RU2804634C1 - Sat-1/kenya/2017 strain of aphtae epizooticae foot and mouth disease virus for the manufacture of biological products for the diagnostics and specific prevention of foot and mouth disease virus of sat-1/nwz genotype - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention concerns a new strain of foot-and-mouth disease virus Aphtae epizooticae genotype SAT-1/I, Picornaviridae family, Aphthovirus genus, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens (GKSM) FGBI "ARRIAH" under registration number No.451 - dep/23-1 — GKShM FGBI "ARRIAH", "SAT-1/Kenya/2017" strain SAT-1/NWZ genotype of foot-and-mouth disease virus. The presented strain is reproduced in transplanted cell cultures of Siberian Mountain Ibex Kidney (PSGK-30), Pig Kidney (IB-RS-2), and Newborn Syrian Hamster Kidney (ВНК-21). In a transplanted suspension culture of Syrian hamster kidney cells ВНК-21/SUSP/ARRIAN for 13.75± 0.63 hours of incubation, the concentration of the 146S component of SAT-1/Kenya/2017 strain of SAT-1/NWZ genotype of the foot and mouth disease virus has an average value of 1.86±0.07 mcg/cm³ (68.68±0.58%), retaining the original characteristics when passaged in ВНК-21/SUSP/ARRIAH cell culture.

EFFECT: presented strain can be used for the manufacture of biopreparations for the diagnostics and specific prevention of FMD of SAT-1/I genotype and to control the antigenic activity of FMD vaccines.

1 cl, 1 dwg, 3 tbl, 5 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used for the development and manufacture of diagnostic tools, specific prevention of foot-and-mouth disease of the SAT-1/NWZ genotype and monitoring the antigenic and immunogenic activity of foot-and-mouth disease vaccines.

Foot and mouth disease is a highly contagious disease that affects artiodactyl animals such as cattle, pigs and small ruminants. The infection is considered a major global threat to animal health. The disease is caused by an RNA virus belonging to the genus Aphthovirus of the Picornaviridae family. There are 7 different serotypes of foot and mouth disease virus: O, A, Asia-1, C and SAT-1, SAT-2, SAT-3. In particular, foot-and-mouth disease virus serotype SAT-1 has sequence variability in the VP ₁ protein gene compared to viruses of serotypes O, A and C [2, 3], for which it is considered as a broad antigenic variant, therefore, in the case of SAT-1, careful selection of an immunodominant vaccine strain [3].

The foot and mouth disease virus genome is represented by a single-stranded RNA molecule (ssRNA(+)) of positive polarity. Virion RNA of different strains of foot-and-mouth disease virus has the same length, about 8500 i.i., encodes 4 structural proteins: VP ₁ , VP ₂ , VP ₃ , VP ₄ [2].

Mutations of the foot-and-mouth disease virus within one serotype lead to the emergence of new isolates that differ in the degree of virulence and immunogenicity from those previously isolated. Each serotype is genetically divided into different topotypes. The differences between them are determined by analyzing the nucleotide sequence of the ID gene (VP ₁ protein), which is characterized by high genomic and antigenic variability [1, 2, 3].

The VP ₁ protein is the most variable, since it accounts for about 90% of the mutations of all structural genes. The most variable regions are areas 40-60, 130-160 and 190-213 aa. [3, 4]. Section of the surface protein VP ₁ in the region 130-160 a.a. is highly variable because it is involved in the process of binding to host cell receptors [4, 5]. The variability of this region allows the foot-and-mouth disease virus to interact with receptors of different types of cells and facilitates the transition from one host species to another.

Foot-and-mouth disease virus serotypes have on average 86% identical sequences, although VP ₁ is much more variable. Nucleotide sequences of the VP ₁ coding region are used for the genetic characterization of foot and mouth disease strains because of their importance for attachment and penetration, protective immunity, and serotype specificity. Phylogenetic analysis based on the VP ₁ (ID gene) sequence is widely used to infer evolutionary dynamics, epidemiological relationships between genetic lineages, and to trace the origin and movement of foot-and-mouth disease virus strains [5].

