RU2551236C1 - STRAIN OF BACTERIA Bacillus thuringiensis, NEUTRALISING INFECTIOUS ACTIVITY OF HUMAN INFLUENZA VIRUS A/H3N2 (VERSIONS) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to novel strains of Bacillus thuringiensis B-1272 and Bacillus thuringiensis B-1273 deposited in the collection of bacteria, bacteriophages and fungi of the Federal State-Funded Institution of Science "State Research Centre of Virology and Biotechnology "Vector". The index of neutralisation of the infectious activity of virus A/H3N2 while using the preparations based on the culture fluid of any of the proposed strains is 0.5-3.2 lg.

EFFECT: strains have the ability to neutralise the infectious activity of the human influenza virus.

2 cl, 1 tbl, 8 ex

Description

The invention relates to new strains of Bacillus thuringiensis (Bt) with antiviral activity, in particular, with respect to the highly pathogenic human influenza virus A / H3N2, for the preparation of drugs aimed at suppressing the virus and can be used in the microbiological industry, biotechnology and medicine.

In the last 10 years, influenza A viruses with surface antigens A (H1N1), A (H2N2), A (H3N2) capable of rapid genetic change have been epidemiologically significant for humans. This property of them creates great problems in the treatment of influenza virus infected not only humans, but also animals and birds. There is a high incidence of the population (up to 40%) with almost the same lesion in all age groups.

Since July 2012, a significant increase in cases of infection with the “variant” H3N2v virus containing the M gene from the influenza virus (H1N1) pdm09 (pandemic 2009 H1N1 virus) was detected. According to the World Health Organization, in 2012, nine US states reported cases of human diseases caused by this virus. In this regard, the search for topical antiviral agents and the creation of drugs that suppress viral activity are currently of particular relevance.

It is known that bacteria of the species Bacillus thuringiensis (Bt) have a selective antagonistic effect against microorganisms [1, 2]. The antiviral activity of Bt strains against phytopathogens, pests of agricultural plants has been shown [3, 4].

Known bacterial strains of Bacillus subtilis and Bacillus licheniformis used as components of the drug against viral and bacterial infections (Patent 2142287, IPC A61K 35/74, publ. 10.12.1999).

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

The closest analogue (prototype) are the strains of B. thuringiensis B-1065 and B. thuringiensis B-1083 (options) with antiviral and anti-candidiasis activity (RF Patent No. 2405035, IPC C12N 1/20, publ. 11/27/2010) .

However, all of the above analogues and prototype do not have an inhibitory effect against human influenza virus A / H3N2.

The technical result of the proposed solution is the identification of bacterial strains with inhibitory activity against human influenza virus A / H3N2.

The specified technical result is achieved by obtaining a spore-forming, forming parasporal crystals, bacterial strain Bacillus thuringiensis AK-1 (the first option), which neutralizes the infectious activity of the human influenza virus A / H3N2 and deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Institution of Science “State Scientific Center for Virology” and biotechnology "Vector" under registration number B-1272.

The indicated technical result is also achieved by obtaining the second variant of the spore-forming, forming parasporal crystals, bacterial strain Bacillus thuringiensis, which neutralizes the infectious activity of the human influenza virus A / H3N2 and deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Under registration number B-1273.

Characterization of strains.

Strains of B. thuringiensis AK-1 and Bacillus thuringiensis AK-2 were obtained by selection for the antiviral activity of Bt strains isolated previously from soil samples from a mud hot boiler (Geyser Valley, Kamchatka).

The morphology of the strains:

Strains of B. thuringiensis AK-1 and B. thuringiensis AK-2 have a similar morphology of colonies and cells. On agri nutrient medium Nutrient Broth with the addition of 18-20% agar (NBA medium) form round, whitish, flat, fine-grained, opaque colonies. The cells of the strains are represented by gram-positive, rods, size 1.2-1.4 × 2-4 microns, with an arrangement of 2 or in chains; form round-shaped parasporal inclusions. The endospores are elliptical, the cell diameter does not exceed, after the destruction of the sporangia, the spores and parasporal inclusions of the studied strains remain paired, they are not connected to the spore tightly, but through their connecting structures. The central axis of the spores and crystal coincide. The cells of strains of B. thuringiensis AK-1 and B. thuringiensis AK-2 are characterized by the presence of a clearly defined macrocapsule (more than 0.2 μm thick). The strain crystals have the same protein composition with a characteristic molecular weight for Bt (130, 125, and 100 kDa).

