RU2708326C1 - Strain n2344/mongolia/2017 virus of foot-and-mouth disease of aphtae epizooticae o type for making biopreparations for diagnosis and specific prevention of foot-and-mouth disease of o type - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. Invention is a virus of foot-and-mouth disease Aphtae epizooticae of type O, fam. Picornaviridae, genus Aphtovirus, type O No. 2344/Mongolia/2017, deposited in the collection of FGBU "VNIIZZh" under registration number No. 116 – dep / 19-6 – MSC FGBU "VNIIZZh".EFFECT: presented strain can be used to control antigenic activity of the anti-feline vaccines and for the manufacture of biologics for the diagnosis and specific prevention of foot-and-mouth disease of the type O.3 cl, 1 dwg, 5 tbl

Description

The invention relates to the field of veterinary virology and biotechnology and can be used to develop and manufacture diagnostic tools and specific prophylaxis of foot-and-mouth disease type O, to control antigenic and immunogenic activity of FMD vaccines.

Foot and mouth disease is a highly contagious viral disease of domestic and wild artiodactyl animals. It belongs to the category of transboundary diseases that can cross borders between states and cause epizootics with serious economic and social consequences. Foot and mouth disease refers to especially dangerous transboundary diseases of animals and is subject to mandatory notification. This pathogen is characterized by a tendency to widespread and epizootic course. The disease is accompanied by large losses of livestock products, complicates commercial operations and economic activity. [sixteen].

The foot and mouth disease virus belongs to the family Picornaviridae, the genus Aphtovirus. There are seven serotypes of the virus: O, A, C, Asia-1, SAT-1, SAT-2, SAT-3. Within each type, there are many genetic variants of the virus. The danger of the disease is due to the high variability and genetic diversity of the virus, the multiplicity of transmission routes of infection, the narrow specificity of immunity in animals within the same serotype.

The causative agent of foot and mouth disease within the same serotype has significant antigenic variability of the strains, which is detected at different time intervals and in different territories, and depends on the species composition of the susceptible population, its immune status and many other various factors. The antigenic variability of foot and mouth disease virus is caused by amino acid substitutions in polypeptide fragments (antigenic epitopes) exposed on the surface of capsid proteins. Changes that persist in the next generation become permanent and are reflected in the modification of the nucleotide sequence of the virus genome [6, 7, 8].

Shifts in the antigenic spectrum, corresponding to the renewal of the structure of the new field isolate, can vary from insignificant ones captured by monoclonal antibodies to significant ones recorded using traditional polyclonal immunoglobulins. Changes in the antigenic properties of the natural isolate cause weakening of specific immunity induced by a non-homologous antigen. At the same time, strain-specific diagnosis of the obtained FMD virus isolates is difficult. The problem of selection and preparation of new production strains of foot and mouth disease virus is one of the main in the system of measures for the prevention of foot and mouth disease. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis.

Known production strains of foot and mouth disease virus type O, previously used for specific prevention of foot and mouth disease in Russia:

A known type O strain No. 1734 / Primorsky / 2000, isolated 13.04.2000 from pigs in the farm "Stepnoe" with. The elite of the Ussuri district of Primorsky Krai [2, 4].

A comparative analysis of the nucleotide sequences showed that strain O No. 1734 / Primorsky / 2000 belongs to the Pan-Asian genetic line of the FMD virus type O. The pan-Asian virus caused a pandemic of foot and mouth disease in 1999-2000 in most countries in Asia, including Vietnam, Japan, Korea, Mongolia, as well as Armenia and Georgia [4]. The devastating epidemic of foot and mouth disease in the UK in 2001 was also caused by a pan-Asian virus. Production strain No. 1734 O / Primorsky-2000-DEPT was previously used in the Russian Federation as an industrial strain in the manufacture of specific prophylaxis and diagnostics used throughout Russia and the CIS countries.

