Strain a (georgia) 1999/no. 1721 of fmd virus type a for the manufacture of diagnostic and vaccine preparations
The invention relates to biotechnology. The original virus to obtain strain And(Georgia)1999/No. 1721 isolated in 1999 from sick cows in the private sector of the village Ude Adigeni district of the Republic of Georgia. The production strain(Georgia)1999/No. 1721 obtained by multiple serial passages in susceptible hetero - and homologous cell cultures. The strain is deposited in the collection of microorganisms VGNKI under registration number And(Georgia)1999/No. 1721. Virus strain A(Georgia)1999/No. 1721-DEPT reproducerea in sensitive cell cultures. Within 18-24 hours of incubation up to 6,0-8,0 Ig TCD50/ml. With a massive infection causes JRS 4 hours. Preserves the original characteristics when passirovannye in cultures of cells within 10 passages. Strain a(Georgia)1999/No. 1721-DEPT has a high biological, antigenic and immunogenic activity in the native form and after inactivation and suitable for the manufacture of means of specific prophylaxis and diagnosis of FMD type a homologous epizootic virus circulating in the countries of the Caucasus and the Middle East. 7 tab., 3 Il.
The invention relates to the field of weeu prevention diagnosis of FMD type A.
Foot and mouth disease is an acute contagious viral disease of cloven-hoofed animals, manifested by fever, vesicular (aphthous) lesions of the mucous membranes of the mouth, hairless skin of the head, udder, Corolla, Milpitas cracks and accompanied the traffic violation. It is characterized by a tendency to widespread epizootic. The disease is accompanied by large losses of milk, meat and other animal products, it is difficult for commercial transactions and economic activities.
Long experience shows that the presence of endemic FMD lowers incomes in the dairy and beef livestock production by 30-40%.
The virus belongs to the genus of aphthovirus, 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 antigenic variability of strains within the same serotype occurring in different time periods in different areas, 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 the change of amino acid which these changes can be minor differences between strains captured only by using subtle methods of molecular analysis to the rise of completely different strains, requiring the use of new tools for serological diagnostics and specific prophylaxis of disease caused by these strains.
Known strains of FMD virus type a, used as production on the territory of the USSR during the last 40 years.
These include the following strains: A7No. 103, dedicated in 1962 in the Kuibyshev region; And7No. 2, dedicated in 1965 in the Tajik SSR; And No. 717/73 allocated in 1973 in the Stavropol region.
These strains were used to obtain diagnostic and FMD vaccines used in different regions of the country.
However, after the elimination of FMD caused similar antigenically strains of the virus, they have been discontinued and are currently supported only in the Museum strains FGI research Institute for the protection of animals(1, 2, 4, 5).
A known strain No. 550 of FMDV And22dedicated in 1964 in Azerbaijan and used in the Russian Federation as production in the manufacture of means of specific prophylaxis and diagnostics, applied on the whole territory of Russia and whinney activity.
This is evidenced by the fact that in 1998 in the Republic of Armenia and in 1999 in the Republic of Georgia were marked foci of FMD type a cattle immunized with bivalent vaccine JSC, which consisted of the antigen of the production strain And22No. 550. However, the vaccine did not provide satisfactory protection of immunized animals. Laboratory studies in the FGI ARRIAH aphthous material isolated from sick animals, was installed the FMD virus type a, having antigenic differences from strain And22No. 550 in the reaction of complement fixation and reaction seroneutralization virus. This testified to the mismatch of the used means of specific prophylaxis and diagnosis of FMD type a, used in Russia and CIS countries, epizootic virus circulating in 1996-1999 in the Caucasus and the Middle East (Turkey, Iran).
Closest to the proposed invention, the essential features is the strain No. 1707/Armenia/98 FMD virus type a, selected 01.08.98, from sick cows on the farm, Kcic Messisbugo district of the Republic of Armenia. Production strain No. 1707/Armenia/98 FMD virus type a obtained by the first strain is used for the manufacture of diagnostic and vaccine preparations (7).
The disadvantages of this strain are its antigenic differences between isolates of FMD virus is selected in the Caucasus in different time intervals.