The observed genetic variation in the foot-and-mouth disease virus genome is the result of two processes. First, these are mutations that arise as a result of viral RNA replication and failure of the encoded RNA-dependent RNA polymerase to repair. Secondly, competitive selection, which acts on the genome of the foot-and-mouth disease virus. In the natural environment, as a result, isolates of new genotypes will predominate, which include in their genome the characteristics required for a given environment [4, 5].

Antigenic variations arise from amino acid mutations that alter how the body's immune system recognizes viral proteins. Superficially exposed structures of the foot and mouth disease virus are especially susceptible to immune attack. The process of emergence of new antigenic forms is determined by the complex interaction of viral factors and host cell factors. Changes in the genes encoding capsid proteins through mutation can lead to antigenic changes and the emergence of new genetic lines [5, 6].

Nucleotide sequencing is usually used to identify genetic relationships between different isolates and strains, which makes it possible to trace the origin of the virus in outbreak settings. Antigenic characteristics of the virus associated with the emergence of new strains are important for analyzing outbreaks and searching for optimal vaccine preparations [4, 6].

Foot and mouth disease virus serotype SAT-1 is characterized by high genetic variability, namely it includes the following 13 topotypes: I (NORTHWEST ZIMBABWE, NWZ), II (SOUTHEAST ZIMBABWE, SEZ), III (WESTERN ZIMBABWE, WZ), IV (EAST AFRICA 1, EA-1), V, VI, VII (EAST AFRICA 2, EA-2), VIII (EAST AFRICA 3, EA-3), IX, X, XI, XII, XIII. Such high genetic and antigenic diversity leads to problems in the specific prevention of foot-and-mouth disease when using culture-inactivated foot-and-mouth disease vaccines, and also complicates the strain-specific diagnosis of isolated foot-and-mouth disease virus isolates. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis against the foot-and-mouth disease virus serotype SAT-1.

In East Africa, in particular in Nigeria, Kenya, Tanzania, and Ethiopia, outbreaks of foot-and-mouth disease serotype SAT-1 are sporadic and caused by the introduction of the pathogen from neighboring countries. It should be noted that trade and economic ties with the countries of the African continent, in particular East Africa, have intensified in recent years. As a result, there is a risk of introducing the foot-and-mouth disease virus of this serotype into the territory of the Russian Federation.

Analyzing the outbreaks that were registered in Africa, it was found that isolates of different topotypes of the SAT-1 serotype were identified, in particular, II (SAT-1 RV 11 37), III (SAT-1 BOT 1 68), IV (SAT-1 UGA BUFF 21 70), V (SAT-1 NIG 11 75), VI (SAT-1 SUD 3 76), VII (SAT-1 UGA 13 74), VIII (SAT-1 UGA 1 97), IX (SAT- 1 ETH 3 2007), X (SAT-1/X), XI (SAT-1 TCH 1/72), XII (SAT-1/ANG/9/74), XIII (SAT 1 MOZP132010 B16). In recent years, starting from 2012, foot-and-mouth disease virus isolates of the SAT-1 topotype NWZ serotype have become widespread. In particular, outbreaks of foot and mouth disease of this genotype were recorded in Kenya, Tanzania, Uganda, and Ethiopia.

There are known production strains of the foot and mouth disease virus type SAT-1, which are used or have been used in the world for the production of specific preventive measures for foot and mouth disease:

- strain “SAT-1 ZIM 23 2003” (genotype SAT-1/NWZ),

- strain “SAT-1 RV 11 37” (genotype SAT-1/II),

- strain “SAT-1 BOT 1 68” (genotype SAT-1/III),

- strain “SAT-1 UGA BUFF 21 70” (genotype SAT-1/IV),

- strain “SAT-1 NIG 11 75” (genotype SAT-1/V),

- strain “SAT-1 SUD 3 76” (genotype SAT-1/VI),

- strain “SAT-1 UGA 13 74” (genotype VII),

- strain “SAT-1 UGA 1 97” (genotype VIII),

- strain “SAT-1 ETH 3 2007” (genotype IX),

- strain “SAT-1/NIG/2015” (genotype X),

- strain “SAT-1 TCH 1/72” (genotype XI),

- strain “SAT-1/ANG/9/74” (genotype XII),

- strain “SAT 1 MOZP132010 B16” (genotype XIII).

