RU2849065C1 - Vaccine against foot-and-mouth disease genotype o/ea-4 culture inactivated emulsion - Google Patents

Abstract

FIELD: veterinary virology; biotechnology.

SUBSTANCE: invention relates to the development of a vaccine against foot-and-mouth disease genotype O/EA-4, which is a cultured inactivated emulsion vaccine. The vaccine embodying the proposed invention is intended as a reserve for use in agriculture, namely in veterinary virology and biotechnology. The feasibility of the invention has been confirmed. This vaccine has high immunogenic activity and is capable of providing effective protection for susceptible animals against the epizootic strain of the foot-and-mouth disease virus genotype O/EA-4. The vaccine against foot-and-mouth disease genotype O/EA-4 is a culture-inactivated emulsion vaccine manufactured using the oil adjuvant Montanide ISA-61 VG.

EFFECT: Based on the results of a control infection, it was established that the vaccine dose contains 55.90 PD50 and 0.04 ImD50 for pigs (high vaccine efficacy).

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 culture-inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype.

One of the most contagious pathogens of infectious diseases belonging to the II pathogenicity group is the foot-and-mouth disease virus, which, according to the classification of the International Committee on Taxonomy, belongs to the Riboviria sphere, Orthornavirae kingdom, Pisuviricota phylum, Pisoniviricetes class, Picornavirales order, Picornaviridae family, Aphthovirus genus, and Foot-and-mouth disease virus species [1]. The genetic material of the foot-and-mouth disease virus is a positive single-stranded RNA approximately 8400-8500 nt long, enclosed in an icosahedral capsid. During the reproduction of the foot-and-mouth disease virus in biological systems, the 146S component (the main immunogenic component) is formed, consisting of one molecule of viral RNA and 60 copies of the polypeptide, each of which is represented by a complex of proteins VP ₁ -VP ₂ -VP ₃ -VP ₄ [2].

Currently, scientists know the following seven characterized serotypes of foot-and-mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, with representatives of the O/EA-4 genotype being the most diverse and widespread, especially in North and East African countries.

More than 60 genetic lineages of foot-and-mouth disease virus are known, the differences between which are determined by analyzing the nucleotide sequence of the highly variable 1D gene, which encodes the amino acid sequence of the surface and most variable viral protein VP ₁ [2]. The VP ₁ protein is the most variable, since it accounts for approximately 90% of mutations in all structural genes. The most variable regions are the 40-60, 130-160, and 190-213 aa regions [3]. The 130-160 aa region of the VP ₁ surface protein is highly variable, since it is involved in the process of binding to host cell receptors [1, 3].

Nucleotide sequences of the _VP1 coding region are used for the genetic characterization of foot-and-mouth disease (FMD) strains due to their importance for viral attachment and entry into cells, protective immunity, and serotype specificity. Phylogenetic analysis based on the _VP1 sequence is widely used to infer evolutionary dynamics, epidemiological relationships between lineages, and to trace the origin and movement of FMD virus strains [4].

Recovery of an animal from foot-and-mouth disease with a single genotype does not provide immune protection against foot-and-mouth disease from another genetic lineage, which differs genetically and antigenically from the vaccine strain [3]. Antigenic variations arise from amino acid mutations that alter the immune system's recognition of viral proteins. Surface-exposed structures of the virus are particularly susceptible to immune attack. The emergence of new antigenic forms is determined by a complex interaction of viral and host cell factors [5].

Since vaccination is used as the main control tool in endemic areas, by analyzing each pool and the genotypes in it, it is possible to select the most specific vaccines corresponding to the topotypes present in that pool [4, 5].

Foot-and-mouth disease serotype O is well-studied and widespread worldwide. Serotype O is divided into 11 topotypes: EAST AFRICA 1-4 (EA-1-4), SOUTHEAST ASIA (SEA), EUROPE-SOUTH AMERICA (EURO-SA), INDONESIA-1 and 2 (ISA-1 and -2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA), and WEST AFRICA (WA) [1, 5]. Such a diversity of topotypes leads to problems in the specific prevention of foot-and-mouth disease using culture-based inactivated foot-and-mouth disease vaccines. As a result, there is a need to create new means of specific prevention against foot-and-mouth disease genotype O/EA-4 [1, 6, 7].

To ensure biological security in the Russian Federation and prevent the emergence of foot-and-mouth disease on farms across our country, a range of measures to combat and prevent it are being implemented. This range of measures aims to prevent the introduction of the virus into the country, systematically vaccinate animals in the buffer zone, and monitor the immune status of cattle and pigs, for which culture-based inactivated foot-and-mouth disease vaccines are used [6-9].

To immunize animals, a vaccine made from a virus homologous to field isolates with a high content of the 146S component should be used, which has been confirmed by studies of many scientists [10-16].

The genetic and antigenic diversity of the O/EA-4 foot-and-mouth disease virus (FMDV) poses challenges in the specific prevention of FMD using culture-based inactivated FMD vaccines and complicates strain-specific diagnostics of isolated FMDV isolates. This necessitates the development of new agents for the specific immunoprophylaxis of FMDV genotype O/EA-4, which is periodically detected and may spread widely across North and East Africa [7, 8].

Given the development and active establishment of trade and economic relations between the Russian Federation and countries of the African continent [8], there is a high risk of introducing isolates of the O/EA-4 genotype into our country, which could seriously threaten Russia's biological security with regard to the emergence of foot-and-mouth disease of the O/EA-4 genotype. In Africa, outbreaks of foot-and-mouth disease of the O/EA-4 genotype are sporadic. Therefore, as a backup vaccine in the event of the spread of foot-and-mouth disease of the O/EA-4 genotype, it is necessary to develop a culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype.

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

- 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 No. 2311/Zabaikalsky/2016" (genotype O/ME-SA/Ind-2001).

- strain "O No. 2241/Ethiopia/2011" (genotype O/EA-3).

