RU2405035C1 - Bacillus thuringiensis BACTERIA STRAIN EXHIBITING ANTIVIRAL AND ANTI-CANDIDA ACTIVITY (VERSIONS) - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: group of inventions relates to biotechnology. Bacillus thuringiensis strains are deposited in the Microorganisms Collection of the Federal State Research Institution 'State Research Centre for Virology and Biotechnology 'Vector' of Federal Service for Supervision of Consumer Rights Protection and Human Welfare' (FGUN GNC VB 'Vector' of Rospotrebnadzor) under collection No.: B. thuringiensis B-1065 and thuringiensis B-1083. The strains show antiviral and anti-candida activity. The preparations made of a culture liquid and lysates of B. thuringiensis B-1065 and B. thuringiensis B-1083 strains effectively inhibit avian influenza virus A/chicken/Kurgan/05/2005 (A/H5N1) replication and show high activity versus Candida activator - pathogenic Candida albicans 620 strain. ^ EFFECT: strain enhancement. ^ 2 cl, 7 ex

Description

The invention relates to the microbiological industry and biotechnology and relates to a new strain of Bacillus thuringiensis (Bt) having antiviral activity, in particular, relatively highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) and anti-candida activity, in particular relative to Candida albicans 620.

It is known that bacteria of the species Bacillus thuringiensis (Bt), capable of forming paraspore crystalline inclusions of protein nature [1], have a selective antagonistic effect against microorganisms [2, 3].

The antibiotic effect of Bt is most apparent with respect to bacteria, yeast, and fungi. The antiviral activity of Bt strains is also known, used mainly against phytopathogens, pests of agricultural plants [4].

A known consortium of microorganisms, including Bacillus licheniformis, strains of the genera Saccharomycetes, Azotobacteria, strains of P. bacteria, K. bacteria and Bacillus thuringiensis, which has antiviral activity (Chinese patent No. 1274702, IPC C12N 1/00, publ. 29.11.2000).

A known bacterial strain Bacillus thuringiensis VKPM B-9790, exhibiting fungicidal activity, insecticidal activity against Colorado potato beetle larvae and leaf beetle larvae and suppressing the virulent properties of phytopathogenic bacteria Erwinia carotovora (RF patent No. 2347809, IPC A01N 63/00, publ. February 27, 2009 )

The closest analogue (prototype) is 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.

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), as well as anti-candida activity.

The technical result of the invention is the identification of new bacterial strains having inhibitory activity against avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) and anti-candida activity.

The specified technical result is achieved by obtaining two bacterial strains of B. thuringiensis Valley of geysers with similar antiviral and anticandidic properties that have an inhibitory effect on the highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) and against Candida albicans candidiasis pathogen 620.

The strains were deposited in the collection of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor (FGUN SSC WB" Vector "Rospotrebnadzor) under the collection numbers: B. thuringiensis B-1065 and B. thuringiensis B-1083.

The detection of the antagonistic activity of the strains of Bacillus thuringiensis B-1065 and Bacillus thuringiensis B-1083 against the pathogenic strain of Candida albicans and against the avian influenza A / H5N1 virus is carried out for the first time, therefore, we can conclude that the proposed strains meet the criteria of the invention of "novelty" and "inventive step ".

Source of strain isolation

Crystal-forming endospore-containing bacteria B. thuringiensis B-1065 and B. thuringiensis B-1083 were isolated from soil samples of the Valley of Geysers (Kamchatka) when they were plated on a complete agar medium with a pH value of 7.0 and at an incubation temperature of 28-30 ° С .

Strain morphology

Strain B. thuringiensis B-1065 on agar medium A forms moist, slimy, whitish, tall, opaque, shiny colonies. The cells of the strain are encapsulated, rod-shaped, 1-1.2 × 2-8 μm in size, straight or slightly curved, located 1-2 or in chains, round-shaped paraspore inclusions tightly fit to the elliptical spore in the form of a mushroom cap. The arrangement of paraspore inclusions on the spore is free, the diameter of the crystal, as a rule, exceeds the diameter of the spore. Unlike most typical Bt strains, spores and parasporal inclusions of the studied strain remain connected after sporangia destruction.

