Attenuated strain of serotype 2 african swine fever virus for developing diagnostic and vaccine preparations

FIELD: medicine.

SUBSTANCE: invention refers to virology and concerns an attenuated strain of serotype 2 African swine fever virus (ASF). The strain is prepared by adaptation to PPK-666 cell culture for 50 passages followed by virus selection by limiting dilution in this culture and CMS cells, and deposited in the collections of microorganism strains of the State Scientific Institution of Russian National Research Institution of Veterinary Virology and Microbiology of the Russian Academy of Agricultural Sciences, No. 183.

EFFECT: presented strain is applicable in the research and diagnostic centres for the purpose of epizootological monitoring, virological, molecular-genetic researches and development of diagnostic and vaccine preparations in ASF.

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The invention relates to the field of Virology, in particular the production of attenuated strain KK-262/With the virus of African swine fever (ASF), and can be used in research and diagnostic centers when conducting Virology, molecular genetic research and development of diagnostics and vaccines at the ASF.

With this purpose, use the virulent and attenuated strains of the virus that multiplies in primary cultures of bone marrow cells pigs (KMC) and leukocytes pigs (LS), and in human cell cultures homologous and heterologous origin (1).

Using response latency haemadsorption (Rshad)proposed for serological typing of strains, installed 8 serotypes of the virus, differing in the immune sample, which leads to the need for attenuated strains for each monography (2).

Abroad obtained and characterized by attenuated strains and variants of ASF virus: Hinde-169, Katanga-105, Madeira-105 1 serotype, etc. that can be grown in primary cell cultures KMC or drugs. These strains are characterized by high reactogenicity and viremia, complications, low immunogenicity, which hampers their use in the development of vaccines.

To eliminate the above disadvantages of the use of alternating passages of the virus in primary and transplantable cell cultures, breeding methods based on immunosorption and selection of temperature-sensitive clones and other strain Katanga-350, HP-72, Kimaks, 155, etc. were characterized by moderate reactogenicity and viraemia and defended 60-70% of immunized animals at 21 days after infection control (SC), the homologous virulent virus (3).

The closest analogue of the proposed invention is selected in Vniivvim strain QC-202 used to get vaccinated raw materials, which thepirouette in Rshad specific reference serum 2nd immunotherapy, and the results genotyping assigned to 2 genotype.

Getting vaccinated raw materials in stationary primary monolayer cell culture KMC. To this end the culture of cells grown in mattresses with a capacity of 1.0 or 1.5 DM3, infect strain QC-202 at a dose of 0,01-0,001 AI50/cell. As the growth medium for growing the virus used Wednesday 0.1% GLA or Needle MEM with 8,0-10,0% blood serum of pigs. The virus cultivation carried out for 5-7 days at a temperature of (37,00,5)C. the Titer of virus in vaccinated raw materials is not less than 6,5-7,0 lg AI50/cm3.

This strain has a high reactogenicity, low immunogenicity and the presence of complications (depression, high thermal response and viremia, bleeding in parenhimatosnoe and others) in vaccinated animals, as well as the inability to use roller technology when getting vaccinated raw materials, as more technological.

In transplantable cell lines kidneys and testicles pig (PP-b, CPD, RC-15, PTP), the virus multiplies.

Strain KK-262/2-th serotype of the virus obtained by adapting to the culture of cells CPD-b strain QC-202 for 50 passages and subsequent breeding of the virus by the method of limiting dilutions in this cell culture and cell culture systems.

Strain KK-262/2-th serotype deposited in the collection of strains of microorganisms wildebeest Vniivvim, inv. No. 1831.

Attenuated strain KK-262/ASF virus has the following properties.

Cultural properties. Breeds in transplantable cell culture CPD-b grown stationary (mattresses) or roller (roller bottles) characteristic of the ASF virus cytoplasmic changes with subsequent destruction of the monolayer. However, it retains the ability to replicate in cell culture KMC and drugs, causing them expressed hemadsorption and subsequent lysis of the cells, which gives the opportunity to use these cell culture to determine the infectious virus activity. The titer of virus grown in cell culture CPD-b when stationary and roller methods of cultivation, reaches 7,0-7,5 lg AI50/cm3.