The isolate "O / Ethiopia 8 / 2013" of the foot-and-mouth disease virus was isolated from cattle in Ethiopia (Debre Zeit (Brishoftu), East Shewa, Oromia, Ethiopia) in 2013 and received by the All-Russian Research Institute of Animal Health for scientific research in 2020. The strain "O / EA-4 / Ethiopia / 2013" was obtained at the All-Russian Research Institute of Animal Health as a result of scientific research by adapting the isolate to reproduction in the primary trypsinized monolayer cell line of pig kidney SP, in continuous cell cultures BHK-21 / SUSP / ARRIAH, IB-RS-2, PSGK-30, in the body of cattle and pigs under the conditions of the All-Russian Research Institute of Animal Health.

According to the results of comparative analysis of nucleotide sequences, the isolated strain of foot-and-mouth disease virus belongs to the O/EA-4 genotype, which differs significantly from the production strains of foot-and-mouth disease virus serotype O.

At the All-Russian Research Institute of Animal Health, this strain was adapted to various sensitive cell lines, including the monolayer and suspension-transplantable BHK-21/SUSP/ARRIAH newborn Syrian hamster kidney cell line. The phylogenetic affiliation of this foot-and-mouth disease virus strain is shown in Fig. 1.

The closest to the proposed invention in terms of the set of essential features is the inactivated emulsion vaccine against foot-and-mouth disease type O [10], which contains avirulent and purified antigen material from a strain homologous to the causative agent of the infection
"O No. 2241/Ethiopia/2011" (genotype O/EA-3) of foot-and-mouth disease virus reproduced in the continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 4.5 μg; 2) oil adjuvant Montanide ISA-61 VG with a content of 60% by weight, providing an increase in the immune response in animals (prototype).

A significant drawback of the prototype vaccine is its weak immunogenic activity against strains/isolates of the foot-and-mouth disease virus genotype O/EA-4.

To address this problem, the present invention was developed, which included the development of a culture-based inactivated emulsion vaccine against O/EA-4 genotype foot-and-mouth disease. This vaccine contains a high content of the 146S immunogenic component of the O/EA-4/Ethiopia/2013 strain and contains the oil adjuvant Montanide ISA-61 VG.

The technical result of using the proposed invention consists in expanding the arsenal of anti-foot-and-mouth disease inactivated culture emulsion vaccines for protecting animals against isolates and strains of the foot-and-mouth disease virus of the O/EA-4 genotype.

The specified technical result has been achieved by creating a culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype, characterized by the following set of features, reflected below.

The developed vaccine in a dose of 2.0 ^cm3 contains the following main components: 1) the active substance in the form of avirulent and purified antigen material from the homologous to the causative agent of the infection strain "O/EA-4/Ethiopia/2013" (genotype O/EA-4) of the foot-and-mouth disease virus, reproduced in the transplantable suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg; 2) oil adjuvant Montanide ISA-61 VG, providing an increase in the immune response in animals with a content of 60% by weight, 3) maintenance medium - up to 2.0 ^cm3 .

The O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus is deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and Other Animal Pathogens (GKShM) of the All-Russian Research Institute of Animal Health, under the registration number: No. 728 - dep / 24 - GKShM of the All-Russian Research Institute of Animal Health.

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

For vaccine production, the BHK-21/SUSP/ARRIAH cell suspension culture is preferably used as the sensitive biological system. Hanks' medium, without serum, is used as the maintenance medium, but with the addition of blood protein hydrolysate at a concentration of 5.0% of the total volume at a pH of 7.50-7.70. Virus inactivation is performed using aminoethylethyleneimine (AEEI) at a concentration of 0.045% of the virus-containing suspension volume. The inactivated virus is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.040% of the total volume.

The avirulent and purified antigen of the O/EA-4/Ethiopia/2013 strain (genotype O/EA-4) of 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 determined using a quantitative complement fixation reaction (CFR) [11-13].

The vaccine is created using viral material containing at least 15.0 μg of inactivated immunogenic 146S particles of the foot-and-mouth disease virus per 2.0 ^cm³ . The stated concentration of the immunogenic component in the vaccine is achieved by concentrating the antigen using a tangential filtration unit.

The O/EA-4 genotype foot-and-mouth disease vaccine, culture-based inactivated emulsion vaccine, is obtained by mixing purified O/EA-4 genotype foot-and-mouth disease virus antigen and Montanide ISA-61 VG oil adjuvant in a ratio of 40% and 60% by weight, respectively.

The resulting vaccine is a stable, white or pale pink water-in-oil reverse emulsion containing the inactivated 146S component of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus.

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

1. Vaccine against foot-and-mouth disease genotype O/EA-4, culture-based inactivated emulsion.

2. An active substance in the form of avirulent and purified antigen material from the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, homologous to the causative agent of the disease, in an effective amount.

3. Targeted supplements.

The essential distinguishing features of the proposed vaccine are that it contains, as an active substance, an avirulent purified antigen of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus in an effective amount.

The proposed invention is also characterized by other distinctive features expressing specific forms of execution or special conditions of its use:

1. An avirulent and purified antigen of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, obtained in the suspension-transplantable cell line BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly the 146S immunogenic component of the foot-and-mouth disease virus in an effective amount.

2. Avirulent and purified antigen of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, obtained in a suspension transplantable cell culture of BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly the 146S immunogenic component of the foot-and-mouth disease virus in an amount of at least 15.0 μg in 2.0 ^cm3 of the finished vaccine preparation.

3. Among the target additives, the vaccine contains the oil adjuvant Montanide ISA-61 VG.

4. The vaccine contains the oil adjuvant Montanide ISA-61 VG in an amount of 60% (by weight) in 2.0 ^cm3 of the finished product.

5. Avirulent and purified antigen of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, obtained in the continuous suspension cell line BHK-21/SUSP/ARRIAH, oil adjuvant Montanide ISA-61 VG, additive in a dose of the finished product of 2.0 ^cm3 in the form of a maintenance medium in the following quantities:

Avirulent and purified antigen of the strain "O/EA-4/Ethiopia/2013" genotype
O/EA-4 foot-and-mouth disease virus not less than 15.0 mcg Oil adjuvant Montanide ISA-61 VG 60% (by weight) Supportive environment up to 2.0 cm ³

The proposed vaccine against foot-and-mouth disease of the O/EA-4 genotype, a culture-based inactivated emulsion vaccine, has high immunogenic activity and provides reliable protection against isolates of the O/EA-4 genotype foot-and-mouth disease virus.

