RU2804803C1 - Culture inactivated emulsion vaccine against fmd of o/sea/mya-98 genotype from o n2383/primorsky/2019 strain - Google Patents

Abstract

FIELD: veterinary virology and biotechnology.

SUBSTANCE: cultural inactivated emulsion vaccine against foot-and-mouth disease of O/SEA/Mya-98 genotype from O No. 2383/Primorsky/2019 strain is described. The vaccine contains an avirulent and purified concentrated antigen of O No. 2383/Primorsky/2019 strain of foot and mouth disease virus of O/SEA/Mya-98 genotype obtained in a suspension transplanted cell line from the kidney of a newborn Syrian hamster (BHK-98/SUSP/ARRIAH), representing a suspension containing predominantly 146S immunogenic component of FMDV and Montanide ISA-71 R VG oil adjuvant in effective ratios. The vaccine is highly immunogenic and can provide effective protection against a homologous infectious agent circulating in Asian countries.

EFFECT: considering the recent increase in trade and economic relations between the Russian Federation and the countries of the Asian continent, the created vaccine is relevant for ensuring the biological safety of our country and, in general, veterinary well-being for FMD in relation to the O/SEA/Mya-98 genotype in the world.

3 cl, 1 dwg, 7 tbl, 14 ex

Description

The invention relates to the field of veterinary virology and biotechnology, namely to the development of a cultural inactivated emulsion vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383 /Primorsky/ 2019”.

Foot-and-mouth disease remains a major barrier to development and poverty reduction throughout the developing world for many reasons, including the cost of control measures, the denial of access to valuable global markets for FMD-free livestock products, loss of production due to decreased milk yields, decreased live weight gain and inability of infected livestock to perform traction. Foot-and-mouth disease virus affects a variety of artiodactyl animals, including cattle, sheep, goats, pigs, and all wild ruminants, with high incidence in adult animals. High mortality may occur in young animals due to myocarditis.

There are seven following serotypes of foot-and-mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, while representatives of serotype O are very common, which determines the relevance of producing vaccines to protect against foot-and-mouth disease of this serotype.

Each serotype is genetically divided into different topotypes, and sometimes into separate genetic lineages and even sublineages. The differences between them are determined by analyzing the nucleotide sequence of the highly variable 1D gene, which encodes the amino acid sequence of the surface protein VP ₁ [1]. Considering that the pathogen

foot and mouth disease is an RNA-containing virus, it is characterized by the formation of a large number of mutations during replication of the viral genome (predominantly in the 5' region) and the phenomenon of recombination (mainly in the 3' region). It is for this reason that more than 60 genetic lines and sublines are distinguished within the serotypes [2]. Recovery from an animal being infected with any one serotype does not provide immune protection against another serotype or even some other genetic lines [3].

Identification of genetic relationships between different isolates and strains is carried out using nucleotide sequencing, which makes it possible to determine the origin of the virus in outbreak conditions. Antigenic characteristics of the virus associated with the emergence of new strains are important for analyzing outbreaks and creating optimal vaccines [4, 6].

Foot and mouth disease virus serotype O is divided into the following 11 topotypes: EAST AFRICA 1-4 (EA-1-4), SOUTHEAST ASIA (SEA), EUROPE-SOUTH AMERICA (Europe-South America) ( EURO-SA), INDONESIA-1 and 2 (ISA-1 and 2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA) and WEST AFRICA (West Africa) (WA). This high genetic and antigenic diversity leads to problems in the specific prevention of FMD when using culture-inactivated FMD vaccines. As a result, there is a need to create new means of specific immunoprophylaxis against foot-and-mouth disease virus serotype O [5-12]. The presented diversity of representatives of serotype O is due to the high degree of variability of RNA-containing viruses and the active movement of this pathogen in the territories of Asian countries, mainly Southeast, Central and Middle Asia.

In order to prevent the occurrence of foot-and-mouth disease on farms in the Russian Federation, a set of measures is used to combat and prevent foot-and-mouth disease, which is aimed at preventing the introduction of the virus into the country, systematically vaccinating animals in the buffer zone, and monitoring the immune status of large and small cattle and pigs.

To immunize animals, a vaccine made from a virus homologous to field isolates should be used, which is confirmed by the studies of many scientists [6, 7, 8, 9, 10, 11].

This infection continues to be an economically significant problem. Today, whole-virion vaccines against foot-and-mouth disease are still recognized as the most effective, which is extremely important for combating the most contagious disease in the world [8, 9, 12, 13].

To conduct an effective vaccination campaign, it is necessary to have an effective and safe vaccine containing as an immunogenic component a viral antigen corresponding to the epizootic spreading isolate of a certain genotype. The creation of such vaccines is still an extremely urgent task. Achieving this goal will make it possible to effectively use the vaccine in disadvantaged regions and threatened areas to build immunity against a specific genotype of the foot-and-mouth disease virus in order to ensure veterinary well-being [14, 15, 16].

On the territory of the Russian Federation, outbreaks of foot-and-mouth disease are sporadic and are caused by the introduction of the pathogen from the territory of neighboring countries, since certain regions of Russia border on Asian countries that are endemic for foot-and-mouth disease and are subject to a very high risk of introducing the infectious agent from the territory of these states. During trade and economic transactions there are serious risks of introducing foot-and-mouth disease virus isolates into the territory of the Russian Federation. This could undermine the veterinary well-being of our country regarding foot-and-mouth disease; therefore, it is necessary to take measures in advance to create a safe and effective foot-and-mouth disease vaccine.

In the Russian Federation, outbreaks of foot-and-mouth disease type O were recorded from 2000 to 2021. on the territory of Primorsky, Khabarovsk, Transbaikal territories, Amur, Orenburg regions, and the Republic of Bashkortostan. Outbreaks of the disease were caused by foot and mouth disease virus type O and genotypes: O/SEA/Mya-98, O/ME-SA/PanAsia, O/ME-SA/Ind-2001.

