RU2396977C1 - Tissue-culture dry goat-pox virus-vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. As an active substance, a virus-vaccine contains antigen material of attenuated "VNIISZH 2003" strain of goat-pox virus (GPV) Variola vims caprmum of Poxviridae fam., Chordopoxvirinae subfam., Capripoxvirus genus deposited in collection of FGU "VGNKI", VNIISZH 2003 registration No. (reference) - DEP, in the effective amount. The strain is reproduced in a Ch-91 cell culture for 5-7 days and is collected in titre 5.5 lg "ТЦД"₅₀/cm³ to 7.0 lg "ТЦД"₅₀/cm³. The strain preserves initial characteristics when passing the Ch-91 cell culture for 35 passages.

EFFECT: virus-vaccine protects immunised animals from contact infection by epizootic goat-pox virus circulating in the territory of Russia.

4 dwg, 8 cl, 7 tbl, 8 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of means for the specific prevention of goat smallpox.

Goat pox is a highly contagious viral disease that proceeds in a severe form, with characteristic clinical signs, often with a fatal outcome, especially among young animals. The economic damage caused by smallpox to goat breeding is extremely large. Direct losses consist of animal waste (death of young animals in severe cases of the disease can reach 50 ÷ 80%, and adult livestock - 5 ÷ 10%), reduced productivity - milk yield, quality and weight gain, loss of hair and down, reduced skin quality.

In accordance with the classification established by the International Committee on Taxonomy of Vimses (ICTV), the virus of goat pox (EQA) belongs to the genus 00.058.1.04 Capripoxvirus, belonging to the subfamily 00.058.1 Chordopoxvirinae of the family 00.058 Poxviridae [1].

The genus Capripoxvirus includes sheep pox virus (BOO), woks and cattle skin tuberculosis virus (nodular dermatitis virus (WB)). Representatives of the genus are closely related, which is proved by immunological analysis and gene structure. So wok is a closely related BOO, but taxonomically independent species. BOO and EQA do not have a clearly defined host specificity: despite the fact that viruses usually cause the most pronounced disease in either sheep or goats, some strains are pathogenic for both types of host animals [2, 3].

In this regard, in practice, as a rule, these two diseases are not differentiated, but are usually designated as a single disease - smallpox of sheep and goats. It is under this name that the disease of sheep and goat smallpox is listed in the list of the International Organization for Animal Health (OIE) diseases that are required to be declared, although this makes it difficult to analyze the real prevalence of sheep and goat smallpox as separate diseases.

According to the OIE, the disease is recorded on all continents, with the exception of Australia and Oceania. In recent years, smallpox has become widespread in countries where it has never been registered before (Vietnam, 2005; Mongolia, 2006; Greece, 2007).

The tense situation remains in the CIS countries of the Central Asian and Caucasian regions (Kazakhstan, Tajikistan, Kyrgyzstan, Azerbaijan, Georgia). In 1996-2003 55 countries were unsuccessful for this disease, including 7 CIS countries. In Tajikistan, for example, smallpox of goats was recorded as separate cases, in 2001 - already in 11 farms, and in 2002 - in almost all zones of the republic. At first, the disease was detected only in goats of the Angora breed, and in 2002 - in goats of local breeds.

For a long time, Russia has remained successful in this disease. In 2002, during the outbreak of sheep pox in the Far East, isolated cases of goat disease were noted, and in 2003 goat smallpox manifested itself as an independent disease.

Due to the fact that Russia has a common border and close economic ties with many disadvantaged smallpox states, the disease poses a great threat to our country regarding the possibility of introducing pathogens.

The current epizootic situation of sheep pox and goat pox indicates the need for highly effective diagnostic drugs and specific prophylaxis against both sheep pox and goat pox, which would quickly identify the causative agents of these diseases and quickly stop the spread of infections.

This circumstance forces us to constantly search for new EQA strains suitable for the manufacture of biological products for the diagnosis and specific prophylaxis of goat smallpox.

In countries where outbreaks of this disease are noted, specific prophylaxis of goat pox is carried out with the help of cultural virus vaccines from attenuated ERA strains and inactivated tissue and culture vaccines. However, inactivated vaccines are inferior to virus vaccines in immunogenic activity.

Known virus vaccine against goat pox, containing the active substance in the form of antigenic material from the attenuated strain “Durg” FOC, obtained in a sensitive biological system, and the target additive in the form of a stabilizer in an effective ratio [4].

Known virus vaccine against goat pox, containing the active substance in the form of antigenic material from the attenuated wok strain “GT ₄ -STV ₄₂ ” obtained in a sensitive biological system, and the target additive in the form of a stabilizer in an effective ratio [5].

Known virus vaccine against goat pox, containing the active substance in the form of antigenic material from the attenuated strain of Mysore wok obtained in a sensitive biological system, and the target additive in the form of a stabilizer in an effective ratio [6].

Known virus vaccine against goat pox, containing the active substance in the form of antigenic material from the attenuated strain "Gorgan" FOC obtained in a sensitive biological system, and the target additive in the form of a stabilizer in an effective ratio [7].

Also known is a strain of cultured goat gonad cells of domestic goat Capra hircus L. - producer of animal viruses. The authors of this invention determined the sensitivity of Ch-91 cells to infection with viruses of foot and mouth disease, Aujeszky's disease, Gumboro disease and African horse plague [8].

The closest to the invention according to the totality of existing signs is a dry culture virus vaccine against goat pox, containing the active substance in the form of antigenic material from the attenuated strain OK / A-04 of the FOC, reproduced in the culture of sheep kidney cells PO-VNIIVViM with the titer of infectious activity 5.5 ÷ 6.0 lg TCD ₅₀ / cm ³ and the target additive in the form of a lactose-peptone stabilizer in the ratio, wt.%, 50.0: 50.0 [9].

