RU2204599C1 - Foot-and-mouth virus strain "primorsky-2000" 1734 of type o1 for diagnostic and vaccine preparation preparing - Google Patents

Abstract

FIELD: veterinary virology, biotechnology. SUBSTANCE: the strain "Primorsky-2000" 1734 is obtained by multiple successive passages in sensitive cell cultures. The strain is deposited in the Collection of microorganisms VGNKI 22.03.2000 at registration 1734 "Primorsky-2000-DEP". Virus of the strain O₁ 1734 "Primorsy-2000" is reproduced in sensitive cell cultures for 20-24 h of incubation and accumulates in the amount up to 7.5-8.0 lg TCO₅₀/ml. In total infection the strain causes ЦПД in 4 h and retains parent indices in passage in cell cultures for 10 passages. Foot-and-mouth virus strain O₁ shows high biological, antigenic and immunogenic activity and useful for preparing agent for specific prophylaxis and diagnosis of foot-and-mouth virus of type O₁ showing homology to epizootic virus distributing in Russia, regions of Asia continent including South-East and Middle Asia. EFFECT: valuable properties of strain. 12 tbl, 2 dwg, 5 ex

Description

 The invention relates to the field of veterinary virology and biotechnology and can be used in the manufacture of agents for specific prophylaxis and diagnosis of foot and mouth disease type O.

 Foot and mouth disease is an acute highly contagious viral disease of artiodactyl animals, manifested by fever, vesicular (aphthous) lesions of the mucous membrane of the oral cavity, hairless areas of the scalp, udder, corolla, inter-hoof gap and accompanied by impaired movement. For this disease, a tendency to widespread and epizootic course is characteristic. This disease is accompanied by large losses of milk, meat and other types of livestock products, thereby complicating commercial operations and economic activity.

 Years of experience show that the presence of endemic foot and mouth disease reduces income in dairy and beef cattle breeding by 35-40%.

 The foot and mouth disease virus belongs to the genus of aphthoviruses, the family of picornaviruses. It includes 7 immunological types and a large number of subtypes [1, 2].

 A well-known feature of the causative agent of foot and mouth disease is the antigenic variation of strains within the same serotype, occurring at different time intervals, in different territories, depending on the species composition of the susceptible population, its immune status and many other factors. It is known that the main cause of antigenic variation is a change in the amino acid sequence in the polypeptides of the viral proteins that make up the virion capsid. Such antigenic changes can be expressed both in insignificant differences between the strains, captured only using subtle molecular analysis methods, and in the appearance of completely different strains requiring the use of new tools for serological diagnostics and specific prophylaxis of the disease caused by these strains [2, 3].

Type O foot and mouth disease virus strains are known that have been used in Russia for the past 34-35 years. These include the following strains: O ₁ 1618 "Chechen-Ingush", isolated in 1966 in the Chechen-Ingush ASSR; O ₁ 194, allocated in 1967 in the Volgograd region and O ₁ 1182/83, allocated in 1983 in the territory of Armenia [4, 5].

 Vaccines from these production strains previously provided fairly intense immunity in vaccinated animals against pathogens that are homologous or antigenically related to them.

 However, in recent years, several outbreaks of foot and mouth disease have been recorded, which in some cases could not be eliminated in the primary foci, despite vigorous veterinary and sanitary measures of a general and special nature.

 This was due to the lack of effectiveness of the vaccines used against the epizootic virus, causing breakthroughs of immunity even in repeatedly vaccinated animals. Therefore, there is a need to prepare a new production strain of the virus for successful specific prophylaxis of agricultural animals from the newly isolated foot-and-mouth disease virus of type O.

The closest to the proposed invention in terms of essential features is strain O ₁ 194 "Volgogradsky", isolated in 1967 and used in the Russian Federation as an industrial one in the manufacture of diagnostic and vaccine preparations used in Russia and the CIS countries [6].

Strain 194 of the virus of foot and mouth disease O ₁ has the following characteristics, are presented in table 1.

 The disadvantages of this strain are its low biological, antigenic and immunogenic activity.