In Southeast, South, and East Asia, outbreaks of foot and mouth disease of the O/SEA/Mya-98 genotype are sporadic. It was found that in Mongolia, foot and mouth disease virus isolates were identified that belong to this genotype in 2004, 2010, 2015, 2017, 2018, 2019. Outbreaks of foot and mouth disease of this genotype were recorded in our country in 2010 (isolates O/Russia/Jul/2010 and O/Russia/Aug/2010), in 2019 (isolate O/Primorskiy/RUS/2/2019). Currently, representatives of this genotype also circulate in the countries of Southeast Asia. It should be noted that since 2019, noticeable changes have appeared in the nucleotide and amino acid composition of foot-and-mouth disease virus isolates of this genotype. This genetic modification is significantly different from its predecessor (vaccine strain “O No. 2212/Primorsky/2014”) and, accordingly, the prototype vaccine against foot-and-mouth disease does not provide complete protection against the O/SEA/Mya-98 genotype. Based on the analysis, the most relevant candidate for creating an effective vaccine that will provide protection against foot and mouth disease of the O/SEA/Mya-97 genotype, taking into account the emerging mutations in the virus genome, is the strain “O No. 2383/Primorsky/2019”.

There are known production strains of foot-and-mouth disease virus serotype O, which are used for the production of specific foot-and-mouth disease prevention products:

- strain “O/Taiwan/1997” (genotype O/CATHAY/CAM 94),

- strain “O No. 2212/Primorsky/2014” (genotype O/SEA/Mya-98),

- strain “O/Primorsky/2012” (genotype O/ME-SA/PanAsia),

- strain “O/Saudi Arabia/2008” (genotype O/ME-SA/PanAsia2),

- strain “O/Zabaikalsky/2016” (genotype O/ME-SA/Ind-2001d).

The foot-and-mouth disease virus isolate “O/Primorskiy/RUS/2/2019” was isolated from pathological material from cattle in the village of Grigoryevka, Mikhailovsky district, Primorsky Territory, Russian Federation in 2019. During scientific research at the Federal State Budgetary Institution "ARRIAH" by adaptation to reproduction in the primary trypsinized monolayer pig kidney cell line SP, in continuous cell cultures BNK-21/SUSP/ARRIAH, IB-RS-2, PSGK-30 was characterized and named - strain “O No. 2383/Primorsky/2019” of serotype O.

According to the results of a comparative analysis of nucleotide sequences, the isolated strain belongs to the O/SEA/Mya-98 genotype of foot-and-mouth disease virus, which differs significantly from the production strains of foot-and-mouth disease virus serotype O (Fig. 1).

Due to the significant increase in trade and economic relations between Russia and Asian countries, in which outbreaks of foot-and-mouth disease of the O/SEA/Mya-98 genotype are consistently recorded, there is a danger of new cases of foot-and-mouth disease in the Russian Federation and the problem of possible emergency prevention of this disease for the formation of immunity in susceptible animals. Thus, there was a need to develop a cultural inactivated emulsion vaccine against foot-and-mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019” to ensure the biological safety and veterinary well-being of the territory of the Russian Federation, neighboring states and countries where foot-and-mouth disease is endemic who will use this drug to prevent the disease.

The closest to the proposed invention in terms of the totality of essential features is an inactivated emulsion vaccine against foot-and-mouth disease serotype O (strain “O No. 2212/Primorsky/2014”, genotype O/SEA/Mya-98) [17], containing the active substance in the form of an avirulent and purified cultural antigenic material from the strain “O No. 2212/Primorsky/2014” homologous to the foot-and-mouth disease pathogen, obtained in a sensitive biological system, and targeted additives in the form of oil adjuvant Montanide ISA-206 VG (“Seppic”, P. France): antigen concentration (inactivated 146S +75S components of the foot and mouth disease virus) not less than 3.0 μg/cm ³ , the content of the oil adjuvant Montanide ISA-206 VG is 50% by weight, the additive in the form of a supporting medium is up to 1.0 cm ³ (prototype).

As a sensitive biological system for the reproduction of the foot and mouth disease virus, a continuous cell line of the kidney of a newborn Syrian hamster BHK-21/2-17 is used; as a supporting medium, Eagle's DMEM medium is used without adding serum, with the addition of enzymatic hydrolyzate of dry muscle, hydrolyzate of dry blood proteins at pH Wednesdays 7.45-7.65.

Aminoethylethylenimine (AEEI) is used to inactivate the virus. When producing an emulsion vaccine, Montanide ISA-206 VG oil adjuvant (Seppic, P. France) is used as an adjuvant. Purification of the virus-containing suspension from ballast impurities is carried out using polyhexamethylene guanidine hydrochloride (PHMG).

A significant drawback of the prototype vaccine is its low immunogenic activity relative to the emerging strains/isolates of the foot-and-mouth disease virus genotype O/SEA/Mya-98.

To solve this problem, the present invention was created, which included the development of a cultural inactivated emulsion vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019”. This vaccine assumes a high content of the 146S immunogenic component in the dose of the drug [19, 20].

The technical result from the use of the proposed invention is to expand the arsenal of anti-foot-and-mouth disease inactivated cultural emulsion vaccines to protect animals against isolates and strains of foot-and-mouth disease virus serotype O and, in particular, genotype O/SEA/Mya-98.

The specified technical result was achieved by creating a cultural inactivated emulsion vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019”, characterized by the following set of characteristics reflected below.

The developed vaccine in 1.0 cm ³ of the drug contains the following main components: 1) the active substance in the form of avirulent and purified antigenic material from the strain “O No. 2383/Primorsky/2019” (genotype O/SEA/Mya-98) of the virus homologous to the causative agent of the infection foot and mouth disease reproduced in the continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; 2) oil adjuvant Montanide ISA-71 R VG, which provides an enhanced immune response in animals with a content of 70% by weight, 3) maintenance medium - up to 1.0 cm ³ .

