Strain no. 1734 "seaside-2000" of fmd virus type o for the manufacture of diagnostic and vaccine preparations
The invention relates to veterinary Virology and biotechnology. Strain 1734 "seaside-2000" obtained by multiple serial passages in susceptible cell cultures. The strain is deposited in the Collection of microorganisms VGNKI 22.03.2000 under registration 1734 "seaside-2000-DEPT". The virus strain O11734 "seaside-2000" reproducerea in sensitive cell cultures and for 20-24 h of incubation up to 7,5-8,0 Ig TCD50/ml. With a massive infection causes JRS after 4 h Preserves the original characteristics when passirovannye in cultures of cells within 10 passages. The strain of FMD virus type O has a high biological, antigenic and immunogenic activity and suitable for the manufacture of means of specific prophylaxis and diagnosis of FMD type O homologous epizootic virus circulating in Russia, regions of the Asian continent, including South-East and Central Asia. 12 tab., 2 Il. The invention relates to the field of veterinary Virology and biotechnology and can be used in the manufacture of tools for specific prophylaxis and diagnosis of FMD type O.Foot and mouth disease is an acute highly contagious mi mucous membrane of the oral cavity, hairless areas of the skin of the head, udder, Corolla, Milpitas cracks and accompanied the traffic violation. This disease tends to be widely distributed and epizootic period. This disease is accompanied by large losses of milk, meat and other livestock products, thereby impedes commercial transactions and economic activities.Long experience shows that the presence of endemic FMD lowers incomes in the dairy and beef cattle by 35-40%.The virus belongs to the genus of aphthovirus, the family of picornaviruses. It includes 7 immunological types and numerous subtypes [1, 2].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 known that the main cause of antigenic variation is a change in the amino acid sequence of the polypeptides of viral proteins that form the capsid of the virion. Such antigenic change can be expressed as minor differences in the Chenin different strains, requiring the use of new tools for serological diagnostics and specific prophylaxis of disease caused by these strains [2, 3].Known strains of FMD virus type O, which are used in Russia in the last 34-35 years. These include the following strains: O11618 "Checheno-Ingushetia", dedicated in 1966 in the Chechen-Ingush ASSR; O1194, dedicated in 1967 in the Volgograd region and O11182/83, dedicated in 1983 on the territory of Armenia [4, 5].Vaccine production of these strains have previously made a rather busy immunity in vaccinated animals against homologous or close to them antigenically pathogens.However, in recent years there have been several reported outbreaks of FMD, which in some cases could not be eliminated in the primary foci, despite vigorous veterinary-sanitary measures General and specific nature.This was due to the lack of efficacy of vaccines used against epizootic virus that causes breakouts immunity even repeatedly vaccinated animals. Therefore there is need to prepare a new production strain of the virus for success is the present invention, the set of essential characteristics is a strain About1194 "Volgograd", dedicated in 1967 and used in the Russian Federation as production in the manufacture of diagnostic and vaccine preparations used on the territory of Russia and member countries of the CIS .Strain 194 of FMDV O1has the following characteristics shown in table 1.The disadvantages of this strain are its low biological, antigenic and immunogenic.This is evidenced by the fact that in 2000 in Russia (Primorsky Krai) and the Caucasus (Georgia) were observed foci of FMD type Of cattle and pigs immunized with bivalent vaccine JSC, which includes the antigen of the production strain O1194. However, the vaccine did not provide satisfactory protection of immunized animals. Laboratory studies in ARRIAH aphthous material isolated from sick pigs, was installed the FMD virus type O having antigenic differences from strain O1194 and O11618 in the reaction of complement fixation (RAC) and the reaction enzyme-linked immunosorbent assay (ELISA). This testified to the mismatch of the used means of specific prophylaxis and diagnosis of FMD t the s region) and the Caucasus (Georgia).In the task of creating the present invention was to obtain a new production strain of FMD virus type O, 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 Russia and the Caucasus.The technical result from the use of the invention is to obtain a new production strain of FMD virus type O, which has high biological, antigenic and immunogenic activity and suitable for the manufacture of diagnostic and vaccines against FMD type O homologous epizootic virus circulating in Russia (Primorsky Krai) and the Caucasus (Georgia).This technical result is achieved by obtaining strain 1734 "seaside-2000 virus of FMD type O. the Strain O11734 "seaside-2000 is a new, previously unknown. The original virus to obtain strain 011734 "seaside-2000" selected 13.04.2000, from sick pigs on the farm OPH "steppe" (C. Elite) Ussuri region of Primorsky Krai. Production strain O11734 "Premantura cells.The resulting strain deposited in the Collection of microorganisms of the