The achievement of the technical result from the use of the invention is ensured by the fact that the proposed anti-foot-and-mouth disease culture inactivated emulsion vaccine contains, as an active substance, an antigen of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, which has high immunogenic activity and creates effective protection of susceptible animals against isolates of the foot-and-mouth disease virus of the presented genotype, which in recent years have caused outbreaks around the world.

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

Fig. 1 - Dendrogram reflecting the phylogenetic relationship of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus with epizootic strains of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP gene₁.

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

SEQ ID NO:1 represents the nucleotide sequence of the 1D gene of the VP ₁ protein of the O/EA-4/Ethiopia/2013 strain of foot and mouth disease virus of the O/EA-4 genotype;

SEQ ID NO:2 represents the amino acid sequence of the 1D gene of the VP ₁ protein of the O/EA-4/Ethiopia/2013 strain of foot and mouth disease virus genotype O/EA-4.

The O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus has the following taxonomy: familyPicornaviridae, genus Aphthovirus, view Foot-and-mouth disease virus, serotype O, genotype O/EA-4. The pathogen strain exhibits the following morphological features characteristic of the foot-and-mouth disease pathogen: the virion is ixahedral in shape and 24 nm in size. The virion consists of a single-stranded, positively charged RNA molecule and 60 copies of a polypeptide, each of which is represented by VP proteins.₄, VP₂, VP₃, VP₁.

Antigenic properties

Antigenically, the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain belongs to serotype O. The virus is reliably neutralized by homologous antiserum and does not exhibit hemagglutinating activity. In animals that have recovered from the disease, specific antibodies are produced in the serum, detectable by enzyme-linked immunosorbent assay (ELISA) and microneutralization assay (MNA).

The primary structure of the 1D gene of the VP protein was determined using nucleotide sequencing.₁strain "O/EA-4/Ethiopia/2013" of the foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "O/EA-4/Ethiopia/2013" of the foot-and-mouth disease virus belongs to the O/EA-4 genotype (Fig. 1).

The antigenic relatedness (r ₁ ) of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain was studied in a virus microneutralization reaction using a cross-test of this foot-and-mouth disease pathogen strain with specific sera obtained for the following production strains of 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 No. 2311/Zabaikalsky/2016" (genotype O/ME-SA/Ind-2001).

- strain "O No. 2241/Ethiopia/2011" (genotype O/EA-3).

The titer of reference bovine blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype O against ¹⁰² _TCID50 of homologous and heterologous virus was determined in RMN by cross-titration, calculating the values using the linear regression equation, and expressed in lg. The _r1 value was determined as the antilogarithm of the difference in lg serum titers against heterologous and homologous virus [7, 9, 14].

The r ₁ value 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.

The maximum relationship is observed when the value of r ₁ in the RMN tends to 1.0.

Antigenic relatedness indices in strain studies
"O/EA-4/Ethiopia/2013" had r ₁ values ranging from 0.01 to 0.14, indicating no apparent antigenic relationship with production strains of foot-and-mouth disease virus serotype O.

Geno- and chemotaxonomic characteristics

The O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular weight of 8.084×10 ⁶ D. The nucleic acid is a single-stranded linear molecule with a molecular weight of 2.90×10 ⁶ D. The virion has a protein membrane consisting of four structural proteins VP ₁ , VP ₂ , VP ₃ and VP _4. The lipoprotein membrane is absent.

The main antigenic protein is _VP1 . The virion contains approximately 31.0% RNA and 69.0% protein. Viral RNA is infectious and participates in the formation of precursor proteins in infected cells. These precursors, in turn, are cleaved to form more stable structural and nonstructural viral polypeptides, including the important enzyme RNA-dependent RNA polymerase (3D gene), which is involved in RNA replication for virion assembly.

Physical properties

The mass of the virion is 8.474×10 ^-18 g. The buoyant density is 1.483 g/cm ³ .

Resistance to external factors

The O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain is resistant to ether, chloroform, and acetone. It is most stable at a pH of 7.50-7.70. Shifts in pH toward acidic and strongly alkaline pH inactivate the virus. It is sensitive to formaldehyde, UV radiation, γ-irradiation, and high temperatures (above 38.1°C).

Additional features and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for cloven-hoofed animals.

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

Stability - retains its original biological properties when passaged in sensitive biological systems for 5 passages (observation period) on transplantable cultures.

Biotechnological characteristics

The O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus is reproduced in continuous cell cultures: Siberian ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), and Syrian hamster kidneys (BHK-21).

Five consecutive passages of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain were conducted in continuous cell cultures of PSGK-30, BHK-21, and IB-RS-2. Biological properties were characterized by determining the infectious activity of the virus at each passage in the continuous cell line IB-RS-2 and in naturally susceptible animals—cattle and pigs.

To reduce and prevent the epizootic risk of foot-and-mouth disease caused by the O/EA-4 genotype and to prevent the emergence of new outbreaks of the disease, timely vaccination is important, which requires the development of a safe and effective vaccine.

A safe and effective culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype has been obtained.

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

Example 1. Genetic characteristics of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus based on PCR and nucleotide sequencing data.

The primary structure of the 1D gene (VP ₁ protein) of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain was analyzed using Sanger nucleotide sequencing and the position of this strain on the phylogenetic tree of O/EA-4 foot-and-mouth disease virus. The method is based on determining the primary structure of the 1D gene (VP ₁ protein) of the test strain, followed by an analysis of its phylogenetic relationship with other isolates/strains of O serotype foot-and-mouth disease virus.

A comparative analysis of the nucleotide sequences of the 1D gene encoding the VP ₁ protein of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus vaccine strain with other strains was required. The nucleotide sequence of the VP ₁ protein gene of the O/EA-4/Ethiopia/2013 strain, genotype O/EA-4 foot-and-mouth disease virus is presented in SEQ ID NO: 1. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the O/EA-4/Ethiopia/2013 strain, genotype O/EA-4 foot-and-mouth disease virus, is shown in SEQ ID NO: 2.

A phylogenetic analysis was performed for the O/EA-4/Ethiopia/2013 strain. The phylogenetic tree was inferred using MEGA and the Neighbor-Joining algorithm [15]. The percentage of repeated branches in which related taxa are grouped together in a bootstrap test is shown next to the branches [16, 17]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [18] and are expressed as units of base substitutions per site. The evolutionary analysis was performed in MEGA 6 [19].

