RU2140452C1 - Foot and mouth virus disease type a strain n 1707 "armenia-98" used for preparing diagnostic and vaccine preparations - Google Patents

Abstract

FIELD: virology, veterinary science. SUBSTANCE: industrial strain N 1707 "Armenia-98" is obtained by multiple successive passages on sensitive hetero- and homologous cell cultures. The strain is deposited in Collection of microorganisms VGNKI at N 1707 "Armenia-98-DEP". The strain N 1707 "Armenia-98" is distinguished from both industrial and early isolated epizootic strains of foot and mouth virus disease type A. Virus of the strain N 1707 "Armenia-98" is reproduced in sensitive cell cultures and its accumulation for 20-24 h of incubation is up to 7.0-8.0 lg TTC₅₀/ml. The strain induces TSPD in 4 h after massive infection, retains the parent indices at passage in cell cultures for 10 passages. New industrial foot and mouth virus disease type A strain shows high biological, antigenic and immunogenic activity and can be used for preparing agents for specific prophylaxis and diagnosis of foot and mouth virus disease type A showing homology to epizootic virus in Transcaucasis and Near East countries. EFFECT: new strain of virus indicated above, improved properties and activity. 2 dwg, 8 tbl, 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 A.

 Foot and mouth disease is an acute 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, and inter-hoof gap and accompanied by impaired movement. It is characterized by a tendency to widespread and epizootic course. The disease is accompanied by large losses of milk, meat and other types of livestock products, complicates 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 30-40%.

 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.

 A well-known feature of the causative agent of foot and mouth disease is the antigenic variability 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 believed 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. In practice, such antigenic changes can be expressed from insignificant differences between the strains, captured only using subtle molecular analysis methods, to the appearance of completely different strains, requiring the use of new tools for serological diagnosis and specific prophylaxis of the disease caused by these strains.

Type A foot and mouth disease virus strains have been used as production strains in the USSR for the past 35 years. These include the following strains: A ₇ N 103, isolated in 1962 in the Kuibyshev region; A _t N 2, allocated in 1965 in the Tajik SSR; AN 717/73, allocated in the Stavropol Territory in 1973

 These strains were used to obtain diagnosticums and FMD vaccines used in various regions of the country. However, after the elimination of foot-and-mouth disease caused by antigenically similar strains of the virus, they were discontinued and are currently only supported in the Museum of strains of the All-Russian Research Institute for Animal Protection [1, 2, 3, 4, 5].

The closest to the invention according to the set of essential features is the strain N 550 of the foot and mouth disease virus A ₂₂ , isolated in 1964 in Azerbaijan and used in the Russian Federation as an industrial product in the manufacture of specific prophylaxis and diagnostics used throughout Russia and the CIS countries [6].

Strain N 550 virus of foot and mouth disease A ₂₂ 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 1998 in the Republic of Armenia foci of FMD type A were detected in cattle immunized with the bivalent AO vaccine, which includes the antigen from production strain A ₂₂ N 550. However, the vaccine did not provide satisfactory protection for the immunized animals . Laboratory studies in ARRIAH of aphthous material isolated from sick animals revealed a type A foot and mouth disease virus with antigenic differences from strain A ₂₂ N 550 in the complement binding reaction and the virus neutralization reaction. This testified to the inconsistency of the means used for specific prophylaxis and diagnosis of type A foot and mouth disease used by Russia and the CIS countries to the epizootic virus circulating in 1998 in the countries of the Caucasus and the Middle East (Turkey, Iran).

 The task of creating the present invention was to obtain a new production strain of foot and mouth disease virus type A, 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 the countries of the Caucasus and the Middle East.

 The technical result from the use of the present invention is to obtain a new production strain of foot and mouth disease virus type A with high biological, antigenic and immunogenic activity and suitable for the manufacture of specific prophylaxis and diagnosis of foot and mouth disease type A homologous to the epizootic virus circulating in the countries of the South Caucasus and the Middle East.

 The specified technical result was achieved by obtaining strain N 1707 "Armenia-98" of FMD virus type A. Strain A N 1707 "Armenia-98" is new, previously unknown. The original virus for obtaining strain A N 1707 "Armenia-98" was isolated on 08/01/98 from sick cows in the farm of Ohchik in the Amassi region of the Republic of Armenia. Production strain A N 1707 “Armenia-98” was obtained by repeated consecutive passages on sensitive hetero- and homologous cell cultures.

 The resulting strain was deposited in the Microorganism Collection of the All-Russian Scientific Research Institute for Control, Standardization and Certification of Veterinary Medicines of the Ministry of Agriculture and Food of the Russian Federation (VGNKI) on 12.11.98 under registration number 1707 "Armenia-98-DEP".

