RU2452511C1 - Attenuated strain of serotype 2 african swine fever virus for developing diagnostic and vaccine preparations - Google Patents

Abstract

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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Description

The invention relates to the field of virology, in particular to the production of an attenuated strain of KK-262 / C of the African swine fever virus (ASF), and can be used in research and diagnostic centers during virological, molecular genetic studies and the development of diagnostic and vaccine preparations with ASF.

For this purpose, virulent and attenuated strains of ASF virus are used, propagating in primary cultures of pig bone marrow cells (KMC) and pig leukocytes (PM), as well as in transplantable cell cultures of homologous and heterologous origin (1).

Using the hemodisorption delay reaction (RHCA), proposed for serological typing of strains, 8 ASF virus serotypes were found that differ in the immune test, which necessitates the production of attenuated strains for each immunogroup (2).

Abandoned strains and variants of the ASF virus were obtained and characterized abroad: Hinde-169, Katanga-105, Madeira-105 of the 1st serotype and others, which are grown in primary cultures of KMC or LS cells. These strains are characterized by high reactogenicity and viremia, complications, low immunogenicity, which complicates their use in the development of vaccine preparations.

To eliminate these drawbacks, intermittent passages of the virus are used in primary and transplantable cell cultures, selection methods based on immunosorption and selection of heat-sensitive clones, etc. The obtained strains Katanga-350, LS-72, Kimaki-155 and others were characterized by moderate reactogenicity and viremia and 60-70% of immunized animals were protected on the 21st day after the challenge (CI) with a homologous virulent virus (3).

The closest analogue of the proposed invention is the strain KK-202 selected in VNIIVViM, used to obtain virus-containing raw materials, which is typed in RZHAD with a specific reference serum of the 2nd immunoserot, and according to the results of genotyping it is assigned to genotype 2.

Obtaining virus-containing raw materials in a stationary monolayer of the primary KMC cell culture. For this purpose, a cell culture grown in mattresses with a capacity of 1.0 or 1.5 dm ³ is infected with strain KK-202 at a dose of 0.01-0.001 GAE ₅₀ / cell. As a growth medium for the cultivation of the virus, 0.1% GLA medium or MEM Needle with 8.0-10.0% pig serum is used. The cultivation of the virus is carried out for 5-7 days at a temperature of (37.0 ± 0.5) ° C. The titer of the virus in the virus-containing raw material is at least 6.5-7.0 lg GAE ₅₀ / cm ³ .

This strain has high reactogenicity, low immunogenicity and the presence of complications (depression, high temperature reaction and viremia, hemorrhages in parenchymal organs, etc.) in vaccinated animals, as well as the inability to use roller technology to obtain virus-containing raw materials, as more technological.

The virus does not multiply in the transplanted lines of kidney cells and piglet testicles (PP-66b, PPK, PK-15, PTP).

Strain KK-262 / C of the 2nd serotype of ASF virus was obtained by adaptation to the cell culture PPK-66b strain KK-202 for 50 passages and subsequent selection of the virus by the method of limiting dilutions in this cell culture and KMC cell culture.

Strain KK-262 / C of the 2nd serotype was deposited in the collection of microorganism strains of GNU VNIIVViM, inv. No. 1831.

The attenuated strain KK-262 / C virus ASF has the following properties.

Cultural properties. Propagated in a transplanted cell culture PPK-66b grown stationary (mattresses) or roller methods (roller bottles) with ASF virus-characteristic cytoplasmic changes followed by destruction of the monolayer. At the same time, it retains the ability to multiply in the KMC and LS cell culture, causing pronounced hemadsorption and subsequent cell lysis in them, which makes it possible to use these cell cultures to determine the infectious activity of the virus. The titer of the virus grown in the cell culture PPK-66b with stationary and roller methods of cultivation reaches 7.0-7.5 lg GAE ₅₀ / cm ³ .

Antigenic properties. When administered intramuscularly to pigs, the strain causes the formation of a complement of binding, precipitating and ZGad antibodies.

Harmlessness. Harmless to pigs when administered intramuscularly at the maximum (10 ^7.0 -10 ^7.5 GAU ₅₀ ) dose. For laboratory animals (rabbits, guinea pigs, mice) is not pathogenic.

Contagiousness and reversibility. The virus is not transmitted when kept together from vaccinated intact pigs.

