RU2798293C1 - Fmd culture inactivated sorbed vaccine from a/afghanistan/2017 strain of a/asia/iran-05far-11 new genotype - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the development of a cultured inactivated adsorbed vaccine against foot-and-mouth disease from A/Afghanistan/2017 strain of A/ASIA/Iran-05^FAR-11 new genotype. The vaccine contains an avirulent and purified concentrated antigen of A/Afghanistan/2017 strain of foot and mouth disease virus of A/ASIA/Iran-05^FAR-11 new genotype, obtained in a suspension transplantable cell line from the kidney of a newborn Syrian hamster (BHK-21/SUSP/ARRIAH), representing is a suspension containing predominantly 146S immunogenic component of FMDV, adjuvant aluminum hydroxide and saponin in effective ratios.

EFFECT: vaccine has a high immunogenicity and is able to provide effective protection against a homologous infectious agent circulating in the countries of Southeast Asia, the Middle East and on the borders of the Russian Federation with these regions.

6 cl, 1 dwg, 9 tbl, 14 ex

Description

The invention relates to the field of veterinary virology and biotechnology, namely the development of a vaccine against foot-and-mouth disease from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated adsorbed.

Foot and mouth disease is a viral, acute disease of artiodactyl animals, characterized by fever and aphthous lesions of the mucous membrane in the mouth, udder skin (mammary gland) and limbs. Young growth is more easily infected and more difficult to tolerate the disease. The source of the disease is animals with foot-and-mouth disease, as well as those kept together with patients for a long time, in the incubation (latent) period of the disease, releasing the virus into the external environment with the contents and walls of aphthae (rounded ulcers) on the tongue along the walls of the oral cavity, with milk, saliva and faeces [1, 2, 3].

There are 7 FMD virus serotypes: O, A, C, Asia-1, SAT-1, SAT-2 and SAT-3. Each serotype is genetically divided into different topotypes, genetic lines and sub-lines. Differences between them are determined by analyzing the nucleotide sequence of the ID gene (VP ₁ protein), which is characterized by high genomic and antigenic variability [1].

Within the seven serotypes, more than 60 genetic lines and sublines have been described. The importance of their knowledge lies in the fact that it is required to select a vaccine very carefully, which must be adapted specifically to the genotype of interest. Recovery after an animal has been ill with any one serotype does not provide immune protection against another serotype and even some other genetic lines [3]. However, FMD viruses may show partial cross-protection within the same serotype.

The observed genetic variation in the FMDV genome is the result of two processes. First, these are errors that result from viral RNA replication and the lack of repair of the encoded RNA-dependent RNA polymerase. Secondly, - competitive selection, which affects the genome. In the natural environment, as a result, isolates of new genetic lines that include in their genome the characteristics required for this environment will prevail [4].

In RNA viruses, genome variation is favored by high mutation rates during viral replication, and emerging lineages and sublines are driven by mutation and recombination. Recombination is another important process that determines the biology and evolution of viruses. It is the exchange of genetic material between two non-segmented RNA genomes. It has been shown that genetic recombination occurs between viruses of the same serotype [5, 6, 7]. Intratypic recombination occurs more frequently than recombination, and recombination events in FMD appear to occur more readily in the 3' portion of the genome than in regions of the genome encoding structural viral proteins. Mutations as a result of recombination lead to the exchange of genetic material and the formation of new antigenic variants, which can avoid immune pressure and lead to changes in cell tropism and host range [7]. One or more amino acid substitutions on the surface of viral particles can lead to altered receptor recognition and utilization. As a result, the same virus can acquire the ability to enter cells using alternative receptors [8].

Antigenic variations arise from amino acid mutations that alter the recognition of viral proteins by the body's immune system. The superficially open structures of the virus are particularly susceptible to immune attack. The process of emergence of new antigenic forms is determined by the complex interaction of host cell factors and the virus. Mutations in the genes encoding capsid proteins can lead to antigenic changes and the emergence of new genetic lines [9].

Nucleotide sequencing is commonly used to identify genetic links between different isolates and strains. Therefore, in an outbreak setting, the origin of the virus can be traced. The antigenic characteristics of the virus associated with the emergence of new strains are important for the characterization of outbreaks and the search for optimal vaccine preparations [6, 10].

Representative strains/isolates for each FMDV topotype are listed below. They can form the main anchor points of the phylogenetic tree. The GenBank database contains nucleotide sequences for various genetic lineages and sublines.

FMD serotype O is the most common throughout the world and has been extensively studied. Serotype O is divided into 11 topotypes: EAST AFRICA 1-4 (East Africa) (EA-1-4), SOUTHEAST ASIA (Southeast Asia) (SEA), EUROPE-SOUTH AMERICA (Europe-South America) (EURO-SA ), INDONESIA-1 and 2 (Indonesia-1 and 2) (ISA-1 and -2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA) (Middle East-South Asia) and WEST AFRICA (Western Africa) (WA).

Serotype A includes 3 characterized topotypes ASIA, AFRICA, EURO-SA and one as yet little studied group of isolates.

Type C has not been isolated anywhere in the world for more than 10 years. Some scientists consider it necessary to declare serotype C eradicated and stop the production of vaccines against it. Type C includes the following 3 topotypes: AFRICA, ASIA, EURO-SA.

According to the WRLFMD (World Reference Laboratory for Foot-and-Mouth Disease) serotype Asia-1 of the FMD virus includes 1 topotype ASIA, which includes 8 characterized genetic lines and some isolates that are currently still not assigned to any line. Divisions into sublines are not noted at present. The ASIA topotype includes the following genetic lines: G-I, G-II, G-III, G-IV, G-V, G-VI, G-VIII, Sindh-08.

The following 13 topotypes are distinguished in the SAT-1 serotype: I (NORTHWEST ZIMBABWE, NWZ), II (SOUTHEAST ZIMBABWE, SEZ), III (WESTERn ZIMBABWE, WZ), IV (EAST AFRICA 1, EA-1), V, VI, VII (EAST AFRICA 2, EA-2), VIII (EAST AFRICA 3, EA-3), IX, X, XI, XII, XIII.

For the SAT-2 serotype, 14 topotypes (I-XIV) are distinguished, within which there is no division into genetic groups.

The SAT-3 serotype includes the following 5 topotypes: I (SOUTHEAST ZIMBABWE, SEZ), II (WESTERn ZIMBABWE, WZ), III (nORTHWEST ZIMBABWE, nWZ), IV, V (East Africa, EA) [7, 8, 9 , 10, 11, 12].

Thus, a wide genetic diversity of foot-and-mouth disease virus is represented in many countries of the world. This diversity is due to the high degree of variability of RNA-containing viruses and the active movement of this pathogen in Asia, Africa and South America.

Due to the easy and rapid spread of the FMD pathogen, this disease can acquire the scope of epizootics [11]. In order to prevent the occurrence of the disease in the farms of the Russian Federation, a system of measures for the control and prevention of foot and mouth disease is used, which is aimed at preventing the introduction of the virus into the country, systematic immunization of cattle and small cattle in the buffer zone, as well as monitoring the immune status of vaccinated animals.