As a result of the study conducted to compare the complete nucleotide sequences of the 1D gene (VP ₁ protein) of the strain "O/EA-4/Ethiopia/2013" 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/EA-4/Ethiopia/2013" belongs to the O/EA-4 genotype, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of the strain “O/EA-4/Ethiopia/2013” of the O/EA-4 genotype to the continuous monolayer culture of Siberian ibex kidney cells PSGK-30.

To infect the continuous monolayer culture of Siberian ibex kidney cells PSGK-30, a 20% aphthous suspension of foot-and-mouth disease virus obtained from pig aphthae (passage 2) was used. The inoculation concentration of cells was 0.20-0.25 million cells/cm ³ , the dose of virus infection was 0.001 TCID ₅₀ /cell. According to the results of the study, the reproduction of foot-and-mouth disease virus of the O/EA-4/Ethiopia/2013 strain occurred with a specific destruction of the monolayer by 90-100% within 20 hours. Over the course of 6 consecutive passages, an increase in the titer of infectious activity of the virus was noted from 5.02 ± 0.10 to 7.04 ± 0.10 lg TCID ₅₀ /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of passage 6 (7.04±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from passages 1 to 6 changed from 0.14±0.02 to 1.02±0.02 μg/ ^cm3 . Moreover, the highest amount of the 146S component was noted at the stage of passage 6 (1.02±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 97.39 to 100.00%. Thus, over the course of 6 consecutive passages, the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus was successfully adapted to the continuous monolayer cell line PSGK-30.

Example 3. Study of conditions for monolayer cultivation of foot-and-mouth disease virus strain “O/EA-4/Ethiopia/2013” genotype O/EA-4 on an industrial scale.

In the process of industrial cultivation of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus in a continuous monolayer culture of PSGK-30 cells, the optimal conditions for culturing the virus were selected based on the supporting medium, temperature factor, and the dose of infection of the cells with the studied virus.

At the first stage of the study, the influence of the composition of the maintenance medium on the reproduction of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain in a monolayer culture of PSGK-30 cells at production scale (at the stage from the 5th to the 6th passage) was assessed. For comparison, three media with the addition of 2.0% fetal calf blood serum were used: 1) Eagle's medium DMEM; 2) RPMI-1640 medium; 3) Hanks' solution with 5% blood protein hydrolysate. The cell inoculation concentration was 0.1 million cells/ ^cm3 , the virus infection dose was 0.1000-0.0001 _TCID50 /cell. Virus cultivation was carried out at temperatures of 35±0.2 - 38±0.2°C until the cell monolayer was destroyed by at least 90%. The results of reproduction of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus with support media of different compositions are shown in Table 1.

The data in the table show that during the reproduction of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain in a monolayer culture of PSGK-30 cells using support media of different compositions, the optimal medium is Hanks' medium, which allows achieving specific destruction of the cell monolayer by 95-100% in 17 hours with the accumulation of the 146S component in the resulting suspension in an amount of 1.11±0.02 μg/cm ³ and an infectious activity titer of 7.40±0.10 lg TCID ₅₀ /cm ^3. When using RPMI-1640 and Eagle's DMEM medium, the virus reproduction rates were lower.

At the next stage of studying the conditions of monolayer cultivation of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain in the PSGK-30 cell culture under production conditions, the effect of temperature on the virus reproduction process was assessed. For this purpose, the virus was cultivated in Hanks' medium at the following temperatures: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2°C. The infection dose of the cell culture with the foot-and-mouth disease virus was 0.010-0.001 _TCID50 /cell. Virus cultivation was 17-32 h (Table 1). The results of the study revealed that for the complete reproduction of the O/EA-4/Ethiopia/2013 strain of foot and mouth disease virus in a monolayer culture of PSGK-30 cells, the optimal temperature of the medium is 37.0±0.2°C, at which, in 17 hours, the development of CPE reached 95-100% with the accumulation of 146S particles equal to 1.11±0.02 μg/ ^cm3 and the titer of infectious activity of the virus of 7.40±0.10 lg _TCID50 / ^cm3 .

The effect of the infection dose of the PSGK-30 cell line monolayer with the O/EA-4/Ethiopia/2013 strain was assessed during cultivation on a production scale. Different virus doses were used for this purpose: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCID ₅₀ /cell. Hanks' medium was used as a maintenance medium. Virus reproduction was carried out at a temperature of 37.0±0.2°C for 17-25 h until specific cell destruction by 90-100%. 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 highest accumulation of the 146S virus component was achieved at an infection dose of 0.01-0.001 TCID ₅₀ /cell. and amounted to 1.11±0.02 μg/cm ³ with a titer of infectious activity of the virus of 7.40±0.10 lg TCID ₅₀ /cm ³ .

Thus, the conditions of monolayer cultivation of the foot and mouth disease virus strain "O/EA-4/Ethiopia/2013" in the PSGK-30 cell culture were studied on an industrial scale. As a result of the study, the following optimal parameters for the reproduction of the foot and mouth disease virus strain "O/EA-4/Ethiopia/2013" in the monolayer cell line PSGK-30 were determined: 1) maintenance medium - Hanks' medium with 5% GBC and 2% bovine blood serum; 2) cultivation temperature - 37.0±0.2 ⁰ C; 3) virus infection dose - 0.01-0.001 TCID ₅₀ /cell.

Example 4. Study of conditions for suspension cultivation of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of foot-and-mouth disease virus on an industrial scale.

During industrial cultivation of the strain "O/EA-4/Ethiopia/2013" genotype O/EA-4 of the foot-and-mouth disease virus in a continuous suspension culture of BHK-21/SUSP/ARRIAH cells, a search was conducted for optimal parameters for the successful reproduction of the foot-and-mouth disease pathogen, using different cell concentrations, temperature conditions and the dose of virus infection.