Between 2014 and 2018 in South Korea, China, on the territory of the Russian Federation, outbreaks of type O foot and mouth disease were caused by the Southeast Asia topotype virus belonging to the Mua-98 genetic line [7].

Known production strain of foot and mouth disease virus type O No. 2212 / Primorsky / 2014. The viral isolate, which served as a source for strain O No. 2212 / Primorsky / 2014, was isolated in May 2014 from a sick pig at Mercy Trade LLC s. Prokhori Spassky district of Primorsky Krai [5, 8].

In December 2017, the type O foot and mouth disease virus isolate taken from cattle in Mongolia was delivered to the All-Russian Research Institute of Folk Medicine.

According to the results of a comparative analysis of the nucleotide sequences, the isolated isolate belongs to the genetic line Ind-2001 of the FMD virus type O and is significantly different from the production strain of the foot-and-mouth disease virus type O of the PanAsia-2 genetic line, which is part of the vaccine used for the prevention of foot and mouth disease in the Russian Federation.

Given the intensive trade relations between the Russian Federation and Mongolia, especially in the border areas of the Trans-Baikal Territory, the Jewish Autonomous Okrug, Khabarovsk Territory, the risk of foot-and-mouth disease entering Russia remains very high. In this regard, it became necessary to obtain a new production strain from the epizootic isolate of foot and mouth disease virus of serotype O to ensure the safety of the territory of the Russian Federation and neighboring countries from the causative agent of foot and mouth disease, genetic line O Ind-2001.

The problem to which the present invention is directed is to expand the arsenal of production strains of foot and mouth disease virus of serotype O, which have high infectious, antigenic and immunogenic activity in their native form and retain antigenic and immunogenic activity after inactivation, suitable for controlling the antigenic and immunogenic activity of vaccines, manufacturing sensitive and highly specific diagnostics and highly immunogenic vaccine preparations.

This problem was solved by obtaining a strain of foot and mouth disease virus of type O No. 2344 / Mongolia / 2017 for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type O.

The foot-and-mouth disease isolate, which served as the source for the type VW strain O type No. 2344 / Mongolia / 2017, was isolated in November 2017 from FMD patients located in Mongolia (examination No. 2344). Production strain VJ type O No. 2344 / Mongolia / 2017 was obtained by successive passages on sensitive hetero- and homologous cell cultures.

The strain of foot and mouth disease virus of type O No. 2344 / Mongolia / 2017 was deposited in the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under the registration number (link): No. 116 - dep / 19-6 - KShM FSBI "ARRIAH".

The experimentally confirmed the possibility of using strain VJ type O No. 2344 / Mongolia / 2017 for the manufacture of diagnostic and prophylactic means of foot and mouth disease type O.

The invention is reflected in the graphic image. A dendrogram is presented that reflects the phylogenetic relationships of strain VJA No. 2344 / Mongolia / 2017 with epizootic and vaccine strains of FMD virus of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

The invention is illustrated by the following lists of sequences:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain O No. 2344 / Mongolia / 2017 of type O foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP1 protein gene of strain O No. 2344 / Mongolia / 2017 of type O FMD virus.

Strain O No. 2344 / Mongolia / 2017 of the foot and mouth disease virus is characterized by the following features and properties.

Morphological features

Strain VIA O No. 2344 / Mongolia / 2017 of type O belongs to the Picornaviridae family, the genus Aphtovirus, O serotype and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the form of the virion is icosahedral, size 23-25 nm. Virion consists of an RNA molecule enclosed in a protein coat. The protein shell consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, the strain VYa O No. 2344 / Mongolia / 2017 of the foot and mouth disease virus belongs to the O serotype. The virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity (GA activity). In sick animals, serum type-specific antibodies are formed that are detected in an enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (PMN).