In accordance with the OIE Guidelines (OIE), the following protective and preventive measures are recommended:

1) mandatory vaccination of susceptible animals;

2) to register all newly acquired animals in the institutions of the state veterinary service and to quarantine animals before entering the main herd;

3) comply with the requirements of zoohygienic norms and rules for keeping animals;

4) to acquire food from a prosperous territory, etc. [3].

For immunization of animals, a vaccine made from a virus homologous to field isolates should be used [6, 7, 8, 9, 10, 11].

The worldwide outbreaks of foot-and-mouth disease demonstrate that this infection continues to be a significant economic problem throughout the world. The debate about the most effective way to respond to FMD outbreaks in disease-free countries continues to focus on the use of vaccines [8, 9, 11]. An effective vaccination campaign requires the availability of an effective, safe vaccine containing, as an immunogenic part, a virus antigen corresponding to an epizootic spreading isolate of a certain genotype.

The creation of such vaccines is an urgent task.

Achieving this goal will make it possible to effectively apply the vaccine in a disadvantaged area and a threatened environment for the formation of immunity against a specific FMDV genotype.

One of several measures that can be taken to control FMD outbreaks is emergency vaccination. Criteria that determine the success of this vaccination include the following characteristics:

1) contains a strain of foot-and-mouth disease virus with sufficient antigenic affinity for isolates circulating in the outbreak;

2) refers to the required species among the developed vaccines;

3) is characterized by acceptable safety and efficacy;

4) has appropriate access, including the volume of consignments and the timeliness of their deliveries.

Contingency planning must include the provision of emergency vaccination and take into account the possibility of complex decisions not only about when, where and how to apply the vaccine, but also the economic feasibility of its use [13].

Considering the danger of foot and mouth disease in the Russian Federation and the high probability in this case of large economic losses, the problem of emergency prevention of this disease is of particular importance for the formation of immunity in susceptible animals.

In order to improve disease control measures in endemic countries, it is important to monitor the current distribution of FMDV field isolates in order to ensure that the biological properties of the isolate of interest can be studied in a short time and an effective vaccine can be developed from the virus strain that caused the outbreak [12, 13, 14, 15 , 16].

On the territory of the Russian Federation, outbreaks of FMD serotype A are sporadic and are caused by the introduction of the pathogen from the territory of neighboring countries. The regions of the Russian Federation bordering China, Mongolia, Azerbaijan and Georgia are subject to a very high risk of FMD introduction [14].

Outbreaks of FMD serotype A in the Russian Federation were recorded in 2013-2014. FMDV serotype A, belonging to the A/ASIA/SEA-97 genotype, was detected in the Trans-Baikal Territory and the Amur Region. On the territory of the Karachay-Cherkess Republic and the Krasnodar Territory, foot-and-mouth disease outbreaks were caused by a virus belonging to the A/ASIA/Iran-05 ^SIS-10 genotype [12].

Currently known industrial strains of FMDV type A, which are used for the production of FMD vaccines:

- strain A No. 550/Azerbaijan/64 (genotype A/ASIA/Iraq-64),

- strain A ₂ 2/Iraq 24/64 (genotype A/ASIA/Iraq-64),

- strain A/Turkey/2006 (genotype A/ASIA/Iran-05),

- strain A No. 2166/Krasnodar/2013 (genotype A/ASIA/Iran-05 ^SIS-10 ),

- strain A No. 2155/Zabaikalskiy/2013 (genotype A/ASIA/Sea-97),

- strain A No. 2269/ARRIAH/2015 (genotype A/ASIA/G-VII).

FMDV isolate A/AFG/14/2017 was transferred to FGBI ARRIAH from Afghanistan in 2017 for basic scientific research. As a result of successive passages on a transplanted cell culture, strain A/Afghanistan/2017 was obtained [17].

According to the results of a comparative analysis of nucleotide sequences conducted at the Pirbright Institute and at the Federal State Budgetary Institution "ARRIAH", the isolated isolate "A/AFG/14/2017" and, accordingly, the strain A/Afghanistan/2017 of foot-and-mouth disease virus belong to serotype A, topotype ASIA, genetic line Iran -05, FAR-11 sublines. This strain differs significantly in its genetics from industrial strains of foot-and-mouth disease virus serotype A.

Given the intensive trade relations between the Russian Federation and the countries of Southeast Asia, as well as the Middle East, the risk of introducing the FMDV of this genotype into the territory of our country is very high. In this regard, it became necessary to develop a new vaccine against foot-and-mouth disease caused by the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11, culturally inactivated, adsorbed to ensure the biological safety of the territory of the Russian Federation and neighboring states.

The closest proposed invention in terms of essential features is an inactivated sorbed vaccine against FMD type A (genotype A/ASIA/Iran-05)) [1], containing the active substance in the form of avirulent and purified cultural antigenic material from strain A/Turkey homologous to the FMD pathogen /2006, obtained in a sensitive biological system, and targeted additives in the form of aluminum hydroxide and saponin (adjuvant): antigen concentration of at least 3.0 µg/cm ³ , content of 10% aluminum hydroxide solution - 50-60% by volume, saponin concentration - 1.5 mg/cm ³ .

As a sensitive biological system for the reproduction of foot-and-mouth disease virus, a transplantable suspension cell line of the kidney of a newborn Syrian hamster BHK-21/SUSP/ARRIAH is used, as a supporting medium, Earle's solution is used without adding serum, with the addition of dry enzymatic muscle hydrolyzate (FGMS), blood protein hydrolyzate dry (GBKS) and antibiotics at pH 7.4-7.6.

Aminoethylethyleneimine (AEEI) is used to inactivate the virus. In the manufacture of an emulsion vaccine, aluminum hydroxide and saponin serve as an adjuvant. Purification of the virus-containing suspension from ballast impurities is carried out using polyhexamethylene guanidine hydrochloride (PHMG).

The avirulent and purified antigenic material from the serotype A strain is a suspension consisting mainly of the 146S component of foot-and-mouth disease virus, which is the immunogenic component of the vaccine preparation.

A significant drawback of the prototype vaccine is its lack of immunogenic activity against FMDV genotype A/ASIA/Iran-05 ^FAR-11 isolates actively circulating in Southeast Asia and the Middle East.

To solve this problem, the present invention was created, which included the development of an inactivated, cultured, adsorbed FMD vaccine that provides effective protection of susceptible animals against field isolates of FMDV genotype A/ASIA/Iran-05 ^FAR-11 , common in Southeast Asia and the Middle East. East.

The technical result from the use of the present invention is to expand the arsenal of inactivated cultural adsorbed vaccines to protect animals against isolates and strains of FMDV genotype A/ASIA/Iran-05 ^FAR-11 .

The specified technical result is achieved by creating a vaccine against foot-and-mouth disease from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 cultural inactivated sorbed, characterized by the following set of features reflected below.