The foot-and-mouth disease virus isolate “SAT-1/KEN/8/2017” was isolated from cattle in the village of Subukia in the Nakuru region of the Republic of Kenya in 2017 and entered the Federal State Budgetary Institution “ARRIAH” for study and scientific research.

According to the results of a comparative analysis of nucleotide sequences, the isolated isolate belongs to the NWZ (Northwest Zimbabwe) topotype of the SAT-1 serotype of the foot-and-mouth disease virus, which differs significantly from the production strains of the foot-and-mouth disease virus type SAT-1, including the strain “SAT-1 T 155 71” (genotype SAT-1/NWZ).

The present invention allows us to expand the arsenal of production strains of foot-and-mouth disease virus serotype SAT-1, which have high infectious, antigenic and immunogenic activity in their native form, suitable for monitoring the antigenic and immunogenic activity of vaccines, manufacturing sensitive and highly specific diagnostic test systems and highly immunogenic vaccine preparations by obtaining strain "SAT-1/Kenya/2017" of the foot-and-mouth disease virus for the production of biological products for the diagnosis and specific prevention of foot-and-mouth disease of the SAT-1/NWZ genotype.

The strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus has been 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. 451 - dep/23-1 - GKSHM FSBI “ARRIAH” .

The possibility of using the “SAT-1/Kenya/2017” strain of foot-and-mouth disease virus for the production of diagnostic and prevention tools for foot-and-mouth disease of the SAT-1/NWZ genotype has been experimentally confirmed.

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 “SAT-1/Kenya/2017” with epizootic strains of the serological type SAT-1. 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 VP ₁ protein gene of the “SAT-1/Kenya/2017” strain of foot and mouth disease virus serotype SAT-1;

SEQ ID NO: 2 represents the amino acid sequence of the VP ₁ protein gene of the “SAT-1/Kenya/2017” strain of foot and mouth disease virus serotype SAT-1.

The foot and mouth disease virus strain “SAT-1/Kenya/2017” is characterized by the following characteristics and properties.

Morphological characteristics

Strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus serotype SAT-1 belongs to the order Picornavirales, family Picornaviridae, genus Aphthovirus, species Foot-and-Mouth Disease virus and has morphological characteristics characteristic of the causative agent of foot-and-mouth disease: the shape of the virion is ixahedral, the size 21-23 nm. A virion consists of a single-stranded, positive-sense RNA molecule enclosed in a protein shell. During the reproduction of the foot-and-mouth disease virus, the main immunogenic component is formed - complete particles, or the 146S component, consisting of one molecule of viral RNA and 60 copies of the polypeptide, each of which is represented by the proteins VP ₂ , VP ₄ , VP ₃ , VP ₁ .

Antigenic properties

According to antigenic properties, the strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus belongs to the SAT-1 serotype. The virus is stably neutralized by homologous antiserum and does not exhibit hemagglutinating activity. In recovered animals, type-specific antibodies are formed in the blood serum, which are detected in enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN).

Using the nucleotide sequencing method, the primary structure of the ID gene encoding the VP ₁ protein of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” was determined. Comparative analysis of nucleotide sequences showed that the strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus belongs to the SAT-1/NWZ genotype (Fig. 1).

