RU2760399C1 - Attenuated asfv/cv60/2020 strain of african swine fever virus of the asfarviridae family, genus asfivirus for the study of immunological reactions, molecular genetic analysis, genetic modification and creation of a prototype vaccine against asf - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. The invention is a strain of the African swine fever virus "ASFV/CV60/2020". The characterized strain was obtained by deep adaptation by sequential passaging of the virus in a continuous culture of African green monkey kidney cells (CV-1) and deposited in the collection of a microorganism strain of the FSBI "ARRIAH" under registration number 334-dep/20-122. The presented strain has a moderate infectious activity, accumulates in the culture of bone marrow and CV-1 cells. The virulence for pigs of all age groups with intramuscular injection does not exceed 12.5%.

EFFECT: presented virus strain can be used to study immunity factors in pigs when creating a prototype vaccine against African swine fever.

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Description

The invention relates to the field of veterinary virology, in particular, to strains of the African swine fever (ASF) virus and can be used in research institutes and laboratories as a strain for studying immunological reactions, molecular genetic analysis, genetic modification of the virus, as well as for production of antigen-containing biological products and creation of a prototype vaccine against ASF.

ASF is a viral disease of pigs characterized by fever, food refusal, lymphadenitis and extensive hemorrhages in the internal organs of infected animals. ASF is characterized by a variety of forms of the course of the disease: from hyperacute and acute with 100% mortality to chronic. At present, taking into account the degree of intensity of the course of the epizootic process and the scale of geographical distribution, we can talk about the development of ASF panzootic disease [1].

In 2014-2020. ASF panzootic disease has spread to many countries of the African continent, Europe, including Ukraine, Belarus, Russia, as well as countries of Asia (China, Korea, India, Vietnam, Laos, etc.) and Oceania. In addition, there is a tendency for the further spread of the disease in the world.

Difficulties in the global eradication of ASF are associated with the lack of specific prevention and treatment tools, as well as the difficulty of controlling its spread in pig farms with a low level of biological safety and in the wild boar population.

Currently, for scientific research carried out to study the virulent properties, genotypic and serotypic belonging of the isolated isolates, two strains are most often used as reference strains of genotype II, serotype VIII: Georgia 2007/01 and Arm07, which initiated the development of the current panzootic disease in the Eurasian continent.

Attenuated strains, as the safest, can be used to obtain hyperimmune sera and antigens for ELISA and immunoblotting, to determine the seroimmunotype of newly isolated natural variants of the ASF virus in the hemadsorption delay reaction (RHAD), as well as as a source of reference DNA when testing new test systems based on PCR, and for the development of specific prophylaxis tools.

However, attempts to use vaccine variants developed according to the classical canons against this especially dangerous pig disease in the 60s of the last century not only did not improve the epizootic situation with ASF, but also exacerbated it [2].

Currently, various scientific groups worldwide are conducting research on the development of an ASF vaccine. As a basis for the development of a vaccine, both highly virulent variants of the ASF virus and its natural or laboratory-attenuated variants are used [3, 4, 5].

According to Lacasta et al., Live attenuated ASF viruses are ideal tools for analyzing the mechanisms involved in viral pathogenesis and immune defense [6].

Due to the formation of ASF-endemic zones on the territory of the Russian Federation, there is an urgent question about the availability of a bank of Russian isolates, the analysis of which will allow not only to find out the nature of the virus virulence, but also to develop a scientifically grounded platform for the development of an effective vaccine against ASF.

At the moment, a number of domestic and foreign strains of the ASF virus are known, which are used for the manufacture of antigen-containing biological products [7, 8, 9].

The attenuated strain KK-262 / C of genotype I, serotype II was used for the development of vaccine preparations. It has the ability to reproduce in primary cultures of porcine bone marrow cells (KMC), and its administration to pigs induces the formation of a protective immune response to infection with a homologous virulent virus I of genotype II serotype [9, 10].

The history of patenting circulating strains of the ASF virus II genotype VIII serotype in our country begins in 2010, when a patent was first registered for the Russian strain Stavropol 01/08 [11]. It is a highly virulent virus that can reproduce only in primary culture of KMC, alveolar macrophages (AMC) or porcine leukocytes (PM).

Subsequently Stavropol 01/08 strain virus was adapted to grow in a continuous culture of cells A ₄ C ₂ (SPEV and splenocyte hybrid pigs - Sus scrofa L) and CV - 1 (African green monkey kidney - Cercopithecus aethiops) [12]. However, the obtained attenuated strain Stavropol 01/08 did not protect animals from infection with a virulent virus, since lost some of the properties necessary for the formation of a protective immune response in ASF.

