RU2412238C2 - Antiviral medication (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to novel antiviral medication, possessing antiviral activity with respect to highly pathogenic virus of avian influenza A/chicken/Kurgan/05/2005 (A/H5N1). Claimed antiviral medication represents culture fluid or lysate of cells of Bacillus thuringiensis strain FSIS SRC VB "Vector" B-1069 or Bacillus thuringiensis strain FSIS SRC VB "Vector" B-1073 or strain of Bacillus thuringiensis FSIS SRC VB "Vector" B-1091.

EFFECT: invention makes it possible to increase efficiency of antiviral medications.

3 cl, 7 ex

Description

The invention relates to the microbiological industry and biotechnology, and relates to a new antiviral agent with antiviral activity, in particular, relatively highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1).

Due to the wide spread of viral diseases caused by the human virus, as well as the bird flu virus (A / H5N1), the search for antiviral agents, an in-depth study of viral infections and the creation of drugs aimed at suppressing them are of particular relevance.

It is known that bacteria of the species Bacillus thuringiensis, capable of forming paraspore crystalline inclusions of protein nature [1], have a selective antagonistic effect against microorganisms [2, 3]. The antiviral activity of Bt strains against phytopathogens, pests of agricultural plants is also known [4].

Known tool based on a consortium of microorganisms, including a strain of Bacillus licheniformis, strains belonging to the genera Saccharomycetes, Azotobacteria, strains of P. bacteria and Bacillus thuringiensis, which has antiviral activity (Chinese patent No. 1274702, IPC C12N 1/00, publ. 29.11 .2000).

Known tool based on a strain of VNPB 17-3 bacteria Bacillus thuringiensis (US patent No. 6528480, IPC A01N 63/00, publ. 04.03.2003), containing a protein toxin with antiviral effect against the tobacco mosaic virus.

The closest analogue (prototype) is a tool based on strains of Bacillus thuringiensis, with the author's name respectively Gi-47, Gi-535, Dg-992, which has antimicrobial and insecticidal activity (Andreeva I. S. Features of the antimicrobial and insecticidal activity of strains of Bacillus thuringiensis isolated from the soil of the geyser valley (Kamchatka) Abstracts of the International Scientific Conference "Microorganisms and the Biosphere", November 19-20, 2007, Moscow. - 2007, p.5-7).

However, all of the above analogues and prototype do not have an inhibitory effect against the highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1).

The technical result of the invention is the creation of 3 variants of a new antiviral agent having inhibitory activity against avian influenza virus A / H5N1.

The specified technical result is achieved in that the antiviral agent is a suspension of metabolic products of the bacterial strain Bacillus thuringiensis B-1069, which has inhibitory activity against avian influenza virus A / H5N1.

The specified technical result is also achieved by the fact that the antiviral agent is a suspension of metabolic products of the bacterial strain Bacillus thuringiensis B-1073, which has inhibitory activity against the avian influenza virus A / H5N1.

The specified technical result is also achieved by the fact that the antiviral agent is a suspension of metabolic products of the bacterial strain Bacillus thuringiensis B-1091, which has inhibitory activity against avian influenza virus A / H5N1.

B. thuringiensis strains, whose metabolic products possess antiviral properties, were deposited in the microorganism collection of the Federal State Institution of Science “State Scientific Center for Virology and Biotechnology“ Vector ”of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (FGUN SSC VB“ Vector ”of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare) under collection numbers: B. thuringiensis B-1091 , D. thuringiensis B-1069, B. thuringiensis B-1073.

Certificates of deposit of strains are attached.

The properties of the metabolic products of strains of B. thuringiensis B-1091, B. thuringiensis B-1069, B. thuringiensis B-1073 with activity against the avian influenza A / H5N1 virus were discovered for the first time, in connection with which we can conclude that the proposed technical solutions meet the criteria inventions of “novelty” and “inventive step”.

Characterization of strains

Crystal-forming, endospore-containing bacteria B. thuringiensis B-1091, B. thuringiensis B-1069, B. thuringiensis B-1073, were isolated by plating on complete agarized media of various samples taken in the Valley of Geysers (Kamchatka). Strains have some differences from the typical strains of B. thuringiensis, consisting in the absence or weakly expressed insecticidal activity and a number of morphological characteristics.

