cattle disease from the village of Nizhny Kurkuzhin, Baksan district, Kabardino-Balkaria Republic (examination No. 2171/2013). Samples of aphthous material were received at the Federal State Budget Scientific Institution VNIIZZH on July 9, 2013.

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 [12].

Biological and virological methods included the isolation of the virus on a culture of primary trypsinized SP cells, transplanted PSGK-30, IB-RS-2 cell lines, followed by adaptation. For staging biological samples in 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 resulting virus-containing material was used for subsequent passages and studies in the CSC for the presence of a viral antigen, using commercial type-specific sera stored in the Museum of strains of the Federal State Budget Scientific Institution VNIIZZH. The virus was considered adapted to cell cultures if, within 18-24 hours, (%) 90-100 CPDs appeared in the monolayer.

A virus adapted to IB-RS-2 cell cultures was used to produce antigen for CSC.

The adaptation of epizootic isolate A No. 2171 / Kabardino-Balkarsky / 2013 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. The results of the adaptation of the virus to cell cultures are presented in table 2.

The data shown in table 2, indicate the high adaptive ability of strain A No. 2171 / Kabardino-Balkarian / 2013 of the disease of foot and mouth disease type A to the used cell cultures.

Isolated using the above methods, the viral preparation was investigated in the reaction: CSC in order to identify its type affiliation (table 3). The strain was tested for the absence of contamination by bacterial and fungal microflora and mycoplasmas, as well as by extraneous viruses in PCR and RT-PCR.

The results shown in table 3 indicate that in the aphthous test material No. 2171 / Kabardino-Balkarsky / 2013 an FMD virus antigen of type A was detected at a 1: 2 dilution in CSC. Contamination by extraneous viruses was not detected.

The resulting strain of FMD virus type A is assigned the copyright name: strain A No. 2171 / Kabardino-Balkarian / 2013.

Example 2

For hyperimmunization of guinea pigs, the antigen from strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus reproduced in a monolayer PSGK-30 cell culture 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. Purified virus and inactivated by aminoethylethyleneimine (AEEI) at a concentration of 0.025-0.05% at a pH of 8.0-8.3.

Inactivated antigen in a mixture with an equal volume of an oil adjuvant such as Freund's incomplete adjuvant type is 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 [12].

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 4.

The data given in table 4 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, a foot and mouth disease virus strain A No. 2171 / Kabardino-Balkarsky / 2013, adapted to the cell culture of the transplantable PSGK-30 and IB-RS-2 lines, 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 data are presented in table 5.

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.

Example 4

For the manufacture of an inactivated sorbed type A vaccine of strain A No. 2171 / Kabardino-Balkarsky / 2013, foot and mouth disease virus is reproduced in a suspension culture of BHK-21 cells. 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 SCC 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. When the number of dead cells (%) 90-95 is reached, 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 pH 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 or polypropylene bottles and sterility is controlled 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 5 days. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

Example 5

Strain A No. 2171 / Kabardino-Balkarian / 2013 of the foot and mouth disease virus Aphtae epizooticae type A, 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 zones of the country where animals have not been vaccinated for the past 2 years against foot and mouth disease. The number of passages of the virus should not exceed 20, starting from the original virus. The virus suspension containing 10,000 ÷ 10000000 PH _50/1 cm ³ with antibiotics administered at 0.2 ÷ 0.3 cm ³ of 20 ÷ 40 channels intradermalingvalno across the 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, 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 ° С ÷ 40 ° С).

Example 6

Monitoring the immunogenic and antigenic activity of the vaccine inactivated adsorbed from FMD virus type A No. 2171 / Kabardino-Balkarian / 2013 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 FMD-free 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 fourth group of 2 goals was left without vaccination.

The vaccine dose volume was 2.0 cm ³ . On the 21st day after vaccination, blood samples were taken from 15 vaccinated and 2 control heads of cattle and the animals were challenged by introducing a type A suspension of foot and mouth disease virus No. 2171 / Kabardino-Balkarsky / 2013 at a dose of 10 ^4.0 ID ₅₀ / 0.2 cm ³ .

Data on the antigenic and protective activity of the vaccine are presented in table 6.

The antigenic activity of the vaccine was monitored by the level of virus-neutralizing antibodies in the blood serum in the pH on the monolayer of the primary trypsinized culture of SP cells and in the microneutralization reaction (PMN) on the IB-RS-2 cell culture. On the 21st day after vaccination, the level of virus-neutralizing antibodies in the pH in cattle vaccinated with the whole vaccine of series No. 1 and No. 2 was 6.8 ± 0.15 log ₂ and 6.5 ± 0.41 log ₂ , the level of virus-neutralizing antibodies in RMN was 7.6 ± 0.44 log ₂ and 5.7 ± 0.28 log ₂ .

8 days after infection, a pathological examination of cattle was performed. All control animals were ill, 4 animals vaccinated with a 1:16 vaccine dilution (series No. 1), and 2 animals vaccinated with a 1:16 vaccine dilution (series No. 2).

ImD ₅₀ vaccines of series No. 1 and No. 2 was 0.19 and 0.11 cm ³ . The vaccine vaccine dose contained 10.56 PD ₅₀ and 18.38 PD _50, respectively, which indicates the effectiveness of the drug. Such a vaccine is considered immunogenic and suitable for practical use.

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.

Information sources

1. Rerer X. Foot and mouth disease: Per. with him. G.A. Surkova / ed. and with the foreword. Cand. Vet.science. 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 p.

3. Viral diseases of animals / V.N. Syurin, A.Ya. Samuilenko, B.V. Soloviev [et al.]. - M.: VNIITIBP, 1998 .-- S.532-548.