all-Russian scientific research Institute of control, standardization and certification of veterinary preparations Ministry of agriculture of the Russian Federation (VGNKI) 22.03.2000, under registration number 1734 "seaside-2000"-DEPT.Compared with the prototype strain O11734 "seaside-2000" has a higher biological, antigenic and immunogenic activity in the native form and after inactivation. Experimentally proved his ability to use for the preparation of diagnostic products and inactivated vaccines. Strain 1734 "seaside-2000 virus type provides About obtaining diagnostic products, which allows serological diagnosis of virus isolates of FMD type O, which caused an outbreak in 2000 in Russia (Primorsky Krai) and the Caucasus (Georgia), and inactivated FMD vaccines that creates the protection of susceptible animals against a specific pathogen.The invention is explained in graphic materials, where: Fig. 1 shows the amino acid sequence of VP1 protein of strain 1734 "seaside-2000" of FMD virus type O is oticheskimi and vaccine strains of FMD virus type O. The dendrogram based on the comparison of the complete nucleotide sequences of the VP1 gene.Strain O11734 "seaside-2000 virus foot and mouth disease is characterized by the following characteristics and properties.Morphological properties of the Strain O11734 "seaside-2000" belongs to the family Picornaviridae, genus Aphtovirus, serotype O 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.Antigenic properties of its antigenic properties of the strain belongs to serotype O. the Virus is stable neutralized homologous anticorodal. The virus does not manifest hemagglutinine activity. The animals recover in serum antibodies are formed, which are identified in the RDP, ELISA and PH. When vaccination of cattle and swine vaccine of inactivated virus induces the formation of 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 1:40-1:80, in ELISA at a dilution of 1: 3000-11 194, O11618, as well as with other strains isolated in the Caucasus and other countries in 1997-2000, was a comparative study of the primary structure of the gene and protein VP1.Method of nucleotide sequencing was determined the primary structure of the VP1 gene of strain 1734 "seaside-2000". It was deduced primary structure of a protein VP1 (Fig.1). Comparative analysis of nucleotide sequences showed that strain 1734 "seaside-2000" belongs to the Pan-Asian genetic lines of FMD virus type O (Fig.2). Pan-Asian virus caused a pandemic of FMD in 1999-2000 in most Asian countries, including Vietnam, China, Taiwan, Japan, Korea, Mongolia, Armenia, Georgia. The devastating epidemic of FMD in the UK in 2001 was caused by the Pan-Asian virus.Analyzed the virus has the highest level of homology (98,74%) with isolates Of1-Vietnam-1999 and O1-Taiwan-1999 (Fig.2).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 from all previous selected isolates, diagnostics and specific prophylaxis of FMD type O.Antigenic relationship of FMDV O11734 "seaside-2000" with the appropriate production and previously isolated strains on the territory of Armenia, Taiwan, Mongolia and Vietnam studied in RAC. The results obtained are presented in table 2.The data presented in table 2, indicate that the epizootic strain O11734 differs from industrial strains O1194 and O11618, but at the same time closely related strains of O1-Taiwan-1999, O11704-Armenia-1997, O1-Vietnam-1999 and O1-Mongolia-2000. Similar results were obtained in the reaction ELISA.Biological characteristics of Strain O11734 "seaside-2000" has a high biological, antigenic and immunogenic activity as in the native form and after inactivation. The strain intended for use as raw material for production of diagnostic products, as well as the manufacture of inactivated FMD vaccines. The virus strain O11734 reproducerea in the monolayer of primary trypsinization culture of kidney cells pigs (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), IB-RS-2, KSS-21 and over 20-24 hours of incubation up to 6,5-7,5 Ig Cepliaetsia to 6,5-7,5 1D TCD50/ml. Preserves the original characteristics when passirovannye in sensitive biological systems within 10 passages.Chemo - and geotectonically characteristic Strain O11734 "seaside-2000" is an RNA-containing virus with mol. mass 7106D.Nucleic acid represented by single-stranded linear molecule mol. weight 2,8108MD. The virion has a protein shell composed of four basic proteins VP1VP2VP3and VP4. Lipoproteina shell is missing. The major antigenic protein is VP1. The virion contains approximately 31.5% of RNA and 68.5% protein. Firiona RNA is infectious and is involved in the formation of protein precursors in infected cells. Predecessors 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 (VP66a) 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. Research results the research Institute of FMD virus type O. Representatives of this line was isolated in recent years in various regions of the Asian continent, including South-East and Central Asia and the far East. The study FMD virus O11734 had a high level of homology (98,74%) with isolates Of1-Vietnam-1999 and O1-Taiwan-1999.At the same time its homology with other genetic lines of FMD virus type O was only 80-90% (Fig.2).Broad geographic distribution of the genetic lines of FMDV belongs to a strain