The foot and mouth disease virus strain “O No. 2383/Primorsky/2019” (genotype O/SEA/Mya-98) was deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Disease Viruses and Other Animal Pathogens (GKSHM) of the Federal State Budgetary Institution “ARRIAH”, under registration number: No. 419 - dep / 22-53 -GKShM FSBI "ARRIAH".

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

To produce a vaccine, it is preferable to use a continuous suspension culture of BHK-21/SUSP/ARRIAH cells as a sensitive biological system, and Hanks’ medium is used as a supporting medium without adding serum, but with the addition of blood protein hydrolysate in an amount of 5.0% of the total volume at pH of the medium is 7.45-7.65. Virus inactivation is carried out using aminoethylethylenimine (AEEI) with a concentration of 0.034% 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 0.014%) of the total volume.

The avirulent and purified antigen of the strain “O No. 2383/Primorsky/2019” (genotype O/SEA/Mya-98) of the foot-and-mouth disease virus is a suspension containing the inactivated 146S immunogenic component of the foot-and-mouth disease virus. The concentration of the component in the product is assessed using a quantitative version of the complement fixation reaction (CRR) [18, 19].

To create a vaccine, viral material is used that contains at least 4.0 μg of inactivated immunogenic 146S particles of foot-and-mouth disease virus per 1.0 cm ³ . The declared concentration of the immunogenic component in the vaccine preparation is ensured by concentrating the antigen using a Centramate 500S Pall tangential filtration unit.

The cultural inactivated emulsion vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019” is prepared by mixing the purified antigen and the oil adjuvant Montanide ISA-71 R VG in a ratio of 30% to 70% by weight, respectively . The resulting vaccine is a stable white or pale pink emulsion containing an inactivated 146S component of the foot and mouth disease virus, which ensures the formation of specific immunity.

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

1. Vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019”, cultural inactivated emulsion.

2. The active substance in the form of avirulent and purified antigenic material from the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” of genotype O/SEA/Mya-98 homologous to the causative agent of the disease in an effective amount.

3. Targeted supplements.

The significant distinctive features of the proposed vaccine are that as an active substance it contains an avirulent purified antigen of the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 of the foot-and-mouth disease virus 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 “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus, obtained preferably in the suspension continuous cell line BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly 146S immunogenic component of the foot and mouth disease virus in an effective amount.

2. Avirulent and purified antigen of the strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus, obtained preferably in a suspension continuous cell culture BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly 146S immunogenic component of the foot and mouth disease virus in an amount of at least 4.0 μg per 1.0 cm ³ of the finished vaccine preparation.

3. Among the targeted additives, the vaccine contains the oil adjuvant Montanide ISA-71 R VG.

4. The vaccine contains the oil adjuvant Montanide ISA-71 R VG in an amount of 70% (by weight) per 1.0 cm ³ of the finished product.

5. Avirulent and purified antigen of the strain “O No. 23 83/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus, obtained preferably in the continuous suspension cell line BHK-21/SUSP/ARRIAH, oil adjuvant Montanide ISA-71 R VG, additive in the finished product in the form of a supporting medium in the following quantities:

Avirulent and purified strain antigen “O No. 2383/Primorsky/2019” genotype not less than 4.0 μg/cm ³ O/SEA/Mya-98 foot and mouth disease virus Oil adjuvant Montanide ISA-71 R VG 70% (by weight) Supportive environment up to 1.0 cm ³

The proposed cultural inactivated emulsion vaccine against foot-and-mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 has high immunogenic activity and provides reliable protection against isolates of the foot-and-mouth disease virus genotype O/SEA/Mya-98.

Achieving a technical result from the use of the invention is ensured by the fact that the antigen of the strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus, which has high immunogenic activity, creating an effective protection of susceptible animals against foot-and-mouth disease virus isolates of the presented genotype, which in recent years have caused outbreaks in the world.

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

Fig. 1. - Dendrogram reflecting the phylogenetic relationship of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” with epizootic strains of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₁ gene.

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

SEQ ID NO: 1 represents the nucleotide sequence of the gene encoding the VP ₁ protein of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus genotype O/SEA/Mya-98;

SEQ ID NO: 2 represents the amino acid sequence of the VP ₁ protein of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus genotype O/SEA/Mya-98.

Strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus is characterized by the following characteristics and properties.

Morphological characteristics

Strain “O No. 2383/Primorsky/2019” of foot-and-mouth disease virus serotype O belongs to the order Picornavirales, family Picornaviridae, genus Aphthovirus, species Foot-and-Mouth Disease virus and has morphological features characteristic of the causative agent of foot-and-mouth disease: the shape of the virion is ixahedral, size 25 nm . The virion consists of a single-stranded positively charged RNA molecule and 60 copies of a polypeptide, each of which is represented by the proteins VP ₄ , VP ₂ , VP ₃ , VP ₁ .

Antigenic properties

According to its antigenic properties, the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus belongs to serotype O. The virus is stably neutralized by a homologous antiserum. In recovered animals, type-specific antibodies are formed in the blood serum, which are detected in an enzyme-linked immunosorbent assay and microneutralization reaction (RMN).

Using the method of nucleotide sequencing, the primary structure of the 1D gene of the VP ₁ protein of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus was determined. Comparative analysis of nucleotide sequences showed that the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus belongs to the genotype O/SEA/Mya-98 (Fig. 1).

The antigenic affinity (r ₁ ) of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus was studied at the Russian Medical Research Institute, in a cross-study of the strain with specific sera obtained for the following production strains of the foot-and-mouth disease virus:

- strain “O/Taiwan/1997” (genotype O/CATHAY/CAM 94),

- strain “O No. 2212/Primorsky/2014” (genotype O/SEA/Mya-98),

- strain “O/Primorsky/2012” (genotype O/ME-SA/PanAsia),

- strain “O/Saudi Arabia/2008” (genotype O/ME-SA/PanAsia2),

- strain “O/Zabaikalsky/2016” (genotype O/ME-SA/Ind-2001d).