4. Industrial regulations for the production of vaccines against foot and mouth disease types A, O, C, Asia-1, Sat-1, Sat-2 and Sat-3 inactivated adsorbed mono- and polyvalent (from a virus grown in BHK-21 cells): approved . Director of the Federal State Institution "ARRIAH" September 11, 2009.- Vladimir, 2009. - 216 p.

5. WRLFMD Quarterly Report July-September 2013 - URL http://www.wrlfmd.org/ref_labs/rei_lab_reports/OIE-FAO

6. Pat. RF No. 2140452, C12N 7/00, A61K 39/135, G01N 33/569, A61K39 / 42, C07K16 / 08.27.10.1999

7. Pat. RF №2242513, C12N 7/00, A61K 39/135, 12.20.2004

8. Foot-and-mouth Disease. Situation worldwide and major epidemiological events in 2005-2006 / K. Sumption, J. Lubroth, T. Murrai, S. De La Rocqe // EMPRESS / Focus on ..., 2007, No. 1, IIP.

9. Pat. RF №2451745, C12N 7/00, A61K 39/135, 05.27.2012

10. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 7th Ed. -, Paris, 2012 - Vol. 1, Chapter 2.1.5. - P.166-169.

11. Selection of foot and mouth disease vaccine strains - a review / D.J. Paton, J.-F. Valacher, J. Bergman [et al.] // Rev. Sci. Tech. OIE. - 2005 .-- Vol.24 (3). - P.981-993.

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

Table 1 Antigenic correspondence (r ₁ ) in the RMN of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus with production of type A foot and mouth disease virus strains Strain Exponent r ₁ A ₂₂ No. 550 0.013 A ₂₂ / Iraq / 64 0.02 A / Iran / 97 0,03 A / Turkey / 06 0.19 A No. 2045 / Kyrgyzstan / 2007 0,03

table 2 Biological properties of the virus of epizootic strain A No. 2171 / Kabardino-Balkarian / 2013 FMD virus type A 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 (lg TCD ₅₀ / cm ³ ) PSGK-30 twenty 5 1: 4 6.45 IB-RS-2 24 5 1: 2 6.0 VNK-21 twenty 5 1: 2 7.5

Table 3 Determination of the type of virus in a 33% suspension of aphthous material in DSC (examination No. 2171 / Kabardino-Balkarsky / 2013) Examination No. 2171 The control Hyperimmune sera in working dilution whole 1: 2 1: 4 1: 8 to / a A / Turkey / 06 A22 / Iraq / 64 OPanAsia2 Asia-1 Shamir 3/89 b / a A / Turkey / 06 four - - - four four - - - A ₂₂ / Iraq / 64 four four - - four - - - O panasia2 - - - - - - four - - Asia-1 Shamir 3/89 - - - - - - four - b / s - - - - - - - - - b / c 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 4 Characterization of the diagnostic serum obtained for the antigen from strain A No. 2171 / Kabardino-Balkarian / 2013 foot and mouth disease The name of the drug Series Volume (cm ³ ) Activity in RSK with homologous antigen with heterologous antigens A / Turkey / About O panasia2 Asia-1 Shamir Hyperimmune Serum one 200 1:30 <1:20 <1:20 <1:20

Table 5 Characterization of diagnostic antigens from strain A No. 2171 / Kabardino-Balkarian / 2013 of foot and mouth disease virus grown in various cell cultures The name of the drug Series Volume (cm ³ ) Activity in RSK with homologous diagnosticum A No. 2171 / Kabardino-Balkarian / 2013 with heterologous diagnosticum A PSGK-30 culture antigen one 40 1: 8 <1: 2 Cultural Antigen IB-RS-2 one 60 1:12 1: 2

Table 6 The test results of the sorbed inactivated monovalent vaccine from strain VYA A No. 2171 / Kabardino-Balkarian / 2013 on cattle Vaccine Series No. 1 Vaccine Series No. 2 Divorced
nie General
n / infected. VNA titer (log ₂ ) BMI ₅₀ Divorced
nie General
n / infected. VNA titer (log ₂ ) IMD50 PH RMN PH RMN Ts - 7.0 7.5 0.19 Ts - 6.5 6.5 0.11 - 6.25 7.5 - 8.0 6.0 - 6.75 7.5 - 6.5 5,0 - 7.0 6.5 - 5.75 5.5 - 7.0 9.0 - 5.75 5.5 M ± m 6.8 ± 0.15 7.6 ± 0.44 M ± m 6.5 ± 0.41 5.7 ± 0.28 1: 4 - 6.25 6.5 1: 4 - 7.25 6.0 - 5,0 5.5 - 5,0 6.0 - 4.25 7.0 - 4.25 5.5 - 4,5 5.5 - 4.0 5.5 - 3,5 5,0 - 6.0 6.0 M ± m 4.75 ± 0.46 5.9 ± 0.41 M ± m 5.3 ± 0.6 5.8 ± 0.13 1:16 + 3.0 4.0 1:16 - 3,5 4.0 - 5,0 6.0 - 3.0 4.0 + 1,5 4.0 + 1.75 4.0 + 2.0 4,5 - 2.25 4.0 + 1.75 4.0 + 2,5 5,0 M ± m 2.65 ± 0.64 4,5 ± 0,43 M ± m 2.6 ± 0.3 4.2 ± 0.22

Claims (1)

The foot and mouth disease virus Aphtae epizooticae type A, fam. Picornaviridae, a genus of Aphtovirus, deposited in the collection of microorganism strains of FSBI “VNIIZZH” under registration number A No. 2171 / Kabardino-Balkarsky / 2013 (production) for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type A and their control.

Images