Antigenic properties. the ri intramuscular swine strain causes the formation of complement binding, precipitating and Shad antibodies.

Harmlessness. Harmless for pigs in intramuscular to the maximum (107,0-107,5AI50) dose. For laboratory animals (rabbits, Guinea pigs, mice) is not pathogenic.

Contagiousness and reversibility. The virus is not transmitted through sharing content from inoculated intact pigs.

Does not restore virulence properties after five consecutive intramuscular passages on the pigs.

Reactogenicity. Laboratorian for pigs with intramuscular injection. Individual pigs inoculated at a dose of 106,0-107,5AI50can call in 3-5 days the body temperature rise to 40.6-40,9C.

Immunogenic properties. After intramuscular injection in a dose of 106,0-107,5AI50invokes the protection of pigs against homologous virulent reference strain K-49 to 21 days after vaccination for at least 4 months.

The main biological properties of strains QC-262/S and AC-202 virus are shown in table 1

Table 1
Comparative characteristics of strains QC-262/S and AC-202 virus
The virus strain ASF 50/cm3)The level of viremia (lg AI50//cm3)ReactogenicityResponse to inoculationShort circuit protection at 21 days (%)
KMCPPK-bTCDuration (days)
QC-262/S7,250,117,50,114.0 to 4.5Laboratorian40.6-of 40.93-580-85
QC-2027,000,18Not breeds5,0-of 5.75Reactogenic40,9-41.76-1165-70

As can be seen from the table, attenuated strain KK-262/ASF virus is superior to the basic biological indicators of strain CC-202 of this serotype.

Contamination by bacteria, fungi, Mycoplasma and extraneous viruses. The virus is not contaminated by bacteria, fungi, Mycoplasma and other microorganisms.

The genetic stability and unbiological signs. The main biological properties of the strain remained stable for 20 passages cultivation in cell culture CPD-b and KMC (observation period).

Way, the retention period, the frequency of passages. Strain KK-262/S stored in a lyophilized state at a temperature not higher than minus 40C and "refresh" passirovannym in cell cultures CPD-b or KMC once in 10 years.

To confirm the advantages of the present invention provides specific examples of its implementation.

Example 1. Adaptation of the virus to transplantable cell culture CPD-b

The adaptation of the virus to the cell culture was performed by inoculation its attenuated strain QC-202, grown in primary culture cells KMC (first passage), and in subsequent passages of the previous material. Infected culture cells grown in mattresses, incubated at (37,00,5)C for 6-9 days. About the replication of the virus in the cells to be judged by the manifestation of the cytopathic effect. Level 6-8-x specific passages degeneration in infected cells (30,0-40,0) % was noted for 4-5 hours, the titer of the virus in 8-9 days of cultivation was 5.5 to 6.0 lg TCD50/cm3. Virus reproduction in the subsequent passages were characterized by specific degenerative changes are noted for 3-4 days, and the accumulation of the virus in the 6-7 day was 7.0 and 7.5 lg TCD /cm3.

Example 2. Cultivation of strain CC-262/ASF virus in human cell culture CPD-b roller way

In the result of the research were worked out the optimal parameters of roller method of cultivation of the strain, which was:

- multiplicity of infection of 0.1-0.01 to AI50/KL;

- content of the blood serum of pigs in the environment 0,1 CHAP or Needle-MEME 8-10,0%;

- the speed of rotation of roller bottles about 12-16/h;

- pH 7,2-7,4;

- the duration of the cultivation of 6-7 days;

- temperature regime (37,00,5)C;

- filling volume three-liter roller bottles 0,3-0,5 DM3.

These modes of cultivation allowed to get vaccinated material with activity 7,0-7,5 lg AI50/cm3. The results of the developments of viral raw material 3 test series based on the strain KK-262/ASF virus is presented in table 2.