 Compared with the prototype, strain A N 1707 "Armenia-98" 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 N 1707 "Armenia-98" of type A foot and mouth disease virus provides diagnostic preparations that allow serological diagnosis of foot and mouth disease type A isolates, causing outbreaks of disease in recent years in the countries of the Caucasus and the Middle East, and FMD inactivated vaccine that protects susceptible animals against this causative agent of the disease.

The invention is illustrated in the graphic materials, where:
FIG. 1 shows the amino acid sequence of protein VP _{1 of} strain N 1707 "Armenia-98" of FMD virus type A ( ^* - amino acid not determined);
FIG. Figure 2 shows a dendrogram reflecting the structural similarity of the VP ₁ gene region of strain 1707 "Armenia-98" of type A FMD virus with similar fragments of genomes of other types of FMD virus A.

 Strain A N 1707 "Armenia-98" of the FMD virus is characterized by the following features and properties.

Morphological properties
The strain AN 1707 "Armenia-98" belongs to the family Picornaviridae, genus Aphtovirus, serotype A 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, strain AN 1707 "Armenia-98" belongs to serotype A. 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 with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA, RDP, 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: 256 and in ELISA at a dilution of 1: 6000.

 When determining the antigenic compliance of strain A N 1707 "Armenia-98" with production strains and field isolates of FMD virus type A, isolated during 1962-1998. in the territory of the CIS countries and a number of other states of the world, a comparative study of the primary structure of the VP1 gene and protein was carried out. A comparative analysis of the established structures of the strain AN 1707 "Armenia-98" and epizootic isolates isolated in recent years in Turkey and Iran (A-Iran-96, A-Turkey-97, A-Turkey-98) showed that both isolates A- Armenia-98 are identical to each other and differ from isolates A-Turkey-97, A-Turkey-98 and A-Iran-96 in three, four and seven nucleotide bases, respectively. The substitution in the 156 codon is significant and leads to a change in threonine to alanine (Fig. 1).

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 significantly differ from all previously isolated isolates, including the A ₂₂ subtype virus, which includes the production strain A ₂₂ N 550 currently used for the production of diagnostics and specific prophylaxis.

 The antigenic affinity of the foot-and-mouth disease virus A N 1707 "Armenia-98" with the corresponding production and previously isolated strains in Turkey and Iran was studied in the RSK. The results are presented in table 2.

 As shown by the data presented in table 2, the virus A N 1707 "Armenia-98" differs from both production and previously isolated epizootic strains.

A similar study of the antigenic affinity of the isolated strain was carried out in a neutralization reaction, where cattle of convalescents of cattle for viruses of strains AN 1707 "Armenia-98", A-Iran-87 and A ₂₂ N 550 were used as immune.

 The results of these studies are presented in table 3.

The data in table 3 confirm the antigenic difference of the isolated strain from the production strain A ₂₂ N 550 and the epizootic strain isolated in Iran in 1987.

Biotechnological characteristics
Strain AN 1707 "Armenia-98" 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 the production of diagnostic sera and antigenic preparations, as well as the manufacture of inactivated FMD vaccine. The virus of strain AN 1707 is reproduced in monolayer cultures of primary trypsinized culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian ibex (PSGK-30), IB-RS-2, BHK-21 and accumulates over 20-24 hours of incubation up to 7.0-8.0 lg TCD ₅₀ / ml. With massive infection, it causes a CPD after 4 hours.

After 3-4 incubation passages, up to 6.0-8.0 lg TCD ₅₀ / ml is accumulated. It retains its original characteristics when passaged in sensitive biological systems for 10 passages.

Chemo- and genotaxonomic characterization
Strain AN 1707 "Armenia-98" is an RNA-containing virus with a molar mass of 7 • 10 ⁶ D.

The nucleic acid is represented by a single-stranded linear molecule of mol. weighing 2.8 • 10 ⁸ MD. The virion has a protein shell consisting of four main proteins VP1, VP2, VP3 and VP4. The lipoprotein membrane is absent.

The main antigenic protein is VP1. 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 (V _pb6a ) 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 research results are presented in figures 1 and 2. A comparative analysis showed that the strain differs from the strain A-Turkey-98, A-Turkey-97 and A-Iran-96 in three, four and seven nucleotide bases, respectively.