In the task of creating the present invention was to obtain a new production strain of FMD virus type a, which has high biological, antigenic and immunogenic activity in the native form and after inactivation and fabricate diagnostic and vaccine homologous epizootic virus, which appeared in the Caucasus and the Middle East.
The technical result from use of the present invention is to expand the Arsenal of industrial strains of FMD virus serological type And of high biological, antigenic and immunogenic activity and suitable for the manufacture of means of specific prophylaxis and diagnosis of FMD type a homologous epizootic virus circulating in the countries of the Caucasus and the Middle East.
This technical result is achieved by obtaining strain (Georgia) 1999/No. 1721 of FMD virus type a Strain (Georgia) 1999/No. 1721 is new, not previously known. The original virus to obtain strain And (Georgia) 1999/the industrial strain (Georgia) 1999/No. 1721 serological type And obtained by serial passages in susceptible hetero - and homologous cell culture.
The resulting strain deposited at the Russian state collection of strains of microorganisms used in veterinary medicine and animal production, Federal state institution "all-Russian state research Institute for control, standardisation and certification of veterinary preparations center quality of veterinary drugs and feed" (FGI VGNKI) of the Ministry of agriculture of the Russian Federation on 19 August 2003 under registration number And (Georgia) 1999/No. 1721-DEPT.
Strain a (Georgia) 1999/No. 1721 - DEPT has a high biological, antigenic and immunogenic activity in the native form and after inactivation.
Experimentally confirmed the possibility of its use for the preparation of diagnostic products and inactivated vaccines. Strain a (Georgia)/1999/No. 1721-DEPT of FMDV type And provides the receive diagnostic products that enable serological diagnosis of virus isolates of FMD type a, causing outbreaks in recent years in the countries of the Caucasus and the Middle East, and inactivated FMD vaccines that creates the protection of susceptible animals against a specific pathogen.
The invention is explained in graphic materials, where:
oricheskogo type A;
Fig.2 shows a dendrogram reflecting the phylogenetic relationships of strain A (Georgia) 1999/No. 1721-DEPT of FMD virus serological type And with epidemic and vaccine strains of FMD virus type A. the Dendrogram based on the comparison of the complete nucleotide sequences of the VP1 gene;
Fig.3 shows the alignment of amino acid sequences of VP1 protein of strains No. 1707/Armenia/98-DEPT and A (Georgia) 1999/No. 1721-DEPT.
Strain a (Georgia) 1999/No. 1721-DEPT of FMD virus type a is characterized by the following characteristics and properties.
Strain a (Georgia) 1999/No. 1721-DEPT belongs to the family Picornaviridae, genus Aphtovirus, serotype a and has morphological features that are specific to the causative agent of foot and mouth disease: a form of icosahedral virion, size 23-25 nm. The virion consists of a molecule of RNA enclosed in a protein shell. The protein shell consists of 32 capsomeres located in the cubic symmetry.
According to their antigenic properties of the strain (Georgia)1999/No. 1721-DEPT of FMDV belongs to serotype A.
The virus is stable neutralized homologous anticorodal. The virus does not manifest hemagglutinine activity. The animals recover in the serum of bresults adowanie specific antibodies, detected in ELISA, RDP and PH. If hyperimmunization Guinea pigs concentrated inaktivirovannye virus induces the formation of virousspecificakih antibodies detected in the RAC at a dilution of 1:128-1:512 and in ELISA at a dilution of 1:6000-1:10000.
When determining antigenic matching strain (Georgia) 1999/No. 1721-DEPT production strains and field isolates of FMDV type And allocated during 1962-1999, on the territory of CIS countries and other countries in the world, was a comparative study of the primary structure of the gene and protein VP1 (Fig.1).
A comparative analysis of the established structures of strain A (Georgia) 1999/No. 1721-DEPT and epidemic isolates in recent years in Turkey, Iran and the Republic of Armenia (A/Iran/96 And/Turkey/97, A/Turkey/98 And/Armenia/98), showed that the isolate A/No. 1721/Georgia/99 differs from isolates of A/Turkey/97, A/Turkey/98, A/Iran/96 and No. 1707/Armenia/98.
