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The morphological and physiological characteristics of Bacillus thuringiensis strains were analyzed and conditions for obtaining culture fluid with maximum yield of secreted RNases were determined. Zymographic analysis showed that culture fluid of B. thuringiensis strains along with low-molecular-weight (15-20 kDa) RNases contained enzymes with a molecular weight ~55 kDa and their content depended on the duration and conditions of culturing. Preparations based on B. thuringiensis culture fluid were effective against human influenza virus A/Aichi/2/68 (H3N2). In experiments on mice infected with 10 LD50 influenza virus strain A/Aichi/2/68 (H3N2), we selected effective variants of preparations based on culture fluid of B. thuringiensi strains for preventive administration that provided reliable protection of infected animals (protection coefficient 50%), close to that of the reference drug Tamiflu.

The results of studies of a newly isolated Serratia species K-57 strain are presented. The strain is characterized by antiviral activity towards human influenza A/Aichi/2/68/H3N2, vaccinia, mouse smallpox, and herpes simplex-2 viruses. The detected characteristics of the strain, including the data on activities on nucleolytic enzymes, recommend it for the development of therapeutic and preventive antiviral drugs.

This is the first study to assess antiviral activity against human influenza virus A/Aichi/2/68 and bird influenza virus A/chicken/Kurgan/05/2005 and phytochemical characteristics of an ethanol extract of Cacalia hastata L. from wild populations growing in the vicinity of Tomsk, Russia (mixed forest). A log10 neutralization index—an indicator of inhibition of virus replication by the extract from C. hastata leaves—was ≤2 lg for each influenza virus. The extract reduced the infectivity of the human influenza virus by 1.5-fold and that of the avian influenza virus by 1.7-fold. The phytochemical characterization of C. hastata (its various organs) showed that the largest amount of the tested biologically active substances is present in leaves and rhizomes.

Background:The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides.Results:The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (−)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions.Conclusion:The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA.

The hairy root culture of halophyte Nitraria schoberi was obtained by transforming seedlings' primary leaves with a wild strain of Agrobacterium rhizogenes 15834 SWISS (transformation frequency was 78.7%). The roots' transgenic status was confirmed by PCR analysis using rolB-, rolC-, virC-, virD1-specific primers. There was a significant increase in the biosynthesis of flavonoids, hydroxycinnamic acids, pectins, protopectins and saponins in hairy roots as compared to intact plant roots. The сatechin content in ethanol extracts of transformed roots' exceeded this factor 3.8 times in plant roots, but the compound composition of catechin nature in the latter was more diverse. The high antiviral activity of N. schoberi hairy root ethanol extracts against influenza virus subtypes A(H5N1), A(H3N2) was revealed, which manifested itself in the effective inhibition of infectivity both in vitro-in MDCK cell culture-and in vivo increasing survival of mice infected with them.

The development of efficient and convenient systems for the delivery of nucleic-acid-based drugs into cells is an urgent task. А promising approach is the use of various nanoparticles. Silica nanoparticles can be used as vehicles to deliver nucleic acid fragments into cells. In this work, we developed a method for the synthesis of silicon–organic (Si–NH2) non-agglomerated nanoparticles by the hydrolysis of aminopropyltriethoxysilane (APTES). The resulting product forms a clear solution containing nanoparticles in the form of low molecular weight polymer chains with [─Si(OH)(C3H6NH2)O─] monomer units. Oligonucleotides (ODN) were conjugated to the prepared Si–NH2 nanoparticles using the electrostatic interaction between positively charged amino groups of nanoparticles and negatively charged internucleotide phosphate groups in oligonucleotides. The Si–NH2 nanoparticles and Si–NH2·ODN nanocomplexes were characterized by transmission electron microscopy, atomic force microscopy and IR and electron spectroscopy. The size and zeta potential values of the prepared nanoparticles and nanocomplexes were evaluated. Oligonucleotides in Si–NH2·ODN complexes retain their ability to form complementary duplexes. The Si–NH2Flu nanoparticles and Si–NH2·ODNFlu nanocomplexes were shown by fluorescence microscopy to penetrate into human cells. The Si–NH2Flu nanoparticles predominantly accumulated in the cytoplasm whereas ODNFlu complexes were predominantly detected in the cellular nuclei. The Si–NH2·ODN nanocomplexes demonstrated a high antisense activity against the influenza A virus in a cell culture at a concentration that was lower than their 50% toxic concentration by three orders of magnitude.