Source of isolation and morphology of strains

Strain B. thuringiensis B-1091 was isolated from a soil sample when it was seeded on a complete agarized fish-peptone agar medium (RPA, pH 7.0, FSUE NPO Microgen of the Ministry of Health of the Russian Federation) and incubated seeding at a temperature of 28-30 ° C. The strain in a dense environment forms a moist, slimy, whitish, tall, opaque, shiny colony. The cells of the strain are rod-shaped, 1 × 2-4 μm in size, arranged in 1-2 or in chains; paraspore inclusions have a rounded shape, fit snugly to an elliptical spore, the diameter of the spore and crystal are close in size.

Strain B. thuringiensis B-1069 was isolated from a water sample from a hot spring in the Valley of Geysers when it was plated on a complete agarized RPA medium (pH 9.0). On an agar medium, the strain forms opaque, fine-grained, opaque, whitish, and humid colonies. The cells of the strain are rod-shaped, 1 × 2-8 μm in size, straight or slightly curved, located 1-2 or in chains, form paraspore inclusions of a rounded or slightly angular shape, tightly adjacent to the elliptical spore in the form of a mushroom cap, significantly exceeding its diameter, the central axis of the spores and crystal coincide.

Strain B. thuringiensis B-1073 was isolated from a sample of sludge from a hot spring in the Valley of Geysers when it was plated on RPA agar medium (1:10, pH 5.0). On an agar medium, the strain forms moist, slimy, whitish, tall, opaque, shiny colonies. The cells of the strain are rod-shaped, 1-1.2 × 2-8 μm in size, straight or slightly curved, arranged in 1-2 or in chains, round-shaped parasporal inclusions tightly fit to the elliptical spore, the crystal arrangement on the spore is free, the crystal size, as a rule, exceeds the diameter of the spores.

Biochemical signs of strains

It was found that all the studied strains are capable of anaerobic growth, form acid from glucose and maltose, but not from arabinose, xylose, mannitol and lactose, do not form indole, do not deaminate phenylalanine, do not possess arginine decarboxylase, do not utilize citrate. Strains produce catalase, lecithinase, have lipase, gelatinase activity, grow in the presence of 0.001% lysozyme. Strain B. thuringiensis B-1069 is negative in amylase, strains of B. thuringiensis B-1091 and B-1073 have amylolytic activity. It is important to note that the studied strains of B. thuringiensis Geyser Valley B-1091, B-1069, B-1073 according to the results of indirect tests for pathogenicity did not have hemolytic, fibrinolytic and plasmocoagulase activities.

Strain storage

Subcultures of the strains were stored by periodic passages on agar medium A (0.7% Difco peptone, 0.4% fish hydrolyzate, 0.5% NaCl, 1.7% agar, pH 7.0-7.2) and 15 % glycerol solution at low temperature freezing at minus 65 ° C [5].

Inoculum is obtained by growing strains on liquid or agarized media: LB (0.25% LB "Difco"), LB with agar added up to 1.7%, medium A; the pH of all media is 7.0-7.2.

Below are examples 1 and 2 of the study of the toxicity and antiviral activity of strains of B. thuringiensis B-1091, B. thuringiensis B-1069, B. thuringiensis B-1073 with respect to avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) .

Example 1. Preparation of preparations based on water-soluble metabolites of Bt strains and their testing for antiviral activity.

Obtaining a preparation based on culture fluid.

Using cultures of the studied strains grown on an agar medium for 18-24 hours at a temperature of 28-30 ° C, prepare cell suspensions with a concentration of 1-5 × 10 ⁷ cells / ml. The prepared suspensions are added in an amount of 1% to flasks with 50 ml of LB medium and cultured for 18 hours using a thermostatically controlled rocking chair (CT 104, Russia). For the preparation of drugs using cell cultures of bacilli, which are in the vegetative stage of development. The absence of the process of sporulation and the formation of paraspore inclusions is monitored by microscopy of cell culture using the phase contrast method (Axioskop 40 microscope, Karl Zeiss, Germany).

To prepare a preparation based on culture fluid (QL), the obtained QL of strains was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). The supernatant is sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm and stored until use at a temperature of minus 20 ° C.

Obtaining a drug based on cell lysates.

After centrifugation and use of the supernatant, the cell pellets are resuspended in 4 ml of distilled H ₂ O and treated on an ultrasonic disintegrator up to 15 times (with an amplitude of 18) for 30 ", with an interval of 30", achieving the maximum possible destruction of cells. The absence of spores and parasporal crystals, the degree of cell destruction in suspension after ultrasonic treatment, was monitored using phase contrast microscopy (Axioskop 40 microscope, Karl Zeiss, Germany). After ultrasonic treatment, the suspension is freed from cell debris by centrifugation at 8000 rpm in a microcentrifuge (Eppendorf, Centrifuge 5415C), the supernatant is removed and sterilized by ultrafiltration. The resulting preparation is stored until use at a temperature of minus 20 ° C.

