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Newly emerged highly pathogenic A/H7N9 viruses with pandemic potential are effectively transmitted from birds to humans and require the development of novel antiviral drugs. For the first time, we studied the in vitro and in vivo antiviral activity against A/H7N9 of oligodeoxyribonucleotides (ODNs), which were delivered into the cells in the proposed TiO2-based nanocomposites (TiO2∼ODN). The highest inhibition of A/H7N9 in vitro (∼400-fold) and efficient, sequence-specific, and dose-dependent protection (up to 100%) of A/H7N9-infected mice was revealed when ODN was targeted to the conserved terminal 3'-noncoding region of viral (-)RNA. After the treatment with ODN, the virus titer values in the lungs of mice decreased by several orders of magnitude. The TiO2∼ODN nanocomposite did not show toxicity in mice under the treatment conditions. The proposed approach for effective inhibition of the A/H7N9 can be tested against other viruses, for example, new emerging influenza viruses and coronaviruses with pandemic potential.

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.

This study describes the effective attack of oligonucleotides on the viral genome of highly pathogenic H5N1 influenza A virus (IAV) in vivo using for the first time the new delivery system consisting of biocompatible low-toxic titanium dioxide nanoparticles and immobilized polylysine-containing oligonucleotides with the native (ODN) and partially modified (ODNm) internucleotide bonds. Intraperitoneal injection of the TiO2•PL-ODN nanocomposite provided 65-70% survival of mice, while intraperitoneal or oral administration of TiO2•PL-ODNm was somewhat more efficient (~80% survival). The virus titer in the lung was reduced by two-three orders of magnitude. The nanocomposites are nontoxic to mice under the used conditions. TiO2 nanoparticles, unbound ODN, and the nanocomposite bearing the random oligonucleotide showed an insignificant protective effect, which indicates the ability of targeted oligonucleotides delivered in mice in the nanocomposites to site-specifically interact with complementary RNAs. The protection of oligonucleotides in nanocomposites by TiO2 nanoparticles and partial modification of the internucleotide bonds provides a continued presence of oligonucleotides in the body for the effective and specific action on the viral RNA. The proposed oligonucleotide delivery system can claim not only to effectively inhibit IAV genes but also to turn off other genes responsible for diseases caused by nucleic acids.

Nucleic-acid-based drugs are a promising class of novel therapeutics; however, their use in medicine is widely limited because of insufficient delivery into cells. This article proposes a new delivery strategy of nucleic acid fragments into cells as components of TiO2-based nanocomposites. For the first time, unmodified Dz molecules were non-covalently immobilized on TiO2 nanoparticles precovered with polylysine (TiO2•PL) with the formation of (TiO2•PL)•Dz nanocomposites. DNAzymes in the proposed nanocomposites were shown to retain their ability to cleave the RNA target in a cell-free system with the same selectivity as unbound Dz molecules. It was shown by confocal laser microscopy that the fluorescein-labelled (TiO2•PL)•DzFlu nanocomposites penetrate into eukaryotic cells, where DzFlu is internalized in the cytoplasm and predominantly in nuclei. Delivery of deoxyribozymes into cells in the proposed nanocomposites permits very efficient interactions with RNA targets inside cells. This was demonstrated by an example of inhibition of H5N1 influenza A virus replication (inhibition by a factor of ca. 3000). This effect was one order of magnitude higher than with using lipofectamine as the transfection agent. The proposed (TiO2•PL)•Dz nanocomposites demonstrated high antiviral activity and are thus potent as nucleic-acid-based drugs.

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.

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.

Development of new antiviral nucleic acid-based drugs and the search for their efficient delivery into cells are an urgent task. We prepared the TiO2·PL-DNA and TiO2·PL±DNA nanocomposites bearing DNA fragments immobilized on TiO2 nanoparticles through polylysine linker (PL) with either covalent or ionic bond between PL and DNA, respectively. The proposed nanocomposites can penetrate into cells without transfection agents. The DNA fragments in both types of the nanocomposites were shown to retain their ability to form complementary complexes. The nanocomposites TiO2·PL-dsDNA and TiO2·PL±dsDNA bearing the double-stranded DNA were also synthesized. The proposed nanocomposites were demonstrated to be efficient antiviral agents against influenza A virus (IAV) in the MDCK cell culture. The TiO2·PL-DNA and TiO 2·PL±DNA nanocomposites bearing the DNA fragments targeted to the 3’-noncoding region of IAV segment 5 inhibited virus replication by four and three orders of magnitude, respectively. Thus, the TiO2 ·PL-DNA nanocomposites appeared to be more efficient than the TiO2·PL±DNA nanocomposites. The antiviral activity of the nanocomposites can be improved by either increasing their capacity for the DNA fragment or using dsDNA. Control samples, i.e., nanoparticles without DNA fragments and vice versa, unbound DNA fragments in the presence of nanoparticles, and nanocomposites bearing DNA fragments with random sequences showed a negligible effect. The presented nanocomposites can be applied in the thriving technology of drug delivery to achieve high therapeutic and biological efficacy.