At the first stage of the work, the influence of the seeding concentration of BHK-21/SUSP/ARRIAH cell line in suspension on the reproduction of the O/EA-4/Ethiopia/2013 strain was determined. foot-and-mouth disease virus on an industrial scale. For analysis, suspensions with the following cell concentrations were prepared: 3.5; 4.0; 4.5; 5.0 million cells/cm³The virus cultivation temperature was 37.0±0.2°C, the virus infection dose was 0.010-0.001 TCID₅₀/cell. Virus reproduction was carried out until specific cell death was 95-100%. Results of suspension cultivation of the strain "O/EA-4/Ethiopia/2013" The results of the analysis of the results of the cell suspensions of the foot-and-mouth disease virus are presented in Table 2.

When cultivating the foot-and-mouth disease virus strain "O/EA-4/Ethiopia/2013" in the suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations, it was determined that the accumulation of the 146S component in a suspension with a cell concentration of 3.5 million cells/cm³amounted to 1.02±0.02 μg/cm³, with a concentration of 4.0 million cells/cm³- 1.26±0.02 μg/cm³, with a concentration of 4.5 million cells/cm³- 1.75±0.02 μg/cm³and with a concentration of 5.0 million cells/cm³- 1.82±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "O/EA-4/Ethiopia/2013" For the foot-and-mouth disease virus, a sufficient concentration of BHK-21/SUSP/ARRIAH cells in suspension is 4.5 million cells/cm³(at a concentration of 5.0 million cells/cm³The concentration is slightly higher, but the production costs per cell number are higher). The infectious activity titer of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain was 8.95±0.10 lg TCD₅₀/cm³.

At the next stage of the work, the influence of temperature on the reproduction of the strain "O/EA-4/Ethiopia/2013" was investigated. foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells 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 TCID₅₀/cell. The virus was cultured to a CPE of at least 85% (specific cell destruction). The analysis revealed that for full reproduction of the O/EA-4/Ethiopia/2013 strain, For the growth of foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells, the optimal temperature is 37.0±0.2°C, at which specific cell death of 95-100% with a high accumulation of the 146S component (1.37±0.02 μg/cm) is observed within 12 hours.³) and a titer of infectious activity of the virus of 8.95±0.10 lg TCID₅₀/cm³.

In the course of studying the conditions of suspension cultivation of the strain "O/EA-4/Ethiopia/2013" The influence of the infection dose on the cells was assessed in the industrial scale production of foot-and-mouth disease virus using the BHK-21/SUSP/ARRIAH cell culture. For this purpose, cell suspensions were infected with the virus at different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD₅₀/cell. Virus reproduction was carried out at a temperature of 37.0 ± 0.2 ° C 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 highest accumulation of the 146S component was observed at an infection dose of 0.010-0.001 TCID.₅₀/cl. (1.75±0.02 µg/cm³) with a titer of infectious activity of the virus equal to 8.95±0.10 lg TCID₅₀/cm³.

Thus, a study of three parameters of suspension cultivation of the strain “O/EA-4/Ethiopia/2013” was carried out. genotype O/EA-4 of the foot-and-mouth disease virus in the suspension cell line BHK-21/SUSP/ARRIAH on an industrial scale. Optimal conditions for the reproduction of the strain "O/EA-4/Ethiopia/2013" in the suspension cell culture BHK-21/SUSP/ARRIAH were identified: 1) cell concentration before infection - 4.5 million cells/cm³; 2) cultivation temperature regime - 37.0±0.2°C; 3) virus infection dose - 0.010-0.001 TCID₅₀/cl.

Example 5. Inactivation of a suspension of foot-and-mouth disease virus strain “O/EA-4/Ethiopia/2013” genotype O/EA-4.

At the end of the reproduction cycle of the foot-and-mouth disease virus, without stopping the thermostatting process (37.0 ± 0.2 ° C), an acidified solution of aminoethylethyleneimine (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 0.045%. Inactivation of the infectious activity of the foot-and-mouth disease virus strain "O / EA-4 / Ethiopia / 2013" genotype O / EA-4 was carried out for 12 hours at a temperature of 37.0 ± 0.1 ° C with periodic stirring.

To determine the time of complete inactivation after the addition of 1,2-AEEI, samples were collected every hour. The resulting samples were tested for the presence of infectivity of the foot-and-mouth disease virus in a primary culture of SP pig kidney cells. The results are presented in Table 3, which demonstrates that the inactivation of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain was 0.0 lg TCID.₅₀/ml occurred 7 hours after the introduction of 1,2-AEEI at a dose of 0.040%, and to a titer of -11.5 lg TCD₅₀/ml - after 12 hours of incubation (Table 3).

The prepared inactivated viral suspensions were analyzed in the RSC to assess their viral component content. The concentration of the 146S component was 1.72±0.02 μg/ ^cm3 .

Example 6. Selection of conditions for purification of a suspension of foot-and-mouth disease virus strain
"O/EA-4/Ethiopia/2013" genotype O/EA-4.

A suspension of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus (FMDV) strain, genotype O/EA-4, obtained by propagation in the BHK-21/SUSP/ARRIAH suspension cell line, was purified. Polyhexamethylene guanidine (PHMG) was used at concentrations of 0.030, 0.040, and 0.050% to obtain purified FMDV antigen. Before and after the purification process of the O/EA-4/Ethiopia/2013 FMDV antigen. The concentration of total viral protein and immunogenic components was determined in the quantitative version of the RSC. The results of the analysis are presented in Table 4, which shows that as a result of the purification of the foot-and-mouth disease virus antigen of the O/EA-4/Ethiopia/2013 strain Using PHMG at a concentration of 0.030%, a decrease in the concentration of ballast protein by 13% and the 146S component by 0.7% was observed. When using PHMG at a concentration of 0.040%, a decrease in the amount of ballast protein by 23% and the 146S component by 1.8% was observed. Using PHMG at a concentration of 0.050%, the content of ballast protein by 29% and immunogenic components by 20.5%.

Thus, having studied the conditions for purifying the antigen of the strain “O/EA-4/Ethiopia/2013”, we came to the conclusion that in order to obtain a purified product of this antigen material, it is optimal to use PHMG with a concentration of 0.040%.

Example 7. Selection of conditions for concentrating the antigen suspension of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus genotype O/EA-4.

The culture inactivated suspension of the strain "O/EA-4/Ethiopia/2013", obtained using the suspension cell line BHK-21/SUSP/ARRIAH, as described in Example 6, was concentrated 12 times by volume. To increase the concentration of the immunogenic components of the foot-and-mouth disease virus antigen, the following techniques were used: 1) adding polyethylene glycol (PEG-6000) at 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) at 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 for 4 hours at an operating pressure on the filter of 2.0-2.3 atm.