Phylogenetic relationships of the studied isolates with previously studied strains isolated in different countries over the last three decades, shown on the dendrogram (Fig.2). It is established that the investigated isolates differ significantly from all previous selected isolates, including strains And22No. 550 and No. 1707/ prevention.
Antigenic relatedness of strain A (Georgia) 1999/No. 1721-DEPT with relevant industrial and previously isolated strains in Turkey and Iran were studied in RAC. The results obtained are presented in table 1.
As the data presented in table 1, strain a (Georgia) 1999/No. 1721-DEPT of FMD virus type a is different from both the production and the previously selected epizootic strains.
On the antigenic properties of a new strain of A (Georgia) 1999/No. 1721-DEPT of FMD virus type a is also different from the previously studied production strain And No. 1707/Armenia/98-DEPT. Bilateral kinship (R) in the RAC between them is 20%.
A similar study of the antigenic relatedness of the selected strain was carried out in PH, where the immune serum was used convalescents cattle virus strains (Georgia) 1999/No. 1721-DEPT, AND/No. 1707/Armenia/98, A/Iran/87 and a22No. 550. The results of these studies are presented in table 2.
In table 2, the data confirm the antigenic difference between isolates from the production strain And22No. 550 and epizootic strains isolated in Iran in 1987 and Uzbekistan in 1989.
Strain a (Georgia) 1999/No. 1721-DEPT VIR is AK and after inactivation. The strain intended for diagnostic sera and antigen preparations, and also for the manufacture of inactivated FMD vaccines. Strain a (Georgia) 1999/No. 1721-DEPT reproducerea in monolayer cultures of primary trypsinization cell culture kidney pig (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), IB-RS-2, KSS-21 and within 18-24 hours of incubation up to 6,0-8,0 Ig TCD50/ml. With a massive infection (1-100 TCD/cell) causes the JRC 4 hours.
After 3-4 passages incubation up to 6,0-8,0 Ig TCD50/ml. Preserves the original characteristics when passirovannye in sensitive biological systems within 10 passages.
Chemo - and geotectonically feature
Strain a (Georgia) 1999/No. 1721-DEPT is an RNA-containing virus with a molecular mass of 7×106D.
Nucleic acid represented by single-stranded linear molecule has a molecular weight of 2.8×10 MD. The virion has a protein shell composed of four basic proteins VP1, VP2, VP3 and VP4. Lipoproteina shell is missing.
The major antigenic protein VP1 is. The virion contains approximately 31.5% of RNA and 68.5% protein. Firiona RNA Awlaki, in turn, are split with the formation of more stable structural and non-structural proteins of the virus. Of the 6 non-structural polypeptides that accumulate in infected cells, one (VP3D) is an RNA-dependent RNA polymerase that is involved in the replication of RNA new virions.
Conducted primary structure of the genome of the virus by PCR and sequence. The results are shown in Fig.1.
It is established that strain a (Georgia) 1999/No. 1721-DEPOT has the highest degree of homology of the primary structure of the gene and protein VP1 with isolates Turkey/99, A/Iran/99 and No. 1707/Armenia/98.
The analyzed isolates significantly different from industrial strains. And22No. 550 (18% difference) and No. 1707/Armenia/98 (20% difference), as well as other variants and strains of the FMD virus A5And10, A12And/№1540/Uzbekistan/89 and others (Fig.2).
Performed alignment of amino acid sequences of VP1 protein of strains A/No. 1707/Armenia/98-DEPT and A (Georgia) 1999/No. 1721-DEPT of FMD virus type A. the Results are shown in Fig.3.
Comparative analysis shows that the strains differ from each other by seven amino acid residues. Two substitutions (G R at position 141 and R by H at position 142) are directly the of adowanie testify, that FMD virus type a even a single substitutions in the VP1 protein structure can lead to substantial changes in antigenic properties.