This is evidenced by the fact that in 2000 in Russia (Primorsky Krai) and the countries of Transcaucasia (Georgia), foci of FMD type O were detected in cattle and pigs immunized with a bivalent AO vaccine, which includes antigen from the production strain O ₁ 194. However, the vaccine did not provide satisfactory protection for the immunized animals. Laboratory studies at ARRIAH of aphthous material isolated from sick pigs revealed the type O foot and mouth disease virus having antigenic differences from strain O ₁ 194 and O ₁ 1618 in the complement binding reaction (CSC) and enzyme-linked immunosorbent assay (ELISA). This testified to the inconsistency of the means used for specific prophylaxis and diagnosis of foot and mouth disease type O used by Russia and the CIS countries, the epizootic virus circulating in 2000 in Russia (Primorsky Territory) and the countries of the South Caucasus (Georgia).

 The task of creating the present invention was to obtain a new production strain of foot and mouth disease virus type O, which has high biological, antigenic and immunogenic activity in its native form and after inactivation and provides for the production of diagnostic and vaccine preparations homologous to the epizootic virus that appeared in Russia and the countries of the Caucasus.

 The technical result from the use of the present invention is to obtain a new production strain of foot and mouth disease virus type O with high biological, antigenic and immunogenic activity and suitable for the manufacture of diagnostic and vaccine preparations against foot and mouth disease type O, homologous to the epizootic virus circulating in Russia (Primorsky Territory) and countries of the Caucasus (Georgia).

The specified technical result was achieved by obtaining strain 1734 "Seaside-2000" of the virus of foot and mouth disease type O. Strain O ₁ 1734 "Seaside-2000" is new, previously unknown. The initial virus for obtaining strain 0 ₁ 1734 "Primorsky-2000" was isolated 04/13/2000 from sick pigs in the farm OPH "Stepnoe" (village Elite) of the Ussuriysky district of Primorsky Krai. The production strain O ₁ 1734 "Primorsky-2000" was obtained by repeated consecutive passages on sensitive hetero- and homologous cell cultures.

 The resulting strain was deposited in the Collection of microorganisms of the All-Russian Research Institute for Control, Standardization and Certification of Veterinary Medicines of the Ministry of Agriculture of the Russian Federation (VGNKI) on March 22, 2000 under registration number 1734 "Primorsky-2000" -DEP.

Compared with the prototype strain O ₁ 1734 "Primorsky-2000" has a higher biological, antigenic and immunogenic activity in its native form and after inactivation. It has been experimentally confirmed that it can be used to prepare diagnostic products and inactivated vaccines. Strain 1734 "Primorsky-2000" of type O virus provides diagnostic preparations that allow for serological diagnosis of FMD virus isolates of type O, which caused an outbreak in 2000 in Russia (Primorsky Krai) and the countries of Transcaucasia (Georgia), and an inactivated FMD vaccine that creates protection of susceptible animals against the specified pathogen.

The invention is illustrated in the graphic materials, where:
FIG. 1 shows the amino acid sequence of VP1 protein of strain 1734 "Primorsky-2000" of type O FMD virus;
FIG. Figure 2 shows a dendrogram reflecting the phylogenetic relationships of strain 1734 "Primorsky-2000" with epizootic and vaccine strains of FMD virus type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

Strain O ₁ 1734 "Seaside-2000" of the FMD virus is characterized by the following features and properties.

Morphological properties
Strain O ₁ 1734 "Primorsky-2000" belongs to the family Picornaviridae, genus Aphtovirus, serotype O 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
By its antigenic properties, the strain belongs to the O serotype. The virus is stably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. In sick animals, antibodies are formed in the blood serum that are detected in RDP, ELISA and pH. When vaccinating cattle and pigs with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA, RDP and pH. During hyperimmunization of guinea pigs with a concentrated inactivated virus, it induces the formation of virus-specific antibodies detected in CSC at a dilution of 1: 40-1: 80, in ELISA at a dilution of 1: 3000-1: 4000. When determining antigenic affinity, strain O ₁ 1734 "Primorsky-2000" With production strains O ₁ 194, O ₁ 1618, as well as with other strains isolated in the countries of the Caucasus and other foreign countries in 1997-2000, a comparative study of the primary structure of the gene and protein VP1 was carried out.