The disadvantages of the above virus vaccines, including the prototype virus vaccine, are that they provide effective protection of animals only from infection with a homologous virus.

The problem to which the present invention is directed is to obtain a dry vaccine virus vaccine against smallpox goats that creates effective protection of susceptible animals from epizootic wok circulating in the Russian Federation.

The technical result from the use of the present invention is to expand the arsenal of virus vaccines against smallpox goats cultured dry, with high antigenic and immunogenic activity and providing effective protection of susceptible animals from the epizootic pathogen of smallpox goats circulating in Russia.

The specified technical result was achieved by the creation of a virus vaccine against smallpox goats culture dry, characterized by the following set of features.

The proposed vaccine virus contains the active substance in the form of antigenic material from the attenuated strain "ARRIAH 2003" (copyright) wok reproduced in the culture of goat gonad cells Ch-91 with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ and the target the additive in the form of a stabilizer in the ratio, wt.%:

Antigenic material 80 ÷ 90 Stabilizer 10 ÷ 20.

The viral isolate, which served as the source for the attenuated wok strain ARRIAH 2003, was isolated in 2003 in the Primorsky Territory of the Russian Federation from goat pox patients.

To obtain a virus with optimal biotechnological properties, various cultivation systems were used, including cell lines: PO, Ch-91, PSGK, MDVK, SPEV, Vero, VNK-21 and Marc-145. In the process of adaptation, it was found that the homologous cultivation system, the transplantable line of goat gonad cells Ch-91, is highly sensitive to woks and turned out to be the optimal culture for growing the isolated virus. As a result of 35 consecutive passages of the isolate on the Ch-91 cell culture, an attenuated strain of ARRIAH 2003, a wok, with stable biotechnological, antigenic and immunogenic properties was obtained.

Depending on the dose of infection and the number of passages under conditions of stationary cultivation, the accumulation of the wok of the strain ARRIAH 2003 in the cell culture of Ch-91 was provided at a level of 6.0 to 7.5 lg TCD ₅₀ / cm ³ .

Compared with the original isolate, the strain "ARRIAH 2003" wok has genetic and phenotypic differences. According to the results of a comparative study of the immunobiological characteristics of various ERA strains, it was established that the ARRIAH 2003 strain is avirulent, has good antigenic and immunogenic activity, and protects against homologous epizootic virus infection control.

The advantage of the strain “ARRIAH 2003” of the wok as compared with the strain “OK / A-04” of the wok is the consistently high accumulation of the virus in the cell culture of Ch-91 and its high biological, antigenic and immunogenic activity.

The strain "ARRIAH 2003" wok was deposited June 6, 2006 in the All-Russian state collection of strains of microorganisms used in veterinary medicine and livestock, Federal state institution "All-Russian state center for the quality and standardization of medicines for animals and feed" (FGU "VGNKI") under registration number (link) - production vaccine strain "ARRIAH 2003" - DEP WOK.

For the manufacture of a vaccine against smallpox goats cultured dry as a sensitive biological system, a cell culture of Ch-91 with a well-formed monolayer of 2–3 days old in wedge mattresses is used.

As an active substance, a suspension of lyophilized or native EQA of the attenuated strain "ARRIAH 2003" with an infectious activity titer of 5.0 to 6.0 lg TCD ₅₀ / cm ^{3 is used} . To infect a culture of Ch-91 cells, a virus is used at a dose of 0.1 TCD ₅₀ / cell. The introduced viral suspension is exposed to a monolayer of Ch-91 cell culture in an incubator for an hour at (37 ± 0.5) ° С. The cultivation of the virus is carried out for 12 days at the indicated temperature. When the virus afflicts a monolayer area of at least 80% (rounding and the appearance of a light-refracting effect), the virus is collected.

Antigenic material from the attenuated strain ARRIAH 2003 "wok obtained in the culture of Ch-91 cells with an infectious activity titer of at least 5.5 lg TCD ₅₀ / cm ^{3 is} used to make a vaccine against smallpox goats.

Dry goat smallpox virus vaccine is obtained by mixing dry antigenic material from the attenuated strain “ARRIAH 2003” of the wok and the stabilizer in the ratio, wt.%, 80: 20 ÷ 90: 10, respectively. As a stabilizer, solutions of sucrose, gelatose and lactalbumin hydrolyzate are used.

The content of antigenic material and the stabilizer in the graft dose of the vaccine against smallpox goats cultured dry in the ratio, wt.%, 80: 20 ÷ 90: 10 is optimal, as it provides a tolerant presentation of the antigen in the body of the immunized animal.

The resulting virus vaccine is Packed in ampoules of 2.0 cm ³ or in vials of 2.0 cm ³ or 4.0 cm ³ , frozen at a temperature of minus 50 ° C and subjected to freeze drying.

The vaccine virus against smallpox goats culture dry, packaged in bottles or ampoules, is a homogeneous porous mass of a cylindrical shape of light yellow color, which dissolves within 1-1.5 minutes in saline. One ampoule contains 2.0 cm ³ , and in the bottle - 2.0 cm ³ or 4.0 cm ^{3 of} dry vaccine, which corresponds to 50 or 100 vaccinated doses of the drug for goats in the ampoule 50.

The resulting preparation is subjected to control on sterility, specificity and immunobiological activity in accordance with indicators established by the standard of the manufacturer.

The present invention includes the following set of essential features that provide a technical result in all cases for which legal protection is sought:

1. Virus vaccine against smallpox goats culture dry.

2. The active substance in the form of antigenic material from the attenuated strain "ARRIAH 2003" wok in an effective amount.

3. Targeted supplement.

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. Virus vaccine against smallpox goats culture dry.

2. The active substance.

3. Targeted supplement.

Compared with the prototype virus vaccine, an essential distinguishing feature of the proposed virus vaccine is that it contains the antigenic material from the attenuated strain ARRIAH 2003 ”of the wok as an active substance, with high antigenic and immunogenic activity, in an effective amount.