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from production strains of foot-and-mouth disease virus serotype O against 10 ² TCD ₅₀ of homologous and heterologous virus was determined in the RMN during cross-titration, calculating the values using a linear regression equation, and expressed in lg. The r ₁ value was determined as the antilogarithm of the difference in serum lg titers against heterologous and homologous viruses [19, 20].

The value of r ₁ in the RMN was interpreted as follows:

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

at <0.3 - the studied sample of the foot-and-mouth disease virus strain differs significantly from the production strain.

Maximum relatedness is observed when the value of r ₁ in the RMN tends to 1.0.

Indicators of antigenic relatedness when studying the strain “O No. 2383/Primorsky/2019” amounted to r ₁ from 0.07 to 0.31, which indicates the absence of obvious antigenic relatedness with production strains of foot-and-mouth disease virus serotype O. The following r ₁ data were obtained for the strains:

- strain “O/Taiwan/1997” (genotype O/CATHAY/CAM 94) - 0.07, - strain “O No. 2212/Primorsky/2014” (genotype O/SEA/Mya-98) - 0.31, - strain “O/Primorsky/2012” (genotype O/ME-SA/PanAsia) - 0.22, - strain “O/Saudi Arabia/2008” (genotype 0/ME-SA/PanAsia2) - 0.17, - strain “O/Zabaikalsky/2016” (genotype O/ME-SA/Ind-2001d) - 0.27.

According to antigenic relatedness data, strain “O No. 2212/Primorsky/2014”

(genotype O/SEA/Mya-98) are related to the strain “O No. 2383/Primorsky/2019”, the r ₁ value is 0.31. Although they belong to the same genotype O/SEA/Mya-98, during phylogenetic analysis (Fig. 1) the strain “O No. 2383/Primorsky/2019” is similar in nucleotide sequences to the strain “O No. 2212/Primorsky/2014” by only 67 %, in other words, the degree of difference between them was 33%. This fact necessitates the creation of a vaccine from the “O No. 2383/Primorsky/2019” strain, which, unlike the original version, is currently widespread in the world.

Geno- and chemotaxonomic characteristics

Strain “O No. 2383/Primorsky/2019” of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular weight of 8.08×10 ⁶ D. Nucleic acid is represented by a single-chain linear molecule with a molecular weight of 2.8×10 ⁶ D. The virion has a protein shell, consisting of four main 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. Viral 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 nonstructural polypeptides of the virus. From 8 non-structural polypeptides that accumulate in infected cells, an RNA-dependent RNA polymerase (3D gene) is isolated, which is involved in RNA replication for the assembly of virions.

Physical properties

The mass of the virion is 8.4×10 ^-18 g. The buoyant density in the sucrose density gradient is 1.44 g/cm ³ .

Resistance to external factors

Strain “O No. 2383/Primorsky/2019” of foot-and-mouth disease virus is resistant to ether, chloroform, and acetone. Most stable at pH 7.45-7.65. Shifts in pH to the acidic and strongly alkaline side lead to inactivation of the virus. Sensitive to formaldehyde, UV irradiation, γ-irradiation, high temperatures (above 37.9°C).

Additional characteristics and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

Stability - retains the original biological properties when passaging in sensitive biological systems for 5 passages (observation period) on continuous crops.

Biotechnological characteristics

The foot and mouth disease virus strain “O No. 2383/Primorsky/2019” is reproduced in continuous cell cultures: Siberian mountain ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (BHK-21).

During the test, 5 consecutive passages of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus were carried out in continuous cell cultures PSGK-30, BHK-21, IB-RS-2. Biological properties were characterized by determining the infectious activity of the virus of each passage in the continuous cell line IB-RS-2 and in naturally susceptible animals - cattle (cattle) and pigs.

To reduce the epizootic risk of foot and mouth disease caused by the O/SEA/Mya-98 genotype and prevent the emergence of new foci of the disease, timely vaccine prevention is important, which requires the development of a new safe and effective vaccine.

A safe and effective vaccine against foot and mouth disease of the O/SEA/Mya-98 genotype from the strain “O No. 2383/Primorsky/2019”, culture inactivated emulsion, has been obtained.

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

Example 1. Genetic characteristics of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” according to PCR and nucleotide sequencing data.

The primary structure of the 1D gene (VP ₁ protein) (639 nt) of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” was analyzed using the Sanger nucleotide sequencing method and the position of this strain was determined on the phylogenetic tree of the foot-and-mouth disease virus serotype O. The method is based on determining the primary structure of the 1D gene (VP ₁ protein) of the test isolate/strain, followed by analysis of the phylogenetic relationship with other isolates (19 representatives) of foot-and-mouth disease virus serotype O.

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 vaccine strain “O No. 2383/Primorsky/2019” with other isolates and strains. The nucleotide sequence of the VP ₁ protein gene of the strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus is presented by SEQ ID NO: 1. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the strain “O No. 2383/Primorsky” /2019" genotype O/SEA/Mya-98 foot and mouth disease virus is reflected in SEQ ID NO: 2.

A phylogenetic analysis was carried out for the strain “O No. 2383/Primorsky/2019”. The phylogenetic tree was inferred using the MEGA6 program and the Neighbor-Joining algorithm. The percentage of replicate branches in which related taxa cluster together in the bootstrap test (increased from the standard 100 to 400 replicates for high confidence) is shown next to the branches [20, 21]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [22] and expressed in units of the number of base substitutions per site. This analysis included 19 nucleotide sequences, including the sequence of the 1D gene of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus. All items containing spaces and missing data were removed (complete deletion option). There were a total of 639 items in the final data set. Evolutionary analysis was carried out in MEGA 6 [23, 24].