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.

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO₂ nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO₂∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO₂∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO₂∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO₂∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.

Antiviral activity of TiO2 · PL · DNA/PNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (H3N2 subtype). A PNA fragment in nanocomposites was electrostatically bound in the form of a DNA/PNA heteroduplex to titanium dioxide nanoparticles precovered with polylysine (TiO2 · PL). It was shown that TiO2 · PL · DNA1/PNA1 nanobiocomposite bearing the PNA1 fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with an efficiency of 99.8%. The 50% cytotoxic concentration (TC50) and 50% effective inhibitory concentration (IC50) of the TiO2 · PL · DNA1/PNA1 nanocomposite were evaluated to be more than 1200 μg/mL and less than 3 μg/mL, respectively. Based on these data, the selectivity index (SI) for TiO2 · PL · DNA1/PNA1 nanobiocomposite defined as the TC50/IC50 ratio, was calculated to be more than 400. Thus, TiO2 · PL · DNA/PNA nanobiocomposites were shown to not only penetrate through cell membranes, but exhibit a high specific antisense activity without toxic effects on the living cells.

The data on insecticidal, antimicrobial, and antiviral activity of Bacillus thuringiensis (Bt) strains isolated in the microbiological study of height and ground atmospheric aerosol samples taken in the South of Western Siberia are presented. Among 36 isolated bacteria determined as related to the Bt species, 15 strains were relegated to the Bt ssp. kurstaki subspecies by results of intraspecific testing and studied for the presence of antagonistic attributes. In spite of long wandering in extreme conditions in the composition of atmospheric aerosols, including higher atmospheric layer (up to 7 km), the isolated Bt ssp. kurstaki strains keep high (inherent to this subspecies) entomopathogenic activity, possessed the ability to inhibit the multiplication of such pathogenic microorganisms as Staphylococcus aureus, Salmonella thyphimurium, Shigella sonnei, Candida albicans, and others, and manifested the ability to effectively neutralize the multiplication of the A/chicken/Kurgan/05/2005 (A/H5N1) avian influenza virus and A/Aichi/2/68 (A/H3N2) human virus. Properties of Bt strains isolated from atmospheric aerosols not only verified their well-known resistance to unfavorable factors of the environment but also exhibited the possibility of using them for developing viricides.

Antiviral activity of the TiO2·PL·DNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). DNA fragments in the nanocomposites are electrostatically bound to titanium dioxide nanoparticles precovered with polylysine. It was shown that TiO2·PL·DNA(v3′) nanocomposite bearing the DNA(v3′) fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with the efficiency of 99.8% and 99.9% (i.e., by factors of ∼400 and 1000, respectively) at a low concentration of DNA(v3′) in nanocomposite (0.1 and 0.2 μM, respectively). The TiO2·PL·DNA(r) nanocomposite containing an oligonucleotide noncomplementary to viral RNA or oligonucleotide DNA(v3′) unbound to the nanoparticles show very low antiviral activity (inhibition by factors of ∼3.5 and 1.3, respectively).

We studied toxicity and antiviral activity of aqueous and ethanol extracts of bioactive substances from the biomass of nematophagous fungus Duddingtonia flagrans prepared by submerged culturing of the mycelium. It is found that both extracts were characterized by low toxicity for cultured Vero cells and inhibited reproduction of DNA-viruses in this cell line. Ethanol extract of the fungus exhibited higher in vitro antiviral activity against Herpes simplex virus type 2, ectromelia virus, and vaccinia virus than water extract, which can be due to higher content of proteins, polysaccharides, flavonols, catechins, or carotenes or more effective their combination. The extracts of cultured mycelium of Duddingtonia flagrans fungus containing a complex of bioactive substances can be used for creation of broad-spectrum antiviral drugs against DNA-viruses.

Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).

Influenza A viruses play a significant role in human and animal pathologies that cause epidemics and epizootics. Therefore, the development of new anti-flu drugs has become increasingly urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently cleave RNA molecules with high specificity. In this study, a number of genomic sequences of the most relevant influenza A virus subtypes, i.e., H5N1, H3N2, and H1N1, were analyzed. Conserved regions were revealed in the five least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with 10–23 deoxyribozymes were determined. We designed and synthesized 46 virus-specific 33-mer deoxyribozymes with the general structure of 5′N8AGGCTAGCTACAACGAN9. Screening of the antiviral activity of these agents in combination with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005(H5N1) revealed 17 deoxyribozymes that suppressed the titer of virus cytopathicity by more than 2.5 logTCID50/mL (i.e. the neutralization index of the virus was more than 300), five of which suppressed the virus titer by a factor of 1000 or more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded NP nucleoprotein.

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.

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.