Before and after the process of concentrating the antigen suspension of foot and mouth disease virus strain "O/EA-4/Ethiopia/2013" The concentration of total viral protein and immunogenic components was determined in the quantitative version of the RSC. The results of the studies are presented in Table 5, which shows that when concentrating the antigen of the strain "O/EA-4/Ethiopia/2013" A 4-hour concentration of PEG-6000-treated foot-and-mouth disease virus revealed a 10-fold increase in component content compared to baseline values (before concentration). Using the same polymer but increasing the exposure time to 12 hours, the concentration of virus components increased by 10.8 times. Using a tangential filtration concentration method, it was possible to increase the amount of immunogenic components of the O/EA-4/Ethiopia/2013 strain antigen. The foot-and-mouth disease virus suspension was 11.9 times more effective. Thus, tangential filtration technology allows for the production of the O/EA-4/Ethiopia/2013 strain antigen with a high concentration of structural viral proteins.

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

For the production of a monovalent emulsion vaccine against foot-and-mouth disease from the antigen of the strain "O/EA-4/Ethiopia/2013" The oil adjuvant Montanide ISA-61 VG was selected as an adjuvant in an amount of 60% by weight.

Example 9. Composition of a vaccine against foot-and-mouth disease from the strain "O/EA-4/Ethiopia/2013" genotype O/EA-4, culture-based inactivated emulsion.

From the obtained concentrate of the foot-and-mouth disease virus antigen of the O/EA-4/Ethiopia/2013 strain, obtained in Example 7, a culture-inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype was prepared using the oil adjuvant Montanide ISA-61 VG. The amount of 146S immunogenic component in 1.0 cm³antigen amounted to 20.11±0.02 μg, and at a dose of (2.0 cm³) - 16.09 mcg (more than 15.0 mcg/dose).

Example 10. Immunization of animals with a culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype .

A series of dilutions with a 5-fold step for pigs were prepared from the obtained culture-inactivated emulsion foot-and-mouth disease vaccine of the O/EA-4/Ethiopia/2013 strain, genotype O/EA-4 (the proposed invention) . Fifteen pigs were divided into 3 groups of 5 pigs each. The immunizing dose was 2.0 cm ³ . The first group of animals (Nos. 1-5) were immunized with the vaccine without dilution, the second group of pigs (Nos. 6-10) were vaccinated with the vaccine diluted 1/5, and the third group of animals (Nos. 11-15) - with the vaccine diluted 1/25. The preparation was administered intramuscularly in the middle third of the neck. Two unvaccinated pigs were left as a virus control.

Example 11. Study of blood serum 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 tested 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]. Testing of the obtained sera was carried out 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 obtained research results are presented in Table 6, which shows 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. Evaluation of the avirulence and harmlessness of the O/EA-4 genotype culture-based inactivated emulsion vaccine against foot-and-mouth disease (proposed invention) .

To determine the avirulence of the swine vaccine, a sample of the vaccine product prepared as described in Example 9 was administered intradermally at a dose of 0.1 ^cm3 . Pigs weighing 30-40 kg were used in the study. Over a 10-day observation period, the animals remained clinically healthy, and pathological examination revealed no changes characteristic of foot-and-mouth disease. This indicated the lack of virulence properties of the developed vaccine.

The safety of the product was tested in animals by intramuscular administration of the vaccine at a dose of 6.0 ^cm3 (a triple dose of 2.0 ^cm3 ). The observation period also lasted 10 days. It should be noted that after immunization, the animal's body temperature may rise to 40.5°C and remain at this level for 1-2 days, which is within the normal range after administration of the vaccine.

Based on the results of the studies, the O/EA-4 genotype culture-based inactivated emulsion vaccine against foot-and-mouth disease (the proposed invention) was found to be harmless; all animals remained clinically healthy during the observation period; no tissue necrosis was detected at the site of vaccine administration during the pathological analysis.

Example 13. Study of the immunogenic properties of the O/EA-4 genotype culture-inactivated emulsion vaccine against foot-and-mouth disease (proposed invention).

The immunogenic properties of the vaccine (the proposed invention) were studied based on its ability to induce virus-neutralizing antibodies in naturally susceptible animals. Thus, 21 days after administration of the foot-and-mouth disease vaccine from the O/EA-4/Ethiopia/2013 strain, genotype O/EA-4, culture-based inactivated emulsion vaccine (the proposed invention) , blood was collected from pigs and the resulting blood serum was tested in a microneutralization reaction [3]. It was found that in pigs immunized with the vaccine in a whole dose (5 heads), the average antibody titer values were 9.50±0.79 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.75±0.25 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 3.80±0.11 log ₂ SN ₅₀ (Table 7). The obtained data from the microneutralization reaction indicate that after the administration of the vaccine in its whole form, 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. Study of the protective ability of the O/EA-4 genotype culture-inactivated emulsion vaccine against foot-and-mouth disease (proposed invention) on naturally susceptible animal species.

The study of the protective ability of the vaccine (the proposed invention) on naturally susceptible animal species was carried out by the method of control infection of naturally susceptible animals.

Fifteen pigs immunized with the vaccine in whole form and in dilutions (example 10) were infected with the control strain "O/EA-4/Ethiopia/2013" of the O/EA-4 genotype 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 ). Seven days after infection, all animals were euthanized and underwent a pathological examination. Animals with no lesions on their extremities 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/EA-4/Ethiopia/2013" of the genotype O/EA-4, culture-inactivated emulsion (the proposed invention) , in whole form, as well as in dilutions of 1/5 and 1/25, administered in a single dose (2.0 ^cm3 ), protects pigs from infection with the homologous strain of all animals (5 out of 5 heads).

Based on the results of the control infection, it was established that the inoculation volume of this vaccine contains 55.90 PD ₅₀ and 0.04 IMD ₅₀ for pigs.