of About11734, lets talk about the possible patootie.The physical properties of the Mass of the virion - 8,410-18, the Coefficient of sentimentali - 146S in the sucrose gradient. Floating density of 1.45 g/mlResistance to external factors, the Virus strain O11734 "seaside-2000" resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7,2-7,6. Changes of pH in acidic and in the alkaline side lead to inactivation of the virus. Sensitive to formaldehyde, UV-irradiation,-radiation and high temperatures.Additional features and characteristics of Immunogenic activity - immunogene the ness - pathogenic for cloven-hoofed animals, newborn mice, rabbits and Guinea pigs.Virulence - virulent for naturally-susceptible animals in contact aerosol and parenteral infection.Stability - maintains the original biological properties when passirovannye in sensitive biological systems within 10 passages.Based on the obtained data, it can be argued that the strain O11734 "seaside 2000 antigenic and immunological spectra is original, in taxonomic relation to new, previously unknown variant of the virus of FMD type O.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 execution.Example 1
The strain of FMDV 011734 "P is travelig in disease FMD, for 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 techniques provided by .Biological and virological methods included the adaptation of the source virus to cultures of primary trypsinization and transplantable cell lines. We used cell culture JV, PSGK-30, IB-RS-2 and KSS-21. For the production of assay primary and transplantable cell cultures were grown in appropriate culture media 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 was 1-10 TCD50a cell prepared in the solution of the Needle with 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 37-38oWith vials were made 5-10 ml supporting nutrient medium and incubated at 37-38oWith until JRS virus. In the presence of JRS (rounding of cells, increasing their optical density, degeneration and separation of the cells from the glass) f is 0g for 15-20 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 cell cultures are presented in table 3.The results in table 3 indicate a good adaptive activity of the strain O11734 used cell cultures.Isolated using the above methods, the virus was investigated in RAC with a set of diagnostics on all types of FMD virus, vesicular exanthema, vesicular disease swine vesicular stomatitis to identify typical facilities and control of purity. The results of typing of the virus in RSK are shown in table 4.In table 4 the results show that isolated the virus belongs to type On; with all other sera was obtained a negative result.Example 2
For hyperimmunization Guinea pigs using the antigen from the virus, strain O11734 "seaside-2000", reproduced in suspension culture cells KSS-21. Virused is 100 times using PEG-6000 (10% by volume) and inactivate aminoethylethanolamine (AAAI) at a concentration of 0.01-0.05% and a pH of 7.8 to 8.0.Inactivated concentrate antigen mixed with an equal volume of oil adjuvant injected Guinea pigs intramuscularly at a dose of 1.0 cm3. After 21 days, immunization repeat twice (interval 7 days) and after 10 days, animals bleed. Individual samples of blood serum test sample specificity and activity in RAC according to .After that prepare a series by mixing typespecific individual samples of the same activity. Strain specificity of serial drug determine in one - and two-way reactions with Homo - and heterologous antigens.After canning sodium azide (1:5000) and incubation at 4oWith within 30 days of the resulting serum is Packed in ampoules of 0.5-1.0 cm3and lyophilizers.The method described in example 2, was prepared 2 series hyperimmune sera, whose characteristics are presented in table 5.The data of table 5 show that the obtained diagnostic serum, specific activity meets the requirements of .Example 3
To obtain antigen for immunochemical reactions using the FMD virus, strain O11734 "seaside-2000", adapted to the cultures of transplantable cells from pigs. The adaptation is carried out in the next 4-5 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 concentrate 100 times using 10% PEG-6000. The obtained concentrate inactivate by heating at 58oC for 40 minutes, Packed in ampoules of 1 cm3and dried 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, show that the obtained diagnostic antigens, specific activity which meets the requirements of .Example 4
For the manufacture of the adsorbate-vaccine virus O11784 "seaside-2000" reproduce in suspension culture cells KSS-21. As a supportive environment using the solution of the Earl without serum with the addition of FGMS, GBX and antibiotics at pH of 7.2 to 7.4. Culture of cells infected by a virus based 0,005-0,1 TCD50on the cell. The virus cultivation is carried out at a temperature of 36-37oC. After 8-10 hours of incubation p is irout for sterility and content 146S+75S components. The number 146S+75S suspension components must meet not less than 0.5 mcg/ml at the end of the cycle of reproduction of the virus, without temperature control, in vaccinated suspension add 15-20% solution AAAI, acidified with glacial acetic acid to a pH of 8.0 to 8.5. The final concentration AEEI in vaccinated suspension must be equal 0,025-0,05%. Inactivation of the