Preparation for testing a strain of virus and cell culture.

To evaluate the antiviral efficacy of the obtained Bt preparations, the highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) from the collection of the Federal State Institution of Science and Education "Vector" Rospotrebnadzor developed on 10-day-old developing chicken embryos (RCE) was used. The virus concentration in the virus-allantoic fluid (IMPORTANT) was 9.5 lg EID ₅₀ / ml (50% of embryonic infectious doses in ml).

To test the toxicity and antiviral activity of Bt preparations, we used a transplantable MDCK cell culture obtained from the collection of cell cultures of the Federal State Pedagogical Research Center of the WB "Vector" of Rospotrebnadzor. Under aseptic conditions, the suspension with a known concentration of cells was diluted with Axcevir-MDCK medium pre-heated to a temperature of 37 ° C from Stem Alpha (France) to a concentration of 1 × 10 ⁵ cells / ml and 100 μl were added to the wells of 96-well plates. Tablets with cells were placed in a thermostat at a temperature of 37 ° C, 5% CO ₂ and 100% humidity for 2-3 days before the formation of a monolayer.

Determination of toxicity of drugs.

To determine toxic doses, the preparations were diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl were added to the corresponding wells of the plate with MDCK cells and incubated in an incubator at a temperature of 37 ° С, 5% СО ₂ and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different concentrations of the preparations was evaluated. To determine the antiviral activity of the drugs used the maximum tolerated concentration (IPC).

Determination of antiviral activity of drugs.

To determine the antiviral activity of the preparations, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. Then, 50 μl of the selected dilution of the preparation and 100 μl of 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

The following are examples 2, 3, 4, 5, 6 and 7 of the specific application of the claimed strains.

Example 2. The antiviral test of the drug No. 11, prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1091.

Preparation of the bacterial preparation No. 11.

Using a culture of a strain of Bacillus thuringiensis B-1091, developed on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1-5 × 10 ⁷ cells / ml was prepared. The prepared suspension was introduced in an amount of 1% into flasks with 50 ml of LB medium and cultured for 18 hours using a temperature-controlled shaker at a temperature of 28-30 ° C and a speed of 190 rpm. To prepare the preparations, QOL of bacilli, which are in the vegetative stage of development, was used. The absence of sporulation and the formation of parasporal inclusions was monitored by microscopy of the culture by the phase contrast method. To prepare the preparation No. 11 based on the culture fluid of strain B. thuringiensis B-1091, the obtained QL of the strain was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). The supernatant was sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation was stored until use at a temperature of minus 20 ° C.

Determination of toxic doses of the drug No. 11.

Preparation No. 11 was diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and incubated in an incubator at a temperature of 37 ° C, 5% CO ₂ , and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different concentrations of the preparations was evaluated. Drug No. 11 was nontoxic on MDCK cell culture with all dilutions used. To assess the antiviral activity of the drug No. 11 used dilution in 2 times.

Determination of antiviral activity of the drug No. 11. Ten-fold dilutions of an IMPORTANT 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. 50 μl / well of preparation No. 11, 2-fold diluted with Axcevir-MDCK medium, and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TC-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgТЦД50 / ml was 9.5), its neutralization under the influence of the preparation No. 11 was 8 lg.

Example 3. The antiviral test of the drug No. 61, prepared on the basis of lysates of cells of the strain Bacillus thuringiensis B-1091.

Preparation of the bacterial preparation No. 61.

Using a culture of a strain of Bacillus thuringiensis B-1091 grown on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1-5 × 10 ⁷ cells / ml was prepared. The prepared suspension was introduced in an amount of 1% into flasks with 50 ml of LB medium and cultured for 18 hours using a temperature-controlled shaker at a temperature of 28-30 ° C and a speed of 190 rpm. For the preparation of drug No. 61, cell cultures of bacilli in the vegetative stage of development were used. The resulting QOL was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 4 ml of distilled water and processed 10 times on an ultrasonic disintegrator (amplitude 18) for 30 "with an interval of 30". Moreover, the degree of destruction of cells of the strain of Bacillus thuringiensis B-1091 was 50%. The absence of spores and parasporal crystals, the degree of destruction of cells in suspension after treatment with ultrasound was monitored using phase contrast microscopy. After ultrasonic treatment, the suspension was freed from cell debris by centrifugation at 8000 rpm on a microcentrifuge (Eppendorf, Centrifuge 5415C), the supernatant was selected, sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 61 was stored until use at a temperature of minus 20 ° C.