Biochemical signs of strains:

By biochemical characteristics, strains of B. thuringiensis AK-1 and B. thuringiensis AK-2 also show great similarity, slightly differing in some alternative characters (hydrolysis of sucrose, esculin) and antibiotic resistance. Strains of B. thuringiensis AK-1 and B. thuringiensis AK-2, when cultured on nutrient media, grow in the presence of 0.001% lysozyme, 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, have lysine carboxylase, do not have arginine decarboxylase, reduce nitrates, hydrolyze casein, are positive in the reaction of MR, FP, in reaction to catalase, oxidase, amylase, lipase, lecithinase and hemolytic (on rabbit erythrocytes hydrolysis) well latin; negative in reaction to plasmocoagulase and fibrinolytic activity, in reaction to urease and phosphatase; do not form indole, hydrogen sulfide, ammonia, do not utilize citrate.

To determine the possibility of using strains of B. thuringiensis together with antibacterial drugs, it is necessary to determine their resistance to antibiotics. The strain B. thuringiensis AK-1 was shown to be resistant to azlocillin, ampicillin, benzylpenicillin, carbenicillin, oxacillin, polymyxin and cefotaxin; sensitive to amikacin, vancomycin, gentamicin, kanamycin, rifampicin, imipenem, chloramphenicol, lincomycin, neomycin, streptomycin, tetracycline and ciprofloxacin. The strain B. thuringiensis AK-2 is resistant to azlocillin, ampicillin, benzylpenicillin, carbenicillin, oxacillin and cefotaxin; slightly sensitive to polymyxin; sensitive to amikacin, vancomycin, gentamicin, kanamycin, rifampicin, imipenem, chloramphenicol, lincomycin, neomycin, streptomycin, tetracycline and ciprofloxacin.

The strains are stored by periodic reseeding on an agarized NBA medium; long-term storage is carried out in a 15% glycerol solution under low-temperature freezing (65 ° C) [6] and in a lyophilized state. The strains of B. thuringiensis AK-1 and B. thuringiensis AK-2 were deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Biological Research Center VB Vektor under collection numbers B-1072 and B-1073, respectively.

Certificates of deposit of strains are attached.

Inoculum was obtained by growing strains on liquid or agarized media: LB (Difco, USA), LB with agar up to 1.7%, NBA medium with 18-20% agar; the pH of all media was 7.0–7.2.

The study of strains of B. thuringiensis AK-1 and B. thuringiensis AK-2 on activity against the human influenza virus A / Aichi / 2/68 (A / H3N2) is carried out for the first time, and therefore we can conclude that the proposed strains meet the criteria of the invention " novelty ”and“ inventive step ”.

1. Preparation of preparations based on water-soluble metabolites of strain B. thuringiensis AK-1 and B. thuringiensis AK-2.

1.1. The accumulation of culture fluid (QOL) of the strain. 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 × 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 paraspore inclusions was monitored by microscopy of the culture by the phase contrast method (Axioskop 40 microscope, Karl Zeiss, Germany).

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

The absence of bacterial cells in the preparation was monitored microscopically and when it was plated on LB agar medium.

2. The study of the toxicity and antiviral activity of strain B. thuringiensis AK-1 and B. thuringiensis AK-2 against human influenza virus A / Aichi / 2/68 (A / H3N2).

2.1. Determination of toxicity and antiviral activity of drugs W. To assess the antiviral efficacy of the obtained Bt preparations, the human influenza virus A / Aichi / 2/68 (A / H3N2) from the Collection of Microorganisms of the Federal State Institution of Biology and Science of the World Bank “Vector” was used. To test the toxicity and antiviral activity of the drugs used transplantable cell culture MDCK obtained from the "Collection of cell cultures FBSI SSC WB" Vector "."

2.2. Determination of toxic doses of drugs. At a sterile site, a suspension with a known concentration of MDCK cells was diluted with RPMI-1640 medium preheated to 37 ° C to a concentration of 1 × 10 ⁵ cells / ml and 100 μl was added into 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.

The preparations were diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 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).

Table Scheme for the preparation of drug samples based on the claimed strains for the study of antiviral activity Strain number Sample No. Features Action against the virus Bacillus thuringiensis AK-1 (B-1272) four 5 times breeding A / Aichi / 2/68 (A / H3N2) 4-10 10 times dilution 4/1 5-fold dilution, storage 3 months at -20 ° C 4 / 1-10 Dilution 10 times, storage 3 months at -20 ° C Bacillus thuringiensis AK-2 (B-1273) 5 5 times breeding A / Aichi / 2/68 (A / H3N2) 5-10 10 times dilution 5/1 5-fold dilution, storage 3 months at -20 ° C 5 / 1-10 Dilution 10 times, storage 3 months at -20 ° C

2.3. Determination of antiviral activity of drugs. Ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 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.

3. Examples of specific applications of the claimed strains.

The preparation of drug samples based on the claimed strains for the study of antiviral activity is shown in the table.