The mass of the virion is 8.4×10-18, the sedimentation Coefficient 146S in the sucrose gradient. Floating density of 1.45 g/ml
Resistance to external factors
Strain a (Georgia) 1999/No. 1721-DEPT of FMDV type And resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.0 and 7.6. Changes of pH in acidic and in the alkaline side lead to inactivation of the virus. Sensitive to formaldehyde, UV-irradiation,-exposure to high temperatures.
Additional characteristics and properties
Immunogenic activity - immunogene comprising inactivated vaccine.
Reactogenicity - reactogenic properties is not.
The pathogenicity of the pathogenic for cloven-hoofed animals, newborn mice, Guinea pigs.
Virulence - virulent for naturally-susceptible animals in contact, aerosol and parentelem infection.
Stability - maintains the original biological properties when passirovannye in sensitive biological systemstheory and immunological spectra is original, in taxonomic relation to new, previously unknown variant of FMD virus type A.
To reduce its epizootic risk need to provide timely laboratory diagnosis and vaccination emerging foci of the disease, which requires a highly active and specific and antigenic or antibody-based test diagnostics and vaccine derived from the use of this strain as a production.
According to the applicant, the proposed strain meets the conditions of patentability of "novelty" and "inventive step".
The essence of the invention is illustrated by examples of its use.
Strain a (Georgia) 1999/No. 1721-DEPT of FMD virus type a was isolated from field material received in ARRIAH in the epithelium of the aft from cattle suspected of disease foot and mouth, when the laboratory diagnosis of this disease and differentiate it from other vesicular diseases. When isolation of the virus used a complex of biological, virological and biochemical methods.
Biological and virological methods included the inoculation material field isolates of cattle and the subsequent adaptation of the virus to the cult of the RS-2 and KSS-21. Primary and transplantable cell culture for the production of assay were grown in appropriate nutrient media under steady-state conditions in bottles with a capacity of 50-100 ml, washed from the growth medium and used to infect 10% suspension aphthous material (multiplicity of infection brought 1-10 TCD50for a cell), prepared in Hanks solution with 0.5% GLA, and antibiotics according to the standard recipe. Removal of microflora and ballast cellular components, the suspension was treated with chloroform in the ratio of 1:10. After a 30 minute incubation at 37°C in vials made in 5-10 ml of maintenance medium and incubated at 37°With until JRS virus. In the presence of JRS (rounding of cells, increasing their optical density, degeneration and separation of the cells from the glass vials were subjected to freeze-thawing, cleaning the cell suspension chloroform and centrifugation at 3000 g for 15 minutes. Received vaccinated material used for the subsequent passages and research in RAC and ELISA for the presence of viral antigen, used a set of commercial typespecification sera and sera stored at the Museum of strains ARRIAH.
The results of the adaptation of the virus to different cellular coy activity of the strain (Georgia) 1999/No. 1721-DEPT of FMD virus type a used cell cultures.
Isolated using the above methods, the virus was investigated in RAC with a set of diagnostics on all types of the virus of foot and mouth disease, vesicular stomatitis, vesicular exanthema and vesicular disease swine to identify typical facilities and control of purity. The results of typing of the virus in RSK are shown in table 4.
Table 4 presents the results suggest that the isolated virus is of type A.
Obtaining and testing of hyperimmune serum for Guinea pigs
For hyperimmunization Guinea pigs using the antigen from the FMD virus type a strain (Georgia) 1999/No. 1721-DEPT reproduced in suspension culture cells KSS-21. Vaccinated suspension clear of ballast impurities by adding 0.05% of polyguanidine. The purified virus is concentrated 100 times by adding 10% of polyethylene glycol (PEG) m 6000 m and inactivate aminoethylethanolamine (AAAI) at a concentration of 0.01 to 0.05%, pH of 7.8 to 8.0.
Inactivated concentrate antigen mixed with an equal volume of oil adjuvant injected Guinea pigs in a volume of 1.0 ml intramuscularly. After 21 days, immunization and repeat after 10 days, animals bleed. Individual serum samples of blood about the specific individual samples of the same activity. Strain specificity of serial drug determine in single and duplex reactions with Homo - and heterologous antigens.