 Using the method of nucleotide sequencing, the primary structure of the VP1 gene of strain 1734 "Primorsky-2000" was determined. The primary structure of the VP1 protein was derived from it (Fig. 1). A comparative analysis of the nucleotide sequences showed that strain 1734 "Primorsky-2000" belongs to the pan-Asian genetic line of the virus of foot and mouth disease type O (figure 2). The pan-Asian virus caused a pandemic of foot-and-mouth disease in 1999-2000 in most countries of Asia, including Vietnam, China, Taiwan, Japan, Korea, Mongolia, Armenia, and Georgia. The devastating epidemic of foot and mouth disease in the UK in 2001 was also caused by the Pan-Asian virus.

The studied virus has the highest level of homology (98.74%) with isolates O ₁ -Vietnam-1999 and O ₁ -Taiwan-1999 (figure 2).

Phylogenetic relationships of the studied isolates with previously studied strains isolated in different countries of the world over the past three decades are indicated on the dendrogram (figure 2). It was established that the studied isolates differ from all previously isolated isolates, including from strains O ₁ 194 and O ₁ 1618, which are currently used for the manufacture of diagnostics and specific prophylaxis of foot and mouth disease type O.

The antigenic affinity of the foot and mouth disease virus O ₁ 1734 "Primorsky-2000" with the corresponding production and previously isolated strains in the territory of Armenia, Taiwan, Mongolia and Vietnam was studied in the RSK. The results are presented in table 2.

The data presented in table 2 indicate that the epizootic strain O ₁ 1734 differs from the production strains O ₁ 194 and O ₁ 1618, but at the same time is closely related to the strains O ₁ -Taiwan-1999, O ₁ 1704-Armenia- 1997, O ₁ Vietnam 1999 and O ₁ Mongolia 2000. Similar results were obtained in the ELISA reaction.

Biological characteristics
Strain O ₁ 1734 "Primorsky-2000" exhibits high biological, antigenic and immunogenic activity both in its native form and after inactivation. The strain is intended for use as a raw material for diagnostic preparations, as well as the manufacture of inactivated FMD vaccine. The virus of strain O ₁ 1734 is reproduced in a monolayer primary trypsinized culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), IB-RS-2, BHK-21 and accumulates over 20-24 hours of incubation up to 6.5-7.5 Ig TCD _{50 / ml} . With massive infection, it causes a CPD after 4 hours.

After 3-5 passages of incubation, up to 6.5-7.5 1d TCD _{50 / ml is} accumulated. It retains its original characteristics when passaged in sensitive biological systems for 10 passages.

Chemo- and genotaxonomic characterization
Strain O ₁ 1734 "Primorsky-2000" is an RNA-containing virus with mol. weighing 7 • 10 ⁶ D.

Nucleic acid is represented by a single-chain linear molecule with a mol. weighing 2.8 • 10 ⁸ MD. 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, cleave to form more stable structural and non-structural virus polypeptides. Of the 6 non-structural polypeptides that accumulate in infected cells, one (VP66a) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

The primary structure of the virus genome region was determined using PCR and sequencing. The results of the studies are presented in figures 1 and 2. A comparative analysis showed that this isolate belongs to the new genetic line of the FMD virus type O. Representatives of this line have been isolated in recent years in various regions of the Asian continent, including Southeast and Central Asia and the Far East . The studied foot and mouth disease virus O ₁ 1734 had the highest homology level (98.74%) with isolates O ₁ -Vietnam-1999 and O ₁ -Taiwan-1999.

 At the same time, its homology with representatives of other genetic lines of the FMD virus type O was only 80-90% (figure 2).

The wide geographical distribution of the genetic line of the foot and mouth disease virus, to which strain O ₁ 1734 belongs, suggests a possible panzootia.

Physical properties
The mass of the virion is 8.4 • 10 ^-18 g. The sentimentation coefficient is 146S in the sucrose gradient. The floating density is 1.45 g / ml.

Resistance to external factors
The virus of strain O ₁ 1734 "Primorsky-2000" is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.2-7.6. PH shifts to both the acidic and alkaline sides lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ radiation and high temperatures.

Additional features and properties
Immunogenic activity - immunogen in the composition of an inactivated vaccine.

 Reactogenicity - does not have reactogenic properties.