3.1. Example No. 1. The antiviral test of the preparation No. 4, prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-1, on the human influenza virus A / H3N2.

Using a culture of a strain of Bacillus thuringiensis AK-1, 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. 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 preparation No. 4, 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 0.2ct; the resulting preparation No. 4 was stored until use at a temperature of minus 20 ° C.

The absence of bacterial cells in the preparation was monitored microscopically and when it was plated on LB agar medium.

To determine toxic doses, preparation No. 4 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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. Drug No. 4 was non-toxic in MDCK cell culture starting with 5-fold dilution. To assess the antiviral activity of drug No. 4, a 5-fold dilution was used.

To determine the antiviral activity of drug No. 4, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 5 times with RPMI-1640 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.

When the A / H3N2 virus was infectious on MDCK cells (titer in lg TCD50 / ml was 6.7), its neutralization under the influence of the studied drug was 0.5 lg.

3.2. Example No. 2. The antiviral test of the preparation No. 4-10, prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-1, on the human influenza virus A / H3N2.

Using a culture of a strain of Bacillus thuringiensis AK-1, 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. 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 preparation No. 4-10, 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, the resulting preparation No. 4-10 was stored until use at a temperature of minus 20 ° C.

The absence of bacterial cells in the preparation was monitored microscopically and when it was plated on LB agar medium.

To determine toxic doses, drug No. 4-10 was diluted 2 times, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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 No. 4-10 was non-toxic in cell culture MDCK starting with a dilution of 5 times. To assess the antiviral activity of drug No. 4-10, dilution of 10 times was used.

To determine the antiviral activity of preparation No. 4-10, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 5 times with RPMI-1640 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 respect to the A / H3N2 virus (titer in lgTCD50 / ml was 6, 7), the neutralization index under the influence of the studied drug was 1.0 lg.

3.3. Example No. 3. Antiviral test of the preparation No. 4/1, prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-1, on the human influenza virus A / H3N2.

Using a culture of strain B. thuringiensis AK-1, which was 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. For the preparation of preparation No. 4/1, cell cultures of bacilli in the vegetative stage of development were used. The resulting culture fluid 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. 4/1 was stored until use at a temperature of minus 20 ° C for 3 months.

To determine toxic doses, preparation No. 4/1 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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 No. 4/1 was non-toxic in the MDCK cell culture starting with a 5-fold dilution. A 5-fold dilution was used to evaluate the antiviral activity of preparation No. 4/1.

To determine the antiviral activity of preparation No. 4/1, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 5 times with RPMI-1640 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.

When the A / H3N2 virus was infectious on MDCK cells (titer in lgТЦД50 / ml was 6.7), its neutralization under the influence of the studied drug was 2.5 lg.

3.4. Example No. 4. The antiviral test of the preparation No. 4 / 1-10, prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-1, on the human influenza virus A / H3N2.

Using a culture of strain B. thuringiensis AK-1, which was 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. To prepare the preparation No. 4 / 1-10, cell cultures of bacilli in the vegetative stage of development were used. The resulting culture fluid 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. 4 / 1-10 was stored until use at a temperature of minus 20 ° C for 3 months.

To determine toxic doses, preparation No. 4 / 1-10 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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 No. 4 / 1-10 was non-toxic in cell culture MDCK, starting with a dilution of 5 times. To assess the antiviral activity of the drug No. 4/1-10, a dilution of 10 times was used.

To determine the antiviral activity of preparation No. 4 / 1-10, ten-fold dilutions of IMPORTANT from 1 to 8 were prepared using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 5 times with RPMI-1640 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.

Relative to the A / H3N2 virus (titer in lgTCD50 / ml was 6.7), the neutralization index under the influence of the studied drug was 3.2 lg.

3.5. Example No. 5. The antiviral test of the preparation No. 5, prepared on the basis of the culture fluid of strain B. thuringiensis AX-2, on the human influenza virus A / H3N2, stored until use at a temperature of minus 20 ° C.

Using a culture of strain B. thuringiensis AK-2, which was 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. For the preparation of preparations, cellular QOL of bacilli, which are in the vegetative stage of development, were 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. 5, 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, the resulting preparation No. 5 was stored until use at a temperature of minus 20 ° C.

To determine toxic doses, preparation No. 5 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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. Drug No. 5 was nontoxic in MDCK cell culture starting with 5-fold dilution. To assess the antiviral activity of drug No. 5, a 5-fold dilution was used.

To determine the antiviral activity of preparation No. 5, dilutions of IMPORTANT from 1 to 8 were prepared in ten-fold steps using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of drugs in a monolayer of MDCK cell culture. 100 μl / well of the preparation diluted 10 times with RPMI-1640 medium and 100 μl from 1 to 8 dilutions of IMPORTANT were added. 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.