After canning sodium azide (1:5000) and incubation at +4°C for 30 days the resulting serum is Packed in ampoules of 1.0 ml and subjected to freeze drying.
The method described in example 2, was prepared 2 series hyperimmune serum, whose characteristics are presented in table 5.
The data of table 5 show that the method described in example 2, the obtained diagnostic serum, specific activity meets the requirements of (8).
Obtaining and testing diagnostic antigen
To obtain antigen for immunochemical reactions using the FMD virus type O strain (Georgia) 11999/No. 1721-DEPT adapted to the culture of cells of transplantable lines PSGC-30 and KSS-21. For adaptation used vaccinated material in the form of a 10% suspension of the aft cattle. Adaptation can be performed during 3-5 consecutive passages. The obtained matrix seed is obtained virus used for producing viral material. Contamination of cell cultures and collection of viral material is conducted according to conventional methods. Received vaccinated liquid the end of the minutes, Packed in ampoules and drying by sublimation under vacuum.
In this way was prepared 2 series diagnostic antigen, whose characteristics are shown in table 6.
The results of the studies are shown in table 6, indicate that the method described in example 3, the obtained diagnostic antigens, specific activity which meets the requirements of (8).
Preparation and testing of inactivated adsorbate-vaccine
Inactivate Arbat-vaccine against FMD type a, prepared from virus strain A (Georgia) No. 199/No. 1721-DEPT grown in suspension culture cells KSS-21.
The resulting virus is subjected to inactivation AEEI at a concentration of 0.025 to 0.05% at 36-37°C for 112-24 hours and a pH of 7.2 and 7.6. After inactivation in suspension add polyhexamethylene guanidine to the concentration of 0,005-0,007% for ballasted flocculation of impurities and inactivation of possible contaminants. The suspension is cooled to 4-8°C. then spend the sedimentation of flocculated proteins and their destruction. The resulting antigen is subjected to the control of the avirulence, content virousspecificakih protein and 146S+75S components of the virus, sterility. The necessary concentration of 146S+75S component is uminia (GOA).
To this cooled suspension of antigen add calculated volume of the gel GOA 3% concentration. After sedimentation GOA merge estimated volume of the supernatant liquid or measure the volume of the remaining suspension. 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 146S+75S components of FMD virus at least 6,0 ág/ml Then add a 10% solution of saponin to a final concentration of 0.075%. The resulting vaccine is filled into glass vials and subjected to the control of sterility. The avirulence and safety of the vaccine tested for 5 heads of cattle, introducing the vaccine first, under the mucous membrane of the tongue at a dose of 2.0 ml, and then subcutaneously at a dose of 10 ml of monitoring the clinical condition of the animals are 10 days. Avirulent, harmless and sterile vaccine tested for immunogenic activity in cattle or Guinea pigs. The resulting adsorbate vaccine against FMD type a has an optimal component composition, ug/ml:
Avirulent and cleared
antigenic material from immunogenic
(146S+75S) components of the virus
FMD type a
strain And (Georgia) 1999/No. 1721-DEPT6,0
Gel GOA 1132,0-2108,0
Supportive environment 997142,0-998112,0
Immune is 999/No. 1721-DEPT was tested in cattle. Its IPD50was equal to 0.15 ml, i.e., one ml of the vaccine contained 6,67 IPD50.
For comparison, the production series of the adsorbate-vaccine virus A22No. 550 had IPD50for Cattle, equal to 0.22±a 0.012 ml, P<0,001, i.e., one ml of the vaccine contained 4,55 IPD50. Volume pravilnoy doses of the vaccine should contain at least 7 IPD50.
Preparation and testing of inactivated emulsion vaccine
The FMD virus type a strain (Georgia) 1999/No. 1721-DEPT grow, inactivate, clear of ballast impurities and possible contaminants, is subjected to the control aurulentus, content virousspecificakih protein, immunogenic components (146S+75S) and sterility as described in example 4. The necessary concentration of 146S+75S components in pravilnoy dose of emulsion vaccine obtained by concentration of the antigen flow ultrafiltration or PEG.