 Pathogenicity - pathogenic for artiodactyl animals, newborn mice, rabbits and guinea pigs.

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

 Stability - preserves the original biological properties when passaged in sensitive biological systems for 10 passages.

Based on the obtained data, it can be argued that strain O ₁ 1734 "Primorsky-2000" in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of foot-and-mouth disease virus type O.

 To reduce its epizootic danger, it is necessary to ensure timely laboratory diagnosis and vaccination of newly emerging foci of the disease, which requires highly active and specific antibody and antigenic diagnostics and a vaccine obtained using this strain as a production one.

 According to the applicant, the proposed strain meets the conditions of patentability "novelty" and "inventive step".

 The essence of the invention is illustrated by examples of its implementation.

Example 1
The foot and mouth disease virus strain 0 ₁ 1734 "Primorsky-2000" was isolated from field material received in ARRIAH as an epithelium of aphthae from pigs suspected of foot and mouth disease during laboratory diagnosis of this disease and its differentiation from other vesicular diseases. When isolating the virus used a complex of biological, virological and biochemical methods provided for [7].

Biological and virological methods included adaptation of the original virus to cultures of primary trypsinized and transplanted cell lines. Cell cultures of SP, PSGK-30, IB-RS-2 and BHK-21 were used. For staging a bioassay, primary and transplantable cell cultures were grown on appropriate nutrient media in 50-100 ml vials, washed from the growth medium and infected with a 10% suspension of aphthous material (infection multiplicity was 1-10 TCD ₅₀ per cell) prepared in solution Needle with antibiotics according to the standard formulation. To remove microflora and ballast cellular components, the suspension was treated with chloroform in a ratio of 1: 10. After 30 minutes of incubation at 37-38 ^o C, 5-10 ml of maintenance medium was introduced into the vials and incubated at 37-38 ^o C until the virus CPD appeared . 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 15-20 min. The obtained virus-containing material was used for subsequent passages and studies in CSC and ELISA for the presence of a viral antigen, and a set of commercial type-specific sera and serums were used, which were stored in the Museum of VNIIZH strains.

 The results of the adaptation of the virus to various cell cultures are presented in table 3.

The results are shown in table 3, indicate good adaptive activity of strain O ₁ 1734 to the used cell cultures.

 The virus isolated using the methods listed above was examined in CSCs with a set of diagnostics for all types of foot-and-mouth disease virus, vesicular exanthema, porcine vesicular disease and vesicular stomatitis in order to identify the type of accessory and control for purity. The results of virus typing in CSCs are presented in table 4.

 The results in table 4 indicate that the selected virus is of type O; with all other sera, a negative result was obtained.

Example 2
For hyperimmunization of guinea pigs, an antigen from foot and mouth disease virus, strain O ₁ 1734 "Primorsky-2000", reproduced in a suspension cell culture of BHK-21, is used. The virus-containing suspension is purified from ballast proteins by the addition of 0.05% polyguanidine. The purified virus is concentrated 100 times with PEG-6000 (10% by volume) and inactivated with aminoethylethyleneimine (AEEI) at a concentration of 0.01-0.05% and a pH of 7.8-8.0.

Inactivated antigen concentrate in a mixture with an equal volume of oil adjuvant is administered to guinea pigs intramuscularly at a dose of 1.0 cm ³ . After 21 days, immunization is repeated twice (interval of 7 days) and after 10 days the animals are bled. Individual serum samples are checked for type specificity and activity in CSCs according to [7].

 After that, prepare a series by mixing type-specific individual samples of the same activity. The strain specificity of a serial preparation is determined in one- and two-sided reactions with homo- and heterologous antigens.

After preservation with sodium azide (1: 5000) and holding at 4 ^o C for 30 days, the resulting serum is Packed in ampoules of 0.5-1.0 cm ³ and lyophilized.

 By the method described in example 2, 2 series of hyperimmune sera were prepared, the characteristics of which are presented in table 5.

 The data in table 5 indicate that diagnostic sera have been obtained that meet the requirements for specific activity [7].