The developed vaccine in 1.0 cm ³ of the preparation contains the following main components: 1) the active substance in the form of avirulent and purified antigenic material from foot-and-mouth disease virus homologous to the pathogen strain A/Afghanistan/2017, reproduced preferably in the continuous suspension cell line BHK-21/SUSP /ARRIAH, in an amount not less than 3.0 μg; 2) aluminum hydroxide 10% colloidal solution with a content of 50-60% by volume (5-6% in terms of dry matter), 3) saponin in the amount of 1.5 mg, 4) supporting medium - up to 1 cm ³ by volume.

The FMD virus isolate A/AFG/14/2 017 was isolated from cattle in the Wonabah province of Afghanistan in the Panjsher settlement in 2017. Strain A/Afghanistan/2017 was obtained from this isolate in the course of fundamental scientific research at the Federal State Budgetary Institution “ARRIAH”, which was deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Virus and Other Animal Pathogens (GKSM) of the Federal State Budgetary Institution “Federal Center for Animal Health” (FGBI "ARRIAH"), under registration number: No. 378 - dep / 22-12 - GKSHM FGBI "ARRIAH".

The strain is adapted to primary trypsinized cells of the porcine kidney (SP) line and transplantable cell lines from the kidney of a newborn Syrian hamster (BHK-21 / SUSP / ARRIAH), the kidney of a pig (IB-RS-2) and the kidney of the Siberian mountain ibex (PSGK-30 ).

For the manufacture of the vaccine, a preferentially transplanted suspension culture of subline cells from the kidney of a newborn Syrian hamster VNK-21/SUSP/ARRIAH is used as a sensitive biological system, and Eagle's medium DMEM is used as a supporting medium without adding serum, but with the addition of hydrolyzed blood proteins in an amount of 5 % of the total volume at pH 7.4-7.6. Virus inactivation is carried out using aminoethylethyleneimine (AEEI) with a concentration of 0.025% of the volume of the virus-containing suspension. The inactivated virus is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.01% of the total volume.

The avirulent and purified antigen of strain A/Afghanistan/2017 is a suspension containing an inactivated 146S immunogenic component of FMDV. The content of the component in the product is estimated using a quantitative variant of the complement fixation reaction (CFR) [18]. To prepare the vaccine, a viral material containing at least 3.0 μg of the inactivated 146S component of the FMD virus is used in 1.0 cm ³ . The required concentration of the immunogenic component in the vaccine preparation is ensured by concentrating the antigen by adsorption on the surface of colloidal particles of aluminum hydroxide.

The vaccine against foot and mouth disease from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 cultural inactivated sorbed is obtained by mixing the purified antigen (inactivated 146S immunogenic component) and a 10% aluminum hydroxide solution in a ratio of 40-50% by 50-60%" by volume, respectively. To enhance the immune response, saponin is used at a concentration of 1.5 mg/cm ³ . The resulting vaccine is a suspension containing particles of water-insoluble aluminum hydroxide carrying on their surface inactivated FMDV virions, which provide the formation of immunity.

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

1. FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated adsorbed.

2. The active substance in the form of an avirulent and purified antigenic material from a new genotype A/ASIA/Iran-05 ^FAR-11 of the FMD virus homologous to the causative agent of the disease strain A/Afghanistan/2017 in an effective amount.

3. Target additives.

The essential distinguishing features of the proposed vaccine are that it contains an avirulent purified antigen of the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 of the foot-and-mouth disease virus as an active substance in an effective amount.

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

1. Avirulent and purified antigen of the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 of foot-and-mouth disease virus, obtained preferably in a suspension continuous cell line BHK-21/SUSP/ARRIAH and which is a suspension containing predominantly 146S immunogenic a component of foot and mouth disease virus in an effective amount.

2. Avirulent and purified antigen of the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 of foot-and-mouth disease virus, obtained preferably in a suspension continuous cell culture of BHK-21/SUSP/ARRIAH and which is a suspension containing predominantly 146S immunogenic a component of foot and mouth disease virus in an amount of at least 3.0 μg per 1.0 cm ³ of the finished vaccine preparation.

3. Of the targeted additives, the vaccine contains an adjuvant.

4. Of the targeted additives, the vaccine contains an adjuvant - aluminum hydroxide and saponin.

5. The vaccine contains an adjuvant - aluminum hydroxide 5-6% in terms of dry residue in combination with saponin at a concentration of 1.5 mg per 1.0 cm ³ of the finished product.

6. Avirulent and purified antigen of strain A/Afghanistan/2017 genotype A/ASIA/Iran-05 ^FAR-11 of foot-and-mouth disease virus, obtained preferably in a continuous suspension cell line BHK-21/SUSP/ARRIAH, adjuvant - aluminum hydroxide and saponin, additive in in the form of a supporting medium in the finished product in the following quantities:

The proposed vaccine has a high immunogenic activity and provides reliable protection against the A/Afghanistan/2017 strain of the new genotype A/ASIA/Iran-05 ^FAR-11 of the foot and mouth disease virus circulating in Southeast Asia and the Middle East.

The achievement of the technical result from the use of the invention is ensured by the fact that the antigen of the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 of the foot-and-mouth disease virus, which has high immunogenic activity and creates effective protection, is introduced into the composition of the proposed FMD vaccine as an active substance. susceptible animals against foot-and-mouth disease virus isolates of the presented genotype, which in recent years have caused outbreaks in the countries of Southeast Asia and the Middle East.

The essence of the invention is reflected in the graphic image:

Fig. 1 - The position of strain A/Afghanistan/2017 on the phylogenetic tree of FMDV serotype A. The dendrogram is based on a comparison of the complete nucleotide sequences of the 1D gene (VP ₁ protein). The strain is highlighted in red flower and designated A/AFG/2017.

The essence of the invention is illustrated by the following sequence listings:

SEQ ID NO: 1 represents the nucleotide sequence of the gene encoding the protein VP ₁ of strain A/Afghanistan/2017 genotype A/ASIA/Iran-05 ^FAR-11 foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP ₁ protein of the A/Afghanistan/2017 strain of the A/ASIA/Iran-05 ^FAR-11 genotype of the foot-and-mouth disease virus.

The A/Afghanistan/2017 strain of FMDV is characterized by the following features and properties.

Morphological features

The A/Afghanistan/2017 strain of FMDV belongs to the family Picornaviridae, genus Aphthovirus, serotype A, genotype A/ASIA/Iran-05 ^FAR-11 and has morphological features characteristic of the FMD pathogen: icosahedral shape of the virion, size 20-25 nm [ 5].

Antigenic properties

According to its antigenic properties, the FMDV strain A/Afghanistan/2017 belongs to serotype A. FMDV is stably neutralized by homologous antiserum. The infectious agent does not show hemagglutinating activity. In sick animals, type-specific antibodies are formed in the blood serum, which are detected in enzyme immunoassay, complement fixation and neutralization reactions.

The primary structure of the VP ₁ protein gene of the A/Afghanistan/2017 strain of foot-and-mouth disease virus was determined by nucleotide sequencing. Comparative analysis of nucleotide sequences showed that the A/Afghanistan/2017 strain belongs to the ASIA topotype, the Iran-05 genetic line, and the FAR-11 subline.

