Strain of fmd virus aphtae epizooticae of type a for controlling antigenic and immunogenic activity and for production of biological preparations for diagnostics and specific prophylaxis of fmd of type a

FIELD: biotechnology.

SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.

EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.

6 tbl, 6 ex

 

The invention relates to veterinary Virology and biotechnology, in particular to a new strain of FMD virus Aphtae epizooticae, and can be used to monitor antigenic and immunogenic activity of FMD vaccines, as well as in the development and manufacture of diagnostics and specific prevention of FMD type A.

FMD is an acute, contagious, viral disease of cloven-hoofed animals, manifested by fever, vesicular (aphthous) lesions of the mucous membrane of the mouth, hairless skin of the head, udder, halo, mezhkopytnoy gap and is accompanied by disturbances of movement. For this pathogen tends to be widespread and epidemic. The disease accompanied by large losses of milk, meat and other animal products makes commercial transactions and economic activity. Long experience shows that with endemic FMD revenues decline in dairy cows and cattle on(%): 30÷40.

The FMD virus belongs to the family Picomaviridae, genus Aphtovirus. It has 7 antigenic types, a large number of subtypes and a variety of strains.

The causative agent of FMD has considerable antigenic variability of strains within the same serotype, which is revealed in different time intervals and n� different areas and depends on the species composition of the susceptible population, his immune status and many other different factors. Antigenic variability of the virus of foot and mouth disease is caused by substitutions of amino acids in polypeptide fragments (antigenic epitopes), exposed on the surface of the capsid proteins.

Antigenic shifts the spectrum corresponding to the update patterns of the new field strains can vary from slight, captured by monoclonal antibodies to the significant recorded using conventional polyclonal immunoglobulins. Significant changes in the antigenic characteristics of the natural strain is likely to cause weakening of specific immunity induced homologous antigen. They also cause difficulties of strain-specific diagnosis.

As a result there is a need for new diagnostic tools and specific immunization.

Known strains of FMD virus type a, used as production on the territory of the USSR and Russia in the past 50 years.

These include the following strains: A7 No. 103, dedicated in 1962 in Kuibyshev region; A7 No. 2, dedicated in 1965 in the Tajik SSR; And No. 717/73, dedicated in 1973 in the Stavropol region.

These strains were used to obtain diagnosticums and FMD vaccines used in different reg�areas of the country.

However, after the elimination of FMD caused by someone close to them antigenically virus strains, they were removed from production and are currently supported only in the Museum strains of the fgbi "ARRIAH" [1, 2, 3].

Known strain No. 550 of FMDV And22allocated in 1964 in Azerbaijan and used in the Russian Federation as a production in the manufacture of means of specific prophylaxis and diagnosis, used throughout Russia and in the countries of post-Soviet space.

In 1990-2000-ies in Central Asia and the middle East was dominated by two genetic lines of FMD virus type a: line a/Iran/96 (to this genetic group includes Russian production strain And/Armenia/98) and A/Iran/2005. Genetic line a/Iran/99 has not received such wide circulation, like the two mentioned above. Strains of group A/Iran/99 and A/Iran/96 are not currently in the priority list recommended by the world reference laboratory OIE/FAO FMD vaccine strains, whereas strain A/Iran/2005 (A/Turkey/06)refers to high-priority [4].

Known strain of FMD virus Aphtae epizooticae A No. 1707 "Armenia-98" for production of diagnostic and vaccine preparations [5].

The selection in 1998 in the household of Kcik Messisbugo region of Armenia from cattle of FMD virus type a and study epoprostenol relationship showed when the comparative study of the primary structure of the gene VPi strain of FMD virus type a No. 1707 "Armenia-98" with production strains and field isolates established that isolates Armenia/98 identical to each other and related strains of type a (Turkey/97, Turkey/98 and Iran/96. Also found that the studied isolates differ significantly from all previously identified isolates, including those from the virus subtype A22(18% difference), which is used for the production of diagnostics and specific prophylaxis of strain And22No. 550.

Production strain And No. 1707 "Armenia-98" was used in the composition of FMD vaccines in the buffer zone of the Caucasus.

Well-known relating to the same lines selected in 1999 from sick cows in the private sector of the village Ude, Adigeni district of the Republic of Georgia strain A (Georgia) 1999/№1721 [6].

Since 2003 in Iran was selected isolates of FMD virus type a, significantly different from the previously studied strains of this type. During 2005-2006 the FMD type a has become widespread in the countries of West Asia-Iran, Turkey, Saudi Arabia and Pakistan. In February 2006, the FMD type a line Iran/05 got to Thrace, the European part of Turkey, which borders with Bulgaria and Greece. 100% vaccination of all ruminants using strain Azz in Thrace in Febr�-March 2006 prevented the spread of FMD in neighbouring Greece and Bulgaria, but after 3-4 months there were fresh cases of the disease [7].

Known strain of FMD virus And 2045/Kyrgyzstan/2007 allocated in the Kyrgyz Republic from FMD patient heifers in 2007, belonging to genetic lineage, known As the Iran/2005 [8].