The present invention is also characterized by other distinctive features expressing specific forms of execution or special conditions for its use:

1. Antigenic material from the attenuated strain "ARRIAH 2003" wok reproduced in a cell culture of homologous origin.

2. Antigenic material from the attenuated strain "ARRIAH 2003" wok reproduced in the culture of goat gonad cells Ch-91.

3. Antigenic material from the attenuated strain "ARRIAH 2003" wok reproduced in the culture of Ch-91 cells with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ .

4. Antigenic material from the attenuated strain "ARRIAH 2003" wok reproduced in the culture of Ch-91 cells with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ , in the amount of 80-90 wt.%.

5. Of the targeted stabilizer additives.

6. The stabilizer in an amount of 10-20 wt.%.

7. Antigenic material from the attenuated strain "ARRIAH 2003" wok reproduced in the culture of Ch-91 cells with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ and a stabilizer in the ratio, wt.%:

Antigenic material 80 ÷ 90 Stabilizer up to 100.

The present invention extends the arsenal of virus vaccines against smallpox cultured dry, with high antigenic and immunogenic activity and providing effective protection of susceptible animals from the epizootic causative agent of smallpox goats circulating in Russia.

The achievement of the technical result from the use of the invention is explained by the fact that as an active substance it contains antigenic material from a new avirulent strain "ARRIAH 2003" wok with high antigenic and immunogenic activity.

The possibility of its use as a part of a virus vaccine against goat pox has been experimentally confirmed. The strain "ARRIAH 2003" VOK provides an effective virus vaccine against goat smallpox, which protects susceptible animals from the causative agent of goat smallpox circulating in Russia.

The invention is illustrated in the graphic materials on which:

figure 1 presents a graphical representation of the change in the titer of the wok strain of the strain "ARRIAH 2003" when it is passaged in cell culture Ch-91 (infection dose of 0.01 ÷ 0.05 TCD ₅₀ / cell);

figure 2 is a photographic image under an electron microscope of the characteristic morphology of the wok strain "ARRIAH 2003" (photo obtained by Ph.D. A.P. Ponomarev), magnification x76000;

figure 3 is a chromatogram of the results of species differentiation of capripox viruses obtained by multiplex PCR with species-specific primers, in which:

1 - negative control;

2 - marker (from bottom to top - 100, 200, 300, 400, 500, 600, 700 bp, etc.);

3 - BOO vaccine strain "ARRIAH";

4 - BOO, epizootic strain "Afghan";

5 - BOO, epizootic strain "Seaside";

6 - BOO, epizootic strain "EAO";

7 - BOO, epizootic strain "Tajik";

8 - EQA, the vaccine strain "ARRIAH 2003";

9 - wok, epizootic strain "Primorsky 2003";

10 - wok, epizootic strain "Pellor";

11 - WB;

12 - a mixture of BOO and wok;

13 - a mixture of BOO, wok and WB;

figure 4 is a chromatogram of the results of species differentiation of BOO and EQA obtained by the method of restriction of the gene "ankyrin-repeat" protein, on which:

1 - marker (from bottom to top - 100, 200, 300, 400, 500, 600, 700 bp, etc.);

2 - BOO vaccine strain "ARRIAH";

3 - BOO, epizootic strain "Afghan";

4 - BOO, epizootic strain "Seaside";

5 - BOO, epizootic strain "EAO";

6 - wok, vaccine strain "ARRIAH 2003";

7 - wok, epizootic strain "Seaside 2003";

8 - BOO, epizootic strain "Tajik";

9 - wok, epizootic strain "Pellor".

The strain "ARRIAH 2003" wok is characterized by the following features and properties.

Morphological features

The strain "ARRIAH 2003" FOCA belongs to the genus Capripoxvirus, the family Poxviridae and has morphological characteristics characteristic of FOCA.

The wok virion consists of a viral core (nucleoid), oval lateral bodies and the outer sheath. The nucleoid is surrounded by a smooth membrane with a thickness of 5 nm. Inside it is a nucleoprotein (DNA bound to a protein), which has an S - shaped or more complex shape. Trypsin-sensitive structures, called lateral bodies, squeeze the nucleoid, giving it the shape of a biconcave disk, which has the appearance of a dumbbell. The nucleoid and lateral bodies are surrounded by an outer shell consisting of lipids and tubular protein structures (10 × 5 nm).

The genome is double stranded DNA. Two DNA chains are connected by hairpin loops, as a result of which one continuous, covalently linked polynucleotide chain is formed. Flip-flop DNA forms (the so-called “hairpins”) are formed by inverted terminal repeats: identical but oppositely oriented DNA sequences, the length of which varies even within the same genus (Classification and nomenclature of vertebrate viruses, 2002).

Antigenic properties

According to its antigenic properties, the strain "ARRIAH 2003" refers to the wok.

The virus is stably neutralized by homologous antiserum in the neutralization reaction (PH) on a Ch-91 cell culture, in the diffusion precipitation reaction (RDP) and in the long-term complement fixation (RDSK) reaction. The strain exhibits antigenic activity in RDP 1: 16 ÷ 1: 32, in RDSK 1: 120 ÷ 1: 160 and in enzyme-linked immunosorbent assay (ELISA) 1: 1000 ÷ 1: 4000.

Tests of goat pox virus vaccine from the ARRIAH 2003 strain in laboratory conditions showed that the immunization of goats is accompanied by the formation of virus-neutralizing antibodies in the blood of animals in a titer of at least 2.0 log ₂ .