Known strain Volgograd / 14s, used for the development of diagnostic drugs and virological, molecular genetic studies [13], which is obtained from the virulent Russian isolate Volgograd / 14c, belonging to the VIII serotype. Although the recombinant Volgograd / 14c virus, after deleting the A238L gene, lost the pathogenicity of the original virus, it does not possess protective properties and does not protect against challenge with a virulent virus of serotype VIII [14]. Consequently, this strain cannot be used to study protective properties and develop protective drugs.

The closest analogue (prototype) of the proposed strain, which has protective properties and the ability to reproduce in a heterologous continuous cell culture, is the attenuated strain BA71V, genotype IV serotype I, adapted to growth in Vero (kidney of the African green monkey - Cercopithecus aethiops), strain BA71V, genotype I, serotype IV [15]. proved the possibility of achieving protection against infection with both homologous (one genotype) and heterologous isolates (different genotypes). In addition, a comparative analysis of the genome of virulent and attenuated strains made it possible to determine the groups of genes that determine the virulence of the ASF virus [16]. However, it belongs to genotype I and serotype IV, so its use in the future as a component of diagnostic systems is inappropriate.

Despite the ongoing control measures, the distribution area of ASF in the world continues to expand. In this regard, the need to conduct a comparative genomic analysis of virulent and avirulent variants and to study the immunogenesis of the disease with the further aim of creating a vaccine is a priority area of research to protect against ASF panzootic disease.

Attenuated ASF virus strains with properties differing in virulence, contagiousness, reactogenicity and severity of clinical signs are necessary both for determining the causes of ASF virus virulence and for developing means of its specific prophylaxis.

The technical problem to be solved by the present invention is to expand the arsenal of attenuated strains of African swine fever virus, which are necessary for studying the immunology of this disease, molecular genetic analysis and genetic modification of the pathogen when creating a vaccine prototype, as well as for diagnostic studies, obtaining antigen-containing drugs.

All the above facts indicate the priority of the task of obtaining a new attenuated strain of the ASF virus, suitable for a detailed study of the structure of its genome, analysis of gene functions and mechanisms of the formation of an immune response in ASF in infected animals, as well as diagnostic studies and modification of the virus when obtaining vaccine preparations.

This problem was solved by obtaining an attenuated strain of the ASF virus II genotype VIII serotype with reduced virulence, stable cultural and biological properties, capable of effective replication in a continuous culture of CV-1 cells, intended for studying the characteristics of immunogenesis in ASF and for the development of diagnostic and protective drugs. ... In addition, the attenuated strain can also be used to obtain avirulent clones of the virus, carry out genetic modifications, both in order to study the functions of individual genes, and to create a prototype of a labeled vaccine based on it.

The initial material, which served as a source for obtaining the ASFV / CV60 / 2020 virus strain, was the ASF / ARRIAH / CV-1 virus strain adapted to growth in a continuous culture of CV-1 cells [17]. In turn, the strain "ASF / ARRIAH / CV-1" was obtained in the process of adaptation to growth in a continuous culture of virus cells with a lethality of 85.7% of the strain "ASF-8 No. 2 / Odintsovo 02/14" of the African swine fever virus, isolated from the spleen of a wild boar shot in the Odintsovo district of the Moscow region in 2014. Virus strain "ASF / ARRIAH / CV-1" accumulates in the culture of bone marrow cells of pigs and CV-1 in a titer of 6.0-7.0 lg GAE ₅₀ / cm ³ , and its lethality for domestic pigs of all age groups does not exceed 37 ,5%.

Compared with the known strains of genotype II, the ASFV / CV60 / 2020 strain has a reduced residual lethality for pigs (12.5%) and is effectively reproduced in the culture of CV-1 green monkey kidney cells, and the virus titer reaches 7, 5-8.0 lg HUDE ₅₀ / cm ³ . In addition, the ASFV strain "ASFV / CV60 / 2020" has a pronounced immunogenicity, since when it is administered to animals, most of the clinical signs of ASF are absent, and the surviving animals acquire resistance to repeated infection with the virulent virus of genotype VIII serotype II, which makes it possible to use it to obtain specific hyperimmune serum.

The attenuated ASFV / CV60 / 2020 strain of African swine fever virus obtained by long-term adaptation (60 passages) was deposited in the Collection of Microorganism Strains of the FGBI ARRIAH under the author's name “ASFV / CV60 / 2020” with registration number No. 334 - dep / 20- 122 - SCSM FGBU "ARRIAH".