The antigenic affinity (r ₁ ) of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” 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 “SAT-1 ZIM 23 2003” (genotype SAT-1/NWZ),

- strain “SAT-1 RV 11 37” (genotype SAT-1/II),

- strain “SAT-1 BOT 1 68” (genotype SAT-1/III),

- strain “SAT-1 UGA BUFF 21 70” (genotype SAT-1/IV),

- strain “SAT-1 NIG 11 75” (genotype SAT-1/V),

- strain “SAT-1 SUD 3 76” (genotype SAT-1/VI),

- strain “SAT-1 UGA 13 74” (genotype VII),

- strain “SAT-1 UGA 1 97” (genotype VIII),

- strain “SAT-1 ETH 3 2007” (genotype IX),

- strain “SAT-1/NIG/2015” (genotype X),

- strain “SAT-1 TCH 1/72” (genotype XI),

- strain “SAT-1/ANG/9/74” (genotype XII),

- strain “SAT 1 MOZP132010 B16” (genotype XIII).

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype SAT-1 against 10 ² TCD ₅₀ of homologous and heterologous virus was determined in the Russian Medical Sciences Department using cross-titration. Values were calculated 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 [7, 8, 9].

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 closely related;

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

Indicators of antigenic relatedness when studying the strain “SAT-1/Kenya/2017” amounted to r ₁ from 0.02 to 0.23, namely for the strain “SAT-1 ZIM 23 2003” (genotype SAT-1/NWZ) - 0. 23, for “SAT-1 RV 11 37” (genotype SAT-1/II) - 0.22, for “SAT-1 BOT 1 68” (genotype SAT-1/III) - 0.20, for “SAT- 1 UGA BUFF 21 70" (genotype SAT-1/IV) - 0.19, for "SAT-1 NIG 11 75" (genotype SAT-1/V) - 0.05, for "SAT-1 SUD 3 76" (genotype SAT-1/VI) - 0.11, for "SAT-1 UGA 13 74" (genotype VII) - 0.10, for "SAT-1 UGA 1 97" (genotype VIII) - 0.15, for “SAT-1 ETH 3 2007” (genotype IX) - 0.12, for “SAT-1/NIG/2015” (genotype X) - 0.06, for “SAT-1 TCH 1/72” (genotype XI) - 0.02, for “SAT-1/ANG/9/74” (genotype XII) - 0.07, for “SAT 1 MOZP132010 B16” (genotype XIII) - 0.21.

The data obtained indicate the absence of antigenic relationship with production strains of foot-and-mouth disease virus serotype SAT-1.

Geno- and chemotaxonomic characteristics

Strain “SAT-1/Kenya/2017” 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 structural 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. Of the 8 non-structural polypeptides that accumulate in infected cells, RNA-dependent RNA polymerase (3D gene), which is involved in the replication of viral RNA, is especially distinguished.

Physical properties

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

Resistance to external factors

The foot and mouth disease virus strain “SAT-1/Kenya/2017” is resistant to detergents and organic solvents such as ether, chloroform, freon, and acetone. Most stable at pH 7.44-7.64. Shifts in pH both to the acidic and alkaline sides lead to inactivation of the virus. Sensitive to formaldehyde, UV irradiation, γ-irradiation, high temperatures (above 38.0°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 “SAT-1/Kenya/2017” is reproduced in continuous cell cultures: Siberian mountain ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (VNK-21).

During the test, 5 consecutive passages of the “SAT-1/Kenya/2017” strain of foot-and-mouth disease virus were carried out in continuous cell cultures PSGK-30, BNK-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.

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

Example 1. Study of the biological properties of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” during reproduction in monolayer continuous cell lines.

When isolating the foot-and-mouth disease virus isolate “SAT-1/KEN/8/2017” in order to obtain its homogeneous population with optimal biotechnological properties, a complex of biological, virological and biochemical methods were used, provided for by the guidelines for the detection and identification of foot-and-mouth disease virus strains [7] .

Biological and virological methods included isolation in cell culture and adaptation of foot-and-mouth disease virus to continuous cell lines.