Example 3
To obtain the antigen for immunochemical reactions, a foot and mouth disease virus, strain O ₁ 1734 "Primorsky-2000", adapted to PSGK-30 and IB-RS-2 transplanted subline cell cultures, is used. For adaptation, virus-containing material is used in the form of a 10% suspension from the walls of pig aphthae. Adaptation is carried out for 4-5 consecutive passages. The resulting matrix virus seed is used to produce viral raw materials. Infection of cell cultures and the collection of viral material is carried out according to standard methods. The resulting virus-containing liquid was concentrated 100 times with 10% PEG-6000. The resulting concentrate is inactivated by heating at 58 ^o C for 40 minutes, Packed in ampoules of 1 cm ³ and dried by freeze-drying under vacuum.

 The specified method was prepared 2 series of diagnostic antigen, the characteristics of which are given in table 6.

 The research results shown in table 6 indicate that diagnostic antigens were obtained, the specific activity of which meets the requirements [7].

Example 4
For the manufacture of an adsorbate vaccine, foot and mouth disease virus O ₁ 1784 "Primorsky-2000" is reproduced in a suspension culture of BHK-21 cells. As a supporting medium, an Earle solution without serum is used with the addition of FGMS, GBKS and antibiotics at a pH of 7.2-7.4. The cell culture is infected with a virus at the rate of 0.005-0.1 TCD ₅₀ per cell. The cultivation of the virus is carried out at a temperature of 36-37 ^o C. After 8-10 hours of incubation, when the number of dead cells reaches 90-95%, the cultivation is stopped and the virus-containing suspension is controlled for sterility and the content of 146S + 75S components. The amount of 146S + 75S components of the suspension should correspond to at least 0.5 μg / ml. Immediately after the end of the virus reproduction cycle, without stopping thermostating, a 15–20% AEEI solution, 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 should be equal to 0.025-0.05%. Inactivation of the infectiousness of the virus is carried out for 12-24 hours at 36-37 ^o C and a pH of 7.2-7.6 with stirring after 5-6 hours for 3-5 minutes. At the end of inactivation, the antigen suspension is cooled to 4-8 ^° C. A 10% solution of polyhexamethylene guanidine (PHMG) is added to the cooled suspension to a concentration of 0.005-0.007% for flocculation of ballast impurities and inactivation of possible contaminants. Flocculated ballast impurities are subjected to sedimentation followed by decantation. The resulting antigen is monitored for avirulence, virus-containing protein content and 146S + 75S virus components and sterility. The required concentration of 146S + 75S components in the vaccine dose of the adsorbed vaccine is obtained by concentration of the antigen with gel of aluminum oxide hydrate (GOA). For this, the calculated volume of GOA gel of 3% concentration is added to the cooled antigen suspension.

 After sedimentation of GOA, the calculated volume of the supernatant is drained or the volume of the remaining suspension is measured. The final concentration of GOA should be in the range of 1.62-0.488 P <0.01 mg / ml n = 10, and the concentration of 146S + 75S components of the FMD virus is at least 6.0 μg / ml. An additional 10% saponin solution is then added to the suspension to a final concentration of at least 0.075%. The resulting vaccine is packaged in glass bottles and subjected to sterility control according to [8].

 The virulence and safety of the vaccine is checked on 5 heads of cattle, introducing the vaccine first under the mucous membrane of the tongue in a dose of 2.0 ml, and then subcutaneously in a dose of 10 ml Monitoring the clinical condition of animals is 10 days. An avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

 The results of the control of the immunogenic activity of the obtained vaccine for cattle are shown in table 7. The results shown in table 7 showed that all six heads of cattle were protected from the generalization of the process after control infection after 21 days.

The immune response of cattle to the administration of an adsorbate vaccine from FMD antigen O ₁ 1734 was also tested on 5 heads of cattle weighing 200-300 kg. The research results are presented in table 8. The data in table 8 indicate that the FMD vaccine from strain O ₁ 1734 induces a good immune response, starting from 21 days after vaccination. The level of virus-neutralizing antibodies (VNA) by 61 days increased by 5.07 log ₂ compared with 21 days after vaccination.

 The resulting vaccine is a light yellow liquid with a loose white precipitate of sorbent, which is formed at the bottom of the vial during storage and is easily broken into a homogeneous suspension with shaking.

 The optimal component composition of the obtained adsorbate vaccine against foot and mouth disease type O are shown in table 9.