Strain B. thuringiensis B-1083 on agar medium A forms opaque, fine-grained, opaque, whitish colonies. The cells of the strain are encapsulated, rod-shaped, as a rule, mobile, straight or slightly curved, 1-1.2 × 2-8 μm in size, 1-2 or in chains. Paraspore inclusions are rounded, not tightly connected to the spore, the presence of the structures connecting them is visible, the central axes of the spore and crystal coincide.

Biochemical signs of strains

Strains of B. thuringiensis B-1065, B. thuringiensis B-1083 are capable of anaerobic growth, secrete acid from glucose and maltose, but not from arabinose, xylose, mannitol and lactose, do not form indole, do not deaminate phenylalanine, do not utilize arginine and citrate . The strains have catalase, lecithinase, gelatinase, lipase, amylolytic activity, grow in the presence of 0.001% lysozyme. It is important to note that the studied strains according to the results of indirect tests for pathogenicity do not have hemolytic, fibrinolytic and plasmocoagulase activities.

Strain storage

Subcultures of the strains are 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”, 1.7% agar) and medium A (0.7% peptone “Difco”, 0.4%) of a fish hydrolyzate, 0.5% NaCl, 1.7% agar). The pH value of all media is 7.0-7.2.

Below is the data on the study of the antagonistic activity of drugs based on water-soluble metabolites of strains of B. thuringiensis B-1065 and B. thuringiensis B-1083 against Candida albicans 620 candidiasis. To study the antagonistic activity of the strains, preparations based on the culture fluid of B. thuringiensis strains were used B-1065 and B. thuringiensis B-1083.

Example 1. Testing the anti-candidiasis action of a preparation prepared on the basis of culture fluid (QL) of the strain Bacillus thuringiensis B-1065

Preparation of a bacterial preparation based on QOL of strain B-1065. Using a culture of the studied strain, which was developed on an agar medium for 18-24 hours at a temperature of 28-30 ° C, a suspension of cells with a concentration of 1-5 × 10 ⁷ cells / ml was prepared. The suspension was introduced in an amount of 1% into flasks with 25 ml of LB medium and cultured for 2 days on a thermostatically controlled shaker (CT 104, Russia) at a temperature of 28-30 ° С and a rotation speed of 190 rpm until the stage of active sporulation of cells. The obtained QOL was kept for 4 days in a refrigerator at a temperature of 4-6 ° C, microscopied by the phase contrast method (Axioskop 40 microscope, Karl Zeis, Germany) and subcultures were selected for antimicrobial testing, where sporulation and crystal formation levels reached 70-95%). After this, the cells were besieged by centrifugation at 10,000 rpm (Beckman centrifuge, USA) for 20 min, the supernatant was taken with a sterile syringe and filtered through a Whatman filter with a pore size of 0.2 μm, and the resulting preparation was stored at minus 20 ° С.

Determination of the antimicrobial action of the drug. Under aseptic conditions, 0.1 ml of a suspension of C. albicans with a cell concentration of 3.9 × 10 ⁷ cells / ml was added to 2.9 ml of the filtered BACillus thuringiensis B-10 QL and incubated at a temperature of 28-30 ° C for 48 hours. The presence of anti-candidiasis activity of the obtained preparation was visually assessed by clarification of the experimental QOL variant relative to the control of C. albicans culture, where the preparation of B. thuringiensis B-1065 strain was not added, and during the titration of the experimental and control QOL on a solid nutrient medium.

As a result of joint incubation of C. albicans strain and QOL of B. thuringiensis B-1065 strain for 24 hours at a temperature of 28-30 ° С, the number of C. albicans cells decreased by 2 orders of magnitude compared to that in the control (from 5.9 × 10 ⁶ to 3.9 × 10 ⁴ cells / ml). After 48 hours of cultivation, viable C. albicans cells in the experimental version were completely absent.