To study the effect of nanoscale titanium dioxide (TiO2) on cell responses, we synthesized four modifications of the TiO2(amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO2were obtained by hydrolysis of TiCl4and Ti(i-OC3H7)4(TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO2nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO2nanoparticles 3–5 nm in size were selected. Examination of different forms of TiO2nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO2. The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell.

Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO(2)·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO(2) nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells. They showed a low toxicity (TC(50) ≈ 1800 μg/ml) and a high antiviral activity (99.9% inhibition of the virus replication). The specificity factor (antisense effect) appeared to depend on the delivery system of DNA fragments. This factor for nanocomposites is ten-times higher than for DNA in the presence of lipofectamine. IC(50) for nanocomposites was estimated to be 1.5 μg/ml (30 nM for DNA), so its selectivity index was calculated as ~1200. Thus, the proposed nanocomposites are prospective for therapeutic application.

Comparative immunogenicity studies of experimental vaccines based onA/Aichi/2/68 neuraminidase peptide fragments (NA) and influenza virus A and B strains produced in MDCK cell culture.Anti-hemagglutinin and virus neutralizing activity of mice sera was determined in MN and HI reactions in accordance with the WHO recommendations.Sera against peptides 136-147 and 154-164 from variable sites, as well as against peptide 314-328 from conservative region of the heavy chain of A/ Aichi/2/68 influenza virus NA showed distinct anti-hemagglutinin and neutralizing activity against homologous influenzavirus. Anti-(314-328) serum was also active in HI and MN reactions against other strains of the H3N2 subtype. Combined administration of peptide sample with an immunomodulator (Immunomax) increased the immunogenicity to the level of the cultural samples based on influenza A virus.The results show higher immunogenicity of cultural vaccines based on influenza virus in comparison to peptide samples. A possibility of peptide vaccine immunogenicity increase was demonstrated by combined administration with the immunomodulator.

Study of possibility of treatment-prophylaxis effect increase during combined administration of ridostin and tamiflu in experiments in mice infected with highly pathogenic influenza virus strain A/chicken/Kurgan/05/2005 (H5N1).Balb/c line mice infected intranasally with influenza virus at 100 and 10 LD50 doses received ridostin and tamiflu as monopreparation or the combined variant before or after the infection. The mice were observed for 16 days, lethality rate, protection coefficient and average life span were evaluated. Virus concentration in lungs was determined by using titration in MDCK cell line.Combined administration ofridostin and tamiflu after the infection increased survivability of the animals when compared with the control group, and reduced influenza virus concentration in lungs.Treatment effect during combined administration of ridostin and tamiflu after influenza virus infection increased.

To study the effect of nanoscale titanium dioxide (TiO(2)) on cell responses, we synthesized four modifications of the TiO(2) (amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO(2) were obtained by hydrolysis of TiCl(4) and Ti(i-OC(3)H(7))(4) (TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO(2) nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO(2) nanoparticles 3-5 nm in size were selected. Examination of different forms of TiO(2) nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO(2). The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell.