Example 5
For the manufacture of an emulsion vaccine, FMD virus type O, strain 1774 "Primorsky-2000", is reproduced in a suspension culture of BHK-21 cells, inactivated and purified from ballast impurities and possible contaminants, subjected to control for avirulence, the content of virus-specific protein, immunogenic components (146S + 75S) and sterility as described in example 4. The required concentration of 146S + 75S components in the vaccine dose of the emulsion vaccine is obtained by concentration of the antigen by flow ultrafiltration or polyethylene glycol. The resulting antigen concentrate is stored at 4-6 ^{° C.} until used in the vaccine. An emulsion vaccine is obtained by dispersing an antigen concentrate and an oil adjuvant in colloidal mills in a ratio of 3: 7-1: 1, respectively.

 The result is an inactivated emulsion vaccine against foot and mouth disease type O, which is a milk-like liquid insoluble in water. The vaccine has a liquid consistency, easily dissolves at the injection site, does not cause abscesses, does not cause a general reaction in the form of a temperature rise and has pronounced immunogenicity for pigs at a dose of 2 ml 1-21 days after vaccination, for cattle at a dose of 5 ml after 3-21 days after vaccination and for sheep - in a dose of 1 ml 3-21 days after administration. The vaccine is administered intramuscularly to pigs, and cattle and sheep subcutaneously. Inoculated volume should contain at least 4 μg of 146S + 75S components. The optimal component composition of the resulting emulsion vaccine against foot and mouth disease type O are shown in table 10.

The immunogenic activity of the emulsion vaccine was tested on pigs weighing 35-40 kg. The research results are presented in tables 11 and 12. ImD ₅₀ = 0.18 ml. A graft volume of 2 ml contains 11.2 ImD5o for pigs. Table 11 shows that the vaccine from foot and mouth disease virus O ₁ 194 induces in the body of gilts 2–3 times less VNA against the heterologous strain of virus O ₁ 1784 "Primorsky-2000" in comparison with the homologous strain.

 The data presented in table 12 show the level of humoral immunity in gilts vaccinated with FMD vaccines with different contents of the immunogenic component.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:
- strain 1734 "Seaside-2000" of the FMD virus type O, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;
- for the proposed invention in the form described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date has been confirmed;
- strain 1734 "Seaside-2000" of the FMD virus type O, obtained in accordance with the invention, has a high biological, antigenic and immunogenic activity in its native form and after inactivation and is suitable for the manufacture of diagnostic and vaccine preparations.

 Therefore, the present invention meets the condition of patentability "industrial applicability".

Sources of information
1. Rerer H. Foot and mouth disease. Per. with him. G.A. Surkova. Ed. and with the foreword. Cand. vet. sciences. P.V. Malaria, M., Kolos, 1971, 432s.

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

 3. Syurin and other viral diseases of animals.-M., VNITIBP, 1998.-c.532-548.

 4. Kruglikov B. A., Stefan M.K. and Tarasenko T.Ya. Study of the infectious properties of foot and mouth disease virus type O strains isolated at different periods of epizootic. - In the book: Act. prob. vet. virusol. - Vladimir, 1988. 1.-s. 77-78.

 5. Auth. testimonial. USSR 1739559, A 61 K 39/135, 10.04.2000

 6. Instructions for the manufacture and control of a monovalent emulsion vaccine against foot and mouth disease type A, type O, type C, type Asia-1 (from a virus grown in BHK-21 cells). Approved by the GUV of the State Commission CM CCP for food and procurement on 01.24.91 (prototype).

 7. GOST 25384-82 "Guidelines for the identification of identification of FMD virus strains." M., Kolos. 1974.

 8. GOST 28085-89.

 9. Shazhko Zh.A., Fomina T.A. et al. Some characteristics of epizootic strains of foot and mouth disease virus isolated in the USSR. Foot and mouth disease (Towards a new strategy for fighting foot and mouth disease), Vladimir, 1992. 1.-s. 82-96.

Claims (1)

 Aphtae epizooticae virus strain, fam. Picornaviridae, genus Aphtovirus, species Aphtae, type O, collection VGNKI 1734 "Primorsky-2000-DEPT", for the manufacture of diagnostic and vaccine preparations.

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