Isolation of the foot and mouth disease virus strain “SAT-1/Kenya/2017” was carried out in monolayer continuous cell lines PSGK-30, IB-RS-2, VNK-21, followed by adaptation for 5 consecutive passages. Cell cultures were grown on appropriate nutrient media under stationary conditions in flasks with a working surface area of 25.0 cm ² , freed from the growth medium and infected with a 33% suspension of aphthous material (multiplicity of infection was 1-10 TCD ₅₀ per cell), prepared in Hanks' solution with 0.50% lactalbumin hydrolyzate according to the standard recipe.

To remove microflora and ballast cellular components, the viral suspension was pre-treated with a 10% chloroform solution. After 30 minutes of contact of the virus with the cell culture at a temperature of 37±0.1°C, 5.0 cm ³ of the supporting medium was added to the vials and incubated at a temperature of 37±0.1°C until a cytopathic effect (CPE) appeared in the cell culture , which is presented in the form of rounding of cells, increasing their optical density, degeneration and separation of cells from the surface of the substrate. When the CPD was at least 95% of cells, the flasks were subjected to freezing-thawing, purification of the cell suspension with chloroform and centrifugation at 3000 g for 15 minutes. The resulting virus-containing material was used for subsequent passages. The virus was considered adapted to cell cultures if 95-100% CPE was observed in a monolayer of cell cultures for at least 24 hours. Adaptation of the epizootic isolate “SAT-1/KEN/8/2017” to various cell lines occurred at the level of the fifth passage. The titer of infectious activity was determined using a previously developed method [10].

The results of adaptation of the virus to various cell cultures are presented in Table 1, the data of which indicate the high adaptive ability of the foot-and-mouth disease virus isolate “SAT-1/KEN/8/2017” to the cell cultures used. In a monolayer culture of PSGK-30 cells, a viral suspension with an activity in RSC of 1:24 and an infectious activity titer of 7.40±0.10 lg TCD ₅₀ /cm ³ was obtained in 17 hours. In a monolayer culture of IB-RS-2 cells, a viral suspension was obtained within 14 hours with an activity in RSC of 1:24 and an infectious activity titer of 7.55±0.10 lg TCD ₅₀ /cm ³ . In a monolayer culture of BHK-21 cells, a viral suspension was obtained within 14 hours with an activity in RSC of 1:24 and an infectious activity titer of 7.50±0.07 lg TCD ₅₀ /cm ³ . As a result, a strain “SAT-1/Kenya/2017” with characterized biological and cultural properties was obtained, which was further used to study its properties.

Example 2. Assessment of the biological properties of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” in cattle.

Infection of cattle with the original strain of foot-and-mouth disease virus was carried out intradermal-lingually (passage I). Adaptation and development of the “SAT-1/Kenya/2017” strain of foot-and-mouth disease virus in cattle was carried out over 2 consecutive passages. In order to determine the titer of infectious activity of the adapted strain from aphthae, at stage 2 of passage on cattle, a 10% viral suspension was obtained, from which serial 10-fold dilutions were prepared using 1/15 M phosphate-buffered saline (PBS). Prepared dilutions from 10 ^-2 to 10 ^-6 were injected intradermolingually into 4 points of 0.1 cm ³ in two heads of cattle. The results of titration in animals were recorded after 24 hours based on the presence of aphthae at the site of injection of dilutions of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017”. The infectious activity titer of the foot and mouth disease virus strain “SAT-1/Kenya/2017” on cattle at the stage of the first passage on cattle was 4.25 lg ID ₅₀ /0.1 cm ³ , the second passage - 5.25 lg ID ₅₀ /0.1 cm ³ . Thus, a 10% aphthous suspension of the foot and mouth disease virus strain “SAT-1/Kenya/2017” (passage 2 on cattle) was obtained with an infectious activity titer of 5.25 lg ID ₅₀ /0.1 cm ³ .

Example 3. Study of the biological properties of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” in pigs.