Using the method of nucleotide sequencing, the primary structure of the VP1 gene of strain VW O No. 2344 / Mongolia / 2017 was determined. A comparative analysis of the nucleotide sequences showed that the strain VJ O No. 2344 / Mongolia / 2017 belongs to the Ind-2001 genetic line of the Middle East - South Asia topotype.

Antigenic affinity of strain VYA O No. 2344 / Mongolia / 2017 with production strains of foot and mouth disease virus O ₁ Manisa; About No. 1734 / Seaside / 2000; About PanAsia-2; About No. 2147 / Primorsky / 2012 and About No. 2212 / Primorsky / 2014 was studied at the RMN.

The results of studies in the RMN are presented in table 1. Antigenic compliance (r ₁ ) was 0.45 for O1 Manisa; О№1734 / Seaside / 2000 - 0.46; About PanAsia2 0.47; About No. 2147 / Primorsky / 2012 - 0.74 and About No. 2212 / Primorsky / 2014 - 0.24.

When r ₁ > 0.3, the field isolate and production strain are antigenogenic. When r ₁ <0.3, the field isolate differs from the production strain.

Biotechnological characteristics

The foot-and-mouth disease virus strain type O No. 2344 / Mongolia / 2017 is reproduced in transplanted cell cultures: Siberian mountain capricorn kidney (PSGK-30), pig kidney (IB-RS-2), Syrian hamster kidney (VNK-21 / 2-17). Within 19-24 hours of incubation, the virus harvest in these cell cultures reaches values from 5.48 to 6.25 lg TCD ₅₀ / cm ³ . It retains its original characteristics when passaged in cell cultures for 5 passages (observation period).

Geno and chemotaxonomic characteristics

Strain O No. 2344 / Mongolia / 2017, type O foot and mouth disease virus is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ D.

The nucleic acid is represented by a single-chain linear molecule with a molecular weight of 2.8 × 10 ⁶ D. The virion has a protein shell consisting of four basic proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The lipoprotein membrane is absent.

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Virionic RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VP _66a ) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

Physical properties

The virion mass is 8.4 × 10 ⁻¹⁸ g. A sedimentation coefficient of 146S in the sucrose gradient. The floating density of 1.45 g / cm ³ .

Resistance to external factors

The foot and mouth disease virus strain No. 2344 / Mongolia / 2017 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.4-7.6. PH shifts to both the acidic and alkaline sides lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-radiation, high temperatures.

Additional features and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

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

During the test, 5 consecutive passages of the strain VYa O No. 2344 / Mongolia / 2017 were conducted in the transplanted cell culture IBRS-2, PSGK-30, VNK-21 / 2-17. To determine the biological activity, each passage virus was titrated in an IBRS-2 transplanted cell culture.

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

Example 1

The viral isolate, which served as the source for the type O foot and mouth disease virus strain No. 2344 / Mongolia / 2017, was isolated at the All-Russian Scientific Research Institute of Animal Welfare from samples of aphthous material obtained in December 2017 from cattle suspected of foot and mouth disease.

When isolating a virus in order to obtain a homogeneous population with optimal biotechnological properties, a complex of biological, virological, and biochemical methods was used, provided for by guidelines for identifying and identifying FMD virus strains [3].

Biological and virological methods included the isolation and adaptation of foot and mouth disease virus.

Virus isolation was carried out on transplanted cell cultures PSGK-30, IB-RS-2, BHK - 21 / 2-17 with subsequent adaptation (5 passages). The transplanted cell cultures were grown on appropriate nutrient media, under stationary conditions in vials with a surface area of 25 cm ² , washed from the growth medium and infected with a 10% suspension of aphthous material (the multiplicity of infection was 1-10 TCD ₅₀ per cell) prepared in Hanks solution with 0.5% lactalbumin hydrolyzate (GLA) and antibiotics according to the standard formulation. To remove microflora and ballast cell components, the suspension was pretreated with a 10% chloroform solution. After 30 minutes of contact at 37 ° C, 5 cm ^{3 of the} support medium was added to the vials and incubated at 37 ° C until the appearance of the CPD of cell cultures. In the presence of CPD (rounding of cells, increasing their optical density, degeneration and separation of cells from glass), the bottles were subjected to freezing, thawing, purification of the cell suspension with chloroform, and centrifugation at 3000 g for 15 min. The resulting virus-containing material was used for subsequent passages. The virus was considered adapted to cell cultures if 90-100% of the CPDs appeared in the monolayer of cell cultures within 19-24 hours. Adaptation of the epizootic isolate About No. 2344 / Mongolia / 2017 to various cell lines occurred at the level of 5 passages. The results of the adaptation of the virus to various cell cultures are presented in table 2.