When the A / H3N2 virus was infectious on MDCK cells (titer in lgTCD50 / ml was 6.7), its neutralization under the influence of the studied drug was 1.2 lg.

3.6. Example No. 6. The antiviral test of the preparation No. 5-10, prepared on the basis of the culture fluid of the strain B. thuringiensis AP-2, on the human influenza virus A / H3N2, stored until use at a temperature of minus 20 ° C.

Using a culture of strain B. thuringiensis AK-2, which was 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. For the preparation of preparations, cellular QOL of bacilli, which are in the vegetative stage of development, were 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. 5-10, 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, the resulting preparation No. 5-10 was stored until use at a temperature of minus 20 ° C.

To determine toxic doses, drug No. 5-10 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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. Drug No. 5-10 was nontoxic on MDCK cell culture starting with 5-fold dilution. To assess the antiviral activity of the drug No. 5-10 used a dilution of 10 times.

To determine the antiviral activity of drug No. 5-10, dilutions of IMPORTANT from 1 to 8 were prepared in tenfold increments using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 10 times with RPMI-1640 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.

Relative to the A / H3N2 virus (titer in lgTCD50 / ml was 6, 7), the neutralization index under the influence of the studied drug was 1.2 lg.

3.7. Example No. 7. The antiviral test of the preparation No. 5/1 prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-2 on the human influenza virus A / H3N2, stored until use at a temperature of minus 20 ° C for 3 months.

Using a culture of strain B. thuringiensis AK-2, which was 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 introduced in an amount of 1% into flasks with 50 ml of LB medium and cultured for 18 hours using a thermostatically controlled rocking chair. For preparation of preparation No. 5/1, cell cultures of bacilli in the vegetative stage of development were used. The resulting culture fluid 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 the resulting preparation No. 5/1 was stored until use at a temperature of minus 20 ° C for 3 months.

To determine toxic doses, preparation No. 5/1 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added into the corresponding wells of the 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 No. 5/1 was non-toxic on the MDCK cell culture starting with a 5-fold dilution. A 5-fold dilution was used to evaluate the antiviral activity of preparation No. 5/1.

To determine the antiviral activity of drug No. 5/1, dilutions of IMPORTANT from 1 to 8 were prepared in ten-fold steps using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 10 times with RPMI-1640 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.

When the A / H3N2 virus was infectious on MDCK cells (titer in lgTCD50 / ml was 6, 7), its neutralization under the influence of the studied drug was 3.0 lg.

3.8. Example No. 8. The antiviral test of the preparation No. 5 / 1-10, prepared on the basis of the culture fluid of the strain Bacillus thuringiensis AK-2, on the human influenza virus A / H3N2, stored until use at a temperature of minus 20 ° C for 3 months.

Using a culture of strain B. thuringiensis AK-2, which was 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. 5 / 1-10, 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). The supernatant was sterilized by ultrafiltration through a Whatman filter, with a pore size of 0.2 μm, and the resulting preparation No. 5 / 1-10 was stored until use at a temperature of minus 20 ° C for 3 months.

To determine toxic doses, preparation No. 5 / 1-10 was diluted 2, 5, 10, 50, 100, 500, 1000, 5000 times with RPMI-1640 medium, 150 μl was added to the corresponding wells of the 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 No. 5 / 1-10 was non-toxic on the cell culture MDCK starting with a dilution of 5 times. To assess the antiviral activity of the drug No. 5 / 1-10, a dilution of 10 times was used.

To determine the antiviral activity of drug No. 5 / 1-10, dilutions of IMPORTANT from 1 to 8 were prepared in tenfold increments using RPMI-1640 medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 100 μl / well of the drug diluted 10 times with RPMI-1640 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.

In relation to the A / H3N2 virus (titer in ^ TCD50 / ml was 6, 7), the neutralization index under the influence of the studied drug was 3.2 lg.

Thus, it was shown that preparations prepared on the basis of the culture fluid of the strains of Bacillus thuringiensis AK-1 and Bacillus thuringiensis AK-2 showed high antiviral activity against human influenza virus A / Aichi / 2/68 (A / H3N2).

Information sources

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Claims (2)

1. The bacterial strain Bacillus thuringiensis, which neutralizes the infectious activity of the human influenza virus A / H3N2 and deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Institution of Science “State Scientific Center for Virology and Biotechnology“ Vector ”under registration number B-1272. 2. The bacterial strain Bacillus thuringiensis, which neutralizes the infectious activity of the human influenza virus A / H3N2 and is deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "under registration number B-1273.