Infection of pigs with the original strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus was carried out intradermally at the stage of the first passage in the area of the corolla of the forelimbs. The adaptation and development of the “SAT-1/Kenya/2017” strain of foot-and-mouth disease virus in pigs was carried out over 2 consecutive passages. In order to determine the titer of infectious activity of the adapted strain from passage 2 in pigs, a 10% viral suspension was prepared, from which serial 10-fold dilutions were prepared using 1/15 M PBS as a solvent. Prepared dilutions from 10 ^-2 to 10 ^-6 were injected intradermally into the corollas of the hooves, 0.1 cm ³ at 4 points on each finger of the limb of one dilution, at the rate of 1 dilution for 2 hooves of 1 limb for each of 2 experimental gilts.

The titration results were recorded after 24 hours based on the presence of aphthae at the site of dilution administration. The titer of infectious activity in pigs for the strain “SAT-1/Kenya/2017” of foot and mouth disease virus in pigs at the stage of the first passage was 4.75 lg ID ₅₀ /0.1 cm ³ , the second - 6.00 lg ID ₅₀ /0.1 cm ³ .

Example 4. Study of the biological properties of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” during reproduction in the continuous suspension cell line BNK-21/SUSP/ARRIAH.

The foot and mouth disease virus strain “SAT-1/Kenya/2017” was reproduced in a suspension continuous culture of cells from the kidney of a newborn Syrian hamster BHK-21/SUSP/ARRIAH. Eagle's medium was used as a supporting medium, with the addition of enzymatic hydrolyzate of dry muscle (FHMS), hydrolyzate of dried blood proteins (GBPS) at a pH of 7.44-7.64. The cell line was infected with the virus at the rate of 0.001-0.100 TCD ₅₀ /cell.

Cultivation of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” was carried out at a temperature of 37.0±0.1°C until the cytopathic effect (CPE) of the virus was achieved, corresponding to 95-100%. The resulting virus-containing suspension was controlled for sterility, and the concentration of the 146S component (the main immunogenic component) and total viral protein (TVP) was determined. The concentration of OM in the suspension was 2.70±0.12 μg/cm ³ . The titer values of the infectious activity of the virus, as well as the percentage of the 146S component of the strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus are reflected in Table 2, from which it can be seen that with an average concentration of BHK-21/SUSP/ARRIAH cells equal to 4, 12±0.13 million cells/cm ³ , infection dose 0.005 TCD ₅₀ /cell. and the duration of virus reproduction was 13.75±0.63 hours, the average titer of infectious activity of the foot-and-mouth disease pathogen was equal to 9.13±0.12 lg TCD ₅₀ /cm ³ . The concentration of the 146S component of the foot-and-mouth disease virus was determined using a previously developed method [11]. The content of this component was 68.68±0.58% (1.86±0.07 μg/cm ³ ).

Example 5. Assessment of the type specificity and activity of the foot and mouth disease virus strain “SAT-1/Kenya/2017”

The assessment of the type specificity and activity of the foot-and-mouth disease virus strain “SAT-1/Kenya/2017” was carried out in the complement fixation reaction (CFR).

To the resulting antigen of the strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus, taken in a volume of 0.4 cm ³ in whole form and in dilutions of 1:2, 1:4, 1:8, 1:16, 1:32 0.1 cm ³ of homologous and heterologous hyperimmune sera obtained for foot-and-mouth disease virus in a working (double) titer and 0.1 cm ³ of complement in a working dilution. The mixture was kept in a water bath for 20 minutes at a temperature of 37±0.1°C. Then 0.2 cm ³ of hemolytic system was added and kept for 30 minutes at a temperature of 37±0.1°C. A positive reaction result corresponded to a 100% delay in hemolysis of sheep erythrocytes (“4 crosses”). In parallel, control reactions were carried out without serum, without complement and reaction components.