Determination of toxic doses of the drug No. 61.

Preparation No. 61 was diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and incubated in an incubator at 37 ° C, 5% CO ₂ and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells with different drug concentrations was evaluated. Drug No. 61 was nontoxic in MDCK cell culture with all dilutions used. To assess the antiviral activity of the drug No. 61 used its dilution in 2 times.

Determination of antiviral activity of the drug No. 61.

Ten-fold dilutions of an IMPORTANT 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. 50 μl / well of the drug 2 times diluted with Axcevir-MDCK medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgTCD 50 / ml was 9.5), its neutralization under the influence of drug No. 61 was 7 lg.

Example 4. The antiviral test of the drug No. 48, prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1069.

Preparation of the bacterial preparation No. 48.

Using a culture of a strain of Bacillus thuringiensis B-1069 grown on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1-5 × 10 ⁷ cells / ml was prepared. The prepared suspension was introduced in an amount of 1% into flasks with 50 ml of LB medium and cultured for 18 hours using a temperature-controlled shaker at a temperature of 28-30 ° C and a speed of 190 rpm. To prepare the drug, QOL of bacilli, which are in the vegetative stage of development, was used. The absence of sporulation and the formation of parasporal inclusions was monitored by microscopy of the culture by the phase contrast method.

To prepare preparation No. 48, the obtained QOL of strain B. thuringiensis B-1069 was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). The supernatant was sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 48 was stored until use at a temperature of minus 20 ° C.

Determination of toxicity of the drug No. 48.

To determine toxic doses, preparation No. 48 was diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and incubated in an incubator at a temperature of 37 ° С, 5% С02 and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells with different drug concentrations was evaluated. The drug No. 48 was toxic in cell culture MDCK when diluted in 2 times and non-toxic with all other dilutions used. To assess the antiviral activity of the drug No. 48 used its dilution 10 times.

Determination of antiviral activity of the drug No. 48.

To determine the antiviral activity of the drug, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. 50 μl / well of the preparation diluted 10 times with Axcevir-MDCK medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TC-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgTCD 50 / ml was 9.5), its neutralization under the influence of drug No. 48 was 7 lg.

Example 5. Test antiviral effect of the drug No. 57, prepared on the basis of lysates of cells of the strain Bacillus thuringiensis B-1069.

Preparation of the bacterial preparation No. 57.

Using a culture of a strain of Bacillus thuringiensis B-1069, prepared on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1 × 10 ⁷ cells / ml was prepared. The prepared suspension was made in an amount of 1% in flasks with 50 ml of LB medium and cultured for 18 hours using a thermostated rocking chair. To prepare the preparation No. 57, we used cell cultures of bacilli that are in the vegetative stage of development. The resulting culture fluid was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 4 ml of distilled water and processed 10 times on an ultrasonic disintegrator (amplitude 18), for 30 ", with an interval of 30". Moreover, the degree of destruction of cells of the strain of Bacillus thuringiensis B-1069 was 90%.

The absence of spores and parasporal crystals, the degree of destruction of cells in suspension after treatment with ultrasound was monitored using phase contrast microscopy. After ultrasonic treatment, the suspension of the destroyed cells was centrifuged at 8000 rpm in a microcentrifuge, the supernatant was taken, sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 57 was stored until use at a temperature of minus 20 ° C.

Determination of toxicity of the drug No. 57.

To determine toxic doses, preparation No. 57 was diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and placed in a thermostat at a temperature of 37 ° С, 5% СО ₂ and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different concentrations of the drug was evaluated. The drug No. 57 was toxic in cell culture MDCK when diluted in 2 times and non-toxic with all other dilutions used. To assess the antiviral activity of the drug No. 57 used its dilution 10 times.

Determination of antiviral activity of the drug No. 57.

To determine the antiviral activity of the drug, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. 50 μl / well of the preparation diluted 10 times with Axcevir-MDCK medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgТЦД50 / ml was 9.5), its neutralization under the influence of drug No. 57 was 7 lg.

Example 6. The test antiviral effect of the drug No. 43, prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1073.

Preparation of the bacterial preparation No. 43.