The African swine fever virus strain "ASFV / CV60 / 2020" is characterized by the following features and properties:

Morphological properties.

The African swine fever virus strain "ASFV / CV60 / 2020" belongs to the Asfarviridae family, genus Asfivirus. It has morphological features characteristic of the ASF causative agent: during electron microscopic examination, virions of the pathogen of an icosahedral shape with a diameter of -200 nm, having several concentric layers, are observed. The central nucleoid of the virion or core, represented by a double-stranded DNA molecule with a dense layer of a protein coat, covers the inner lipid coat, enclosed in a capsid. The capsid consists of 1892-2172 capsomeres and is surrounded by a supercapsid (outer) lipoprotein sheath.

Antigenic properties.

The mature virions of the ASF virus contain more than 50 proteins. The main antigenic properties of the strain "ASFV / CV60 / 2020" correspond to those of the virus of VIII serotype: virus-specific antigens are detected by specific antibodies in enzyme-linked immunosorbent assay and immunoblotting; in the reaction of delayed hemadsorption and immunoassay, properties characteristic of the corresponding serotype appear, and in the reaction of direct immunofluorescence, its antigen interacts with specific antibodies to the ASF virus.

Genotaxonomic characteristics.

Using phylogenetic analysis based on nucleotide sequencing of the B646L gene fragment, ASFV / CV60 / 2020 strain was assigned to ASFV genotype II (100% homology with reference strain II of Georgia 2007/01 genotype).

The genetic affiliation of the variant of the virus proposed for patenting is illustrated on a dendrogram reflecting the phylogenetic relationship of the ASFV / CV60 / 2020 strain with the epizootic strains of the African swine fever virus genotype II (the dendrogram is based on a comparison of the complete nucleotide sequences of the B646L region encoding the main capsid protein of the virus) (Fig. 2).

The essence of the invention is explained in graphic images, which show:

FIG. 1 - Dynamics of changes in the level of antibodies in the blood serum of four groups of pigs infected with the virus of the attenuated strain "ASFV / CV60 / 2020" in the enzyme-linked immunosorbent assay (ELISA);

FIG. 2 - Position of the ASFV strain "ASFV / CV60 / 2020" on the phylogenetic tree of the Asfarviridae family of the genus Asfivirus;

FIG. 3 - Specificity of the interaction of antibodies to the virus of the strain "ASFV / CV60 / 2020" with the antigen of the ASF virus of different serotypes (a - blottogram of proteins of the preparation of the purified cytoplasmic antigen (a1-I serotype, a2-IV serotype) after electrophoresis in 10% separating gel; b - reaction of indirect immunofluorescence with a virus of a heterologous serotype (b1 - IV serotype, b2 - control of uninfected cell culture).

The serotype of the ASF virus was determined in the RHAD (hemadsorption delay reaction) and in the immunoassay according to the method developed at VNIIVViM [18, 19].

The serotypic affiliation of the ASFV / CV60 / 2020 strain of the ASF virus was established in the RZHAD using specific sera for VIII and IV serotypes.

Primary cell culture (KMC, AMC) was grown in 96-well plates at a seeding concentration of 6-7 × 10 ⁶ / cm ³ . After sorption of cells, unattached cells and excess erythrocytes were removed, and 200 μl of growth medium was added to the wells and the panels were incubated for 48 hours at 37 ° C and 3-5% CO ₂ . The reaction was carried out in a volume of 250 μL of a supporting medium of the following composition:

- 200 μl DMEM medium,

- 25 μl vaccinated suspension,

- 25 μl of specific or control serum.

The reaction was taken into account after 24-72 hours with pronounced hemadsorption in the control samples.

It was found that the specific serum obtained for the "Arm07" strain of the VIII serotype of the ASF virus causes a delay (absence of GAD) in dilutions of 1:20, while the introduction of negative porcine serum and reference serum of the IV serotype obtained from the Reference Laboratory of the European Union "CISA INIA" (Spain), did not cause a delay in the appearance of specific hemadsorption. The results of strain typing are shown in Table 1.

According to the results of staging the GAD delay reaction using reference sera of different serotypes of the ASF virus, attenuated and adapted to growth in a continuous culture of CV-1 cells, the ASFV / CV60 / 2020 strain was assigned to African swine fever virus of VIII serotype (Table 1).