Does not restore virulent properties after five consecutive intramuscular passages in pigs.

Reactogenicity. Weakly reactive for pigs by intramuscular injection. In individual pigs vaccinated in a dose of 10 ^6.0 -10 ^7.5 GAE ₅₀ , it can cause an increase in body temperature to 40.6-40.9 ° C within 3-5 days.

Immunogenic properties. After intramuscular administration at a dose of 10 ^6.0 -10 ^7.5 Gai ₅₀ causes the protection of pigs against homologous virulent reference strain K-49 to 21 days post inoculation for at least 4 months.

The main biological properties of strains KK-262 / C and KK-202 of the ASF virus are shown in Table 1

Table 1 Comparative characteristics of strains KK-262 / C and KK-202 ASF virus ASF virus strain Accumulation in cell cultures (lg GAE ₅₀ / cm ³ ) The level of viremia (lg GAE ₅₀ // cm ³⁾ Reactogenicity Vaccine response Short circuit protection for 21 days (%) Kmc PPK-66b T ° C Duration (days) KK-262 / S 7.25 ± 0.11 7.5 ± 0.11 4.0-4.5 Weakly Reactogenic 40.6-40.9 3-5 80-85 KK-202 7.00 ± 0.18 Does not breed 5.0-5.75 Reactogenic 40.9-41.7 6-11 65-70

As can be seen from the table, the attenuated strain KK-262 / C of the ASF virus surpasses the strain KK-202 of this serotype in the main biological indicators.

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

Stability of genetic and immunobiological traits. The main biological properties of the strain are stably maintained for 20 passages of cultivation in the cell culture PPK-66b and KMC (observation period).

Method, shelf life, frequency of passages. Strain KK-262 / C is stored in a lyophilized state at a temperature not exceeding minus 40 ° C and is “freshened” by passaging in cultures of PPK-66b or KMC cells once every 10 years.

To confirm the advantages of the present invention, examples of its specific implementation are given.

Example 1. Adaptation of ASF virus to transplantable cell culture PPK-66b

The virus was adapted to the cell culture by inoculating it with an attenuated strain KK-202 grown in the primary KMC cell culture (first passage), and in the subsequent passages with the material of the previous one. The infected cell culture grown in mattresses was incubated at (37.0 ± 0.5) ° C for 6-9 days. The reproduction of the virus in the cells was judged by the manifestation of a cytopathic effect. At the level of 6–8 passages, specific degeneration in infected cells (30.0–40.0)% was observed on days 4–5, the virus titer on days 8–9 of growth was 5.5–6.0 lg TCD ₅₀ / cm ³ . Reproduction of the virus in subsequent passages was characterized by specific degenerative changes, the occurrence of which was noted on 3-4 days, and virus accumulation on 6-7 days was 7.0-7.5 lg TCD ₅₀ / cm ³ .

Example 2. The cultivation of strain KK-262 / C virus ASF in transplantable cell culture PPK-66b roller method

As a result of the studies, the optimal parameters of the roller method of culturing the strain were worked out, which were:

- the multiplicity of infection of 0.1-0.01 GAE _{50 /} CL;

- the serum content of pigs in the environment of 0.1 GLA or Igla-MEM 8-10.0%;

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

- pH of the medium 7.2-7.4;

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

- temperature condition (37.0 ± 0.5) ° С;

- the volume of filling roller three-liter bottles of 0.3-0.5 DM ³ .

These cultivation modes allowed to obtain virus-containing material with an activity of 7.0-7.5 lg GAE ₅₀ / cm ³ . The results of the production of viral raw materials of 3 experimental series based on the strain KK-262 / C of the ASF virus are presented in Table 2.

table 2 Characterization of virus-containing raw materials obtained in the cell culture PPK-66b. Batch number Cell culture Number of bottles Infecting Dose, GAE ₅₀ / cl Duration of cultivation (days) Series Volume (L) Virus titer (lg GAE ₅₀ / cm ³ ) one 10 0.1 5 3.0 7.5 2 PPK-66b 10 0.01 6 3.0 7.25 3 10 0.001 8 3.0 7.0

As can be seen from table 2, the activity of 3 series of virus-containing raw materials obtained by the roller method in the cell culture PPK-66b, was 7.0-7.5 lg GAE ₅₀ / cm ³ .