The data shown in table 2 indicate a high adaptive ability of a strain of foot and mouth disease virus type O No. 2344 / Mongolia / 2017.

Inactivated material was examined in RSK and ELISA for the presence of FMD virus antigen, using type-specific serums for serological reactions and the Kit for detection of FMD virus antigen in ELISA, manufactured by FSBI ARRIAH.

The results shown in table 3 indicate that in the aphthous test material No. 2344, an FMD virus antigen of type O was detected in CSC at a dilution of 1: 2.

The strain was tested for sterility, the absence of contamination with bacterial, fungal microflora and mycoplasmas, as well as extraneous viruses in PCR and RT-PCR.

Example 2

Evaluation of the biological properties of the type O foot and mouth disease virus strain No. 2344 / Mongolia / 2017 during reproduction in the transplantable suspension cell line BHK-21 / 2-17.

The strain of foot and mouth disease virus type O No. 2344 / Mongolia / 2017 was reproduced in suspension transplanted cell culture from the kidney of a newborn Syrian hamster VNK-21 / 2-17. Earl solution without serum was used as a supporting medium, with the addition of dry muscle enzymatic hydrolyzate (FGMS), dry blood protein hydrolyzate (GBKS) and antibiotics at pH 7.4-7.6. The cell line was infected with a virus at the rate of 0.007-0.05 TCD ₅₀ / cell.

The cultivation of the virus was carried out at a temperature of 35-37 ° C. The cytopathic effect of the virus, corresponding to 90-95%, was achieved after 19-21 hours. After that, the reproduction of foot-and-mouth disease virus was stopped, the resulting suspension was monitored for sterility and the content of 146S component and total viral protein (BIA). The concentration of 146S component of foot and mouth disease virus in suspension was 1.96-2.42 μg / cm ³ depending on passage. The values of the titer of the infectious activity of the virus, as well as the percentage of 146S component of the strain of foot and mouth disease virus No. 2344 / Mongolia / 2017 are shown in table 4, the data of which show that with an average cell concentration of BHK-21 / 2-17 equal to 2.98 ± 0.15 million cells / cm ³ , the infection dose of 0.007-0.05 TCD ₅₀ / cell and the duration of the reproduction of the virus 20.10 ± 0.28 h, the titer of infectious activity of the causative agent of foot and mouth disease was in the range of 6.00-6.50 lg TCD ₅₀ / cm ³ (the average value is 6.25 ± 0.05 log TTCD ₅₀ / cm ³ ), the concentration of the 146S component is 86.24 ± 1.08%.

Example 3

Study of the effect of inactivation and purification of antigenic material of strain O No. 2344 / Mongolia / 2017 using AEEI and PHMG on the immunogenic (146S and total viral protein) components of foot and mouth disease virus.

At the end of the virus reproduction cycle, without stopping the thermostating process, a 15-20% solution of aminoethylethyleneimine (AEEI) acidified with glacial acetic acid to pH 8.0-8.5 is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension was 0.025-0.050%. Inactivation of foot and mouth disease virus was carried out for 16-24 hours at a temperature of 36-37 ° C and a pH of 7.2-7.6. The aminoethylethyleneimine residue was neutralized by the addition of sodium thiosulfate. For flocculation of ballast impurities and inactivation of possible contaminants, a 10% solution of polyhexamethylene guanidine hydrochloride (PHMG) was added to the suspension to a concentration of 0.005-0.007%. Flocculated ballast impurities were sedimented followed by decantation.