As a result of the work carried out by comparing the complete nucleotide sequences of the 1D gene (VP ₁ protein) of the strain “O No. 2383/Primorsky/2019” and other isolates/strains of foot-and-mouth disease virus serotype O, the position of the studied strain on the phylogenetic tree was determined (Fig. 1).

Thus, the studied strain “O No. 2383/Primorsky/2019” belongs to the genotype O/SEA/Mya-98, which is confirmed by nucleotide and amino acid analysis data.

Example 2. Adaptation of the strain “O No. 2383/Primorsky/2019” of genotype O/SEA/Mya-98 to a continuous monolayer cell culture of the kidney cells of the Siberian mountain ibex PSGK-30.

To infect a continuous monolayer cell culture of Siberian mountain ibex kidney cells PSGK-30, a 10% aphthous suspension of foot-and-mouth disease virus obtained from pig aphthae (passage 2) was used. The inoculating cell concentration was 0.20-0.25 million cells/cm ³ , the virus infection dose was 0.001 TCD ₅₀ /cell. According to the results of the study, the reproduction of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” took place with a specific destruction of the monolayer by 90-100% in 18-20 hours. Over the course of 6 consecutive passages, an increase in the titer of infectious activity of the virus from 6.00 ± 0 was noted ,10 to 7.50±0.10 lg TCD ₅₀ /cm ³ .

The maximum titer of infectious activity of the virus was achieved at stage 6 of passage (7.50±0.10 lg TCD ₅₀ /cm ³ ). The concentration of 146S particles from passages 1 to 6 changed from 0.41±0.02 to 1.02±0.02 μg/ ^cm3 . In this case, the largest amount of the 146S component was noted at the stage of passage 6 (1.02±0.02 μg/cm ³ ). The degree of reliability (R ² ) of the study results in the quantitative version of RT-PCR-RT ranged from 96.58 to 99.79%. Thus, over the course of 6 consecutive passages, the adaptation of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” to the continuous monolayer cell line PSGK-30 was successfully carried out.

Example 3. Study of the conditions for monolayer cultivation of foot and mouth disease virus strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 on an industrial scale.

In the process of industrial cultivation of the strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus in a continuous monolayer cell culture PSGK-30, the optimal conditions for cultivating the virus were selected according to the supporting medium, temperature factor and dose of infection of cells with the studied virus .

At the first stage of the study, the effect of the composition of the supporting medium on the reproduction of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” in a monolayer culture of PSGK-30 cells was assessed on a production scale (at the stage from the 5th to the 6th passages). For comparison, three media were used with the addition of 2.0% fetal calf serum: 1) Eagle's medium DMEM; 2) RPMI-1640 environment; 3) Hanks solution. The inoculating cell concentration was 0.20 million cells/cm ³ , the dose of virus infection was 0.1000-0.0001 TCD ₅₀ /cell. Cultivation of the virus was carried out at temperatures of 35±0.2 - 38±0.2°C until the cell monolayer was destroyed by at least 85%. The results of the reproduction of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” with supporting media of different compositions are shown in Table 1.

The table data shows that when reproducing the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in a monolayer culture of PSGK-30 cells using supporting media of different compositions, Hanks’ medium is optimal, allowing for specific destruction of the cell monolayer in 18-20 hours by 95-100% with accumulation of the 146S component in the resulting suspension in an amount of 1.02±0.02 μg/cm ³ and an infectious activity titer of 7.50±0.10 lg TCD ₅₀ /cm ³ . Viral reproduction rates were lower when using RPMI-1640 and Eagle DMEM media.

At the next stage of studying the conditions for monolayer cultivation of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in the PSGK-30 cell culture under production conditions, the influence of the temperature factor on the virus reproduction process was assessed. To do this, the virus was cultivated in Eagle's medium DMEM with 2% bovine serum at the following temperatures: 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 foot-and-mouth disease virus was 0.010-0.001 TCD50/CL. The virus was cultivated until the cell monolayer was destroyed by 85-100% within 18-28 hours (Table 1). Based on the results of the study, it was revealed that for complete reproduction of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in a monolayer cell culture PSGK-30, the optimal temperature is 37.0 ± 0.02 ° C, at which the development of The CPE reached 95-100% with the accumulation of 146S particles equal to 1.02±0.02 μg/cm ³ and the titer of infectious activity of the virus 7.50±0.10 lg TCD ₅₀ /cm ³ .

We assessed the effect of the dose of infection of a monolayer of cells of the PSGK-30 line with the strain “O No. 2383/Primorsky/2019” during cultivation on a production scale. For this, different doses of the virus were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCD ₅₀ /cl. Eagle's MEM medium with 2% cattle blood serum was used as a supporting medium. Virus reproduction was carried out at a temperature of 37.0±0.2°C for 18-32 hours until specific cell destruction was 85-100%). Based on the results of cultivation, the concentration of the 146S component and the titer of infectious activity of the foot-and-mouth disease virus were determined. As follows from the data in Table 1, the greatest accumulation of the 146S component of the virus was achieved at an infection dose of 0.01-0.001 TCD ₅₀ /cell. and amounted to 1.02±0.02 μg/cm ³ with a titer of infectious activity of the virus of 7.50±0.10 lg TCD ₅₀ /cm ³ .

Thus, the conditions for monolayer cultivation of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” in PSGK-30 cell culture on an industrial scale were studied. As a result of the study, the following optimal parameters for the reproduction of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in the monolayer cell line PSGK-30 were determined: 1) maintenance medium - Hanks’ medium; 2) cultivation temperature - 37.0±0.02°C; 3) dose of virus infection - 0.01-0.001 TCD ₅₀ / cell.

Example 4. Study of the conditions for suspension cultivation of the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 of the foot-and-mouth disease virus on an industrial scale.

During the industrial cultivation of the strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus in a continuous suspension cell culture BHK-21/SUSP/ARRIAH, the search for optimal parameters for the successful reproduction of the foot-and-mouth disease pathogen was carried out, using different concentrations of cells, temperature conditions and dose of virus infection.