Antigenic relatedness (ri) of strain A/Afghanistan/2017 of foot-and-mouth disease virus was studied in a cross-sectional study in the microneutralization reaction (RMN) of the strain with specific sera obtained for the following production strains of foot-and-mouth disease virus:

- strain A No. 550/Azerbaijan/64 (genotype A/ASIA/Iraq-64),

- strain A ₂₂ /Iraq 24/64 (genotype A/ASIA/Iraq-64),

- strain A/Turkey/2006 (genotype A/ASIA/Iran-05),

- strain A No. 2166/Krasnodar/2013 (genotype A/ASIA/Iran-05 ^SIS-10 ),

- strain A No. 2155/Zabaikalsky/2013 (genotype A/ASIA/SEA-97),

- strain A No. 2269/ARRIAH/2015 (genotype A/ASIA/G-VII)

The titer of reference blood serum of cattle obtained by immunization of animals with monovalent vaccines from industrial strains of FMD virus serotype A, against 10 ² TCD ₅ about homologous and heterologous virus was determined in the microneutralization reaction during cross titration, calculating the values using the linear regression equation, and expressed in lg . The ri value was determined as the antilogarithm of the difference between lg serum titers against heterologous and homologous virus [19].

The value of ri in RMN was interpreted as follows:

at >0.3 - the studied and production strains of foot-and-mouth disease virus are closely related;

at <0.3 - the studied sample of the FMDV strain differs from the production strain.

The indicators of antigenic relationship in the study of strain A/Afghanistan/2017 amounted to ri from 0.06 to 0.24, which indicated the absence of antigenic relationship with industrial strains of FMDV serotype A (Table 1).

Biotechnological characteristics

The A/Afghanistan/2017 strain of foot and mouth disease virus is reproduced in a suspension continuous cell culture from the kidney of a newborn Syrian hamster BHK-21/SUSP/ARRIAH. As a supporting medium, Eagle's medium is used with the addition of dry enzymatic muscle hydrolyzate (FGMS), dry blood protein hydrolyzate (GBKS) at a medium pH of 7.4-7.6. The cell line is infected with the virus at the rate of 0.001-0.100 TCD ₅₀ /cell. The concentration of total viral protein in the suspension according to the results of the study is 2.40±0.11 μg/cm ³ . The values of the titer of the infectious activity of the virus, as well as the percentage of the 146S component of the strain A/Afghanistan/2017 of the foot-and-mouth disease virus are shown in Table 2, from the data of which it can be seen that with an average concentration of BHK-21/SUSP/ARRTAH cells equal to 4.05±0 ,10 million cells/cm ³ , infection dose of 0.005 TCD ₆₀ /cl. and duration of virus reproduction 10.65±0.53 h, the average titer of infectious activity of the FMD pathogen is 7.22±0.06 lg TCD ₅₀ /cm ³ . The concentration of the 146S component of the FMD virus is determined using the previously developed RSC method [18]. The content of this component is 72.74±2.42% (1.74±0.08 µg/cm ³ ).

Geno- and chemotaxonomic characteristics.

The A/Afghanistan/2017 strain of foot-and-mouth disease virus is an RNA-containing virus with a molecular weight of 7×10 ⁶ D.

The nucleic acid is represented by a single-stranded linear positive RNA molecule with a molecular weight of 2.8×10 ⁶ D and a length of 8500 n. The virion has a protein coat consisting of four major proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The lipoprotein membrane is absent.

The main antigenic protein is the VP ₁ protein. The virion contains approximately 31.5% RNA and 68.5% protein. Virion 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.

When reproduced in a biosystem, FMDV forms 4 variants of components: 146S particles (virions) consisting of one viral RNA molecule and 60 copies of the polypeptide, each of which is represented by a complex of proteins VP ₁ -VP ₂ -VP3-VP ₄ ; 75 S component, devoid of RNA and including 60 copies of the polypeptide VP ₁ -VP ₃ -VP ₀ ; 12S particles represented by proteins VP ₁ , VP ₂ , VP ₃ ; 3,8S subunits, consisting of the non-structural protein VP _g . The protein shell consists of 32 capsomeres arranged in cubic symmetry

Physical Properties

The mass of the virion is 8.4×10 ^-18 g. The floating density is 1.45 g/cm ³ .

Resistance to external factors

The A/Afghanistan/2018 strain of foot-and-mouth disease virus is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.5-7.8. Shifts in pH, both in the acidic and alkaline directions, lead to virus inactivation. Sensitive to alkalis, acids, formaldehyde, UV irradiation, y-irradiation, high temperatures.

Additional features and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

Stability - retains the original biological properties when passaged in sensitive biological systems for 15 passages on a transplanted subline cell culture from the kidney of a newborn Syrian hamster BHK-21/SUSP/ARRIAH (observation period).

Based on the data obtained, it can be argued that the A/Afghanistan/2017 strain of foot-and-mouth disease virus, according to antigenic and immunological spectra, is an original, taxonomically new, previously unknown variant of foot-and-mouth disease virus of the A/ASIA/Iran-05 ^FAR-11 genotype.

To reduce the epizootic risk of foot and mouth disease caused by the A/ASIA/Iran-05 ^FAR-11 genotype and to prevent the emergence of new foci of the disease, timely vaccination is important, which requires the development of a highly immunogenic vaccine.

A highly immunogenic vaccine against foot-and-mouth disease was obtained from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated adsorbed.

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

Example 1. Genetic characterization of strain A/Afghanistan/2017 of foot-and-mouth disease virus according to PCR and nucleotide sequencing

The conducted studies consisted in studying the primary structure of the 1D gene (VP ₁ protein) (652 n.b.) of the A/Afghanistan/2017 strain of foot-and-mouth disease virus by nucleotide sequencing using the Sanger method and determining the position of this strain on the phylogenetic tree of type A foot-and-mouth disease virus. The method is based on determination of the primary structure of the 1D gene (VP ₁ protein) of the tested isolate/strain, followed by analysis of phylogenetic relationship with other FMDV type A isolates.

It was necessary to conduct a comparative analysis of the nucleotide sequences of the 1D gene encoding the VP ₁ protein of the foot-and-mouth disease virus of the reference vaccine strain A/Afghanistan/2018 with other isolates and strains. The nucleotide sequence of the VP ₁ protein gene of the strain A/Afghanistan/2017 of the genotype A/ASIA/Iran-05 ^FAR-11 of the foot-and-mouth disease virus is presented by SEQ ID NO:1. The amino acid sequence of the VP1 protein gene of the A/Afghanistan/2017 strain of the A/ASIA/Iran-05 ^FAR-11 genotype of the foot-and-mouth disease virus is shown in SEQ ID NO:2.

A phylogenetic analysis of the A/Afghanistan/2017 strain was carried out. The phylogenetic tree was derived using the MEGA6 program and the Neighbor-Joining algorithm [20]. The percentage of repetitive branches in which related taxa are grouped together in the bootstrap test (100 repetitions) is shown next to the branches [21, 22]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [23] and expressed in units of base substitutions per site. This analysis included 32 nucleotide sequences, including the FMDV strain A/Afghanistan/2017 ID gene sequence. All positions containing spaces and missing data have been removed (complete removal option). There were only 632 positions in the final data set. Evolutionary analysis was carried out in MEGA 6 [24].