Seroimmunity was determined by setting an immunoassay on pigs (7 heads) infected with the virus of the ASFV / CV60 / 2020 strain at a dose of 10 HAU / head. 35 days after infection, all pigs were injected with 1000 HAU / head of the virulent ASF virus strain "Arm07". The animals acquired resistance to re-infection with the virulent ASF virus serotype VIII. Thus, the immunoassay confirmed that the virus of the ASFV / CV60 / 2020 strain belongs to the VIII serotype.

Cultural properties.

The virus strain "ASFV / CV60 / 2020" multiplies in primary cell cultures AMC, LS, CC (pig spleen) and KMC, moreover, its reproduction is accompanied by the appearance of "loose" hemadsorption, when no more than 20-30 pig erythrocytes are attached to one infected cell [23]. The titer of virus accumulation on the 5th day reaches values of 7.0-7.5 lg HADU ₅₀ / cm ³ .

In a culture of green monkey kidney cells (CV-1), the titers of virus accumulation are 7.5-8.0 lg HAE ₅₀ / cm ³ during reproduction for 7 days at a temperature of 36-37 ° C (Table 2). It has been established that the virus of the ASFV / CV60 / 2020 strain retains these properties for 15 serial passages (observation period).

Pathogenic properties.

The virus strain "ASFV / CV60 / 2020" is weakly pathogenic for pigs when injected intramuscularly, surviving animals have only a short-term temperature rise up to 41.0 ° C, and clinical signs characteristic of ASF are absent in most animals.

Contamination with bacteria, fungi, mycoplasmas and foreign viruses.

The strain is not contaminated with bacteria, fungi, mycoplasmas and heterologous viruses.

Method of obtaining and storage conditions.

The strain is stored at minus 70 ° C in the form of a lyophilized vaccinated suspension, which is obtained by culturing the virus in a monolayer of CV-1 cells at 37 ° C for 7 days, with the addition of 10% fetal cattle serum. The frequency of refreshing the strain is once every 10 years.

Additional characteristics.

Virulence - low virulence (12.5%) for naturally susceptible animals with intramuscular infection.

Antigenic activity - the introduction of the virus of the attenuated strain "ASFV / CV60 / 2020" to pigs induces the formation of virus-specific antibodies, which are detected in the enzyme immunoassay, immunoblotting and the reaction of indirect immunofluorescence with test antigens of the virus I, IV and VIII serotype (Fig. 3). This image shows:

M - protein molecular weight marker;

BA I - viral antigen of serotype I, detected using antibodies to the virus "ASFV / CV60 / 2020";

BA IV - viral antigen of the IV serotype, detected using antibodies to the virus "ASFV / CV60 / 2020";

- стрелкой отмечено специфическое свечение комплекса антигена вируса АЧС IV серотипа с антителами к вирусу «ASFV/CV60/2020», мечеными ФИТЦ.

- the arrow marks the specific luminescence of the ASFV serotype IV antigen complex with antibodies to the ASFV / CV60 / 2020 virus labeled with FITC.

Immunogenic activity - control infection of the surviving pigs made it possible to determine that 100% of the animals acquired persistent protective immunity.

Stability - stably active during reproduction in a continuous culture of CV-1 cells, retains its original biological properties when passaged in sensitive biological systems for 15 consecutive passages (observation period).

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

Example 1.

The study of the stability and reproductive properties of the ASF virus of the attenuated strain "ASFV7CV60 / 2020" was carried out in a CV-1 cell culture for 15 consecutive passages. The presence of hemadsorbing activity was determined in the KMC cell culture. The main characteristics of the ASF virus of the attenuated strain "ASFV / CV60 / 2020" in KMC and CV-1 cell cultures during passaging are presented in Table 2.

The data given in Table 2 indicate that during 15 (from 61 to 75 passages) consecutive passages the virus of the attenuated ASFV / CV60 / 2020 strain of the ASF virus effectively multiplied in CV-1 cells and retained the ability to reproduce in culture KMC cells, moreover, the reproduction of the virus in the culture of KMC cells was accompanied by the appearance of "loose" hemadsorption in the presence of 0.01% pig erythrocytes in the nutrient medium.

After 7 days of cultivation in a CV-1 cell culture at a temperature of 37.0 ° C, the titer of virus accumulation was 7.5-8.0 lg HAU ₅₀ / cm ³ .

Example 2.

In the study of the population structure and biological properties of the virus of the attenuated strain "ASFV / CV60 / 2020", intramuscular infection of 8 heads of large white pigs weighing ~ 15-18 kg with a virus-containing suspension at a dose of 10 GAU / bird was carried out. Such a minimum dose of an infectious virus administered to animals makes it possible to establish a clear difference in the biological properties of individual components of the viral population.