Evaluate reactogenicity, safety and immunogenicity in phase 2 clinical trials of 2 immunization schedules with Ultragrivac--an allantoic intranasal life influenza vaccine based on A/17/ duck/Potsdam/86/92 [17/H5] reassortant strain.4 groups of volunteers participated in the study: group 1--40 individuals were vaccinated twice with a 10 day interval; group 2--40 individuals were vaccinated twice with a 21 day interval; group 3 (control)--10 individuals received placebo twice with a 10 day interval; group 4 (control)--10 individuals received placebo twice with a 21 day interval. Local (secretory IgA), cellular and humoral immune response were evaluated. Humoral immunity was evaluated by the intensity of increase of geometric mean antibody titers against 2 influenza virus strains A/17/duck/Potsdam/86/92 [17/H5] and A/chicken/Suzdalka/Nov-1 1/2005 (H5N1), and by the level of significant (4 times or more) antibody seroconversions after the vaccination.After the use of Ultragrivac the level of secretory IgA in the nasal cavity of vaccinated volunteers in the groups with revaccination intervals of 10 and 21 days increased significantly. The second immunization with 10 or 21 day intervals significantly increased postvaccinal humoral immune response. Humoral immune response induction after 2 vaccinations with 10 day interval was no less effective than with 21 day interval.Ultragrivac allantoic intranasal live influenza vaccine is areactogenic, harmless for vaccinated individuals, safe for those around, and has immunogenic properties against not only homologous virus A(H5N2), but also against influenza strain A(H5N1).

Study in CBA line mice of immunogenicity of cold adapted reassortant influenza virus H5N2 vaccine strain samples produced in rollers in MDCK and Vero cell cultures by using plant derived components.Antibody levels in blood sera and nasal swabs, lungs and small intestine of experimental vaccine strain sample immunized mice were evaluated by using HI reaction in accordance with WHO recommendations.Reassortant vaccine strain A/17/duck/Potsdam/86/92 (H5N2) produced in MDCK and Vero cells by using plant derived components (rice and soy flour hydrolyzate and plant protease based nutrient medium) after intranasal immunization of mice induced local and humoral antibodies, and the latter not only against homologous virus, but also against highly pathogenic avian influenza virus strains A/ Chicken/Suzdalka/Nov-11/2005 and A/Chicken/Kurgan/05/2005.Immunogenicity studies of reassortant influenza virus A/17/duck/Potsdam/86/92 (H5N2) vaccine strain samples cultivated in MDCK and Vero cells by using media with plant derived components in mice show high levels of humoral and secretory immunity.

To study the optimal conditions for roller cultivation of cold-adapted reassortant vaccine strain of influenza virus A/17/Duck/ Potsdam/86/92 (H5N2) in MDCK and Vero cell cultures grown on nutrient medium based on soy and rice flour hydrolysates obtained using trypsin and bromeline.Vaccine strain was cultivated on MDCK and Vero cells in rollers in the presence of plant proteases. Obtained culture samples of vaccine strains were lyophilized and their infectivity was assessed.Cultivation of vaccine strain on MDCK and Vero cells grown in experimental media containing reduced quantity (2 and 3% respectively) of fetal calf serum ("Gibco", USA) resulted in high titers of the virus in the presence of plant proteases (4 mcg/ml of papain and 20 mcg/ml bromeline).Use of plant enzymes and nutrient media based on enzymic plant hydrolysates, including those obtained with bromeline, for cultivation of vaccine strain on MDCK and Vero cell cultures in rollers could make the manufacturing process of live influenza vaccines safer and more cost effective.

MDCK and Vero (B) cell cultures were propagated during 10 passages in the experimental nutrient media containing the soybean powder hydrolyzate prepared using trypsin and bromelain enzymes and the rice powder hydrolysate prepared with trypsin and in the control DMEM and SFM4 MegaVir media. The karyological, morphological, and proliferative characteristics of continuous cultures were examined and compared. The experimental media supplied with 3% fetal bovine serum (FBS) (Gibco, U.S.A.) showed high growth-enhancing properties and failed to affect their morphology. After propagated during 10 passages in the experimental media preserved a stable karyotype. MDCK cell cultures in the nutrient media based on rice and soybean powder hydrolyzates low (2%) in FBS caused no substantial changes in the proliferation indices and morphological and karyological characteristics of cell cultures.