At the first stage of the work, we determined the effect of the seed concentration of cells of the BHK-21/SUSP/ARRIAH line in suspension on the reproduction of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” on an industrial scale. Suspensions with the following cell concentrations were prepared for analysis: 2.5; 3.0; 3.5; 4.0 million cells/ ^cm3 . The pH value was maintained in the range of 7.45-7.65. The virus cultivation temperature was 37.0±0.02°C, the dose of virus infection was 0.010-0.001 TCD ₅₀ /cell. Virus reproduction was carried out until specific cell death was 95-100%, which was carried out within 11-23 hours. The results of suspension cultivation of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” in suspension with different cell concentrations are presented in Table 2.

As follows from Table 2, when cultivating the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” in the suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations, it was determined that the accumulation of the 146S component in the suspension with a cell concentration of 2.5 million cells/cm ³ was 0.75±0.02 μg/cm ³ , with a concentration of 3.0 million cells/cm ³ -1.10±0.02 μg/cm ³ , with a concentration of 3.5 million cells ./cm ³ - 1.59±0.02 μg/cm ³ , and with a concentration of 4.0 million cells/cm ³ - 1.65±0.02 μg/cm ³ . As follows from the data obtained, for the reproduction of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus, the concentration of BHK-21/SUSP/ARRIAH cells in suspension is equal to 3.5 million cells/cm ³ (at a concentration of 4.0 million cells/ ^cm3 , the concentration is slightly higher, but the production costs for the number of cells are higher; based on this, it is economically feasible to use a cell concentration equal to 3.5 million cells/ ^cm3 ). The titer of infectious activity of the virus was 9.25±0.10 lg TCD ₅₀ /cm ³ .

At the next stage of the work, we studied the influence of the temperature factor on the reproduction of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in a suspension culture of cells of the BHK-21/SUSP/ARRIAH line on an industrial scale. For this purpose, virus reproduction 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 0.010-0.001 TCD ₅₀ /cell. The pH value of the viral suspension was maintained in the range of 7.45-7.65. Cultivation of the virus was carried out until the CPD was equal to at least 80% (priority was given to complete specific destruction of cells), for 11-23 hours. Based on the results of the analysis, it was determined that for complete reproduction of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in suspension For the BHK-21/SUSP/ARRIAH cell culture, the optimal temperature is 37.0±0.02°C, at which 95-100% specific cell death is observed within 13-14 hours with a high accumulation of the 146S component (1.59 ±0.02 μg/cm ³ ) and the titer of infectious activity of the virus is 9.25 ± 0.10 lg TCD ₅₀ / cm ³ .

In the course of work to study the conditions for suspension cultivation of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” on an industrial scale using cell culture BHK-21/SUSP/ARRIAH, the effect of the dose of cell infection was assessed. To do this, cell suspensions were infected with the virus in different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD ₅₀ /cl. Virus reproduction was carried out at a temperature of 37.0±0.2°C for 11-22 hours until the cytopathic effect was at least 90%.” Based on the results of cultivation, the concentration of 146S particles and the titer of infectious activity of the foot-and-mouth disease virus were determined. As can be seen from the data in Table 2, the greatest accumulation of the 146S component was observed at an infection dose of 0.010-0.001 TCD ₅₀ /cell. (1.59±0.02 μg/cm ³ ) with a titer of infectious activity of the virus equal to 9.25±0.10 lg TCD ₅₀ /cm ³ .

Thus, we studied three parameters of suspension cultivation of the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 of foot-and-mouth disease virus in the suspension cell line BHK-21/SUSP/ARRIAH on an industrial scale. The optimal conditions for the reproduction of the strain “O No. 2383/Primorsky/2019” in a suspension culture of BHK-21/SUSP/ARRIAH cells were identified: 1) cell concentration before infection - 3.5 million cells/cm ³ ; 2) cultivation temperature -37.0±0.02°C; 3) dose of virus infection - 0.010-0.001 TCD ₅₀ / cell.

Example 5. Inactivation of a suspension of foot and mouth disease virus strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98.

At the end of the reproduction cycle of the foot-and-mouth disease virus, without stopping the thermostatting process (37.0±0.02°C), an acidified solution of aminoethylethylenimine (AEEI) with a pH of 8.2-8.5 was added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to 0.034%. Inactivation of the infectious activity of the foot and mouth disease virus strain "O No. 2383/Primorsky/2019" genotype O/SEA/Mya-98 was carried out for 12 hours at a temperature of 37.0±0.1°C and pH 7.45-7.65 with periodic stirring.

To determine the time of complete inactivation after the addition of 1,2-AEEI, samples were taken every hour. The obtained samples were tested for the presence of infectious activity of the foot-and-mouth disease virus in the primary culture of pig kidney cells SP. The results are presented in Table 3, from which it can be seen that complete inactivation of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” reproduced in cultivators occurred 6 hours after the addition of 1,2-AEEI.

The prepared inactivated viral suspensions were examined at the RSC to assess the content of virus components in them. The concentration of the 146S component was 1.55±0.02 μg/cm ³ , and the 146S+75S component was 2.04±0.02 μg/cm ³ .

Example 6. Selection of conditions for purification of a suspension of foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98.

A suspension of foot and mouth disease virus strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98, obtained during reproduction in the suspension cell line BHK-21/SUSP/ARRIAH, was subjected to inactivation and purification. To obtain purified foot-and-mouth disease virus antigen, polyhexamethylene guanidine (PHMG) was used with concentrations of 0.010, 0.014 and 0.018%. Before and after the process of purifying the antigen of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” in the quantitative version of the RSC, the concentration of the total viral protein and immunogenic components was determined. The results of the analysis are reflected in Table 4, from which it follows that as a result of purification of the antigen of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019” using PHMG at a concentration of 0.010%, a decrease in the concentration of ballast protein by 12% and immunogenic components by 2% was noted . When using PHMG at a concentration of 0.014%, a decrease in the amount of ballast protein by 38% and the 146S component by 3.2% was observed. Using PHMG at a concentration of 0.018%, the content of ballast protein decreased by 45%, and immunogenic components by 24%.