Based on the analysis of clinical manifestations and the level of viremia in infected animals, the ambiguity of clinical manifestations in infected animals was revealed, as a result, based on experimental data, all experimental animals were divided into 4 groups (Table 3).

Out of 8 infected animals, only one showed clinical signs characteristic of the subacute form of ASF with a maximum body temperature of up to 41.7 ° C, and death occurred on the 17th day after infection (d.p.) (group I). The titer of the virus in the blood reached values of 7.2 lg HAU ₅₀ / cm ³ .

Two pigs (group II) showed clinical signs characteristic of the chronic form of ASF with a maximum body temperature of up to 41.0 ° C, expressed in short-term rises in temperature, necrotic skin lesions, swelling of the joints, the titer of the virus in the blood did not exceed 6.25 lg HAU ₅₀ / cm.

Two of the 8 infected pigs (group III) had no clinical signs of African swine fever disease, and the virus titer in the blood was kept within the range of 2.0-3.8 lg GAADE50 / cm.

In the IV group of pigs (3 heads), not only clinical signs of the disease were not recorded, but the presence of viremia was not established either in PCR or in RGAD throughout the entire experiment (35 days) prior to challenge with a virulent strain.

The results obtained indicate the heterogeneity of the population structure of the virus of the strain "ASFV / CV60 / 2020", which manifested itself in the ambiguity of clinical manifestations of the disease caused by individual variants of the virus or the absence of such in infected animals.

Blood was taken from the surviving experimental pigs, in the serum of which, when performing an enzyme-linked immunosorbent assay (ELISA), virus-specific antibodies were detected. As a result of analysis using a TF ELISA kit manufactured by Ingenaza, the presence of detectable post-infectious virus-specific antibodies was determined, the titer of which was 27 bp. reached in pigs III and IV groups values of 4.0 log ₂ / cm ³ , and in pigs of group II - 7.0 log ₂ / cm ³ .

Example 3.

The study of the dynamics of antibody production in pigs infected with the attenuated ASFV / CV60 / 2020 strain of the ASF virus was carried out by determining the antibody titer in the blood serum sampled 1-32 days after infection from animals of groups I-IV (presented in example 2).

The level of antibodies in the blood was determined by titration of serum with two-fold dilutions with further analysis of samples in TF ELISA using a commercial kit Ingenasa-Ingezim PPA Sotras K3 (Spain).

As a result, it was found that the dynamics of antibody production differs in all 4 groups of pigs, the features of which are revealed both at the early stage of their appearance in the blood (7-9 days after infection - d.p.), and at the stage from 11 to 25 d.c.z. (Fig. 1), and the level of antibodies reached its maximum at 28 DPH. and remained unchanged up to 35 days in three groups of animals (observation period).

The analysis of the results obtained confirmed the difference in the induced immunological manifestations in animals of groups I-IV upon administration of low doses of the attenuated ASFV / CV60 / 2020 strain of the ASF virus.

Thus, the obtained attenuated ASFV / CV60 / 2020 strain of the ASF virus has infectious activity for cell culture and pigs at a dose of 10 GAU / head, a mortality rate of 12.5% and can be used in experimental studies to study the dynamics of antibody formation and protective properties. , as well as exploratory research work.

Example 4.

Determination of the main clinical signs of the course of the disease and a comparative analysis of the level of the virus in the blood and nasal washings are important characteristics of the pathogenicity / immunogenicity of the ASF virus. Infection was carried out by intramuscular inoculation of a viral suspension at a dose of 10 GAE ₅₀ / head of the attenuated strain ASFV / CV60 / 2020 virus to a group of 7 pigs, and the second group of 7 pigs was infected with virus-containing material of the ASF-8 No. 2 / Odintsovo 02 / 14 "using the same dose of the virus and the method of infection [20]. 2 pig heads were used as control, i.e. remained uninfected (control group).

Further, the dynamics of the development of clinical signs of the disease, viremia and virus shedding in ASF was studied by comparative analysis of temperature indicators and parameters of virus accumulation in the blood. For this purpose, the rectal temperature was measured in the experimental animals and samples of blood and nasal washings were taken with an interval of 2 days during the entire experiment (32 days).