As reagents, the RSC used foot-and-mouth disease type-specific hyperimmune guinea pig sera obtained for foot-and-mouth disease virus strains “SAT-1/Kenya/2017”, “SAT-1/ZIM 23 2003”, “SAT-2/SAU/6/2000”, “SAT-3/Bech/65”, “Asia-1/Tajikistan/2011”, “A/Turkey/06”, dry complement for RSC, hemolysin (hemolytic serum), sheep red blood cells 2% (suspension in saline solution, pH =7.05-7.15). Antigens of the foot and mouth disease virus strains “SAT-1/Kenya/2017”, “SAT-1/ZIM 23 2003”, “SAT-2/SAU/6/2000”, “SAT-3/Bech/65”, “Asia-1/Tajikistan/2011”, “A/Turkey/06”.

The results of the study are reflected in Table 3, from which it follows that the strain “SAT-1/Kenya/2017” of the foot-and-mouth disease virus has a pronounced type specificity, belongs to the foot-and-mouth disease virus of the SAT-1 serotype and is active in RSC at a dilution of 1:16.

Sources of information taken into account when compiling a description of the invention for the application for a patent of the Russian Federation for the invention “Strain “SAT-1/Kenya/2017” of foot-and-mouth disease virus Aphtae epizooticae for the manufacture of biological products for the diagnosis and specific prevention of foot-and-mouth disease genotype SAT-1/NWZ” .

1. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7th ed. Paris, 2018. - Ch. 3.1.8

2. Ponomarev A.P., Uzyumov V.L. Foot and mouth disease virus: structure, biological and physicochemical properties. Vladimir: Foliot, 2006. - 250 p.

3. Alexandersen, S. The pathogenesis and diagnosis of foot and mouth disease / S. Alexsandersen, Z. Zhang, A.L. Donaldson // J. Compr. Pathol. - 2003. - V. 129. - P. 268-282.

4. Zhiltsova M.B. Biological properties of epizootic isolates of foot and mouth disease virus types A, O and Asia-1: Abstract of thesis. can. Sci. - Vladimir: 2008. - 23 p.

5. Burdov A.N., Dudnikov A.I., Malyarets P.V. and others. Foot and mouth disease. / Ed. A.N. Burdova. - M., Agropromizdat, 1990, 320 p.

6. Analysis of the epizootic situation regarding foot and mouth disease in Russia from 2010 to March 2019 / V.P. Semakina, T.P. Akimova, V.A. Mishchenko, A.K. Karaulov // Veterinary medicine. - 2019. - No. 11. - pp. 16-20.

7. Methodological recommendations for the isolation and identification of foot-and-mouth disease virus strains / A.A. Gusev, V.M. Zakharov, Zh.A. Shazhko et al.; FGU "ARRIAH" - Vladimir. 2002. - 31 p.

8. Methodological recommendations for determining antigenic correspondence between epizootic isolates and industrial strains of foot-and-mouth disease virus in a cross-microneutralization reaction / S.R. Kremenchugskaya; FSBI "ARRIAH". - Vladimir, 2012. - 36 p.

9. Epizootological features of foot and mouth disease type A caused by heterologous strains of the virus / A.V. Mishchenko, V.A. Mishchenko, V.V. Drygin [and others] // Veterinary medicine. - 2014. - No. 11. - pp. 20-24.

10. Patent No. 2674076 C1 Russian Federation, IPC A61K 39/135 C12Q 1/68. A method for determining the titer of infectious activity of the foot-and-mouth disease virus in non-inactivated raw materials for a vaccine using the method of reverse transcription and polymerase chain reaction in real time: No. 2017145889: application. 12/25/2017: publ. 12/04/2018 / D.A. Lozovoy, D.V. Mikhalishin [and others]; applicant Federal State Budgetary Institution "Federal Center for Animal Health" (FSBI "ARRIAH").

11. Patent No. 2712769 C1 Russian Federation, IPC G01M 33/58, C12Q 1/68. Method for spectrometric determination of the concentration of 146S particles of foot-and-mouth disease virus in non-inactivated raw materials for a vaccine by assessing the number of viral RNA molecules isolated after immune capture of virions: No. 2019116272: application. 05/27/2019: publ. 01/31/2020 / D.A. Lozovoy, D.V. Mikhalishin [and others]; applicant Federal State Budgetary Institution "Federal Center for Animal Health" (FSBI "ARRIAH").