Biotechnological characteristics

The strain "ARRIAH 2003" wok shows high biological, antigenic and immunological activity in lyophilized form and in the form of a culture of virus-containing suspension. The strain is intended for the manufacture of vaccine and diagnostic products. The virus of the ARRIAH strain 2003 is reproduced in monolayer cell cultures: the primary trypsinized sheep kidney cell culture and the transplanted Ch-91 cell culture and accumulate at least 5.5 lg TCD ₅₀ / cm ³ in the titer of 5–7 days of incubation. It retains its original characteristics when passaged in sensitive biological systems for 35 passages.

Genotaxonomic characteristic

The EQA genome is not infectious and is represented by a linear double-stranded DNA molecule with a central less variable region of about 145 kb in length and more variable ends.

Physical properties

Electron microscopy of the strain "ARRIAH 2003" EQA revealed viral particles, the structure and size of which are characteristic of capricox viruses.

Smallpox virions have linear double-stranded DNA with a length of 45 ÷ 60 μm, a molecular weight of 80 ÷ 240 MDa. The floating density of the DNA of smallpox viruses in cesium chloride and sucrose is equal to 1,676 ÷ 1,723 g / cm ³ . An important feature of smallpox virus DNA is the unusually strong structure of the double-stranded molecule. Both strands of the DNA molecule are covalently linked together.

Resistance to external factors

The wok of the strain “ARRIAH 2003” is resistant to drying, ether, not resistant to high temperatures, various chemicals, such as formalin (0.5-1%), weak solutions of iodine (1: 10000), hydrochloric, sulfuric acid (2, 5%), alcohol, its infectious activity persists at pH 6.0 ÷ 7.4.

Additional features and properties

Immunogenic activity - immunogen as part of a live lyophilized vaccine.

Reactogenicity - at the site of subcutaneous administration of the native virus in a volume of 5 cm ³ , a skin cord up to 4 cm long is formed.

Pathogenicity - when administered intramuscularly to animals, the disease does not cause. When testing a goatpox virus vaccine from the ARRIAH 2003 strain on goats, it was found that it did not provoke a local response to subcutaneous administration in a volume of 5 cm ³ (125 vaccination doses), was harmless and areactogenic to goats.

Virulence - avirulent for small ruminants with contact, aerosol and parenteral infection.

Antigenic properties - the virus induces the formation of virus-neutralizing antibodies in goats and sheep.

Stability of biological properties. It retains its original characteristics when passaged on a culture of gonadal cells of the goat Ch-91 for 35 passages. The strain is irreversible for 5 passages on goats.

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

Example 1

The viral isolate, which served as a source for obtaining the strain "ARRIAH 2003", was isolated in 2003 from patients with smallpox goats in the Primorsky Territory of the Russian Federation. The preparation of the material was carried out by the conventional method.

Biological and virological methods included adaptation of the virus isolated from diseased goats to cultures of primarily trypsinized and transplanted cell lines and its attenuation. For the growth of woks, different cell culture lines were tested: PO, Ch-91, PSGK, MDBK, SPEV, Vero, BHK - 21, Marc-145. In the process of adaptation, it was found that the transplanted Ch-91 cell line is highly sensitive to EQA and turned out to be the optimal culture for obtaining a seed strain for the manufacture of a virus vaccine.

Subsequently, to obtain a brood, the viral suspension was introduced onto a monolayer of cell culture freed from the growth medium and was exposed for an hour in an incubator at a temperature of (37 ± 0.5) ° С. After that, a support medium PSP or Needle was added with the addition of 1 ÷ 2% of serum of cattle. The infected culture was incubated at (37 ± 0.5) ° C until the appearance of the virus CPD, which was characterized by rounding of cells, their accumulation in the form of groups of 10 ÷ 20 cells and further total destruction of the cell monolayer. When the monolayer area was affected by at least 70–80% (rounding and the appearance of a light-refracting effect, peeling off the glass) by 3-fold freezing of the culture mattresses, the virus was collected, followed by sampling to exclude microbial contamination and determine the infection activity of the virus by titration in Ch-91 cell culture .

The isolated virus had a cytopathic effect on the Ch-91 cell culture with an initial titer of 3.0 ÷ 4.0 lg TCD ₅₀ / cm ³ . In subsequent passages on the same cell culture, the virus titer increased in the 6-7th passages to 5.0 ÷ 5.5 lg TCD ₅₀ / cm ³ , in the 8 ÷ 10th passages to 6.0 lg TCD ₅₀ / cm ³ .

Depending on the dose of infection, which comprised 0.01 ÷ 1.0 TCD ₅₀ / cell, the accumulation of the attenuated strain ARRIAH-2003 "FOC in the cell culture Ch-91 was provided at the level of 5.33 ÷ 7.17 lg TCD ₅₀ / cm ³ for 4 ÷ 7 days at 50 ÷ 90% damage to the cell monolayer. The optimal infectious dose of the virus was considered to be 0.01 ÷ 0.05 TCD ₅₀ / cell, which, during stationary cultivation for 5-7 days, virus accumulation in the titer from 6.67 ± 0.08 to 7.17 ± 0.10 lg TCD ₅₀ / cm ³ at 80 ÷ 90% damage to the cell monolayer. The research results are presented in table 1. The stability of the cultural properties of the lyophilized virus when determining the degree of its accumulation during 35 passages in the Ch-91 cell culture at an infectious dose of 0.01-0.05 TCD ₅₀ / cell is presented in figure 1.

From the data presented in figure 1, it follows that in the process of passage under conditions of stationary cultivation of the wok with an infection dose of 0.01-0.05 TCD ₅₀ / cell infectious activity at the level of 6.0 ÷ 7.50 lg TCD ₅₀ / cm ³ was noted during the first 13 passages. With an increase in the number of passages, the infectious activity of the virus gradually decreased. The results of the study of the timing of the cultivation of EQA in cell culture Ch-91 are presented in table 2.