Thus, having studied the conditions for purifying the antigen of the strain “O No. 2383/Primorsky/2019”, we came to the conclusion that to obtain a purified product it is optimal to use PHMG with a concentration of 0.014%.

Example 7. Selection of conditions for concentrating the antigen suspension of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus genotype O/SEA/Mya-98.

A cultural inactivated suspension of the strain “O No. 2383/Primorsky/2019”, obtained using the suspension cell line BHK-21/SUSP/ARRIAH, was concentrated 5 times by volume. To increase the concentration of immunogenic components of the foot-and-mouth disease virus 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 a tangential filtration unit Centramate 500S Pall for 6 hours at an operating pressure on the filter of 2.2-2.3 atm.

Before and after the process of concentrating the antigen suspension of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019,” the concentration of the total viral protein and immunogenic components was determined in the quantitative version of the RSC. The research results are presented in Table 5, from which it can be seen that when concentrating the antigen of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus using PEG-6000 for 4 hours, an increase in the content of components was noted compared to the initial values (before concentration) in 3.5 times. Using the same polymer, but increasing the exposure time to 12 hours, we increased the concentration of virus components by 3.9 times. Using the concentration method using tangential filtration, it was possible to increase the amount of immunogenic components of the antigen of the strain “O No. 2383/Primorsky/2019” of the foot-and-mouth disease virus in suspension by 4.9 times. Thus, dance filtration technology made it possible to obtain the antigen of the strain “O No. 2383/Primorsky/2019” with a high concentration of structural viral proteins.

Example 8. Selection of adjuvant and antigen-adjuvant ratio.

To produce an anti-foot-and-mouth disease monovalent emulsion vaccine from the antigen of the strain “O No. 2383/Primorsky/2019,” aluminum hydroxide in combination with saponin was chosen as an adjuvant. In the manufacture of this vaccine preparation, the oil adjuvant Montanide ISA-71 R VG was used in an amount of 70% by weight.

Example 9. Composition of a vaccine against foot-and-mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 in a culture-inactivated emulsion.

From the resulting concentrate of the antigen of the foot-and-mouth disease virus strain “O No. 2383/Primorsky/2019,” a cultural inactivated emulsion vaccine against foot-and-mouth disease genotype O/SEA/Mya-98 was prepared using aluminum hydroxide and saponin as adjuvants. The amount of 146S immunogenic component per 1.0 cm ³ of antigen was 7.35 ± 0.02 μg/cm ³ (Table 5).

Example 10. Immunization of animals with a vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98, cultural inactivated emulsion.

From the resulting vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98, a cultural inactivated emulsion (the proposed invention), a series of dilutions were prepared for pigs in 5-fold increments. 15 pigs were divided into 3 groups of 5 pigs each. The immunizing dose was 2.0 cm ³ . The first group of animals (No. 1-5) were immunized with the vaccine without dilution, the second group of pigs (No. 6-10) were vaccinated with the vaccine diluted 1/5, the third group of animals (No. 11-15) - with the vaccine diluted 1/25 . The drug was administered intramuscularly into the middle third of the neck. As a control for the virus, 2 heads were left without vaccination.

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

Blood sera from pigs obtained before immunization and 14 days after immunization 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 OIE recommendations [3]. The obtained sera were tested using the ELISA test system “Prio CHECK ^® FMDV NSP ELISA for in vitro detection of antibodies against Foot and Mouth Disease Virus in serum of cattle, sheep and pigs” (Prionics Lelystad BV, the Netherlands). The research results obtained are reflected in Table 6, from which it can be seen that the animal blood sera did not contain antibodies to non-structural proteins of the foot-and-mouth disease virus (PI values for pig blood sera <50%).

Example 12. Assessment of the aerulence and harmlessness of a vaccine against foot-and-mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 cultural inactivated emulsion (the proposed invention).

To determine the avirulence of the vaccine for pigs, a selected sample of the vaccine preparation was injected intradermally at 0.1 cm ³ . The study used pigs weighing 30-40 kg. During 10 days of observation, the animals remained clinically healthy, and the pathological examination did not reveal any 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 in animals was carried out by intramuscular injection of the vaccine at a dose of 6.0 cm ³ (triple dose of 2.0 cm ³ ). The observation period was also 10 days. It should be noted that after immunization, the animal’s body temperature can rise to 41.5°C and remain at this level for 1-2 days, which does not go beyond the norm after administration of the vaccine preparation.

According to the results of the research, the vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98, cultural inactivated emulsion (the proposed invention) was found to be harmless, all animals during the observation period remained clinically healthy, with postmortem analysis of tissue necrosis No vaccine was found at the injection site.

Example 13. Study of the immunogenic properties of the vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 cultural inactivated emulsion (the proposed invention) in terms of the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 21st day after the introduction of the vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 cultural inactivated emulsion (the proposed invention), blood was taken from pigs and the resulting blood sera were examined in a microneutralization reaction [3] . It was revealed that in pigs immunized with the vaccine in a whole dose (5 heads), the average antibody titer was 7.85±0.22 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.75±0.18 log ₂ SN ₅₀ , with breeding 1/25 (5 heads) - 3.50±0.31 log ₂ SN ₅₀ . (Table 7). The data obtained from the microneutralization reaction indicate that after administration of the vaccine in its entirety, 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 vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 cultural inactivated emulsion (the proposed invention) on naturally susceptible animal species.