Example 2. Testing the anti-candidiasis effect of the drug prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1083

Preparation of a bacterial preparation based on the culture fluid of strain B-1083. Using a culture of the studied strain, which was developed on an agar medium for 18-24 hours at a temperature of 28-30 ° C, cell suspensions were prepared with a concentration of 1-5 × 10 ⁷ cells / ml. The prepared suspension was introduced in an amount of 1% into flasks with 25 ml of LB medium and cultured for 2 days on a thermostatic shaker at a temperature of 28-30 ° C and a rotation speed of 190 rpm to the stage of active sporulation of cells. The obtained culture fluid was kept for 4 days in a refrigerator at a temperature of 4-6 ° C, microscopied by phase contrast and subcultures were selected for antimicrobial testing, where sporulation efficiency and crystal formation efficiency reached 70-95%. After this, the cells were besieged by centrifugation at 10,000 rpm for 20 min, the supernatant was taken with a sterile syringe and filtered through a Whatman filter with a pore size of 0.2 μm.

Determination of antimicrobial action of the drug. Under aseptic conditions, 0.1 ml of a suspension of C. albicans (3.9 × 10 ⁷ cells / ml) was added to 2.9 ml of filtered QoL of Bacillus thuringiensis B-1083 and incubated at a temperature of 28-30 ° C for 48 hours . The presence of anti-candidiasis activity of the obtained preparation was visually assessed by clarification of the experimental QOL variant relative to the control of C. albicans culture, where the preparation of B. thuringiensis B-1083 strain was not added, and when QL was titrated on a solid nutrient medium.

As a result of joint incubation of the strain C. albicans and QOL of the strain B. thuringiensis B-1083 for 24 hours at a temperature of 28-30 ° C, the titer of C. albicans cells decreased by 3 orders of magnitude compared with the titer in the control (from 6.0 × 10 ⁶ to 3.0 × 10 ³ cells / ml). After 48 hours of cultivation, viable C. albicans cells in the experimental version were completely absent.

Thus, preparations based on the culture fluid of the studied strains of Bacillus thuringiensis B-1083 and Bacillus thuringiensis B-1065 Geyser Valley showed higher activity against the pathogen Candida albicans compared to the analogue strain of Bacillus thuringiensis.

The following are data from a study of the toxicity and antiviral activity of water-soluble metabolites of B. thuringiensis B-1065 and B. thuringiensis B-1083 strains against avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1).

Example 3. The study of the toxicity and antiviral activity of drugs based on water-soluble metabolites of strains of B. thuringiensis B-1065 and B. thuringiensis B-1083

Obtaining a preparation based on culture fluid. Using cultures of the studied strains developed on an agar medium for 18-24 hours at a temperature of 28-30 ° C, cell suspensions were prepared with a concentration of 1 × 10 ⁷ cells / ml. 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 thermostated rocking chair (CT 104, Russia). 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 cultures by the phase contrast method.

To prepare the preparation based on the culture fluid, the obtained QL 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 and used for testing as an antiviral drug. The drug was stored until use at a temperature of minus 20 ° C.

Obtaining a drug based on cell lysates. Cell pellets obtained after centrifugation and freed from the supernatant were resuspended in 4 ml of distilled H ₂ O and processed up to 15 times on an ultrasonic disintegrator (amplitude 18) for 30 ”with an interval of 30”, achieving the most complete destruction of cells. 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 ultrasound treatment, the destroyed cells were besieged by centrifugation at 8000 rpm in a microcentrifuge, the supernatant was selected and 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.

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 lgEID ₅₀ / ml (50% of embryonic infectious doses in ml). To test the toxicity and antiviral activity of the preparations, we used a transplantable culture of MDCK cells obtained from the collection of cell cultures of the Federal State Institution of Science and Science of the World Bank “Vector” of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare. In a sterile place, a suspension with a known cell concentration 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 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 the drug. 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 placed in a thermostat 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. 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. 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 4, 5, 6 and 7 of the specific application of the claimed strains.

Example 4. The antiviral test of the drug No. 35, prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1065

Preparation of the bacterial preparation No. 35. Using a culture of the Bacillus thuringiensis B-1065 strain, which was 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 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 the drug used cell cultures 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, the obtained QOL of strain B. thuringiensis B-1065 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. 35 was stored until use at a temperature of minus 20 ° C.