Infectious activity of the strain "ARRIAH-2003" wok at a dose of infection of 0.01 ÷ 0.05 TCD ₅₀ / CL depended on the number of passages. At the level of 8–13 passages, the duration of wok cultivation did not exceed 7 days. During this time, the most pronounced (80–90%) virus CPD and its maximum accumulation in the Ch-91 cell culture were achieved. Subsequently, virus accumulation was established at a lower level and the infectious activity of the virus of the following passages was characterized by lower values with an increase in the duration of cultivation.

As a result of 35 consecutive passages of the selected EQA on the Ch-91 cell culture, an attenuated strain “ARRIAH 2003” (author's name) was obtained, which has stable biotechnological, antigenic and immunogenic properties that allow it to be used to obtain antigenic material in the manufacture of diagnosticums and anti-virus vaccines smallpox goats.

Subsequently, the Ch-91 cell culture was used to produce wok seedlings in the manufacture of a virus vaccine.

The resulting wok strain was tested in laboratory and then under epizootic conditions. As a result of the studies established the following:

1. The sterility test was carried out by seeding samples of virus-containing liquid on bacterial nutrient medium MPB, MPA, MPPB, Saburo agar (liquid) and thioglycol medium, as well as Edward or Marten medium to detect mycoplasmas. Bacterial or fungal contamination of the strain “ARRIAH 2003” wok was absent, as well as contamination with other viruses.

2. When electron microscopy of the strain "ARRIAH 2003" EQA were detected viral particles, the structure and size of which are characteristic of capripox viruses (figure 2).

3. The species affiliation of the strain "ARRIAH 2003" wok is confirmed by two molecular genetic methods: multiplex PCR with species-specific primers (figure 3) and restriction analysis of the gene fragment "ankyrin-repeat" protein (figure 4).

4. The specificity of the strain "ARRIAH 2003" wok was confirmed in the RDP, RDSK and ELISA.

The resulting strain was deposited June 6, 2006 in the All-Russian State Collection of microorganism strains used in veterinary medicine and animal husbandry, FGU "VGNKI" under the registration number (link) - production vaccine strain "ARRIAH 2003" - DEP WOK.

Example 2

During the development of multiplex PCR for the indication and species differentiation of capripoxviruses as a result of the analysis of the complete nucleotide sequences of the capripoxvirus genome presented in the GenBank database, species-specific regions for BOO, EQA and WB were established. Based on these data, species-specific primers S1 and S2 were designed for BOO, G1 and G2 for FOC, L1 and L2 for WB. Species-specific primers were designed so that the amplicons synthesized with their participation had different lengths: 415 bp for wok, 311 bp for BOO and 254 bp for WB. This made it possible to use all three pairs of primers in one reaction and to distinguish mPCR products by size using agarose gel electrophoresis.

During the species differentiation of capricoxviruses, 11 samples of virus-containing cell cultures with the VNIIZH vaccine strain and with the epizootic strains Afghan, Primorsky, EAO BOO, with the VNIIZH 2003 vaccine strain and with epizootic strains Primorsky 2003 were studied. , "Pellor" wok, with WB, as well as with isolates "EAO" and "Tajik" in the form of a suspension of skin of sick animals.

Figure 3 shows the results of PCR with species-specific primers. In samples containing the DNA of the ARRIAH strain 2003, a 415 bp fragment was synthesized, the amplification of which involved species-specific primers for EQA.

Using the restriction analysis method, one can also differentiate BOO and EQA and determine the strain BOO. Using genus-specific primers, an amplification of the 564 bp protein section of the ankyrin-repeat gene is performed. Then amplifications are treated with restriction enzyme Dra I. Within the amplified fragment of the genome of epizootic isolates of BOO, there is one restriction site of Dra I, vaccine strains of BOO have two sites, and FOCs do not. Analysis of restriction products on an agarose or polyacrylamide gel makes it easy to distinguish between BOO and EQA and vaccine strains of BOO from epizootic isolates by a characteristic set of DNA fragments.

When this method was carried out, in the ARRIAH vaccine strain BOO, the amplicon was split into three fragments as a result of restriction enzyme treatment with Dra I, in all the epizootic isolates BOO, it was split into two fragments, and in the vaccine strain ARRIAH 2003, the wok and two epizootic wok isolates of the amplicon did not occurred (figure 4).

Thus, when the ankyrin-repeat gene fragment of the protein of the ARRIAH 2003 strain was processed by the Don I endonuclease, no DNA cleavage was observed, indicating that the strain belongs to the wok.

Additionally, the specificity of the strain "ARRIAH 2003" wok was confirmed by serological methods in the RDP and RDSK. RDP and RDSK were positive only between a specific virus with homologous serum (Table 3, Table 4).

Example 3

The technological process for the production of goatpox virus vaccines includes two stages: obtaining a seed wok and manufacturing a series of virus vaccines.

To obtain the inoculum virus, a Ch-91 cell culture is used with a well-formed monolayer of 2–3 days old in wedge mattresses and a suspension of lyophilized or native wok strain “ARRIAH 2003” with an infectious titer of 5.0 to 6.0 lg TCD ₅₀ / cm ³ . The average infectious dose of the virus is 0.1 MTC ₅₀ / cl. The introduced viral suspension is exposed to a monolayer pre-washed from the growth medium residues with a Hanks solution in an incubator for an hour at (37 ± 0.5) ° С, then the supporting nutrient medium PSP is added in the required volume. The cultivation of the virus is carried out for 12 days at the indicated temperature.

If the virus affects the monolayer area of at least 80% (rounding and the appearance of a light-refracting effect), the virus is collected. To do this, the contents of the wedge mattresses are frozen at a temperature of minus 40 ° C and thawing is carried out at room temperature, shaking vigorously.

Forty liters of virus-containing suspension are needed to receive one million doses of the vaccine.