The disadvantages of the above strains that prepared on the basis of their vaccines provide effective protection of animals against infection with homologous virus.

The strain relating to the present invention, previously on the territory of the Russian Federation are not met.

An outbreak of FMD due to virus type a, was awarded on September 22, 2013 in cattle (3 heads) and 4 pigs. The disease is reported in 2 yards. Pigs were not vaccinated against FMD. Ruminants in 2013 were immunized 3 times, including: 2 April 2013, 10 June 2013 - the vaccine containing the production strain (A22No. 550.

Third time the ruminants were vaccinated universal vaccine adsorbed from FMD virus type a, containing the production strain "A/Iran/2005".

All this testified to the lack of effectiveness of vaccines of the production strains.

In this connection there was a necessity to get a new production strain of epizootic VIR�sa FMD serotype And to ensure the security of Russia and adjacent States of this pathogen.

The problem to be solved by the present invention, is to expand the Arsenal of field strains of FMDV serotype A, with high infection, antigenic and immunogenic activity in the native form and retains antigenic and immunogenic activity after inactivation, suitable for monitoring the antigenic and immunogenic activity of vaccines, the fabrication of sensitive and highly specific diagnostics and vysokomanevrennyh vaccine preparations, homologous epizootic virus, which appeared on the territory of Russia.

This problem is solved by obtaining the strain And No. 2187/Kuti/2013 (author's name) of FMD virus type a for the control of antigenic and immunogenic activity, as well as for the manufacture of biological products for diagnostics and specific prevention of FMD type A.

Viral isolate, which served as the source for obtaining strain And No. 2187/Kuti/2013, was selected in September 2013 from diseased pigs in the village of Kuti Priargunsky district of the Zabaikalsk region (examination No. 2187). Strain And No. 2187/Kuti/2013 of FMD virus type a is obtained by successive passages on sensitive hetero - and homologous cell cultures.

Strain And No. 2187/Kuti/2013 of FMD virus type a, deposited on 22 January 2014 in the Collection of microorganisms of Federal state budget�CSOs institution "Federal centre for animal health" (fgbi "ARRIAH"), under registration number (reference): strain of FMD virus And No. 2187/Kuti/2013 (production).

Compared with known strains strain And No. 2187/Kuti/2013 has a higher infectious, antigenic and immunogenic activity in the native form and after inactivation.

Experimentally confirmed the possibility of using the FMD virus And No. 2187/Kuti/2013 to monitor antigenic and immunogenic activity, as well as for the manufacture of biological products for diagnostics and specific prevention of FMD type A.

The invention is explained on the dendrogram reflecting the phylogenetic relationships of the strain of FMD virus And No. 2187/Kuti/2013 epidemic and vaccine strains of FMD virus serological type A. Dendrogram based on the comparison of complete nucleotide sequences of the VP1 gene.

The invention is explained in the sequence listing in which:

SEQ ID NO:1 represents the nucleotide sequence of the gene VP1 protein of strain And No. 2187/Kuti/2013 of FMD virus type A;

SEQ ID NO:2 represents the amino acid sequence of the gene VP1 protein of strain And No. 2187/Kuti/2013 of FMD virus type A.

Strain And No. 2187/Kuti/2013 of FMD virus type a is characterized by the following characteristics and properties.

Morphological features

Strain And No. 2187/Kuti/2013 of FMD virus type a belongs to the family Picomaviridae, genus Aptovims, the serotype A and has morphological features characteristic of the causative agent of foot and mouth disease: the shape of the virion is icosahedral, size 23-25 nm. The virion consists of a molecule of RNA enclosed in a protein shell. The protein coat is composed of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to their antigenic properties of the strain And No. 2187/Kuti/2013 FMDV belongs to serotype A. the Virus is consistently neutralized the homologous antiserum. The virus does not show hemagglutinin activity (HA activity). I had been ill animals in the serum formed antibodies detected in the ELISA assay (ELISA) and the reaction microneutralization (RMN).

If hyperimmunization Guinea pigs concentrated antigen inactivated FMD virus type a No. 2187/Kuti/2013 induces the formation of virus-specific antibodies detected in DGCs in a dilution of 1:40.

Method of nucleotide sequencing was determined the primary structure of VP1 gene of FMDV type a No. 2187/Kuti/2013 and deduced primary structure protein VP1. Comparative analysis of nucleotide sequences showed that the strain of FMD virus type a No. 2187/Kuti/2013 is significantly different from industrial strains of type A. the Degree of nucleotide differences between sequences of strain And No. 2187/Kuti/2013 with strains of the FMD virus serology�ical type And amounted to: A 22No. 550 - 21,49% And22/Iraq/64 - To 21.73%, A/Iran/96 - 22,03%, And/Armenia/98 - 22,39%, A/Turkey/06 - 22,11%, A/Iran/05 - 22,32%.

Thus, the phylogenetic analysis showed that strain And No. 2187/Kuti/2013 belongs to a genetic lineage Southeast Asia 97 (Sea-97) topotype Asia.