As a result of the comparison of the complete nucleotide sequences of the 1D gene (VP ₁ protein) of strain A/Afghanistan/2017 and other isolates/strains of FMDV serotype A, the position of the studied strain on the phylogenetic tree was determined (Fig. 1).

Based on the results of the entire analysis, we conclude that the studied strain A/Afghanistan/2017 belongs to the serotype A topotype ASIA of the genetic line Iran-05, subline FAR-11, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of the strain A/Afghanistan/2017 to a continuous monolayer cell culture of the kidney of the Siberian mountain ibex PSGK-30.

A 10% aphthous suspension of foot-and-mouth disease virus obtained from aft cattle (2nd passage) was used to infect a continuous monolayer culture of Siberian ibex kidney cells PSGK-30. The inoculum concentration of cells was 0.15-0.20 million cells/cm ³ , the virus infection dose was 0.01 TCD ₆₀ /cell. According to the results of the study, the reproduction of FMDV strain A/Afghanistan/2017 took place with a specific destruction of the monolayer by 90-100% in 15-17 hours. During 6 consecutive passages, an increase in the values of the titer of infectious activity of the virus was noted from 5.95 ± 0.10 to 7 .80 ± 0.10 lg TCD ₅₀ / cm ³ .

The maximum value of the titer of infectious activity of the virus was reached at the stage of passage 6 (7.80±0.10 lg TCD ₅₀ /cm ³ ). The concentration of 146S particles from passages 1 to 6 changed from 0.34±0.02 to 1.14±0.02 µg/cm ³ . At the same time, the largest amount of the 146S component was noted at the stage of the 6th passage (1.14±0.02 µg/cm ³ ). The degree of reliability (R ² ) of the results of the study in the quantitative version of RT-PCR-RT ranged from 95.98 to 99.14%. Thus, over the course of 6 consecutive passages, the A/Afghanistan/2017 strain of foot-and-mouth disease virus was successfully adapted to the continuous monolayer cell line PSGK-30.

Example 3. Study of the conditions for monolayer cultivation of FMDV strain A/Afghanistan/2017 on an industrial scale

In the process of industrial cultivation of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in a continuous monolayer cell culture PSGK-30, the optimal conditions for cultivating the virus were selected by analyzing the different composition of the supporting medium, the temperature factor, and the dose of cell infection.

At the first stage of the study, the influence of the composition of the supporting medium on the reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in a monolayer cell culture of PSGK-30 was evaluated at the production scale (at the stage from the 5th to the 6th passages). Three media supplemented with 2% fetal calf serum were used for comparison: 1) MEM Needle medium; 2) RPMI-1640 medium; 3) Hank's solution with 2.5% HBA. The inoculum concentration of cells was 0.15-0.20 million cells/cm ³ , the dose of infection with the virus was 0.1000-0.0001 TCD ₅ o/cell. The cultivation of the virus was carried out at temperatures of 35±0.2 - 38±0.2°C until the destruction of the cell monolayer by 90-100% within 15-30 hours. in table 3.

From the data of Table 3, it can be seen that when the A/Afghanistan/2017 strain of foot and mouth disease virus is reproduced in a monolayer culture of PSGK-30 cells using supporting media of different composition, the Eagle's MEM medium is optimal, which makes it possible to achieve a specific destruction of the cell monolayer by 95% in 15-17 hours. -100%) with the accumulation in the resulting suspension of the 146S component in the amount of 1.14±0.02 μg/cm ³ and the infectious activity titer of 7.80±0.10 lg TCD ₅₀ /cm ³ . When using RPMI-1640 medium and Hanks' solution with the addition of 2.5% blood protein hydrolysate, the virus reproduction rates were significantly lower.

At the next stage of studying the conditions for monolayer cultivation of the FMDV strain A/Afghanistan/2017 in PSGK-30 cell culture under production conditions, the influence of the temperature factor on the reproduction process of the virus was evaluated. To do this, the cultivation of the virus was carried out in Eagle's MEM medium with the addition of 2% bovine serum at the following temperatures: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2°С. The dose of infection of the cell culture with foot-and-mouth disease virus was 0.010-0.001 TCD ₅₀ /cell. The cultivation of the virus was carried out until the destruction of the cell monolayer by 80-100% for 15-30 h (table 3). According to the results of the study, it was found that for the complete reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in a monolayer cell culture PSGK-30, the optimal medium temperature is 37.0±0.02°C, at which the development of CPP reached 95- 100%) with the accumulation of 146S particles equal to 1.14±0.02 μg/cm ³ and the titer of infectious activity of the virus 7.80±0.10 lg TCDzo/cm ³ .

The influence of the dose of infection of a monolayer of PSGK-30 cells with strain A/Afghanistan/2017 during cultivation on a production scale was evaluated. For this, different doses of the virus were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCD ₅₀ / class. Eagle's MEM medium was used as a support medium. The reproduction of the virus was carried out at a temperature of 37.0±0.2°C for 15-30 hours until the specific destruction of cells by 90-100%. Based on the results of cultivation, the concentration of the 146S component and the titer of FMDV infectious activity were determined. As follows from the data in Table 3, the greatest accumulation of "complete" virus particles was achieved at an infection dose of 0.1-0.01 TCD ₆₀ /cell. and amounted to 1.14±0.03 μg/cm ³ with a titer of infectious activity of the virus 7.80±0.10 lg TCD ₅₀ /cm ³ .

Thus, the conditions for monolayer cultivation of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in PSGK-30 cell culture on an industrial scale were studied. As a result of the study, the following optimal reproduction parameters of the A/Afghanistan/2017 strain of FMDV in the monolayer cell line PSGK-30 were determined: 1) supporting medium - Eagle MEM medium; 2) temperature regime of cultivation -37.0±0.02°C; 3) the dose of virus infection - 0.1-0.01 TCD ₆₀ /cl.

Example 4. Study of the conditions for suspension cultivation of the strain A/Afghanistan/2017 of FMDV on an industrial scale.

During the industrial cultivation of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in a continuous suspension culture of BHK-21/SUSP/ARRIAH cells, we searched for the optimal parameters for successful reproduction of the foot-and-mouth disease pathogen using different cell concentrations, temperature, and dose of infection.

At the first stage of the work, the influence of the seeding concentration of BHK-21/SUSP/ARRIAH cells in suspension on the reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus on an industrial scale was determined. For analysis, suspensions were prepared with the following cell concentrations: 2.5; 3.0; 3.5; 4.0 million cells/cm ³ . The pH was maintained in the range of 7.45-7.60. The virus cultivation temperature was 37.0±0.02°C, the virus infection dose was 0.10-0.01 TCD ₅₀ /cell. The reproduction of the virus was carried out until specific cell death by 95-100%, which was carried out within 10-12 hours. The results of suspension cultivation of the FMDV strain A/Afghanistan/2017 in suspension with different cell concentrations are presented in Table 4.