Goat smallpox virus vaccine is obtained by mixing the obtained antigenic material from the ARIH strain 2003 of the wok reproduced in the Ch-91 cell culture with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ and a stabilizer in the ratio, wt.%: 80 : 20 ÷ 90: 10 respectively. As a stabilizer, solutions of sucrose, gelatose and lactalbumin hydrolyzate are used.

From the obtained antigenic material, 2 series of goatpox virus vaccine are prepared, which differ in the percentage of stabilizer in the preparation.

Series No. 1 of a virus vaccine against smallpox goats from the attenuated strain "ARRIAH 2003" wok contains, wt.%:

Antigenic material 80.0 Stabilizer 20.0.

Series No. 2 of a virus vaccine against smallpox goats from the attenuated strain "ARRIAH 2003" wok contains, wt.%:

Antigenic material 90.0 Stabilizer 10.0

The resulting virus vaccine is Packed in 2.0 cm ³ ampoules or in bottles of 2.0 cm ³ or 4.0 cm ³ , frozen at a temperature of minus 50 ° C and subjected to freeze drying. After sublimation, the vials (ampoules) are filled with dry sterile air or inert gas, closed with rubber stoppers and sealed with metal caps, the ampoules are sealed.

The vaccine virus against smallpox goats culture dry, packaged in bottles or ampoules, is a homogeneous porous mass of a cylindrical shape of light yellow color, dissolved within 1 ÷ 1.5 minutes in saline. One ampoule contains 2.0 cm ³ , and in a vial of 2.0 cm ³ or 4.0 cm ^{3 of a} dry vaccine containing, respectively, in an ampoule of 50 and in a bottle of 50 or 100 vaccinated doses of the drug for goats.

The resulting preparation is subjected to control for sterility, specificity and immunogenic activity in accordance with indicators established by the standard of the manufacturer.

Example 4

The infectious activity of the strain “ARRIAH 2003” EQA in the virus vaccine after lyophilization is determined by titration of ten-fold dilutions of the viral suspension in a culture of Ch-91 cells in vials. Analysis of the results is carried out within 12 days by the presence of a cytopathic effect. The results of titration of 2 series of vaccines are presented in table 5.

The virus titer in the vaccine is defined in the values from 5.11 ± 0.09 to 5.41 ± 0.15 log TCD ₅₀ / cm ³ . When stored for 1.0 ÷ 1.5 years, the titer of the virus decreased by no more than 0.5 lg TCD ₅₀ / cm ³ .

However, it should be noted that storing the vaccine for 2–3 weeks at room temperature reduced the infectious activity of the wok by 1.0–1.5 lg TCD ₅₀ / cm ³ . Therefore, the transport of goatpox virus vaccines should be carried out at low temperatures.

Example 5

To determine the harmlessness and reactogenicity, the goat pox virus vaccine from the ARRIAH strain 2003 was administered to the goats at 5.0 cm ³ in the initial dilution (125 vaccination doses) subcutaneously in the area of the hairless area of the inner surface of the forelimb. The animals were monitored for 21 days.

When examining the vaccine for harmlessness and reactogenicity in isolated cases in animals at the injection site, the initial dilution established the formation of inflammatory edema on the day 4–6 days in the form of a small strand with a size of 1 to 3 cm, which disappeared 3–14 days after the appearance did not affect the clinical condition of animals. Throughout the entire observation period (21 days), the goat body temperature remained within the physiological norm. This allowed us to conclude that the vaccine against goat pox from the attenuated strain "ARRIAH 2003" wok is a harmless and non-reactive drug.

Example 6

The antigenic activity of the virus vaccine against goat pox from the strain "ARRIAH 2003" series No. 1 and No. 2 was determined on the 7th, 14th and 21st day after immunization of goats with various dilutions of the drug. Blood samples were taken from animals at the indicated times in order to obtain blood serum, which was studied in a neutralization reaction (PH) in a Ch-91 cell culture for the presence of virus-neutralizing antibodies (VNA) with a constant dose of VOK 100 TCD ₅₀ / cm ³ and serial double dilutions of serum from 1: 2 to 1:32. The results were taken into account for 12 days with a change in the supporting medium after 3-4 days. The antibody titer was determined by the method of Reed and Mench and expressed in logarithms with base 2 (log ₂ ).

The level of antibodies in the blood of immunized animals depended on the dose of the vaccine administered and the timing after vaccination (tables 6 and 7). So, the VNA titer after administration of vaccine of series No. 1 in a volume of 5 cm ³ in the initial dilution of 125 vaccination doses, on the 7th day was 2.34 ± 0.46 log ₂ , on the 14th day - 2.53 ± 0.19 log ₂ and on the 21st day - 3.00 ± 0.50 log ₂ , in one vaccination dose - 1.50 ± 0.31, 2.02 ± 0.30 and 2.50 ± 0.12 log ₂ respectively. In all cases, the presence of the maximum level of antibodies was established on the 21st day after immunization of animals. After immunization at a dose of 0.00001 LDPE (vaccine dilution 1: 100000), the presence of a low antibody titer was found in animal blood compared to other dilutions, which corresponded to 21.20 ± 0.15 log ₂ on the 21st day.

After the introduction of the vaccine of series No. 2 on the 7th day, the titers of antibodies in the blood of goats vaccinated in doses from 10 ² to 10 ⁴ TCD ₅₀ and in their entire form were in the range from 1.08 ± 0.11 to 1.63 ± 0 , 20 log ₂ ; in doses of 10 and 1 TCD ₅₀ from 0.20 ± 0.18 to 0.63 ± 0.32 log _2, respectively. On the 14th day, the activity of the blood serum of goats vaccinated in the indicated doses ranged from 0.75 ± 0.35 to 1.96 ± 0.19 log ₂ . On the 21st day after immunization of goats with a vaccine in whole form, the antibody titer in the blood of animals reached 2.33 ± 0.9, at a dilution of 1: 25-2.54 ± 0.22; 1: 100-1.83 ± 0.24; 1: 1000-1.75 ± 0.12; 1: 10000-1.36 ± 0.13 and 1: 100000-1.13 ± 0.18 log ₂ .