Antigenic relatedness of strain VYA AND No. 2187/Kuti/2013 with existing production strains VYA type And investigated serological method in the cross-reaction of microneutralization (RMN). Strain And No. 2187/Kuti/2013 antigenically different from industrial strains And22No. 550 And No. 2179/Shchelkovo biocombine And/Iran/97, A/Kyrgyzstan/07 (A/Iran/05). Strain VYA AND No. 2187/Kuti/2013 a poorly marked antigenic relatedness with strain A/Turkey/06 (A/Iran/05).

The results of studies in RMN presented in table 1. Antigen matching (r1amounted to A22No. 550 - 0,13, And No. 2179/Shchelkovo biocombine compared to 0.11 A/Iran/97 - 0,004, A/Turkey/06 - 0,37, And No. 2045/Kyrgyzstan/07 - 0,13. When the value of r1≥0,3 field isolates and production strain are closely related, and the vaccine production strain will protect against epizootic virus, a value of r1<0,3 field isolate differs from the production strain, and the vaccine from the strain does not protect against epizootic virus [9, 10].

Biotechnological characteristics

Strain And No. 2187/Kuti/2013 reproduced in primary�repsonsiveness monolayer cell culture of pig kidney (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), BHK-21 and IB-RS-2, and within 18÷24 hours of incubation of the virus in these cell cultures accumulated from 6.0 to 7.0 lg TCD50about us/cm3. At high multiplicity of infection (1-10 TCD/cell) causes the JRS in 5 hours. Preserves the original characteristics during passaging in cell cultures for 5 passages (observation period).

Geotectonically feature

Strain And No. 2187/Kuti/2013 of FMD virus type a is an RNA-containing virus with a molecular weight of 7×106Yes.

A nucleic acid represented odnozadachnoy linear molecule of a molecular weight of 2.8×106Yes. The virion has a protein shell consisting of four major proteins VP1VP2VP3and VP4. The lipoprotein shell.

The main antigenic protein is VP1. The virion contains approximately 31.5% of RNA and 68.5% protein. Virion RNA is infectious and is involved in the formation of proteins-precursors in infected cells. Predecessors, in turn, is cleaved to form a more stable structural and non-structural polypeptides of the virus. 8 non-structural polypeptides that accumulate in infected cells, one (VP66a) is an RNA-dependent RNA polymerase, me�setup portion in the replication of RNA for new virions.

Physical properties

The mass of the virion is 8.4×10-18G. the sedimentation Coefficient of the 146S in the sucrose gradient. Floating density of 1.45 g/cm3.

Resistance to external factors

Strain And No. 2187/Kuti/2013 resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7,2÷7,6. Changes of pH in the acidic and in the alkaline side lead to the inactivation of the virus. Sensitive to formaldehyde, UV-irradiation, γ-irradiation, high temperatures.

Additional features and properties

Immunogenic activity - immunogene consisting aktivirovannoi vaccine.

Antigenic activity - introduction of inactivated antigen Guinea pigs induces the formation of virus-specific antibodies.

Reactogenicity - reactogenic properties do not possess.

Pathogenicity pathogenic for cloven-hoofed animals, newborn mice, Guinea pigs.

The virulence virulent for naturally-susceptible animals in contact, aerosol, and parenteral exposure.

Stability - maintains the original biological properties under passaging in sensitive biological systems for 5 passages (observation period).

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

Example 1

Viral isolate, which served as the source for obtaining strain And No. 2187/Kuti/2013, was highlighted in the fgbi "ARRIAH" samples aphthous material obtained in September 2013 from suspected cases of FMD pigs from the village of Kuti Priargunsky district of the Zabaikalsk region (examination No. 2187/2013). Sample canker material received in the fgbi "ARRIAH" 26 September 2013.

When isolation of the virus with the aim of obtaining homogeneous populations with optimal biotechnological properties, used the complex biological, virological and biochemical methods provided methodological guidelines for the detection and identification of strains of FMD virus [11].

Biological and virological methods include virus isolation, which was performed on primary culture trypsinization SP cells, transplantable cell lines PSGC-30, IB-RS-2 with the subsequent adaptation (passage 3). For the production of the bioassay on primary and transplantable cell cultures were grown in appropriate nutrient media under stationary conditions in vials with a surface area of 25 cm2was washed from the growth medium and used to infect 10% suspension of canker material (multiplicity of infection was 1÷10 TCD50on the cell), prepared in Hanks solution with 0.5% lactalbumin hydrolysate (GLA) and Antibes�Omikami according to the standard recipe. For removal of microbial and cellular components of the ballast suspension was treated with 10% chloroform. After 30 minutes of contact at 37°C in vials made of 5 cm3supportive environment and incubated at 37°C until the JRS virus. In the presence of JRS (rounding of cells, increase their optical density, degeneration and detachment of cells from glass) vials were subjected to freezing-thawing, treatment of cell suspension with chloroform, and centrifuged at 3000 g for 15 min. Obtained virussecurity the material used for subsequent passages and research in RSC for the presence of viral antigen, using a commercial type-specific serum preserved in the Museum of strains of the fgbi "ARRIAH". The virus was considered to be adapted to cultures of cells, if within 18÷24 hours was manifested (%) 90÷100 JRS in the monolayer.