It was established that the Armenian FMD virus isolate has the highest degree of homology of the primary structure of the VP1 gene and protein with A-Turkey-98 isolates. Two substitutions (in 67 and 174 codons) of the three that distinguish the FMD virus A-Armenia-98 and A-Turkey-98 are silent, while the substitution in the 156 codon is significant and leads to a change in threonine to alanine. The studied isolate significantly (18% differences) differs from the production strain A ₂₂ N 550 and other variants and strains of foot and mouth disease virus A ₅ , A ₁₀ , A ₁₂ , A ₂₄ , AN 1640 Uzbekistan-91, etc.

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

Resistance to external factors
The virus of strain AN 1707 "Armenia-98" is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.0-7.6. 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
Immunogenic activity - immunogen in the composition of an inactivated vaccine.

 Reactogenicity - does not have reactogenic properties.

 Pathogenicity - pathogenic for artiodactyl animals, newborn mice, 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 data obtained, it can be argued that strain A N 1707 "Armenia-98" in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMD virus type A.

 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
Getting strain
The foot-and-mouth disease virus strain AN 1707 "Armenia-98" was isolated from field material received at ARRIAH as an epithelium of aphthae from cattle 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 intradermolingual inoculation of cattle field isolate material (1 passage) and subsequent adaptation of the virus isolated from the diseased animal to cultures of primary trypsinized and transplanted cell lines. Cell cultures of SP, PSGK-30, IB-RS-2 and BHK-21 were used. Primary and transplantable cell cultures for bioprobe production 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 Hanks solution with 0.5% GLA and antibiotics according to the standard formulation. To remove microflora and ballast cell components, the suspension was treated with chloroform in a ratio of 1:10. After 30 minutes of incubation at 37 ^° C, 5-10 ml of maintenance medium was added to the vials and incubated at 37 ^° C until the appearance of the virus CPP. 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 and studies in the CSC for the presence of a viral antigen, using a set of commercial type-specific sera and serums stored in the Museum of strains ARRIAH.

 The results of the adaptation of the virus to various cell cultures are presented in table 4.

 The data shown in table 4 indicate a good adaptive activity of strain A N 1707 to the used cell cultures.

 The virus isolated using the above methods was studied in CSC with a set of diagnostics for all types of foot-and-mouth disease virus, vesicular stomatitis, vesicular exanthema and porcine vesicular disease in order to identify the type of accessory and control for purity. The results of virus typing in CSCs are presented in table 5.

The results shown in table 5 indicate that the isolated virus belongs to type A, however, with hyperimmune serum from the production strain A ₂₂ N 550 in CSC, it gives a negative result.

Example 2
Production and testing of hyperimmune serum in guinea pigs
For hyperimmunization of guinea pigs, an antigen from foot and mouth disease virus, strain AN 1707 "Armenia-98", reproduced in a suspension culture of BHK-21 cells, is used. The virus-containing suspension is purified from ballast impurities by the addition of 0.05% polyguanidine. The purified virus is concentrated 100 times by adding 10% polyethylene glycol (PEG) m.m. 6000 and inactivate aminoethylethyleneimine (AEEI) at a concentration of 0.01-0.05% pH 7.8-8.0.

 Inactivated antigen concentrate in a mixture with an equal volume of oil adjuvant is administered to guinea pigs in a volume of 1.0 ml intramuscularly. After 21 days, immunization is repeated 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 keeping at +4 ^o C for 30 days, the resulting serum is Packed in 1.0 ml ampoules.

 By the method described in example 2, 2 series of hyperimmune serum were prepared, the characteristics of which are presented in table 6.

 The data in table 6 indicate that by the method described in example 2, diagnostic sera were obtained that meet the requirements for specific activity [7].

Example 3
Obtaining and testing diagnostic antigen
To obtain the antigen for immunochemical reactions, FMD virus, strain AN 1707 "Armenia-98", adapted to the cell culture of the inoculated PSGK-30 and BHK-21 lines, is used for adaptation. Virus-containing material is used in the form of a 10% suspension of cattle aft. Adaptation is carried out for 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 is concentrated 100 times by adding 8-10% PEG. The resulting concentrate is inactivated by heating at 56 ^o C for 40 minutes, Packed in ampoules and dried by vacuum sublimation.

 The specified method was prepared 2 series of diagnostic antigen, the characteristics of which are given in table 7.

 The research results are shown in table 7, indicate that the method described in example 3, obtained diagnostic antigens, the specific activity of which meets the requirements [7].