Example 7

The immunogenic activity of the virus vaccine against smallpox goats from the strain "ARRIAH 2003" was tested by control infection. On the 21st day after immunization, the animals vaccinated with various dilutions of the preparation of series No. 1 and series No. 2, as well as control goats, were injected intracutaneously in the area of the papillary fold with a virulent strain “Primorsky 2003” FOC at a dose of 1000 ID ₅₀ / cm ³ . After infection, animals were observed for 14 days with daily thermometry and inspection.

During the period of immunization and after control infection, the body temperature of goats immunized with various doses of goat pox virus vaccine remained at the physiological norm. Control infected animals fell ill with smallpox in a generalized form and died 8-10 days after infection.

The results of the study of the immunogenic activity of the virus vaccine against smallpox goats from the strain "ARRIAH 2003" are presented in tables 6 and 7.

Although various specific activity of blood serum of animals immunized with various doses virusvaktsiny (10 ⁴ to 1 TCD _50), all the goats were protected against smallpox in experimental infection with virulent "Seaside 2003" POC at a lethal dose (1000 ID _50/0 5 cm ³ ).

Thus, data were obtained that testify to the disease immunity of animals vaccinated with the vaccine against goat pox from the strain "ARRIAH 2003".

Example 8

Tests of the effectiveness of the vaccine against smallpox goats cultured dry (series No. 3) from the strain "ARRIAH 2003", obtained as described in example 3, and containing, wt.%:

Antigenic material 80.0 Stabilizer 20.0

in threatened farms in several areas of the Republic of Tajikistan in connection with the occurrence of smallpox goats.

A total of 5,000 goats were immunized with goat pox virus vaccine. Goats were immunized in accordance with the instructions for use of the drug. After application of the vaccine, no clinical complications were observed in animals. Vaccination of goats in smallpox-threatened farms ensured the formation of active immunity in animals and their full protection against smallpox. There were no cases of breakthrough immunity after immunization of animals.

To determine the level of post-vaccination immunity from 10 animals for each of the animal groups before immunization, as well as 10, 20 and 30 days after vaccination, serum samples were sent that were sent to the Federal State Institution “ARRIAH”.

It was found that the virus vaccine against goat pox from the ARRIAH 2003 strain, used in threatened smallpox farms in the Republic of Tajikistan, had high immunobiological activity, was harmless to animals and protected vaccinated animals from smallpox.

Information sources

1. http://www.virustaxonomyonline.com.

2. Kitching R.P. The control of sheep and goats pox // Rev. Sci. Tech. Off. Int. Epiz. - 1986. - V.5, No. 2. - R. 503-511.

3. Gershon P.O., Black D.N. A comparison of the genomes of capripoxvirus isolates of sheeps, goats and cattle // Virology. - 1988. - V.164. - P.341-349.

4. Joshi R.K. Physico-chemical characterization of "Durg" isolate of goat pox virus // Indian Vet. J. - 2001. - V.78, No. 5. - P.369-370.

5. Wang Shaohua et al. The pathomorphological studies on immunological effect of attenuated goat pox cellular virus // Acta vet. Zootechn. Sinica. - 1988. - V.19, No. 2. - P.111-116.

6. Kitching RP, Cam VM Sheep pox and goat pox // OIE Manual of standarts for diagnostic test and vaccines; 4 ^th edn. - Paris: World organization for animal health, 2000. - P.168-177.

7. Tulman E.R. et al. The genomes of pox and goat pox viruses // J.of Virol. - 2000. - V.76, No. 12. - R. 6054-6061.

8. Pat. RF №2061753; C12N 5/06, 7/00; 06/10/1996

9. Grachev D.V. Immunobiological properties of goat pox virus isolated in the Republic of Tajikistan // Abstract. diss. for the competition step. Cand. vet. sciences. - Pokrov, 2006 .-- 26 p. (prototype).

Table 1 Influence of the dose of infection on the reproduction of the wok strain of ARRIAH 2003 in the culture of goat gonad cells Ch-91 Dose (TCD ₅₀ / cell) The cultivation period (days) The lesion area of the cell monolayer (%) Virus titer (lg TCD ₅₀ / cm ³ ) 0.01 6 ÷ 7 80 ÷ 90 7.17 ± 0.10 0.05 5 ÷ 6 80 ÷ 90 6.67 ± 0.08 0.1 5 ÷ 6 60 ÷ 70 6.17 ± 0.72 one 4 ÷ 5 50 ÷ 60 5.33 ± 0.17

table 2 The biological activity of the strain "ARRIAH 2003" wok in the culture of Ch-91 cells depending on the number of passages and the timing of cultivation in stationary conditions Passage number The virus titer (log TCD ₅₀ / cm ³ ) at a dose of infection of 0.01-0.05 TCD ₅₀ / CL Cultivation days one 2 four 5 6 8 8 2,50 ± 0,25 3.25 ± 0.23 4.75 ± 0.25 5.25 ± 0.13 6.75 ± 0.16 6.33 ± 0.23 9 3.00 ± 0.11 4.00 ± 0.22 5.50 ± 0.25 6.00 ± 0.28 6.66 ± 0.15 6.25 ± 0.61 10 2.33 ± 0.13 2.59 ± 0.18 3,50 ± 0,40 5.55 ± 0.34 6.55 ± 0.25 6.00 ± 0.23 13 2.00 ± 0.31 2.33 ± 0.13 3.75 ± 0.41 4.25 ± 0.28 6.00 ± 0.31 5.50 ± 0.25 fourteen 1.50 + 0.31 2.03 + 0.28 3.03 ± 0.30 4.25 ± 0.43 5.33 ± 0.14 5.77 ± 0.20 19 1.00 ± 0.12 1.51 ± 0.35 2,50 ± 0,24 3.51 ± 0.45 4.50 ± 0.26 5.00 ± 0.25