The virus adapted to cultures of cells IB-RS-2, was used to obtain antigen for CRC.

Epizootic isolates And No. 2187/Kuti/2013, adapted to cell culture PSGC-30, was used to infect suspension of BHK-21 cells with the purpose of producing antigen for production of pilot lots of vaccine, and to hyperimmunization Guinea pigs.

The results of adaptation of the virus to various cell cultures are presented in table 2.

The data are shown in table 2, SV�, which have been tested on a high adaptive ability of the strain And No. 2187/Kuti/2013 FMDV type a to used cell cultures.

Isolated using the methods of the viral preparation was studied in RSK to identify its model facilities (table 3). Audited strain on the absence of contamination by extraneous viruses by PCR and RT-PCR.

In table 3 the results indicate that in aphthous material examination No. 2187/Kuti/2013 revealed the antigen of FMD virus type a in a dilution of 1:2 in RSK. Contamination of bacterial and fungal microflora, Mycoplasma and extraneous virus detected.

The obtained strain of FMD virus type a has the author's name: strain And No. 2187/Kuti/2013.

Example 2

For hyperimmunization Guinea pigs used antigen from strain And No. 2187/Kuti/2013 of FMD virus type a, reproduced in monolayer cell culture PSGC-30. Virussolaris the suspension is concentrated 100-fold by adding 8÷10% polyethylene glycol (PEG) M. M. 6000, purified from ballast impurities by adding 10% of chloroform. The purified virus is inactivated by aminoethylethanolamine (AAAI) at a concentration of 0.025÷0,05% at pH 8,0÷8,3.

Inactivated concentrate of antigen mixed with an equal volume of oil adjuvant incomplete type of adjuvant freind injected Guinea pigs in a volume of 1.0 cm3intramuscularly. In 21 and 7 days repeat immunization and 10 days after the last injection EN�Egan animals exsanguinated. Individual samples of serum check on model specificity and activity in RSC in accordance with the methodological recommendations for the detection and identification of strains of FMD virus [11].

After this, prepare a series by mixing typespecific individual serum samples of the same activity. The strain specificity of serial drug is determined in single and duplex reactions with Homo - and heterologous antigens.

After concentrating with sodium azide (1:5000) and incubation at 4°C for 30 days the resulting serum is Packed in vials of 0.5÷1.0 cm3and dried by sublimation under vacuum.

The method described in example 2, was prepared 1 series hyperimmune serum which characteristic is presented in table 4.

The data in table 4 indicate that the received diagnostic serum, specific activity meets the requirements of GOST 25384-82.

Example 3

To obtain the antigen for serological and immunochemical reactions, used the FMD virus type a strain And No. 2187/Kuti/2013, adapted to cell culture, continuous cell lines PSGC-30 and IB-RS-2. To adapt use virussecurity material in the form of 10% aphthous suspension. Passaging was carried out for 3-5 consecutive passages. Received VIR�to use for producing viral material. Contamination of cell cultures and collection of viral material is carried out by conventional methods.

Received virussolaris the suspension is concentrated 100-fold by the addition (%) 8÷10 PEG. The resulting concentrate inactivate AAAI, Packed in vials and dried by sublimation under vacuum. The data presented in table 5.

Example 4

For the manufacture of a vaccine inactivated adsorbed type And from strain And No. 2187/Kuti/2013, the FMD virus reproduce in suspension culture cells KSS-21. The supportive environment of use a solution of Earl without serum with the addition of PGMS, GBX and antibiotics at pH 7,2-7,4. Culture of cells infected with viruses the rate of 0,001÷0,05 TCC50on the cage. Virus culture is carried out at a temperature of 36°C)÷(37°C). After 8÷10 hours of cultivation shall count the living and dead cells by staining trypan blue. If the number of living cells (%) 15÷20, the cultivation is continued for 2÷3 hours. When you reach the number of dead cells (%) 90÷95 cultivation was stopped and virussolaris suspension control for sterility and content of 146S and 75 S components.

The quantity of 146S and 75S components in suspension shall not be less than 0.5 μg/cm3. Immediately after the reproduction cycle of the virus, not stopping for temperature control in virussolaris suspension DOB�make 15÷20% solution AAAI, acidified with glacial acetic acid to pH 8,0÷8,5. The final concentration AEEI in virusdatabase suspension must be equal to (%) 0,025÷0,05. Inactivation of infectivity of the virus was carried out for 12÷24 hours at (36°C)÷(37°C) and pH 7.2÷7,6 with stirring after 5÷6 hours for 3÷5 minutes. After inactivation of the antigen suspension was cooled to (4°C)÷(8°C). To the cooled suspension was added 10% solution of pgmg to concentrations of 0.005÷0,007% for ballast flocculation of impurities and inactivation of possible contaminants. Flocculated ballast impurities is subjected to sedimentation followed by decantation. The resulting antigen in control of the avirulent, the content of virus-specific protein, 146S and 75S components of the virus and sterility. The necessary concentration of 146S and 75S components in pravilnoe dose of adsorbed vaccine is produced by concentration of antigen gel hydrate of aluminum oxide (GOA).