Using a culture of a strain of Bacillus thuringiensis B-1073, developed on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1x10 ⁷ cells / ml was prepared. The prepared suspension was made in an amount of 1% in flasks with 50 ml of LB medium and cultured for 18 hours using a thermostated rocking chair. For the preparation of preparations used cell cultures of bacilli, which are in the vegetative stage of development. The absence of sporulation and the formation of parasporal inclusions was monitored by microscopy of the culture by the phase contrast method. To prepare the preparation No. 43 based on the culture fluid, the obtained QL of the strain was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). The supernatant was sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 43 was stored until use at a temperature of minus 20 ° C.

Determination of toxicity of the drug No. 43.

To determine toxic doses, preparation No. 43 was diluted 2, 10, 100, 1000, 10,000, 100,000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and placed in a thermostat at a temperature of 37 ° С, 5% СО ₂ and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different concentrations of the preparations was evaluated. Drug No. 43 was nontoxic on MDCK cell culture with all dilutions used. A 2-fold dilution was used to evaluate the antiviral activity of the drug.

Determination of antiviral activity of the drug No. 43.

To determine the antiviral activity of drug No. 43, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. Then, 50 μl / well of the drug diluted 2 times with Axcevir-MDCK medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgTCD 50 / ml was 9.5), its neutralization under the influence of preparation No. 43 was 8 lg.

Example 7. The antiviral test of the drug No. 58, prepared on the basis of cell lysates of the strain Bacillus thuringiensis B-1073.

Preparation of the bacterial preparation No. 58.

Using a culture of a strain of Bacillus thuringiensis B-1073, prepared on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a cell suspension of 1 × 10 ⁷ cells / ml was prepared. The prepared suspension was made in an amount of 1% in flasks with 50 ml of LB medium and cultured for 18 hours using a thermostated rocking chair. To prepare the preparation No. 58, we used cell cultures of bacilli that are in the vegetative stage of development. The obtained culture fluid was centrifuged at 6000 rpm for 30 min in a JA-21 centrifuge (Beckman, USA). After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 4 ml of distilled water and processed 10 times on an ultrasonic disintegrator (amplitude 18), for 30 ", with an interval of 30". Moreover, the degree of destruction of cells of the strain of Bacillus thuringiensis B-1073 was 90%. Using phase contrast microscopy in the suspension after the ultrasonic treatment, the absence of spores and parasporal crystals and the degree of cell destruction were monitored. After ultrasonic treatment, the suspension of the destroyed cells was centrifuged at 8000 rpm in a microcentrifuge, the supernatant was taken, sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 58 was stored until use at a temperature of minus 20.

Determination of toxicity of the drug No. 58.

To determine toxic doses, drug No. 58 was diluted 2, 10, 100, 1000, 10000, 100000, 1,000,000 times with Axcevir-MDCK medium, 150 μl was added to the corresponding wells of the plate and placed in a thermostat at a temperature of 37 ° С, 5% СО ₂ and 100% humidity for 2 days. After 2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different concentrations of the preparations was evaluated. Drug No. 58 was nontoxic in MDCK cell culture with all dilutions used. To assess the antiviral activity of the drug No. 58, a 2-fold dilution was used.

Determination of antiviral activity No. 58.

To determine the antiviral activity of the drug, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using Axcevir-MDCK medium containing 2 μg / ml trypsin. 50 μl / well of the drug 2 times diluted with Axcevir-MDCK medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added to the monolayer of the MDCK cell culture. Cells were incubated for 2 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

With a high infectivity of the virus on MDCK cells (titer in lgTCD 50 / ml was 9.5), its neutralization under the influence of drug No. 58 was 7 lg.

Thus, for the first time, it was shown that preparations prepared on the basis of the culture fluid of the strains of Bacillus thuringiensis B-1091, Bacillus thuringiensis B-1069 and Bacillus thuringiensis B-1073 of the Geyser Valley, as well as preparations prepared on the basis of cell lysates of the same strains, showed high antiviral activity against the highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1).

Sources of scientific and technical information

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Claims (3)

1. The antiviral agent for inhibiting the avian influenza virus A / H5N1 is a culture fluid or a cell lysate of the bacterial strain Bacillus thuringiensis FSHI SSC VB "Vector" V-1069. 2. Antiviral agent for inhibiting avian influenza A / H5N1 virus. represents a culture fluid or a cell lysate of a bacterial strain of Bacillus thuringiensis FGUN SSC VB "Vector" V-1073. 3. The antiviral agent for inhibiting the avian influenza virus A / H5N1 is a culture fluid or a cell lysate of the bacterial strain Bacillus thuringiensis FGUN SSC VB Vektor V-1091.