--->

<?xml version="1.0" encoding="UTF-8"?>

<!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing

1.3//EN" "ST26SequenceListing_V1_3.dtd">

<ST26SequenceListing dtdVersion="V1_3" fileName="FMDV genotype

SAT-1 NWZ_1674726391255 xml.xml" softwareName="WIPO Sequence"

softwareVersion="2.1.2" productionDate="2023-01-26">

<ApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

<ApplicationNumberText>0</ApplicationNumberText>

<FilingDate>2023-01-26</FilingDate>

</ApplicationIdentification>

<ApplicantFileReference>483</ApplicantFileReference>

<EarliestPriorityApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

<ApplicationNumberText>0</ApplicationNumberText>

<FilingDate>2023-01-26</FilingDate>

</EarliestPriorityApplicationIdentification>

<ApplicantName languageCode="ru">FSBI "Federal Security Center"

animal health" (FSBI "ARRIAH")</ApplicantName>

<ApplicantNameLatin>FGBI &quot;ARRIAH&quot;</ApplicantNameLatin>

<InventorName languageCode="ru">Doronin Maxim

Igorevich</InventorName>

<InventorNameLatin>Doronin Maksim Igorevich </InventorNameLatin>

<InventionTitle languageCode="en">SAT-1/Kenya/2017 virus strain

foot and mouth disease Aphtae epizooticae for the production of biological products for

diagnosis and specific prevention of foot and mouth disease genotype

SAT-1/NWZ</InventionTitle>

<SequenceTotalQuantity>2</SequenceTotalQuantity>

<SequenceData sequenceIDNumber="1">

<INSDSeq>

<INSDSeq_length>663</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..663</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<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>accacctcggcgggtgagggcgcagaacccgtgaccaccgatgcatcgc

aacacggtggtgggcgccgcgccacacgcaggcaacacactgacgtgtctttccttcttgaccggttcac

actggtcggaaagactgccaacaacaagctgacactggatctgctccagaccaaggagaaggcgctagtg

ggcgcaatcctgcgcgctgctacgtactacttctcggacctggaggtggcatgtgttggaacgaacaagt

gggttggctggacaccgaatggtgcgcctgaactcagtgaggtcggcgacaacccagtcgtcttctctca

caacgggaccacccgctttgctctaccatacactgccccgcacagatgtcttgccactgcctacaatggc

gactgcaagtacaagccagatagtgaggcaccacggacgcacattcgtggagacctcgcgacgctcgctg

agcgcatcgctagcgagacacacatcccaaccactttcaactacggcaggatctacacggaggcggaagt

cgacgtgtacgtgcggatgaagcgagcggagctctactgcccccgcccggttctgactcactatgaccac

caaggtaggaatcgctacaaagtggccctgacaaagcctgccaagcaactgtgc</INSDSeq_sequen

ce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>221</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..221</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>TTSAGEGAEPVTTDASQHGGGRRATRRQHTDVSFLLDRFTLVGKTANNK

LTLDLLQTKEKALVGAILRAATYYFSDLEVACVGTNKWVGWTPNGAPELSEVGDNPVVFSHNGTTRFALP

YTAPHRCLATAYNGDCKYKPDSEAPRTHIRGDLATLAERIASETHIPTTFNYGRIYTEAEVDVYVRMKRA

ELYCPRPVLTHYDHQGRNRYKVALTKPAKQLC</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (1)

Strain “SAT-1/Kenya/2017” of foot-and-mouth disease virus Aphtae epizooticae genotype SAT-1/NWZ, family Picornaviridae, genus Aphthovirus, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and other Animal Pathogens (GKSM) of the Federal State Budgetary Institution “ARRIAH” under registration number No. 451 - dep / 23-1 - GKSHM FSBI "ARRIAH" and intended for the production of biological products for the diagnosis and specific prevention of foot and mouth disease of the SAT-1/NWZ genotype.