Determination of toxicity of the drug No. 35. To determine toxic doses, the preparation 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 with MDCK cells and placed in a thermostat at 37 ° С, 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. The drug No. 35 was non-toxic on the cell culture MDCK with all dilutions used. To assess the antiviral activity of the drug No. 35 used its dilution in 2 times.

Determination of antiviral activity of the drug No. 35. 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. 35 was 8 lg.

Example 5. Test antiviral effect of the drug No. 59, prepared on the basis of lysates of cells of the strain of Bacillus thuringiensis B-1065

Preparation of the bacterial preparation No. 59. Using a culture of a strain of Bacillus thuringiensis B-1065, developed 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. 59, 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 minutes in a centrifuge. After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 4 ml of distilled water and processed 15 times on an ultrasonic disintegrator (amplitude 18) for 30 ”with an interval of 30”. Moreover, the degree of destruction of the cells of the strain of Bacillus thuringiensis B-1065 was 80%. 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 ultrasound treatment, the destroyed cells were besieged by centrifugation at 8000 rpm in a microcentrifuge, the supernatant was selected and 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 toxicity of the drug No. 59. To determine toxic doses, the preparation 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 with MDCK cells and placed in a thermostat at 37 ° С, 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. The drug No. 59 was nontoxic on the MDCK cell culture with all dilutions used; its dilution was used 2 times to evaluate antiviral activity.

Determination of antiviral activity of the drug No. 59. 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 diluted in 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 drug No. 59 was 7 lg.

Example 6. The test antiviral effect of the drug No. 37, prepared on the basis of the culture fluid of a strain of Bacillus thuringiensis B-1083

Preparation of the bacterial preparation No. 37. Using a culture of a strain of Bacillus thuringiensis B-1083, which was 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 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. 37 based on the culture fluid, the obtained QL of the strain was centrifuged at 6000 rpm for 30 min in a centrifuge (JA-21, 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 toxicity of the drug. To determine toxic doses, preparation No. 37 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 an MDCK cell plate and placed in a thermostat 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. The drug was nontoxic in MDCK cell culture with all dilutions used. To assess the antiviral activity of the drug No. 37 used its dilution in 2 times.

Determination of antiviral activity of the drug No. 37. To determine the antiviral activity of drug No. 37, 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. 37 was 7 lg.

Example 7. The antiviral test of the drug No. 53, prepared on the basis of cell lysates of the strain Bacillus thuringiensis B-1083

Preparation of the bacterial preparation No. 53. Using a culture of a strain of Bacillus thuringiensis B-1083, which was 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 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. 53, 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 minutes in a centrifuge. After centrifugation, the supernatant was removed, and the cell pellet was resuspended in 4 ml of distilled water and processed 10 times on a U3-disintegrator (amplitude 18) for 30 ”with an interval of 30”. Moreover, the degree of destruction of the cells of the strain of Bacillus thuringiensis B-1053 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 the U3 treatment, the suspension of the destroyed cells was centrifuged at 8000 rpm in a microcentrifuge, the supernatant was collected, sterilized by ultrafiltration through a Whatman filter with a pore size of 0.2 μm. The resulting preparation No. 53 was stored until use at a temperature of minus 20 ° C.

Determination of toxicity of the drug. To determine toxic doses, preparation No. 53 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% 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. The drug was nontoxic in MDCK cell culture with all dilutions used. To assess the antiviral activity of the drug No. 37 used its dilution in 2 times.

Determination of antiviral activity of the drug No. 53. 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. 37 was 7 lg.

Thus, it was shown for the first time that preparations prepared on the basis of culture fluid and lysates of strains of Bacillus thuringiensis B-1065, Bacillus thuringiensis B-1083 Geyser Valley, effectively suppressed the multiplication of the highly pathogenic avian influenza virus A / chicken / Kurgan / 05/2005 (A / H5N1) on MDCK cells and showed high activity against the pathogenic strain of Candida albicans 620.

Information sources

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Claims (2)

1. The bacterial strain Bacillus thuringiensis, which has antiviral and anti-candidiasis activity, deposited in the collection of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor" under registration number B-1065. 2. The bacterial strain Bacillus thuringiensis, which has antiviral and anti-candidiasis activity, deposited in the collection of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor" under registration number B-1083.