The presence of the viral genome in nasal washes and blood was determined by real-time PCR in accordance with the instructions for the ASF test system for detecting African swine fever virus by polymerase chain reaction (FBSI Central Research Institute of Epidemiology, Rospotrebnadzor, Moscow) [21]. The titer of the virus in the blood was determined in the hemadsorption reaction according to the Methodological Recommendations for the Isolation and Titration of African Swine Fever Virus, FGBI ARRIAH [22].

To determine the number of infected cells in the blood of animals, a staining method was used using the FITC conjugate of anti-ASF antibodies to the structural protein p72 (Ingenaza, Spain). After staining, the samples were analyzed using flow cytometry.

Comparative analysis of the main characteristics of the ASF virus of the strains "ASF-8 No. 2 / Odintsovo 02/14" and "ASFV / CV60 / 2020" when setting up a bioassay is shown in Table 4.

As can be seen from the results shown in Table 4, the maximum rectal temperature in animals infected with the virus strain "AShS-8 No. 2 / Odintsovo 02/14" reached 42 ° C, and the duration of the course of the disease was 7-9 days, while in pigs infected with the attenuated strain ASFV / CV60 / 2020 virus, a slight temperature reaction was recorded in the absence of other signs of ASF disease in most animals.

Comparative analysis of the levels of viremia and virus secretion in nasal washings revealed significant differences in both indicators. Thus, the titer of the virus in the blood of pigs infected with the virus of the attenuated strain "ASFV / CV60 / 2020" was 100 or more times lower, and it was not detected in nasal washings, in contrast to animals infected with the virus of the strain "ASFV-8 No. 2 / Odintsovo 02/14 ".

Thus, the use of a comparative analysis of the biological properties of various isolates and strains of the ASF virus with those of the attenuated strain "ASFV / CV60 / 2020" with the determination of the main clinical signs of the course of the disease and the levels of the virus in the blood and nasal washings makes it possible to characterize the pathogenicity and / or immunogenicity of the newly isolated ASF virus isolates.

Example 5.

To study the protective properties and immune response in pigs infected intramuscularly at a dose of 10 HAU ₅₀ / head with the attenuated ASFV / CV60 / 2020 strain of the ASF virus, they were challenged with the virulent Arm07 virus at a dose of 1000 HAU ₅₀ / head. All 7 animals that survived the infection with the ASFV / CV60 / 2020 virus acquired 100% protection against infection with the homologous virulent virus Arm07, with the death of all non-immune control animals (2 pigs - control group). It was found that the acquired immunity completely protected pigs from the development of clinical signs characteristic of ASF, such as fever, refusal to feed, and skin redness. At the same time, short-term viremia was found in vaccinated animals (no more than 7 days). Nasal excretion of the virus was not observed.

At postmortem autopsy 28 days after challenge, all surviving animals showed no pathological changes characteristic of ASF.

At the same time, the control animals showed clinical signs and pathological changes characteristic of ASF, as well as high values of the virus titer (in the blood 7.0-7.5 lg HADE ₅₀ / cm ³ and in nasal washings 2.0-4.5 lg HUDE ₅₀ / cm ³ ), which were detected starting from 3 DPZ.

Sources of information taken into account in the preparation of the description of the invention for the application for the grant of a patent of the Russian Federation for the invention "Attenuated strain" ASFV760CV / 2020 "of the African swine fever virus of the Asfarviridae family of the genus Asfivirus for studying immunological reactions in ASF, molecular genetic analysis and genetic modification when creating vaccine prototype ":

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Claims (4)

1. Strain "ASFV / CV60 / 2020" of African swine fever virus II genotype VIII serotype of the Asfarviridae family of the genus Asfivirus, deposited in the Collection of microorganism strains of the FGBI "ARRIAH" under the registration number: No. 334-dep / 20-122 - GKShM FGBU "ARRIAH" , to study immunity factors in pigs when creating a prototype vaccine against African swine fever. 2. The strain according to claim 1, characterized in that it was obtained during 60 serial passages in a CV-1 cell culture, the titer of virus accumulation is 7.5-8.0 lg HADE ₅₀ / cm ³ , is stable when passaged in cell culture CV-1 for 15 consecutive passages. 3. The strain according to claim 1, characterized in that it is obtained within 60 serial passages in a CV-1 cell culture with a residual virulence for pigs of not more than 12.5% with intramuscular infection, the infectious activity is 6.5-7.0 lg HADE ₅₀ / cm ³ . 4. The strain according to claim 1, characterized in that it induces in pigs the formation of post-infectious virus-specific antibodies detected in TF ELISA, the titer of which reached values in the range of 4.0-7.0 log ₂ / cm ³ .

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