infectivity of the virus is carried out in 12-24 hours at 36-37oC and a pH of 7.2 and 7.6 with stirring over 5-6 hours for 3-5 minutes. After inactivation of the antigen suspension is cooled to 4-8oC. To the cooled suspensio add a 10% solution of guanidine (phmg) to the concentration of 0,005-0,007% for ballasted flocculation of impurities and inactivation of possible contaminants. Flocculated ballast impurities is subjected to sedimentation, followed by decantation. The resulting antigen control for avirulence, content vaccinated protein and 146S+75S components of the virus and sterility. The necessary concentration of 146S+75S components in pravilnoy dose adsorbed vaccine is obtained by concentration of the antigen by gel hydrate of aluminum oxide (GOA). To this cooled suspension of antigen add calculated volume of the gel GOA 3% catalase 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 the virus, at least, of 6.0 µg/ml Then in the slurry, add an additional 10% solution of saponin to a final concentration of at least 0,075%. The resulting vaccine is filled into glass vials and subjected to control for sterility by .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 results of the control immunogenic derived vaccine for cattle is shown in table 7. In table 7 the results showed that all six heads of cattle were protected from the generalization process after control of infection after 21 days.The immune response of cattle to the introduction of the adsorbate-vaccine yasunaga antigen O11734 checked also on 5 heads of cattle weighing 200-300 kg the Results are shown in table 8. In table 8 suggests that FMD adsorbate-vacationwhich antibodies (VNA) to 61 days increased by 5.07 log2compared to 21 days after vaccination.The resulting vaccine is a liquid light amber with a loose white solid sorbent, which is formed on the bottom of the vial during storage and can be easily broken in a homogeneous suspension with shaking.The optimal composition of the resulting adsorbate-vaccines against FMD type O are shown in table 9.Example 5
For the manufacture of emulsion vaccine FMD virus type O strain 1774 "seaside-2000", reproduce in suspension culture cells KSS-21, inactivate and clear of ballast impurities and possible contaminants, is subjected to the control of the avirulence, 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 polyethylene glycol. The obtained concentrate antigen stored at 4-6oWith until use in the vaccine. Emulsion vaccine is produced by dispersion concentrate antigen and an oil adjuvant on colloid mills in the ratio 3:7-1:1, respectively.The result is here, insoluble in water. The vaccine has a liquid consistency that is easily absorbed in 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 2 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 4 µg 146S+75S components. Optimal component composition of the obtained emulsion vaccine against FMD type O are given in table 10.Immunogenic activity of emulsion vaccine tested on pigs weighing 35-40 kg the Results are shown in tables 11 and 12. IPD50= 0,18 ml previfem volume of 2 ml containing 11,2 Imdo for pigs. Table 11 shows that the vaccine of the virus O1194 induces in the body of the pigs in 2-3 times fewer VNA against heterologous virus strain O11784 "seaside-2000" in comparison with the homologous strain.Table 12 describes the data demonstrate the level of humoral immunity in pigs vaccinated FMD emulsion shows the implementation of the use of the present invention, the following cumulative conditions:
a strain 1734 "seaside-2000" of FMD virus type O, embodying the invention, intended 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 techniques;
a strain 1734 "seaside-2000" of FMD virus type O, obtained in accordance with the invention, it has high biological, antigenic and immunogenic activity in the native form and after inactivation and suitable for the manufacture of diagnostic and vaccine preparations.Therefore, the present invention meets the condition of patentability "industrial applicability".Sources of information
1. Rarer H. Foot And Mouth Disease. TRANS. with it. G. A. Surkova. Ed. and Annot. Kida. wet. Sciences. P. C. of Malarze, M, ear, 1971, 432s.2. Burdov A. N., Dudnikov A. I., Malaret P. C. and other FMD. Ed. by A. N. Burdova.-M, Agropromizdat, 1990, s.3. Syurin and other Viral diseases of animals.-M, UNITEMP, 1998.-c.532-548.4. Kruglikov B. A., Stefan M. K., and Tarasenko I. J. Study of infection is - ladimir, 1988. -1 o'clock. -S. 77-78.5. Auth. mon. The USSR 1739559 And 61 To 39/135, 10.04.2000,6. Instruction for production and control of vaccines monovalent emulsion against FMD type a, type O, type C, type Asia-1 (from virus grown in cell KSS-21). Approved BS Commission (CM CCP food and procurement 24.01.91, (prototype).7. GOST 25384-82 "guidelines for the allocation of identification of strains of FMD virus". M, Spike.-1974.8. GOST 28085-89.9. Saiko J. A., Fomin T. A. and Some other characteristics of epizootic strains of FMD virus, isolated in the USSR. Foot and mouth disease (Towards a new strategy for FMD control), Vladimir, 1992.-h 1.-C. 82-96.
The virus strain Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, type Aphtae, the type Of the collection VGNKI 1734 "seaside-2000-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