As follows from Table 4, when cultivating foot-and-mouth disease virus strain A/Afghanistan/2017 in a suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations, it was determined that the accumulation of the 146S component in suspension with a cell concentration of 2.5 million cells/ cm ³ was 1.04±0.03 µg/cm ³ , with a concentration of 3.0 million cells/cm ³ - 1.30±0.02 µg/cm ³ , with a concentration of 3.5 million cells/cm ³ - 1.78±0.03 µg/cm ³ , and with a concentration of 4.0 million cells/cm ³ -1.80±0.05 µg/cm ³ . As follows from the data obtained, for the reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus, the optimal concentration of BHK-21/SUSP/ARRIAH cells in suspension is 3.5 million cells/ ^cm3 . The value of the titer of infectious activity of the virus in this case amounted to 8.25±0.15 lg TCD ₅₀ /cm ³ .

A high concentration of cells made it possible to obtain the same product yield with a slight increase. From an economic point of view, therefore, for the reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus, it is optimal to use a suspension of BHK-21/SUSP/ARRIAH cells with a concentration of 3.5 million cells/ ^cm3 .

At the next stage of the work, the influence of the temperature factor on the suspension cultivation of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in the BHK-21/SUSP/ARRIAH cell line on an industrial scale was studied. To do this, the reproduction of the virus was carried out under the following temperature conditions: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2°C. The dose of infection of the cell culture with the virus was 0.10-0.01 TCD ₅₀ /cell. The pH of the viral suspension was maintained in the range of 7.45-7.60. The cultivation of the virus was carried out until the CPP, equal to 90-100%, for 10-28 hours. According to the results of the analysis, it was determined that for the complete reproduction of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in the suspension culture of BHK-21/SUSP/ARRIAH cells, the temperature is optimal medium 37.0 ± 0.02 ° C, at which within 10-12 hours specific cell death is observed by 95-100% with a high accumulation of the 146S component (1.78 ± 0.03 μg / cm ³ ) and a titer of infectious activity virus 8.25±0.15 lg TCD ₅₀ /cm ³ .

In the course of work on the study of the conditions of suspension cultivation of the FMDV strain A/Afghanistan/2017 on an industrial scale using the BHK-21/SUSP/ARRIAH cell culture, the effect of the cell infection dose was evaluated. To do this, cell suspensions were infected with the virus at different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD ₅₀ / class. The reproduction of the virus was carried out at a temperature of 37.0 ± 0.2°C for 10-30 h according to the cytopathic effect (CPE) equal to 90-95%. Based on the results of cultivation, the concentration of 146S particles and the titer of FMDV infectious activity were determined. As can be seen from the data in Table 4, the greatest accumulation of the 146S component was noted at an infection dose of 0.10-0.01 TCD ₆₀ /cell. (1.78±0.03 μg/cm ³ ) with a titer of infectious activity of the virus equal to 8.25±0.15 lg TCD ₅₀ /cm ³ .

Thus, we studied the parameters of suspension cultivation of the FMDV strain A/Afghanistan/2017 in the suspension cell line BHK-21/SUSP/ARRIAH on an industrial scale. The optimal conditions for the reproduction of the strain A/Afghanistan/2017 in the suspension culture of VNK-21 cells were revealed: 1) the concentration of cells before infection - 3.5 million cells/ ^cm3 ; 2) temperature regime of cultivation -37.0±0.02°C; 3) the dose of virus infection is 0.10-0.01 TCD ₆₀ /cell.

Example 5. Inactivation of a suspension of foot-and-mouth disease virus strain A / Afghanistan / 2017

At the end of the FMDV reproduction cycle, without stopping the thermostating process (37.0±0.02°C), an acidified solution of aminoethylethyleneimine (AEEI) with a pH of 8.2-8.6 was added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be 0.05%. Inactivation of the infectious activity of FMDV strain A/Afghanistan/2017 was carried out for 12 hours at a temperature of 36-37°C and pH 7.2-7.6 with stirring.

To determine the time of complete inactivation after the addition of AEEI, samples were taken every hour. The obtained samples were tested for the presence of FMD virus infectivity in the primary culture of porcine kidney cells SP. The results are presented in table 5.

As can be seen from Table 5, the complete inactivation of the infectious activity of the cultural foot-and-mouth disease virus of strain A/Afghanistan/2017, reproduced in cultivators, occurred 6 hours after the introduction of the inactivant.

Ready-made viral inactivated suspensions were examined in the complement fixation test to assess the content of virus components in them. The concentration of the 146S component was 1.76±0.10 µg/cm ³ , and the 146S+75S component was 2.35±0.08 µg/cm ³ .

Example 6. Selection of conditions for purification of a suspension of foot-and-mouth disease virus strain A / Afghanistan / 2017

The suspension of foot and mouth disease virus strain A/Afghanistan/2017 obtained during reproduction in the suspension cell line BHK-21/SUSP/ARRIAH is subjected to inactivation and purification. To obtain a purified foot-and-mouth disease virus antigen, polyhexamethylene guanidine (PHMG) was used at concentrations of 0.010 and 0.007%. Before and after the process of purification of the antigen of foot-and-mouth disease virus strain A/Afghanistan/2017, the concentration of total viral protein and immunogenic components was determined in the quantitative version of the complement fixation reaction. The results of the analysis are shown in table 6.

As follows from the data in Table 6, as a result of the purification of the FMDV antigen of strain A/Afghanistan/2017 using PHMG at a concentration of 0.010%, a slight decrease in the content of the 146S component of the virus was noted compared to the initial values (before purification) by 2.3% in the finished product . At the same time, the concentration of ballast protein decreased by 46%. When using PHMG at a concentration of 0.007%), the content of 146S particles decreased by 1.7%, and the concentration of ballast protein only by 36%). Thus, studying the conditions for purification of the antigen of strain A/Afghanistan/2017, we came to the conclusion that it is optimal to use PHMG with a concentration of 0.010% to obtain a purified product.

Example 7. Selection of conditions for concentrating the antigen of strain A/Afghanistan/2017 of foot and mouth disease virus

Cultural inactivated suspension of the strain

A/Afghanistan/2017, obtained using the suspension cell line BHK-21/SUSP/ARRIAH, was concentrated 9 times by volume. To increase the concentration of the immunogenic components of the FMDV antigen, the following methods were used: 1) adding polyethylene glycol (PEG-6000) with a concentration of 50%) to the antigen in a ratio of 1:4, with an exposure of 4 hours; 2) adding polyethylene glycol (PEG-6000) with a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 12 hours; 3) concentration using aluminum hydroxide (10%) colloidal solution 50-60%) of the total volume, virus antigen - 40-50%).

Before and after the process of concentrating the antigen of foot-and-mouth disease virus strain A/Afghanistan/2017, the concentration of total viral protein and immunogenic components was determined in the quantitative variant of RSC. The research results are presented in table 7.

As follows from the data in Table 7, when concentrating the antigen of the strain A/Afghanistan/2017 of FMDV using PEG-6000 for 4 hours, an increase in the content of components compared to the initial values (before concentration) was noted by 5 times. Using the same polymer, but increasing the exposure time to 12 h, the concentration of virus components was increased by 6 times. Using the method of sorption using aluminum hydroxide, it was possible to increase the amount of immunogenic components of the antigen of the A/Afghanistan/2017 strain of foot-and-mouth disease virus in suspension by 9 times (without loss). Thus, the best way to increase the concentration of the components of the FMDV strain A/Afghanistan/2017 is the sorption method using a colloidal solution of aluminum hydroxide.