Thus, the above information indicates that the O/EA-4 genotype foot-and-mouth disease vaccine, a culture-based inactivated emulsion vaccine embodying the proposed invention, is intended for use in agriculture, namely in veterinary virology and biotechnology; the possibility of implementing the presented invention has been confirmed; the vaccine is made from the O/EA-4/Ethiopia/2013 strain. genotype O/EA-4 in accordance with the proposed invention, has high immunogenic activity and is capable of providing effective protection to susceptible animals against the epizootic strain of foot-and-mouth disease virus of genotype O/EA-4.

Sources of information taken into account when preparing the description of the invention for the application for the issuance of a Russian patent for the invention "Vaccine against foot-and-mouth disease of the O/EA-4 genotype, culture-based inactivated emulsion vaccine":

1. Foot-and-mouth disease. Preventive measures. URL: http://89.rospotrebnadzor.ru/directions/epid_nadzor/146902 (Accessed: 04.10.2024).

2. The danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Accessed: 03.10.2024).

3. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed. - Paris, 2022 - Vol. 1, Chapter 2.1.5. - P. 166-169.

4. Burdov A.N., Dudnikov A.I., Malyarets P.V. et al. 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: Foliant, 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. Mugezi I, Kimaanga M, Namwabira A, Chevanne E, Nekouei O, Mclaws M, Motta P, Dulu T, Sumption K. Risk of foot and mouth disease spread through cattle movements in Uganda. Rev Sci Tech. 2020 Jan;39(3):847-861.

8. González-Gordon L, Porphyre T, Muwonge A, [et.al.]. Identifying target areas for risk-based surveillance and control of transboundary animal diseases: a seasonal analysis of slaughter and live-trade cattle movements in Uganda. Sci Rep. 2023 Oct 30;13(1):18619.

9. Muleme M, Barigye R, Khaitsa ML, Berry E, Wamono AW, Ayebazibwe C. Effectiveness of vaccines and vaccination programs for the control of foot-and-mouth disease in Uganda, 2001-2010. Trop Anim Health Prod. 2013 Jan;45(1):35-43.

10. Russian Federation Patent No. 2816264, July 31, 2023. Culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-3 genotype from the strain “O No. 2241/Ethiopia/2011” // Application No. 2013120224.

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

12. Methodological recommendations for determining the concentration of 146S, 75S, 12S components of vaccine strains of culture foot-and-mouth disease virus in the complement fixation reaction (CFR) / FGBU "ARRIAH". - Approved. 21.09.17.

13. Guidelines for determining the antigenic match between epizootic isolates and production strains of foot-and-mouth disease virus in a cross-microneutralization reaction: approved by Rosselkhoznadzor on September 13, 2017 / FGBU "ARRIAH". - Vladimir: 2017. - 24 p.

14. Barnett P.V. A review of emergency foot-and-mouth disease (FMD) vaccines / Vaccine. - February 2002. - V. 20. - P. 1505-1514 Longjam N., Tayo T. Antigenic variation of Foot and Mouth Disease Virus // Vet. World. - 2011. - Vol. 4(10). - P. 475-479.

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

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

17. 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.

18. 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.

19. 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.

Table 1

Results of adaptation of the O/EA-4/Ethiopia/2013 foot-and-mouth disease virus strain during cultivation in a continuous monolayer culture of PSGK-30 cells under different conditions

(n=3, M ± m, p<0.01)

Parameter Cultivation conditions Reproduction time, h CPD, % Concentration of 146S particles according to RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCID ₅₀ /cm ³ Composition of the supporting environment Medium Needle DMEM+ 2% S _cattle 19 95-100 0.72±0.02 6.32±0.10 RPMI-1640 medium + 2% S _cattle 20 95-100 0.84±0.02 6.75±0.10 Hanks' solution with 5% HBC + 2% S _cattle 17 95-100 1.11±0.02 7.40±0.10 Temperature, °C 35.0±0.2 32 90-95 0.21±0.02 4.86±0.10 36.0±0.2 28 90-95 0.49±0.03 5.98±0.10 37.0±0.2 17 95-100 1.11±0.02 7.40±0.10 38.0±0.2 21 90-95 0.85±0.02 6.74±0.10 Dose of infection 0.1-0.01 20 90-95 0.87±0.02 6.79±0.10 0.01-0.001 17 95-100 1.11±0.02 7.40±0.10 0.001-0.0001 25 90-95 0.44±0.02 5.91±0.10

Note: S _KRS - bovine blood serum,

GBC - blood protein hydrolysate,

DMEM is the name of Eagle's medium,

RPMI-1640 is the name of the culture medium,

CPE - cytopathic effect.

Table 2

Results of reproduction of the O/EA-4/Ethiopia/2013 strain of foot-and-mouth disease virus in the continuous suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations

(n=3, M ± m, p<0.01)

Parameter Cultivation conditions Reproduction time, h CPD, % Concentration of 146S particles according to RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCID ₅₀ /cm ³ Cell concentration before infection, million cells/ ^cm3 3.5 11 95-100 1.02±0.02 7.74±0.10 4.0 11 95-100 1.26±0.02 8.02±0.10 4.5 12 95-100 1.75±0.02 8.95±0.10 5.0 13 95-100 1.82±0.02 9.03±0.10 Temperature, °C 35.0±0.2 23 85-90 0.44±0.02 5.91±0.10 36.0±0.2 21 90-95 0.59±0.02 6.29±0.10 37.0±0.2 12 95-100 1.75±0.02 8.95±0.10 38.0±0.2 17 95-100 1.12±0.02 7.86±0.10 Dose of infection 0.1-0.01 17 95-100 1.15±0.02 7.90±0.10 0.01-0.001 12 95-100 1.75±0.02 8.95±0.10 0.001-0.0001 20 90-95 1.12±0.02 7.85±0.10

Note: RT-PCR-RV - reverse transcription and polymerase chain reaction,

TCD - tissue cytopathic dose,

CPE - cytopathic effect.