To study antiviral activity of extracts obtained from basidial fungi against influenza viruses of different subtypes.Antiviral activity of extracts obtained from basidial fungi against influenza virus A/chicken/Kurgan/05/2005 (H5N1) was determined in in vitro experiments. Changes in infectiousness of pandemic influenza virus A/Moscow/226/2009 (HIN1)v caused by extracts of basidial fungi was studied in experiments in vitro and in vivo.Seventy water extracts of basidial fungi were studied, of which 10 were able to inhibit infectiousness of influenza virus strain A/ chicken/Kurgan/05/2005 (H5N1) in MDCK cell culture. Also, several studied extracts decreased infectiousness of pandemic influenza virus strain A/ Moscow/226/2009 (H1N1)v in MDCK cells and inhibit its reproduction in lungs of infected mice.High antiviral activity of extracts obtained from basidial fungi against influenza viruses opens perspectives for development of drugs with preventive and treatment effects.

To develop nutrient medium for MDCK and Vero cells based on soy hydrolysate obtained using bromeline and to assess of growth characteristics of influenza virus vaccine strains cultivated on them.Physico-chemical characteristics of hydrolysate were assessed according to FS 42-3874-99. Growth characteristics of nutrient medium based on soy hydrolysate and vaccine strains of influenza virus A/Solomon Islands/03/06 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004 were studied on MDCK and Vero cells.MDCK and Vero cells grew well on medium based on soy hydrolysate obtained using bromeline with decreased (to 2% and 3% respectively) content of fetal calf serum and allowed effective production of vaccine strains of influenza virus.Technology for producing of nutrient medium based on hydrolysate of soy flour obtained using bromeline was developed. This medium could successively used for cultivation of continued cell cultures MDCK and Vero used as substrate for tissue culture-based vaccines against influenza.

Seeding and working banks of the continuous MDCK cell culture suitable for the production of cultured influenza vaccine were created and deposited at liquid nitrogen temperature at the "Vector" State Research Center of Virology and Biotechnology. The MDCK cell culture was shown to have morphology typical of the discussed cell line; it does not have any alien agents and is oncogenically safe; its enzimogram and karyotype are typical of the donor line; finally, its biological properties are stable during a long period of cultivation and its sensitivity to influenza virus is high, therefore, it can be recommended for the production of influenza vaccine. The continuous MDCK cell line was certified at Tarasevich Committee and was recommended by the MIBP Committee, Russia's Health Ministry, for its use as a substrate in the production of diagnostic and preventive immunoglobulins.

C-terminal peptide fragments of the heavy chain (HA1) of influenza virus, subtypes H1 and H3, hemagglutinins were synthesized. Rabbits and guinea pigs immunized with peptides H3 (314-328) and H1 (314-328) conjugates with BSA developed high immune response to conjugated peptides. Solid-phase RIA demonstrated interaction of antisera to peptide H3 (314-328) with A/Aichi/2/68 virus which was inhibited by peptides H3 (314-328) and H3 (317-328) but not by H3 (320-328), that is, antibodies interacted predominantly with amino acids 317-323 HA1. Anti-H3 (314-328)-BSA showed a marked activity in H1 and NT with a number of H3 subtype viruses. Antisera to peptide H1 (314-328) reacted with A/PR/8/34 virus insignificantly and were also inactive in H1 and NT with subtype H1 viruses.

Immunogenic properties of synthetic peptides corresponding to areas 122-133, 136-147, and 154-164 of the heavy chain of A/Aichi/2/68 virus HA were studied in two biological models: rabbits and guinea pigs. Peptides 122-123 and 136-147 together comprise the nearly compete antigenic determinant A, and peptide 154-164 is a part of determinant B. Both animal species immunized with peptide conjugates with bovine serum albumin produced a high immune response to all hapten-peptides. Comparative studies of rabbit and guinea pig sera by hemagglutination-inhibition tests revealed differences in antibody titres (1:10-1:40 for rabbits and 1:512-1:1024 for guinea pigs), the highest titres in both models being observed with peptide 136-147. Examinations of the virus-neutralizing capacity of the sera showed this property to be possessed by anti-(136-147) and anti-(154-164)E, the anti-(136-147) being slightly more active both for rabbits and for guinea pigs.