Example 8 Adjuvant Selection and Antigen-Adjuvant Ratio

Aluminum hydroxide in combination with saponin was chosen as an adjuvant for the manufacture of an FMD monovalent adsorbed vaccine from the A/Afghanistan/2017 strain antigen. In the manufacture of a monovalent adsorbed vaccine against FMDV used 5-6% aluminum hydroxide in terms of dry matter and saponin in the amount of 1.5 mg/cm ³ .

Example 9. The layout of the FMD vaccine from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR ' ⁿ culture inactivated adsorbed

From the obtained concentrate of FMD virus antigen of strain A/Afghanistan/2017, an FMD monovalent sorbed inactivated vaccine was prepared using aluminum hydroxide and saponin as adjuvants. The amount of 146S immunogenic component in 1.0 cm ³ of the prepared antigen was 15.69 μg/cm ³ (table 7).

Example 10. Immunization of animals with a FMD vaccine from strain A/Afghanistan/2017 of a new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated adsorbed.

From the obtained FMD inactivated sorbed vaccine, a series of dilutions for cattle was prepared with a 4-fold step. 15 heads of cattle were divided into 3 groups of 5 heads each. The immunizing dose was 2.0 cm ³ . The first group of cattle (No. 1-5) was immunized with the vaccine without dilution, the second group of cattle (No. 6-10) was vaccinated with the vaccine diluted 1/4, the third group of cattle (No. 11-15) was vaccinated with the vaccine diluted 1/16 . The drug was administered intramuscularly in the middle third of the neck. As a virus control, 2 heads were left without vaccination.

Example 11. Examination of the blood sera of vaccinated animals for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus.

2 heads of cattle were immunized with the FMD vaccine from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 cultured inactivated adsorbed at a dose of 2 cm ³ .

Blood sera from cattle obtained before immunization and on the 14th day after immunization of animals were examined for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus using a blocking enzyme-linked immunosorbent assay (ELISA) in accordance with the recommendations of the OIE (OIE) [3]. The obtained sera were tested using the PrioCHECK®FMDVNSELISA for in vitro detection of antibodies against Foot and Mouth Disease Virus in serum of cattle, sheep and pigs ELISA test system (Prionics Lelystad B.V., the Netherlands). The results of the studies are shown in Table 8, which shows that the blood sera of animals did not contain antibodies to non-structural proteins of the foot-and-mouth disease virus (PI values for bovine blood sera<50%).

Example 12. Evaluation of the avirulence and safety of the FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture-inactivated adsorbed ³ . In the study, cattle weighing 250-300 kg were used. Within 10 days of observation, the animals remained clinically healthy, and the postmortem examination did not reveal changes characteristic of foot and mouth disease. This indicated the absence of virulent properties of the developed vaccine.

Control of the safety of the product was carried out by intramuscular injection of cattle vaccine at a dose of 6.0 cm ³ . The observation period was also 10 days. It should be noted that after immunization, the animal's body temperature can rise to 41°C and it is possible to stay at this level for 1-3 days.

According to the results of the studies, the vaccine was found to be harmless, all animals remained clinically healthy during the observation period, and no tissue necrosis at the site of vaccine administration was detected during the pathoanatomical analysis.

Example 13. Study of the immunogenic properties of the FMD vaccine from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated sorbed by the ability to induce virus-neutralizing antibodies in naturally susceptible animals

On the 21st day after the introduction of the FMD vaccine from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11, cultured inactivated sorbed blood was taken from cattle and the obtained blood sera were studied in the microneutralization reaction [3]. It was found that in cattle immunized with the vaccine in a whole dose (5 heads), the average values of antibody titer were 7.00±0.18 log2 SN50, with a dilution of 1/4 (5 heads) - 4.90±0.14 log ₂ SN ₅₀ , with a dilution of 1/16 (5 heads) - 3.55 ± 0.21 log ₂ SN50. (Table 9). The data obtained from the microneutralization reaction indicate that after the administration of the whole vaccine, the formation of humoral immunity with protective titers of strain-specific antibodies (5.5 or more log ₂ SN ₅₀ ) is ensured, which meets the requirements of international standards [3].

Example 14 Challenge of naturally susceptible animals. Study of the protective ability of the FMD vaccine from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR ' ^U cultured inactivated sorbed on naturally susceptible animal species.

Cattle, immunized in the amount of 15 heads, with various dilutions of the vaccine, were infected with the control strain A/Afghanistan/2017 of foot and mouth disease virus adapted to these animals into the mucous membrane of the tongue at a dose of 10 ^4.0 ID ₅₀ /0.2 cm ³ (in two points of 0.10 ± 0.05 cm ^{3 each} ). 7 days after infection, all animals were euthanized and postmortem examination was performed. Animals were considered protected from foot-and-mouth disease, in which there were no lesions on the limbs. Primary aphthae were not taken into account.

The results of the control infection are presented in Table 9, from which it follows that the FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11, culture-inactivated sorbed in whole form and at a dilution of 1/4, introduced in a single dose (2.0 cm ³ ) protects cattle from infection with a homologous strain. All 10 animals remained alive and without visible lesions. The vaccine, administered at a dilution of 1/16, protected 4 out of 5 animals.

According to the results of the control infection, it was found that the inoculation volume of this vaccine contains 24.25 PD ₅₀ and 0.08 IMD ₆₀ for cattle.

Thus, the above information indicates that the FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 cultural inactivated adsorbed, embodying the proposed invention, is intended as an experimental backup for use in agriculture , namely in veterinary virology and biotechnology; confirmed the possibility of implementing the presented; the vaccine made from the strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 in accordance with the invention, has a high immunogenic activity and is able to provide effective protection of susceptible animals against the epizootic strain of FMDV type A circulating in countries Southeast Asia and the Middle East.

Sources of information taken into account when compiling the description of the invention to the application for a patent of the Russian Federation for the invention "FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated sorbed":

1. Foot and mouth disease. Prevention measures. URL: http://89.rospotrebnadzor.ru/directions/epid nadzor/146902 (Date of access: 05/03/2022).

2. Danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Date of access: 04/14/2022).

3. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed.-Paris, 2015-Vol.1, Chapter 2.1.5. - P. 166-169.

4. Burdov A. N., Dudnikov A. I., Malyarets P. V. et al. - M.: Agropromizdat, 1990. - 320 p.

5. Ponomarev A. P., Uzyumov V. L., Gruzdev K. N. Foot-and-mouth disease virus: structure, biological and physico-chemical properties. - Vladimir: Folio, 2006. - 250 p.

6. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M/ Garland, R.P. Kitching [et.al.] // Comparative Immunology, Microbiology and Infectious Diseases. - October 2002. - V. 25. - P. 345-364.