The calculated volume of gel GOA 3% concentration is added to a chilled suspension of antigen when operating the mixer. Stirring was continued for 30 minutes. After sedimentation of the gel GOA is drained, the estimated size of the remaining suspension. The final concentration of GOA should be within 1,62±0,488 mg/cm3P<0.01, n=10, and the concentration of 146S and 75S components of FMD virus at least 3.0 g/cm3. Then the suspension was added an additional 1% solution of saponin to a final concentration of not less than 0,075%.

The vaccine is packaged in glass or polypropylene bottles and conduct monitoring sterility according to GOST 28085-89.

The avirulent and harmless vaccine check for 5 heads of cattle, introducing the vaccine first, under the mucous membrane of the tongue at a dose of 2 cm3then subcutaneously at a dose of 10 cm3. Monitoring the clinical condition of the animals was conducted for 5 days. Avirulent, harmless and sterile vaccine check for immunogenic activity in cattle or Guinea pigs.

Example 5

Strain And No. 2187/Kuti/2013 virus Aphtae epizooticae type And designed to control the immunogenic activity of FMD vaccines in cattle, was prepared as follows. The resulting virus pre-refresh on cattle, infecting 1-2 animals weighing 250÷300 kg, delivered from wealthy FMD zones of the country, where for the past 2 years not vaccinate animals against FMD. The number of passages of the virus should not exceed 20, starting from the source of the virus. The virus suspension containing 10000÷10000000 EID50/1 cm3with antibiotics, administered 0.2÷0.3 cm320÷40 channels intraterminal across the surface of the tongue. Infected animals do not feed until the removal of the virus. After 20÷-30 hours after infection thrush off as they Mature. Lymph is collected by syringe. On the basis of the collected aphthous material prepared 20%-well� suspension of the virus, that is clear from the ballast impurities followed by the addition of antibiotics and an equal volume of glycerol. Received 10% suspension of the virus is estimated at RSC on typespecification and RT-PCR followed by nucleotide sequencing on the original virus is the primary structure of the VP1 gene. The activity of the virus in RSC shall be not less than 1:4.

The infectious activity of the virus is determined on cattle and in primary cultures of SP cells. The control virus must have a titer of infectious activity of not less than 104,0EID50/0.1 cm3. Bottles of suspension of the virus in the presence of glycerol was stored in a refrigerator at a temperature of minus (70°C÷40°C).

Example 6

Control immunogenic and antigenic activity of vaccine inactivated adsorbed from strain of FMD virus type a No. 2187/Kuti/2013 performed as follows. To check the immunogenic activity of the drug used 17 head of cattle weighing 200÷250 kg, delivered from a safe for FMD control zones. The first group of animals from 5 heads of cattle introduced the vaccine subcutaneously in solid form. The second group of animals from 5 heads of cattle were injected subcutaneously dilution of the vaccine in phosphate-buffer solution in a ratio of 1:4 and the third group of 5 goals in a dilution of 1:16. The fourth group of 2 goals were left without vaccination.

Volume pravilnoe dose was 2.0 sup> 3. On day 19 after vaccination, 15 vaccinated and control 2 head of cattle took blood samples and had a test the introduction of infected animals under mucous language suspension of FMD virus type a No. 2187/Kuti/2013 at a dose of 104,0EID50/0.2 cm3.

The results of the test vaccine inactivated adsorbed against FMD type a strain of FMD virus type a No. 2187/Kuti/2013 to cattle are presented in table 6.

Antigenic activity of the vaccine is controlled by the level of neutralizing antibodies in the serum in PH at primary monolayer trypsinization culture of SP cells in response microneutralization (RMN) on a cell culture IB-RS-2. On day 19 after immunization, the level of virus-neutralizing antibodies in PH in cattle vaccinated with a solid vaccine, was 6.2±0,27 log2the level of neutralizing antibodies in RMN - 8,4±0,30 log2.

8 days after infection held PATOLOGOANATOMICHESKOE a study of cattle. Sick 2 control animals, 1 animal inoculated with the dilutions of vaccine 1:4, and 4 animals inoculated with the dilutions of vaccine 1:16.

IPD50the vaccine was 0.25 cm3. Prepuna dose of the vaccine contained 8,0 PD50in previfem volume 2.0 cm3that indicates the effectiveness of the drug. This vaccine is considered to be immunogenic and suitable for practical application.

Sources �nformation

1. Rarer X. FMD: the lane with it. G. A. surkovoy / edited and with a Pref. Cand. vet. Sciences. P. V. Malarze. - M.: Kolos, 1971. - 432 p.

2. FMD / A. N. Burdov, A. I. Dudnikov, P. V. Malaret [and others]. - M.: Agropromizdat, 1990. - 320 p.

3. Viral animal diseases / V. N. Syurin, A. J. Samuilenko, B. V. Soloviev [and others]. - M.: VNITIBP, 1998. Pp. 532-548.