The results of studies on the effect of inactivation and purification of antigenic material using AEI and PHMG on the immunogenic (146S and total viral protein) components of the type 1 FMD virus strain No. 2344 / Mongolia / 2017 are shown in Table 5.

The data of table 5 indicate that the concentration of 146S component of the strain of foot and mouth disease virus type O No. 2344 / Mongolia / 2017 decreases from 2.07 ± 0.10 μg / cm ³ (after the end of the cultivation of the virus) to 1.69 ± 0.10 μg / cm ³ (after the end of the inactivation process).

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention “Strain O No. 2344 / Mongolia / 2017, foot and mouth disease virus Aphtae epizooticae type O for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type O”:

1. Burdov A.N., Dudnikov A.I., Malyarets P.V. and other foot and mouth disease. / Ed. A.N. Burdova. - M., Agropromizdat, 1990, 320 p.

2. Immunobiological properties of the epizootic strain of FMD virus type O No. 1734 "Primorsky-2000" / V.K. Spirin, A.I. Egorova, S.R. Kremenchug [et al.] // Proceedings of the Federal Center for Animal Health. - Vladimir, 2007.- T. 5. - S. 52-57.

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

4. Pat. RF No. 2204599, C12N 7/00, A61K 39/135, 05/20/2003

5. Pat. RF №265068, C12N 7/00, A61K 39/125, G01N 33/569, 04.17.2018

6. Syurin V.N., Samuilenko A.Ya., Soloviev B.V. and other viral diseases of animals. - M., VNIITIBP, 1998, S. 532-548.

7. Phylogenetic analysis of FMD virus isolates identified in the post-Soviet space and in Mongolia in 2016 / A.M. Timina, N.G. Zinyakov, A.V. Shcherbakov, D.A. Lozova // Veterinary medicine today. - 2017. - No. 4. - S. 3-8.

8. Phylogenetic characteristics of FMD virus isolates of type O isolated in the Russian Federation / D.A. Lozova, A.V. Shcherbakov, V.M. Zakharov, S.N. Fomina // Veterinary medicine. - 2018. - No. 5. - S. 3-8.

Claims (4)

1. The strain of FMD virus Aphtae epizooticae type O, fam. Picornaviridae, genus Aphtovirus, deposited in the collection of microorganism strains of FSBI "VNIIZH" under registration number No. 116 - dep / 19-6 - KShM FSBI "VNIIZZH", strain O No. 2344 / Mongolia / 2017 of foot and mouth disease virus for the manufacture of biological products for diagnosis and specific prevention type O foot and mouth disease, characterized by the fact that it was obtained during sequential passage in cell cultures of homologous and heterologous origin, has high biological activity in its native form and retains high antigenic and immunogenic activity by after inactivation. 2. The strain according to claim 1, characterized in that it was obtained for 5 consecutive passages in a primary trypsinized cell culture of the kidneys of the Siberian mountain ibex (PSGK-30) with an infectious activity titer of at least 5.70 ± 0.25 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (CSC) 1: 2. 3. The strain according to claim 1, characterized in that it was obtained for 5 consecutive passages in a transplanted culture of pig kidney cells (IB-RS-2) with an infectious activity titer of at least 5.60 ± 0.25 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (CSC) 1: 2. 4. The strain according to claim 2, characterized in that it was obtained for 5 consecutive passages in a transplanted culture of Syrian hamster kidney cells (BHK-21 / 2-17) with an infectious activity titer of at least 6.25 ± 0.05 lg TCD ₅₀ / cm ³ and antigenic activity in the complement binding reaction (CSC) 1: 2.

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