7. Brown F. A brief history of FMD and its casual agent. FMD: Control Strateies: Proc. Jnt. Symp.2-5. June 2002, Lyons, France. - Paris, 2003. - p. 13-21.

8. Vaccination against foot-and-mouth disease virus confers complete clinical protection in 7 days and partial protection in 4 days: Use in emergency outbreak response / T.G. William, J.M. Pachecoa, T. Doel [et.al.] // Vaccine. - December 2005. -V. 23.- P. 5775-5782.

9. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M/ Garland, R.P. Kitching [et. al.] // Comparative Immunology, Microbiology and Infectious Diseases. - October 2002. - V. 25. - P. 345-364.

10 Barnett P.V. A review of emergency foot-and-mouth disease (FMD) vaccines / Vaccine. - February 2002. - V. 20. - P. 1505-1514.

11. Salt I.S. Emergency vaccination of pigs against foot-and-mouth disease: protection against disease and reduction in contact transmission / Vaccine. - April 1998. - V. 16.- P. 746-754.

12. Rakhmanov A.M. The current epizootic situation in the world on foot and mouth disease and measures for its control // Veterinary medicine mizhvid. topics. Sciences. Kharyuv, 2013. - S. 37-38.

13. Longjam N., Tauo T. Antigenic variation of Foot and Mouth Disease Virus // Vet. World. - 2011. - Vol. 4(10). - P. 475-479.

14. Melnik R.N., Khaustova H.V., Melnik H.V., Samuylenko A.Ya., Grin S.A., Svyatenko M.S., Litenkova I.Yu. Antigenic variability of foot-and-mouth disease virus serotype A and justification for the need to obtain new relevant industrial strains / Veterinary medicine and feeding. - 2019. - No. 3. - S. 29-31.

15. Paton D.J., Sumption K.J., Charleston B. Options for control of foot-and-mouth disease: knowledge, capability and policy / Phil. Trans. R. Soc. In (2009) 364. - P. 2657-2667.

16 Barnett P.V. A review of emergency foot-and-mouth disease (FMD) vaccines / Vaccine. - February 2002. - V. 20. - P. 1505-1514.

17. Official website of The FAO World Reference Laboratory for Foot-and-Mouth Disease - URL: http://www.wrlfmd.org/ref_labs/find ref lab reports.htm (Accessed October 15, 2020).

18. Guidelines for determining the concentration of 146S, 75S, 12S components of the vaccine strains of the cultured foot-and-mouth disease virus in the complement fixation test (CFR) / FGBU "ARRIAH". - Approved. 09/21/17.

19. Guidelines for determining the antigenic correspondence between epizootic isolates and industrial strains of foot-and-mouth disease virus in the cross-reaction of microneutralization: approved. Rosselkhoznadzor 13.09.2017 / FGBU "ARRIAH". - Vladimir: 2017. - 24 p.

20. Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-42.

21. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.

22. Tamura K., Nei M., and Kumar S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101:11030-11035.

23. Kumar S., Stecher G., Li M., Knyaz C, and Tamura K. (2018). MEGA 6: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35:1547-1549.

24. Barnett P. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M / Garland, R.P. Kitching [et.al.] // Comparative Immunology, Microbiology and Infectious Diseases. - 2002. - V. 25. - P. 345-364.

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agtaagtactccacaactggtggcggtagaaggggtgacctgggctctcttgcggcgcgggtcgccgcac

agctgcccagctctttcaattttggtgcaattcgggccacgaccatccacgagcttctcgtgcgtatgaa

acgtgccgagctctactgcccccgaccactgctggcagtggaagtgtcgtcgcaggacagacacaagcag

aagatcattgcacctgcaaaacagctcctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>213</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..213</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>FMDV</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TTTAGESADPVTTTVENYGGETQTQRRHHTDVGFIMDRFVKITPVNPTH

VIDLMQTHQHALVGALLRAATYYFSDLEIVVRHEGNLTWVPNGAPEGALANTSNPTAYHRQPFTRLALPY

TAPHRVLATVYNGVSKYSTTGGGRRGDLGSLAARVAAQLPSSFNFGAIRATTIHELLVRMKRAELYCPRP

LLAVEVSSQDRHKQKIIAPAKQLL</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (8)

1. The FMD vaccine from strain A/Afghanistan/2017 of the new genotype A/ASIA/Iran-05 ^FAR-11 culture inactivated adsorbed containing an active substance and an adjuvant, characterized in that it contains avirulent and purified antigenic material from A/Afghanistan/2017 strain of the Aphtae epizooticae virus, fam. Picornaviridae, genus Aphthovirus, serotype A, genotype A / ASIA / Iran-05 ^FAR-11 , deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Virus and Other Animal Pathogens (GKSHM) of the Federal State Budgetary Institution "Federal Center for Animal Health" (FGBU "ARRIAH"), under registration number: No. 378 - dep / 22-12 - GKShM FGBI "ARRIAH", in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains an avirulent and purified antigenic material from a strain A/Afghanistan/2017 homologous to the infectious agent of the new genotype A/ASIA/Iran-05 ^FAR-11 , obtained in a sensitive biological system and representing suspension containing predominantly the 146S immunogenic component of FMDV serotype A. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from the strain A/Afghanistan/2017 homologous to the infectious agent of the new genotype A/ASIA/Iran-05 ^FAR-11 , preferably obtained in a continuous suspension kidney cell line newborn Syrian hamster BHK-21/SUSP/ARRIAH and is a suspension containing predominantly 146S immunogenic component of FMDV serotype A. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from the strain A/Afghanistan/2017 homologous to the infectious agent of the new genotype A/ASIA/Iran-05 ^FAR-11 , preferably obtained in a continuous suspension kidney cell line newborn Syrian hamster BHK-21/SUSP/ARRIAH and is a suspension containing predominantly 146S immunogenic component of foot-and-mouth disease virus serotype A in an amount of at least 3.0 μg per 1.0 cm ³ of the finished product. 5. The vaccine according to claim 1, characterized in that it contains aluminum hydroxide in combination with saponin as an adjuvant. 6. The vaccine according to claim. 5, characterized in that it contains an adjuvant aluminum hydroxide, preferably in an amount of 5-6% in terms of dry matter and saponin with a content of 1.5 mg per 1.0 cm ³ of the finished product. 7. The vaccine according to any one of paragraphs. 1-6, characterized in that it contains an avirulent and purified antigenic material from a strain A/Afghanistan/2017 homologous to the infectious agent of the new genotype A/ASIA/Iran-05 ^FAR-11 obtained preferably in a continuous suspension cell line of the kidney of a newborn Syrian hamster BHK -21 / SUSP / ARRIAH and is a suspension containing mainly the 146S immunogenic component of FMD virus serotype A, an adjuvant - aluminum hydroxide and saponin, an additive in the form of a supporting medium in the finished product in the following quantities: Avirulent and purified strain antigen A/Afghanistan/2017 genotype A/ASIA/Iran- 05 ^far-11 FMD virus not less than 3.0 µg/cm ³ aluminum hydroxide 5-6% (in terms of dry matter) Saponin 1.5 mg / cm ³ Supportive environment up to 1.0 cm ³

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