4. WRLFMD Quarterly Report July-September 2013 - URL http://www.wrlfmd.org/rei_labs/ref_lab_reports/OIE-FAO

5. Pat. Of the Russian Federation No. 2140452, C12N 7/00, A61K 39/135, G01N 33/569, A61K 39/42, C07K 16/08, 27.10.1999.

6. Pat. Of the Russian Federation No. 2242513, C12N 7/00, A61K 39/135, 20.12.2004.

7. Foot-and-mouth Disease. Situation worldwide and major epidemiological events in 2005-2006 / K. Sumption, J. Lubroth, T. Murrai, S. De La Rocqe // EMPRESS / Focus on..., 2007, No. 1, IIP.

8. Pat. Of the Russian Federation No. 2451745, C12N 7/00, AC/135, 27.05.2012.

9. OIE. Manual of diagnostic tests and vaccines for terrestrial animals, 7th Ed. -, Paris, 2012 - Vol.1, Chapter 2.1.5. - P. 166-169.

10. Selection of foot and mouth disease vaccine strains - a review / D. J. Paton, J.-F. Valacher, J. Bergman [et al.] // Rev. Sci. Tech. OIE. - 2005. - Vol. 24(3). - P. 981-993.

11. Guidelines for the detection and identification of strains of FMD virus / Gusev, A. A., Zakharov V. M., Saiko J. A. [and others]. Vladimir, 2002, 31 p.

Table 1
Antigen matching (r1in RMN strain And No. 2187/Kuti/2013 FMDV type And production strains of FMD virus type a
StrainThe rate r1
'an No. 5500,13
And No. 2179/Shchelkovo biocombine0,11
A/Iran/970,004
A/Turkey/06 (A/Iran/05)0,37
And No. 2045/Kyrgyzstan/2007 (A/Iran/05)0,13

Table 2
Biological properties of the virus of epizootic strain And No. 2187/Kuti/2013 of FMD virus type a
System of virus reproductionThe existence of biological activity (hours)The amount of adaptation passagesFeature adapted material
Activity in RSCThe titer of infectious activity(lg TCD50/cm3)
PSGC-302051:26,0
IB-RS-2245 1:46,23
BHK-211851:27,0

Table 3
Determining the type of the virus in 33% suspension aphthous material in RSK (examination No. 2187/Kuti/2013)
Examination No. 2187Control
Hyperimmune serum in the working dilutionsolid1:21:41:8to/aA/Turkey/ 06A22/Iraq/64O PanAsia2Asia-1 Shamir 3/89b/a
A/Turkey/06+---44---
And22/Iraq/6444--4---
O PanAsia2------4--
Asia-1 Shamir 3/89------4-
b/s---------
b/C 444444444
Note: 4 - 100% delay of hemolysis;
"- " complete hemolysis;
b/C without serum;
b/a - without antigen;
b/C - without complement

Table 4
Characteristics of diagnostic serum derived antigen from strain And No. 2187/Kuti/2013 of FMD virus type a
Name of productSeriesVolume (cm3)Activity in RSC
with homologous antigenwith heterologous antigens
A/Turkey/06O PanAsia2 Asia-1 Shamir 3/89
Hyperimmune serum12001:40<1:20<1:20<1:20

Table 5
Characteristics of diagnostic antigens from strain And No. 2187/Kuti/2013 of FMD virus type a, grown on different cell cultures
Name of productSeriesVolume (cm3)Activity in RSC
with homologous diagnosticum And No. 2187/Kuti/2013with heterologous diagnosticum And
Culture antigen PSGC-301401:8<1:2
Culture antigen IB-RS-21601:81:2

Table 6
The results of the test of inactivated vaccine against FMD type a from strain No. 2187/Kuti/2013 on cattle
BreedingThe number of virus-neutralizing antibodies (log2) in the serum on day 19 after immunizationThe number of cattle that are protected in the control of infectionIPD50
PHRMN
CThe 6.259,05/50.25 cm3
6,58,0
7,08,5
5,58,5
Of 5.758,0
M±m6,20±0,278,4±0,30
1:44,06,04/5
3,05,0
6,07,5
4,757,5
4,756,0
4,06,0
M±m4,5±0,497,6±0,54
1:163,255,51/5
1,755,0
2,55,0
2,256,0
2,754,5
3,255,5
M±m2,5±0,256,3±0,28
Control 1<4,00/2
Control 2<4,0

the Strain of FMD virus Aphtae epizooticae type a, SEM. Picornaviridae, genus Aphtovirus deposited in the collection of strains of microorganisms of the fgbi "ARRIAH" under registration number strain of FMD virus And No. 2187/Kuti/2013 (manufacturing) for control of antigenic and immunogenic activity and for the manufacture of biological products for diagnostics and specific prevention of FMD type A.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.

EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.

EFFECT: increase in accuracy.

2 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.

EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.

22 cl, 24 dwg, 47 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.

EFFECT: presented group of inventions can be used in medicine.

18 cl, 44 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.

4 tbl

FIELD: veterinary medicine.

SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.

EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with a strain of influenza virus. The vaccine strain A/17/Indiana/2011/72 (H3N2v) - reassortant, obtained by crossing of the "wild" virus A/Indiana/10/2011 (H3N2v) with the cold-adapted temperature-sensitive virus A/Leningrad/13 4/17/57 (H2N2) - attenuation donor. The strain A/17/Indiana/2011/72 (H3N2v) is deposited in the State Collection of Viruses FSBI D. I. Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health, under No 2739, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature-sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (NA) and neuramindase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza disease morbidity among adults and children.

4 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medical virology and concerns a vaccinating influenza virus strain. The characterised strain B/60/Wisconsin/2010/125 is a reaccortant produced by crossing the epidemic virus B/Wisconsin/1/2010 with the cold-adapted heat-sensitive virus B/USSR/60/69 that is an attenuation donor. The strain B/60/Wisconsin/2010/125 is characterised by heat-sensitivity and cold-adaptation. The reassortant has inherited genes coding the surface antigens, haemagglutinine (HA) and neuraminidase (NA), from the epidemic virus and rest six genes coding internal non-glycosylated proteins, from the attenuation donor. The strain is deposited in the State Collection of Viruses of Federal State Budgetary Institution D. I. Ivanovskiy Research Institute of Virology, under No. 2723.

EFFECT: invention can be used in practical health care for preventing the influenza rate in adults and children.

1 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine, namely to biopharmaceutics, and can be applied for the preservation of an immunogenic composition in the suitable condition for introduction to an animal. For this purpose the claimed composition contains an antigen of foot-and-mouth disease (FMD) and an emulsion, which is a single emulsion at the first temperature lower than the temperature of the animal body, and the said emulsion is used at the second temperature between the first temperature and the body temperature. The method includes: provision of the composition, freezing the composition and storing the frozen composition until it is required for introduction to the animal. A group of inventions also relates to a method of testing the immunogenic composition.

EFFECT: group of inventions makes it possible to reach an increase of the storage term of a vaccine against FMD, based on the emulsion as an adjuvant, and it is possible to apply the process of slow freezing, without rendering an impact on adjuvant properties of the emulsion.

13 cl, 6 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary virology and biotechnology and concerns a type A foot-and-mouth disease vaccine. The vaccine contains an avirulent and purified antigen material of the viral strain Aphtae epizooticae, of the family Picornaviridae, of the genus Aphtovirus, of the serotype A, deposited in the collection of FGU VGNKI (Federal State Institution Russian National Centre for Quality and Standartisation of Drugs and Feed for Animals), registration No. 2045/Kirghizia/2007-DEPA No. 2045/Kirghizia/2007-DEP prepared in a passaged cell culture VNK-21, representing s suspension containing preferentially the immunogenic viral components 146S and 75S of foot-and-mouth disease, the adjuvants aluminium hydroxide with saponin and a supporting medium in effective proportions.

EFFECT: vaccine possesses high immunogenicity and is able to provide the effective protection against a foot-and-mouth disease agent circulating in the countries of Transcaucasus, Central Asia, Near and Far East.

12 cl, 14 tbl, 1 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: method of production of vaccine against foot and mouth disease comprises culturing virus antigen in suspension culture of cells BHK-21 at a temperature of 36-37°C, purification of the viral suspension from ballast impurities, inactivation, concentration of the obtained foot and mouth disease virus antigen, and connection of the antigen concentrate with an adjuvant. Purification of the virus suspension from ballast impurities is carried out by adding derived polyguanidines at a final concentration of 0.005-0.01%, or the mixture of chloroform and derivatives of polyguanidines taken in weight ratios of (40-160):1, respectively, at a final concentration of 0.4-0.8. As derivatives of polyguanidines dihydrochloride 1,12 diguanidinohexane or dihydrochloride bis (3-guanidinopropyl) piperazine, or dihydrochloride 3,6-dioxaoctane-1,8-diguanidine, or dihydrochloride 4,9-dioxadodecane-1,2-dibiguanide.

EFFECT: improvement of purification level of virus suspension from ballast impurities and increase in immunogenicity of the target product.

2 cl, 1 tbl, 16 ex

FIELD: biotechnology.

SUBSTANCE: application of aethonium as the adjuvant for production of sorbed foot-mouth disease vaccine is proposed, where aethonium is used as a 10% aqueous solution which is introduced into the composition of foot-mouth disease vaccines in an amount of 750 mcg per 1 cm3 of the preparation.

EFFECT: invention extends the list of adjuvants for production of foot-mouth disease vaccines.

4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to a polyepitope vaccine protein applicable for immunisation against murrain virus, nucleic acid coding this protein, recombinant plasmid pA7248-AMV-H-PE for production of said protein in plants and to a vaccine preparation for prevention and protection against infection caused by murrain virus. The polyepitope vaccine protein has the amino acid sequence SEQ ID NO: 1 which contains the amino acid sequence of B-cell epitope of VP4 protein from 21-th to 40-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 135-th to 160-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 200-th to 213-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 2C protein from 68-th to 76-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 3D protein from 1-st to 115-th amino acid starting from N-terminal, and the amino acid sequence of T-cell epitope of 3D protein from 421-st to 460-th amino acid of murrain virus serotype O/ Taiwan/99. Nucleic acid coding such protein has the nucleotide sequence SEQ ID NO: 2. Recombinant plasmid pA7248-AMV-H-PE has a physical map presented in dwg. 5.