To do this, use ultrafilter BTU 0.5 to 2. Filtering are under pressure of 1.5 ATM. The obtained concentrate antigen stored at 4-6°C until use in the vaccine. Emulsion vaccine is produced by dispersion in colloidal mills concentrate antigen and an oil adjuvant at a ratio of 3:7-1:1 sootvetstvyet a molokopodobnye liquid, not soluble in water.
The vaccine has a liquid consistency, easily resolved with the introduction, does not cause formation of abscesses, does not cause the overall reaction in the form of a high temperature and has a pronounced immunogenicity for pigs at a dose of 1 ml through 1-21 days post-vaccination for cattle at a dose of 5 ml through 3-21 days post-vaccination for sheep at a dose of 1 ml through 3-21 day after injection. The vaccine is administered to pigs intramuscularly, and cattle and sheep subcutaneously. In previfem volume must contain at least 2 µg 146S+75S components.
The obtained emulsion vaccine against FMD type a has an optimal component composition, ug/ml:
Avirulent and cleared
antigenic material from immunogenic
(146S+75S) components of FMDV
type a strain (Georgia)
Oil adjuvant 500000,0-700000,0
Supportive environment 299998,0-499998,0
Immunogenic activity of emulsion vaccine tested on pigs weighing 35-50 kg the Results are shown in table 7.
In previfem volume of 1 ml of vaccine contains 7,1 IPD50for pigs.
Thus, the above information shows the implementation using the proposed izobreteniya the invention, designed for use in agriculture, namely in veterinary Virology and biotechnology;
for the present invention in the form as it is described in the independent claim, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and methods;
- strain And (Georgia) 1999 No. 1721-DEPT of FMD virus type a, obtained in accordance with the invention, has a high specific immunogenic and antigenic activity and suitable for the manufacture of diagnostic kits and inactivated vaccines.
Therefore, the present invention meets the condition of patentability "industrial applicability".
Sources of information
1. Darda P. I. and other Antigenic properties of the virus strain And22(Aand). Veterinary. - 1966, 1, 20-22.
2. Bojko A. A. Declaration sur I'apparition en URSS d'un virus type aphteux different des soucfes de type A anterieurement etudies dans le Pays. BOIE, 1965, 64, 2, 1075-1077.
4. Burdov A. N., Dudnikov A. I., Malaret P. C. and other FMD/Ed. by A. N. Burdova. - M, Agropromizdat, 1990, 320 S.
5. Syurin Century. N. and other Viral diseases of animals. - M, VNITIBP, 1998, 532-548.
6. Instruction for production and control of vaccines monovalent, emulsio the mission of the USSR food and procurement 24.01.91.
7. RF patent №2140452; IPC612 N 7/00, a 61 K 39/135, G 01 N 33/569, And 61 To 39/42, 07 K 16/08; 27.10.99, (prototyp).
8. Guidelines for the detection and identification of strains of FMDV. Vladimir, 2002. 31 C.
The virus strain Apthae epizooticae, SEM. Picomaviridae, genus Aphtovirus, type a collection VGNKI(Georgia)1999/No. 1721-DEPT, for the manufacture of diagnostic and vaccine preparations.
FIELD: veterinary virology, biotechnology.
SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721 obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus. Moreover, the vaccine contains maintenance medium and butyric adjuvant in efficient ratios. The strain has been deposited in collection of FGU VGNKI under registration number - industrial culture strain of foot-and-mouth disease virus A (Georgia) 1999/N1721-DEP of serotype A. As maintenance medium it is necessary to apply serum-free Earle's solution at addition of FGMS, GBCS and antibiotics at pH being 7.4-7.6. Out of butyric adjuvants the vaccine contains butyric adjuvant of the All-Russia Research Institute of Animal Protection (VNIIZZH) or butyric adjuvant of Montanide ISA-70 or Montanide ISA-260 marks by "Seppic" (France). The vaccine provides efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.
EFFECT: higher efficiency.
11 cl, 1 dwg, 5 ex, 8 tbl