Example 4
Preparation and testing of inactivated adsorbate vaccine
The inactivated adsorbate vaccine against foot and mouth disease type A is prepared from the virus strain N 1707 "Armenia-98" grown in a suspension culture of BHK-21 cells. The resulting virus is subjected to inactivation AEEI at a concentration of 0.025-0.05% at 36-37 ^o C for 12-24 hours and a pH of 7.2-7.6. At the end of inactivation, polyhexamethylene guanidine is added to the suspension to a concentration of 0.005-0.007% for flocculation of ballast impurities and inactivation of possible contaminants. The suspension is cooled to 4-8 ^o C. After this, sedimentation of flocculated proteins is carried out and their removal. The resulting antigen is subjected to control for avirulence, the content of virus-specific protein and 146S + 75S components of the virus, 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 FMD virus is at least 6.0 μg / ml. Then add a 10% solution of saponin to a final concentration of 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. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

The obtained adsorbate vaccine against foot and mouth disease type A has the optimal component composition, μg / ml:
Avirulent and purified, antigenic material from immunogenic (146S + 75S) components of foot and mouth disease virus of strain AN 1707 "Armenia-98" - ≥6.0
Gel GOA - 1132.0-2108.0
Saponin - ≥ 750
Support Environment - Else
The immunogenic activity of one of the production series of the adsorbate vaccine from foot and mouth disease virus AN 1707 "Armenia-98" was tested for cattle. Her IMD ₂₂ was 0.15 ml, i.e. one ml of the vaccine contained 6.67 imd ₅₀ .

For comparison, the production series of the adsorbate vaccine from foot and mouth disease virus A ₂₂ N 550 had an IMD of ₅₀ cattle equal to 0.22 ± 0.012 ml P <0.001, i.e. in one ml the vaccine contained 4.55 ImD ₅₀ . The volume of the vaccine dose should contain at least 7 ImD ₅₀ .

Example 5
Preparation and testing of inactivated emulsion vaccine
Type A foot-and-mouth disease virus of strain N 1707 "Armenia-98" is grown, inactivated, cleaned from ballast impurities and possible contaminants, subjected to control for avirulence, virus-specific protein content, 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.

For this, BTU 0.5-2 ultrafilters are used. Filtering is carried out under a pressure of 1.5 atm. The resulting antigen concentrate is stored at 4-6 ^o C until used in the vaccine. An emulsion vaccine is obtained by dispersing antigen concentrate and oil adjuvant in a ratio of 3: 7-1: 1, respectively, in colloid mills.

 The result is an inactivated emulsion vaccine against foot and mouth disease type A, which is a milk-like liquid insoluble in water. The vaccine has a liquid consistency, easily resolves from the injection site, does not cause abscesses, does not cause a general reaction in the form of a temperature increase and has pronounced immunogenicity for pigs at a dose of 1 ml 1-21 days after vaccination, for cattle at a dose of 5 ml after 3 -21 days after vaccination and for sheep at 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 2 μg of 146S + 75S components.

The resulting emulsion vaccine against foot and mouth disease type A has the optimal component composition, μg / ml:
Avirulent and purified antigenic material from immunogenic (146S + 75S) components of foot-and-mouth disease virus of strain A N 1707 "Armenia-98" - ≥ 2.0
Oil adjuvant - 500,000 - 700,000
Support Environment - Else
The immunogenic activity of the emulsion vaccine was tested on pigs weighing 35-50 kg. The research results are presented in table 8.

The graft volume of 1 ml of the vaccine contains 7.1 ImD ₅₀ for pigs.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:
- strain N 1707 "Armenia-98" of FMD virus type A, 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 N 1707 "Armenia-98" of type A foot and mouth disease virus obtained in accordance with the invention, has a high specific, immunogenic and antigenic activity and is suitable for the manufacture of diagnosticums and inactivated vaccines.

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

Sources of information
1. Darda P.I. et al. Antigenic properties of foot and mouth disease virus strain A ₂₂ (A _and ). Veterinary Medicine - 1966, 1, 20-22.

 2. Bojko A. A. Declaration sur l'apparition en URSS d'un type virus aphteux different des souches de type A anterieurement etudies dans le Pays. BOIE, 1965, 64, 2, 1075-1077.

 3. Pepep X. Foot and mouth disease. Per. with him. G.A. Surkova / ed. and with the foreword. Cand. Vet.science P.V. Malaria.-M .; Kolos, 1971, 432 p.

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

 5. Syurin V. N. and other Viral diseases of animals. -M .; VNITIBP, 1998, 532-548.

 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 of the Council of Ministers of the USSR for Food and Procurement on 01.24.91.

 7. Guidelines for the isolation and identification of foot and mouth disease virus strains GOST 25384-82 M .; Kolos, 1974.

 8. GOST 28085-89.

Claims (1)

 The strain of the virus Aphtae epizooticae, family Picornaviridae, genus Aphtovirus, type A, collection VGNKI N 1707 "Armenia-98-DEPT", for the manufacture of diagnostic and vaccine preparations.

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