Example 3. Verification of the reversibility of strain KK-262 / C virus ASF

For this purpose, 5 passages of strain KK-262 / C of the ASF virus were performed on susceptible animals. During the first passage, the culture virus was injected with a gum of 3 months of age at a dose of 7.25 lg GAE ₅₀ intramuscularly in the middle third of the neck. On the 6th day, the gilt was killed, bronchial and prelobe lymph nodes were taken from him, from which a 10.0% suspension in physiological saline was prepared. The resulting material was twice frozen at minus 40.0 ° C and after clarification (1500 rpm for 15 minutes) was administered intramuscularly to the next animal in a volume of 1.0 cm ³ (second passage). Subsequent passages were performed similarly.

In experimental animals there was no clinical response to vaccination, as well as a reaction at the site of the virus. Gilt of the 5th passage did not develop ASF within 21 days after the introduction of the material (observation period). This indicated that strain KK-262 / C of the ASF virus was not reversible.

Example 4. Verification of the immunobiological properties of the strain

The strain safety was studied on 3 gilts of 3-4 months of age. The strain was administered intramuscularly in the middle third of the neck. Two gilts were vaccinated with the virus at a dose of 7.5 lg GAE ₅₀ , and one at a dose of 7.0 lg GAE ₅₀ . For 15 days, the immunized gilts remained clinically healthy. In vaccinated animals, an increase in body temperature to 40.6–40.7 ° C for 2–3 days was noted on days 7–9.

The immunogenicity of the strain was determined by the results of the control infection of vaccinated animals with a homologous virulent strain K-49 at a dose of 1000 GAE (LD) ₅₀ / cm ³ .

For this purpose, 2 gilts of 3-4 months of age were inoculated with the virus at a dose of 7.0 and 6.0 GAE _50, respectively. In vaccinated animals, a temperature increase of up to 40.7 ° C was noted for 3 days.

21 days after the introduction of the virus, all vaccinated animals and one control were infected with a homologous virulent virus. At the same time, these animals were infected with pigs used in the experiment to determine the safety (Table 3).

Table 3 Immunobiological properties of strain KK-262 / C virus ASF ASF virus strain Grafting dose (lg GAE ₅₀ / cm ³ ) Qty Vaccine response Short Results Duration pace. reactions, (days) Max tem pa (° C) Duration pace. reactions (days) Max temperature (° C) KK-262 / S 7.5 2 3 40.7 3 40.9 7.0 one 2 40.6 5 40.8 7.0 2 3 40.7 four 40.9 6.0 2 2 40.6 3 41.0 The control one - - 9 41.8

The control gilt died on the 9th day after infection with signs of ASF (body temperature up to 41.8 ° C, cyanosis of the skin, depression, anorexia, diarrhea, lymph node hyperplasia, enlargement and plethora of the spleen, etc.), while the vaccinated animals remained clinically healthy during the entire observation period.

The data obtained show that the KK-262 / C strain adapted to the transplanted cell culture PPK-66b creates 21 days later in animals vaccinated with a dose of 6.0-7.0 lg GAE ₅₀ , protection against the virulent ASF virus. The strain is weakly reactogenic, not reversible and harmless to pigs.

Information sources

1. V.A. Sergeev. Viral vaccines. 1993 year

2. V.M. Balyshev. Immunobological and molecular genetic properties of African swine fever virus isolates isolated in the Russian Federation. / V.M. Balyshev, Yu.F. Kalantaenko, A.N. Zhukov, S.Zh. Tsybanov, I.M. Kalabekov / Oriented fundamental research and their implementation in the agro-industrial complex of Russia: materials of the All-Russian Scientific Conference. - M., 2010 .-- S. 94-98.

3. Cheryatnikov L. L. Obtaining attenuated variants from various ASF virus isolates and their comparative evaluation. / L.L. Cheryatnikov, Yu.I. Petrov, N.I. Mitin / Issues of Veterinary Virology, Microbiology and Epizootology 1992. - Part 1. - S. 56.

Claims (1)

The attenuated strain of the virus of African swine fever of the 2nd serotype, the Asfarviridae family, the genus Asfivirus, deposited in the Collection of microorganisms of the All-Russian Research Institute of Veterinary Virology and Microbiology of the Russian Academy of Agricultural Sciences, No. 1831 for the development of diagnostic and vaccine preparations.