Pigs immunized in the amount of 15 heads with the vaccine in whole form and in dilutions were infected with the control strain “O No. 2383/Primorsky/2019” of the genotype O/SEA/Mya-98 of the foot-and-mouth disease virus, adapted to these animals at a dose of 10 ^4.0 ID ₅₀ /0.20 cm ³ (in two points of 0.10 ± 0.05 cm ³ ). 7 days after infection, all animals were euthanized and a postmortem examination was performed. Animals that had no lesions on their limbs were considered protected from foot and mouth disease. Primary aphthae were not taken into account.

The results of the control infection are presented in Table 7, from which it follows that the vaccine against foot and mouth disease from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 is a cultural inactivated emulsion (the proposed invention) in whole form, as well as in dilutions 1/5 and 1/25, administered in a single dose (2.0 cm ³ ), protects pigs from infection with a homologous strain of all animals (5 out of 5 heads).

Based on the results of the control infection, it was found that the vaccination volume of this vaccine contains 40.516 PD ₅₀ and 0.049 IMD ₅₀ for pigs.

Thus, the above information indicates that the cultural inactivated emulsion vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019”, embodying the proposed invention, is intended as a reserve for use in agriculture, namely in veterinary virology and biotechnology; the possibility of implementing the presented has been confirmed; the vaccine made from the strain “O No. 2383/Primorsky/2019” genotype O/SEA/Mya-98 in accordance with the proposed invention has high immunogenic activity and is capable of providing effective protection of susceptible animals against the epizootic strain of foot-and-mouth disease virus serotype O, genotype O/ SEA/Mya-98.

Sources of information taken into account when compiling a description of the invention for the application for a RF patent for the invention “Vaccine against foot-and-mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019”, cultural inactivated emulsion”:

1. Foot and mouth disease. Prevention measures. URL: http://89.rospotrebnadzor.ru/directions/epid_nadzor/146902 (Access date: 02/13/2023).

2. The danger of foot and mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Access date: 02/01/2023).

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. and others. Foot and mouth disease. -M.: Agropromizdat, 1990. - 320 p.

5. Ponomarev A.P., Uzyumov V.L., Gruzdev K.N. Foot and mouth disease virus: structure, biological and physicochemical 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 Strategies: 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. Official website of The FAO World Reference Laboratory for Foot-and-Mouth Disease - URL: http://www.wrlfmd.org/ref_labs/fmd_ref_lab_reports.htm (Access date 02/02/2023).

12. 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.

13. Rakhmanov A.M. Current epizootic situation in the world regarding foot and mouth disease and measures for its control // Veterinary medicine of the interspecies. topics Sci. Kharkiv, 2013. - pp. 37-38.

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

15. Melnik R.N., Khaustova N.V., Melnik N.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 current production strains / Veterinary medicine and feeding. - 2019. -№3. - pp. 29-31.

16. 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.

17. Patent RU 2 665 849 Inactivated emulsion vaccine against foot and mouth disease type O / Lozovoy Dmitry Anatolyevich, Mikhalishin Dmitry Valerievich, Starikov Vyacheslav Alekseevich, Borisov Alexey Valerievich, Doronin Maxim Igorevich, application: 2017144155, 2017.12.15, published o: 2018.09.04.

18. Methodological recommendations for determining the concentration of 146S, 75S, 12S components of vaccine strains of cultural foot-and-mouth disease virus in the complement fixation reaction (FRR) / Federal State Budgetary Institution "ARRIAH". - Approved 09.21.17.

19. Guidelines for determining antigenic correspondence between epizootic isolates and production strains of foot-and-mouth disease virus in a cross-microneutralization reaction: approved. Rosselkhoznadzor 09/13/2017 / FSBI "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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cttgggtcccgaatggggcgcctgaaaacgcgttggataacaccaccaacccaacggcataccacaaggc

accactcacccggcttgcattgccgtacacggcaccacaacgtgtgttggcaaccgtttacaacgggaac

tgcaagtacggtgatggttcggtgaccaacataagaggtgacctacaagtgttggcccagaaggcggcga

gaacgctgcctacctccttcaactacggtgccatcaaagctactcgggtgactgaactgctttaccgcat

gaagagggctgagacgtactgcccccggcctcttttggccattcacccgaacgaggccagacacaaacag

aagattgtggcacctgtgaagcagctcctg</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>TTSTGESADPVTATVENYGGETQVQRRQHTDVSFILDRFVKVTPKDQIN

VLDLMQTPAHTLVGALLRTATYYFADLEVAVKYEGNLTWVPNGAPENALDNTTNPTAYHKAPLTRLALPY

TAPQRVLATVYNGNCKYGDGSVTNIRGDLQVLAQKAARTLPTSFNYGAIKATRVTELLYRMKRAETYCPR

PLLAIHPNEARHKQKIVAPVKQLL</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (3)

1. Vaccine against foot and mouth disease genotype O/SEA/Mya-98 from the strain “O No. 2383/Primorsky/2019” is a cultural inactivated emulsion containing an active substance and an adjuvant, characterized in that it consists of an active substance in the form of avirulent and purified antigenic material from homologous to the infectious agent of the strain “O No. 2383/Primorsky/2019” of genotype O/SEA/Mya-98, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and other Animal Pathogens (GKSHM) of the Federal State Budgetary Institution “ARRIAH”, under registration number: No. 419 - dep / 22-53 - GKSHM FSBI "ARRIAH"; reproduced in the continuous suspension cell line BNK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; from oil adjuvant Montanide ISA-71 R VG containing 70% by weight and supporting medium - up to 1.0 cm ³ . 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the strain “O No. 2383/Primorsky/2019” of genotype O/SEA/Mya-98 homologous to the infectious agent, obtained in a sensitive biological system and representing suspension containing predominantly the 146S immunogenic component of foot and mouth disease virus serotype O. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from the strain “O No. 2383/Primorsky/2019” of genotype O/SEA/Mya-98, homologous to the infectious agent, obtained in a continuous suspension cell line of the kidney of a newborn Syrian hamster BHK-21/SUSP/ARRIAH and is a suspension containing predominantly the 146S immunogenic component of foot-and-mouth disease virus serotype O.