EFFECT: invention provides creating polyepitope vaccine protein which is applicable for immunisation against murrain virus.

5 cl, 7 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: polyepitope proteins consisting of two or more protein fragments of the foot-and-mouth disease virus, particularly serotype O/Taiwan/99, connected by linker amino acid sequences, are constructed. The vaccine polyepitope protein can be characterised by general formula VP4(X1 -X2)-GRL-VP1(X3-X4)-GRL-VP1 (X5-X6)GRL-2C(X7-X8)-GRL-3D(X9-X10)-GRL-3D(X11-X12), where GRL is a glycine-rich linker, Xn-Xm are integers denoting the number of amino acid residues of the corresponding foot-and-mouth disease virus protein, VP4, VP1, 2C, 3D are names of foot-and-mouth disease proteins. The invention discloses a nucleotide sequence (NS) which codes peptides included in the polyepitope protein, a recombinant plasmid which facilitates synthesis of a hybrid polyepitope protein in procaryote (E.coli) and eucaryote (plant) cells, and a solvent composition for a foot-and-mouth disease vaccine based on the polyepitope protein.

EFFECT: polyepitope protein has high immunising power and can be considered a potential recombinant vaccine against foot-and-mouth disease.

15 cl, 7 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to veterinary virology and biotechnology, as well as the foot-and-mouth disease virus Aphtae epizooticae type A Picornaviridae, genus Aphtovirus, which is deposited in the Russian State Centre for Quality and Standardisation of Veterinary Drugs and Feed under registration number A No.2045/Kyrgyzstan/2007 - DEP. The disclosed strain is reproduced in a monolayer swine kidney cell culture, passaged Siberian mountain goat kidney cell cultures (PSGK-30), VHK-21 and IB-RS-2. After 18-24 hours of incubation, virus yield in said cell cultures reaches 6.0-7.5 Ig TCD50/cm3. For multiplicity of infection (1-10 TCD/cell), the strain causes CPD after 5 hours, while preserving the initial characteristics when passaging in cell cutures for 10 passages.

EFFECT: disclosed strain can be used to control antigenic and immunogenic performance of foot-and-mouth disease vaccines and to produce biopreparations for diagnosis and specific prevention of foot-and-mouth disease type A.

6 cl, 1 dwg, 10 tbl, 12 ex

FIELD: medicine; veterinary science.

SUBSTANCE: method involves preparation of antigen material from avian influenza virus strain thereafter cleaned from ballast impurity, virus inactivation, mixing of antigen material and oil adjuvant with controlling the end product. The avian influenza virus strain is represented with the strain "Primorsky" of avian influenza virus referred to Orthomyxoviridae family, Influenzae virus avicum type of serotype A, subtype H5N2, collection Federal State Institurion VGNKI, serial №129 - "ДЕП". The vaccine made under the given method, contains cleaned and avirulent antigen material from strain " Primorsky" and oil adjuvant in ratio (wt %): antigen material - 30.0-40.0, oil adjuvant - 60.0-70.0.

EFFECT: decreased labour and power inputs in making the vaccine and improved quality of antigen material, high antigen activity of the vaccine and effective protection of susceptible poultry from epizootic virus of subtype H5.

14 cl, 5 tbl, 6 ex

FIELD: medicine; veterinary.

SUBSTANCE: method of vaccine obtaining involves virus antigen cultivation in BHK-21 suspension cell culture at the temperature of 36-37°C, purification of virus suspension from ballast admixtures, inactivation, concentration of foot-and-mouth disease virus antigen obtained and combination of antigen concentrate with adjuvant. After 6-8 hours of virus antigen cultivation, percentage of dead cells is registered every 2 hours. When the level of dead cells reaches 95% or more, further cultivation is performed for 2-8 hours depending on the virus type. Foot-and-mouth disease vaccine obtained by this method contains antigen material of a-type and/or O-type and/or Asia-1 type foot-and-mouth disease viruses in effective quantity of 146S-component, aluminium hydroxide gel, saponin and maintenance medium.

EFFECT: increased volume of antigen material during foot-and-mouth disease virus cultivation and obtaining harmless immunogenic vaccine.

13 cl, 1 tbl, 14 ex

FIELD: biotechnology, virology.

SUBSTANCE: claimed strain is obtained by passaging of epizootic isolate on sensitive hetero- and homologous cell cultures. Virus strain is reproduced in sensitive cell cultures and after incubation for 18-24 h 6.0-7.66 lg TCD50/ml is accumulated. In case of mass infection strain takes cytopathic effect over 5 hours, and retains starting characteristics for 10 passages.

EFFECT: strain of high biological, antigenic and immunogenic activity.

1 dwg, 7 tbl, 5 ex

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

Up!