Table 2
Description vaccinated raw material obtained in the cell culture CPD-b.
The series numberCell cultureNumber of bottlesInfecting dose, the GUY50/KLThe duration of cultivation is s (d) Volume of the series (l)The titer of virus (lg AI50/cm3)
1100,153,07,5
2PPK-b100,0163,07,25
3100,00183,07,0

As can be seen from table 2, activity 3 series vaccinated raw material sourced roller method in cell culture CPD-b, was 7.0 and 7.5 lg AI50/cm3.

Example 3. Check reversibility strain KK-262/ASF virus

To this end spent 5 passages strain KK-262/ASF virus to susceptible animals. When performing the first passage culture the virus was introduced to the gilt 3 months of age at a dose of 7.25 lg AI50intramuscular injection in the middle third of the neck. On the 6th day of gilt killed, he was selected bronchial and predkomatozny lymph nodes, of which goto is or 10.0% suspension in physiological solution. The resulting material was twice promarijuana at minus 40.0C and after purification (1500 rpm for 15 minutes) was administered intramuscularly to the next animal in a volume of 1.0 cm3(second passage). Similarly performed subsequent passages.

In the experimental animals had no clinical response to vaccination, and the reaction to the injection of the virus. The gilt 5-th passage, not ill ASF within 21 days after the injection of the material (observation period). It was indicated that the strain KK-262/ASF virus is not reversible.

Example 4. Check immunobiological properties of strain

The harmless strains was studied on 3 pigs 3-4 months of age. The strain was injected intramuscularly in the area of the middle third of the neck. Two pigs were inoculated with virus at a dose of 7.5 lg AI50and one dose of 7.0 lg AI50. Within 15 days of the immunized pigs remained clinically healthy. In vaccinated animals for 7-9 days noted increased body temperature to 40.6 40.7 inC for 2-3 days.

The immunogenicity of the strain was determined based on control of infection of vaccinated animals homologous virulent strain K-49 at a dose of 1000 GAJ(LD)50/cm3.

To this end, 2 gilt 3-4 months of age were inoculated with virus at a dose of 7.0 and 6.0 AI50respectively. In vaccinated animals were observed temperature rise to 40.7C during the course the e 3 days.

After 21 days after administration of the virus all vaccinated animals and one control were infected homologous virulent virus. At the same time these animals were infected pigs used in the experiment for the determination of harmlessness (table 3).

Table 3
Immunobiological properties of strain CC-262/ASF
The virus strain ASFPrivigna dose (lg AI50/cm3)Qty W-xResponse to inoculationThe results of short circuit
Length. the pace. reaction (d)IGMASS. the-pa (C)Length. the pace. reaction (d)Max temperature (C)
QC-262/S7,52340,7340.9
7,01240.65 40,8
7,02340.74of 40.9
6,02240,6341,0
Control1--941.8

Reference gilt died on the 9th day after infection with signs of fever (body temperature up to 41.8C, blueness of the skin, depression, anorexia, diarrhea, hyperplasia lymph node, and increase the plethora spleen and other), whereas the vaccinated animals remained clinically healthy throughout the observation period.

The data obtained show that adapted to the transplantable cell culture CPD-b strain KK-262/S constructs after 21 days the animals, inoculated at a dose of 6,0-7,0 lg AI50protection against virulent virus. Strain laboratorian, not reversible and harmless for pigs.

Sources of information

1. Vasiliev. Viral vaccines. 1993

2. Vmmouse. Immunobiologicheskie and molecular genetic properties of isolates VIR the sa of African swine fever selected in the Russian Federation. / Vmmouse, Upholsters, Angelkov, Srzyban, IM the Kalabekov / Oriented fundamental research and their implementation in the agro-industrial complex of Russia: materials of all-Russian scientific conference. - M., 2010. - S-98.

3. Cherednikov L.L. Getting attenuated variants of different isolates of the virus and their comparative evaluation. / LTC, Whitecrow, Nieten / veterinary Virology, Microbiology and epizootology 1992. - Part 1. - P.56.

Attenuated strain of African swine fever virus 2nd serotype, the family Asfarviridae, genus Asfivirus, deposited in the Collection of microorganisms of the GSI all-Russian scientific research Institute of veterinary Virology and Microbiology of the RAAS, No. 1831 for the development of diagnostics and vaccines.



 

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