Table 4 Differentiation of BOO and EQA in RDSK Name of material Activity in RDSK SSBOK SSBOO organ-tissue suspension wok strain "ARRIAH 2003" 1: 30 ÷ 1: 240 1:10 wok concentrated antigen 1: 60 ÷ 1: 320 1:10 organ-tissue suspension BOO strain "ARRIAH" nonspec. 1:40 Concentrated BOO Antigen nonspec. 1:40

Table 5 The biological activity of the virus vaccine against smallpox goats from the strain "ARRIAH 2003" Series No. Repetition Vaccine dilutions Title, TCD ₅₀ / cm ³ The average titer, TCD ₅₀ / cm ³ 10 ^-4 10 ^-5 10 ^-6 10 ^-7 Series No. 1 one ++ ++ ++ - - - - - 5,0 5.11 ± 0.09 2 ++ + - ++ + - - - - - 5.17 3 ++ + - ++ + - ++ + - - - 5.17 Series No. 2 one ++ ++ ++ - + - - - - 5.23 5.41 ± 0.15 2 ++ ++ ++ + - + - - - - 5.50 3 ++ ++ ++ + - + - - - - 5.50

Table 6 Antigenic and immunogenic activity of the virus vaccine against smallpox goats culture dry from strain "ARRIAH 2003" series No. 1 No. p / p Vaccine Dilution and Dose Number of animals VNA titer, log ₂ Infection result before vaccination after 7 days after 14 days after 21 days one One-piece, 5 cm ³ 2 - 1.42 ± 0.59 1.92 ± 0.59 2.33 ± 0.95 2/0 2 1:25 (10 ⁴ TCD ₅₀ ) 7 - 1.63 ± 0.20 1.96 ± 0.19 2.54 ± 0.22 7/0 3 1: 100 (10 ³ TCD ₅₀ ) 3 - 1.22 ± 0.42 1.53 ± 0.22 1.83 ± 0.24 3/0 four 1: 1000 (10 ² TCD ₅₀ ) 2 - 1.08 ± 0.11 1.42 ± 0.14 1.75 ± 0.12 2/0 5 1: 10000 (10 TCD ₅₀ ) four - 0.63 ± 0.32 1.08 ± 0.24 1.36 ± 0.13 4/0 6 1: 100000 (1 TCD ₅₀ ) 2 - 0.20 ± 0.18 0.75 ± 0.35 1.13 ± 0.18 2/0 7 The control 10 - - - - 10/10 Note: “-” - serum is not active. In the column “infection result” - in the numerator - the number of animals in the experiment, in the denominator - the number of sick animals.

Table 7 Antigenic and immunogenic activity of the virus vaccine against smallpox goats culture dry from strain "ARRIAH 2003" series No. 2 No. p / p Vaccine Dilution and Dose Number of animals VNA titer, log2 Infection result after 7 days after 14 days after 21 days one One-piece, 5 cm ³ four 2.34 ± 0.46 2.53 ± 0.19 3.00 ± 0.50 0/4 2 1:25 (10 ⁴ TCD ₅₀ ) 7 1,50 ± 0,31 2.02 ± 0.30 2,50 ± 0,12 0/7 3 1: 100 (10 ³ TCD ₅₀ ) 3 1.18 ± 0.42 1.52 ± 0.15 1.97 ± 0.15 0/3 four 1: 1000 (10 ² TCD ₅₀ ) 2 1.12 ± 0.23 1.41 ± 0.20 1.75 ± 0.12 0/2 5 1: 10000 (10 TCD ₅₀ ) 3 0.56 ± 0.10 0.94 ± 0.36 1.45 ± 0.27 0/3 6 1: 100000 (1 TCD ₅₀ ) 2 0.26 ± 0.12 0.79 ± 0.15 1.20 ± 0.15 0/2 7 The control 2 - . - 2/2 Note: “-” - serum is not active. In the column “infection result” - in the numerator - the number of animals in the experiment, in the denominator - the number of sick animals

Claims (8)

1. The vaccine virus against goat pox is culture dry, containing the active substance and the target additive, characterized in that it contains antigenic material from the attenuated strain "ARRIAH 2003" goat smallpox virus (VOI) Variola vims caprmum family. Poxviridae, subfamily. Chordopoxvirinae, of the genus Capripoxvirus, deposited in the collection of FGU VGNKI under the registration number (link) VNIIZZh 2003 - DEPT, in an effective amount. 2. The vaccine virus according to claim 1, characterized in that it contains antigenic material from the attenuated strain "ARRIAH 2003" wok, reproduced in a cell culture of homologous origin. 3. The vaccine virus according to claim 2, characterized in that it contains antigenic material from the attenuated wok strain ARRIAH 2003 ”of the wok, reproduced in the goat gonad cell culture Ch-91. 4. The vaccine virus according to claim 3, characterized in that it contains antigenic material from an attenuated wok strain "ARRIAH 2003" wok reproduced in the culture of goat gonad cells Ch-91 with an infectious activity of at least 5.5 lg TCD ₅₀ / cm ³ . 5. The vaccine virus according to claim 4, characterized in that it contains antigenic material from the attenuated wok strain ARRIAH 2003 ”in an amount of 80-90 wt.%. 6. The vaccine virus according to claim 1, characterized in that it contains a stabilizer from the target additives. 7. The vaccine virus according to claim 6, characterized in that from the target additives it contains a stabilizer in an amount of 10-20 wt.%. 8. The vaccine virus according to any one of claims 1 to 7, characterized in that it contains antigenic material from the attenuated strain "ARRIAH 2003" of the wok and the stabilizer in the ratio, wt.%:
Antigenic material 80-90 Stabilizer Up to 100

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