Table 3

Study of the kinetics of inactivation of a suspension of foot-and-mouth disease virus strain "O/EA-4/Ethiopia/2013" genotype O/EA-4

(n=3, M ± m, p<0.05)

Sampling time from the moment of inactivation, h Titer of infectious activity of foot-and-mouth disease virus, lg TCID ₅₀ /cm ³ 0 8.95±0.10 1 7.23±0.10 2 5.90±0.10 3 4.30±0.10 4 3.00±0.10 5 2.03±0.10 6 1.02±0.10 7 0 12 -11.50

Table 4

The content of components of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of foot-and-mouth disease virus in suspensions before and after purification

(n=3, M ± m, p<0.005)

Before cleaning Cleaning conditions After cleaning Ballast protein concentration, mg/cm ³ Concentration of 146S component, μg/cm ³ concentration of ballast protein, mg/cm ³ Concentration of 146S component, μg/cm ³ 8.25±0.02 1.72±0.02 PGMG, 0.030% 7.18±0.02 1.70±0.02 PGMG, 0.040% 6.35±0.02 1.69±0.02 PGMG, 0.050% 5.86±0.02 1.37±0.02

Note: The concentration of ballast protein was determined using the Kalkar formula:

C=1.45 × A ₂₈₀ -0.74 × A ₂₆₀ ,

where A ₂₈₀ is the optical density value at a wavelength of 280 nm,

A ₂₆₀ is the optical density value at a wavelength of 260 nm,

PHMG - polyhexamethylene guanidine (flocculant).

Table 5

The content of antigen components of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of foot-and-mouth disease virus in suspensions before and after concentration

(n=3, M ± m, p<0.01)

Concentration of 146S component before concentration, μg/cm ³ Concentration methods (12 times) Concentration of 146S component after concentration, μg/cm ³ 1.69±0.02 PEG-6000, 4 h 16.91±0.02 PEG-6000, 12 h 18.25±0.02 Tangential filtration, 4 h 20.11±0.02

Note: PEG - polyethylene glycol,

OVB - total viral protein.

Table 6

Results of a study of pig blood serum for antibodies to non-structural proteins of the foot-and-mouth disease virus before and after vaccination with a preparation (proposed invention) manufactured using the antigen of the foot-and-mouth disease virus strain "O/EA-4/Ethiopia/2013" genotype O/EA-4

Animal No. Optical density value of the sample Percentage of inhibition (PI), % sample type reference values before vaccination 14 days after vaccination sample type reference values before vaccination 14 days after vaccination SPK 0.412 -//- SPK 59,127 -//- PC 0.065 PC 93,552 OK 1,008 OK 0.00 1 -//- 0.956 0.923 -//- 5,159 8,433 2 0.953 0.919 5,456 8,829

Note: SPC - weak positive control,

PC - positive control,

OK - negative control, serum is negative at PI less than 50%.

Table 7

Study of humoral immunity and immunogenic activity of the O/EA-4 genotype culture-inactivated emulsion vaccine against foot-and-mouth disease in pigs (proposed invention)

(n=5, p<0.01, M ± m)

Name of the strain for challenge infection Dilution of the administered vaccine No. of animals Antibody titer in RMN, log ₂ SN ₅₀ Results of challenge infection IMD ₅₀ / PD ₅₀ O/EA-4/Ethiopia/2013 without dilution 1 10.50 - 0.04/55.90 2 9.50 - 3 8.50 - 4 9:00 - 5 10.00 - M±m 9.50±0.79 0/5 1/5 6 4.50 - 7 4.75 - 8 5.00 - 9 4.50 - 10 5.00 - M±m 4.75±0.25 0/5 1/25 11 4.00 - 12 3.75 - 13 3.75 - 14 3.75 - 15 3.75 - M±m 3.80±0.11 0/5 control 1 - + control 2 - +

Note: "-" absence of generalization of the foot-and-mouth disease process,

RMN - microneutralization reaction,

ImD ₅₀ - immunogenic dose,

PD ₅₀ is a protective dose.

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<INSDQualifier_value>genomic DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q1">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>FMDV</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>accacctccccaggtgaatcagctgaccccgtgactgccactgttgagaact

acggtggcgagacacaggtccagaggcgtcaacacaccgacgtctcgttcatccttgacaggtttgtgaa

agtaacaccaagggaaaacctgattaatgtcttggacctgatgcagatccctgcccacacactggttggg

gcgctccttcgcactgccacctactattttgctgacttggaggtggcggtcaaacacgaagggaacctca

catgggtcccgaacggagcgcccgagtcagcactggacaacaccaccaacccaacggcataccataaagc

accactcacccgtcttgctctgccctacacagcgcctcaccgcgtgttggcaaccgtttacaacgggaac

tgcaagtacggaagtgcaccagtgactaacgtgaggggtgatctccaagtgttggcccagaaggcaacca

ggacgctgcctacctccttcaactacggtgccatcaaggccacccgggtgaatgaactgctctaccgcat

gaagaggggccgagacatactgtcccagacctcttctggctattcacccaaacgaggccaggcacaaacag

aagatcgtggcgcctgtgaagcaacttttg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>214</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..214</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>TTSPGESADPVTATVENYGGETQVQRRQHTDVSFILDRFVKVTPRENLINVL

DLMQIPAHTLVGALLRTATYYFADLEVAVKHEGNLTWVPNGAPESALDNTTNPTAYHKAPLTRLALPYTA

PHRVLATVYNGNCKYGSAPVTNVRGDLQVLAQKATRTLPTSFNYGAIKATRVNELLYRMKRAETYCPRPL

LAIHPNEARHKQKIVAPVKQLL</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (3)

1. A culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-4 genotype, containing an active substance and an adjuvant, characterized in that it consists of an active substance in the form of avirulent and purified antigen material homologous to the causative agent of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and Other Animal Pathogens (GKShM) of the All-Russian Research Institute of Animal Health under the registration number: No. 728 - dep/24 - GKShM of the All-Russian Research Institute of Animal Health, reproduced in the continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg in a vaccine dose of 2.0 ^cm3 ; from the oil adjuvant Montanide ISA-61 VG with a content of 60% by weight and a supporting medium - up to 2.0 cm ³ . 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus, homologous to the infectious agent, obtained in a sensitive biological system and representing a suspension containing predominantly the 146S immunogenic component of the O/EA-4 genotype of the foot-and-mouth disease virus. 3. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigen material of the O/EA-4/Ethiopia/2013 strain of the O/EA-4 genotype of the foot-and-mouth disease virus homologous to the infectious agent and the oil adjuvant Montanide ISA-61 VG in an effective ratio of 40% and 60% by weight, respectively.