Antiviral activity against type A influenza virus of birds A/chicken/Kurgan/05/2005 (H5N1) and humans A/Aichi/2/68 (H3N2) was investigated for aqueous extracts from mycelium of 11 basidial fungi species collected in the Altai Mountains (Altai Republic, Russia). The most perspective strains for producing antiviral medicines are studied strains Daedaleopsis confragosa, Datronia mollis, Ischnoderma benzoinum, Trametes gibbosa, T. versicolor, Laricifomes officinalis, and Lenzites betulina.

Immunogenic properties of synthetic peptides corresponding to regions 122–133, 136–147, 154–164, and 314–328 of the heavy chain (HA1) of A/Aichi/2/68 virus hemagglutinin were studied. Peptides 122–133 and 136–147 together form a nearly complete antigenic determinant A, peptide 154–164 is a part of determinant B, and peptide 314–328 corresponds to the C-terminal HA1 fragment. In a model influenza A/Aichi/2/68 infection in CBA mice, a protective effect of conjugates of BSA with peptides 136–147 and 314–328 was shown. Immunization of animals with conjugates BSA-(136–147) and BSA-(314–328) in combination with interferon inducers (larifan and ridostin) and a plant immunomodulator (immunomax) intensified the protection of mice against the influenza infection.

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).

Titanium dioxide (TiO2) in a suspension or absorbed on a film possesses bactericidal and virucidal properties caused by photocatalytic reactions. Our electron microscopic examinations showed that titanium dioxide nanoparticles destroyed the influenza virus after 30 min incubation. Virological studies revealed the inactivation of the influenza virus by TiO2, depending on the concentration of nanoparticles and the incubation period. The inactivation effect was observed when the incubation was performed in darkness, unlike the TiO2 suspension. We propose that the virus-inactivation properties of TiO2 are mainly based on the direct contact between nanoparticles and virus particles.

The impact of culturing conditions (multiplicity of cell culture infection with influenza virus, composition of growth and maintenance nutrient media) for the efficiency of multiplication of cold-adapted reassortant vaccine strains of influenza A and B viruses was evaluated. Soybean hydrolysate protein-based biological additive to nutrient medium provided effective reproduction of influenza A virus in MDCK cells in the presence of 2 microg/ml trypsin. The use of soybean peptone-based stabilizer provided retention of infectious titer of influenza virus grown in MDCK culture after its lyophilization to a level of 8.5 lg EID50/ml.

The development of new antiviral nucleic acid-based drugs and the search for their efficient delivery into cells are an urgent task. We developed methods of immobilizing DNA fragments to titanium dioxide nanoparticles with the formation of TiO2∼DNA nanocomposites. It is shown that DNA fragments in nanocomposites retain their ability to form complementary complexes and can be delivered into cells without transfection agents and other methods of exposure. The proposed nanocomposites were shown to be efficient agents to affect target nucleic acids inside cells with an example of inhibition of influenza A virus (IAV) reproduction. The nanocomposites bearing the DNA1 fragment targeted to the 3′-noncoding region of segment 5 (-)vRNA showed a low toxicity (TC50 ≥ 1500 µg/ml) and a high antiviral activity against different IAV subtypes (H1N1, H5N1, and H3N2) in infected MDCK cells (the virus replication was inhibited by up to four orders of magnitude). The IC50 value for nanocomposites was estimated to be 1.5 µg/ml (30 nM for DNA), so its selectivity index was calculated as ∼1000. Control samples, i.e., nanoparticles without DNA fragments and vise versa as well as unbound DNA fragments in the presence of nanoparticles were much less active, if at all. A high site-specificity of the action of designed nanocomposites (∼3000-fold difference between the effect of the nanocomposites containing target-directed DNA and DNA with a random sequence) indicates a pronounced antisense effect. The presented types of nanocomposites can be applied in the thriving technology of drug delivery to achieve high therapeutic and biological efficacy.

SiO2 nanoparticles as platform for delivery of 3′-triazole analogues of AZT-triphosphate into cells were proposed. The results confirmed the successfulness of the Huisgen's 1,3-dipolar cycloaddition reaction for the preparation of the SiO2∼dNTP nanocomposites. It was shown that polymerase can recognize nucleoside triphosphates in the proposed nanobiocomposites and incorporate them into the growing DNA chain. The proposed SiO2∼dNTP nanocomposites were demonstrated to penetrate into eukaryotic cells. The tentative results indicate that these nanocomposites at low concentrations can inhibit the reproduction of POX and Herpes viruses. This makes it possible to use the nanobiocomposites bearing nucleoside triphosphate analogues as promising therapeutic drugs.

Therapeutic and preventive treatment of mice intranasally infected with a lethal dose of A/Aichi/2/68 (H3N2) influenza virus with anaferon (pediatric formulation) demonstrated an antiviral effect of the drug (increased percent of survivors and prolonged lifespan).

Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).

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.

Results of phase II of a clinical trial of the influenza allantoic intranasal live vaccine "Ultragrivac" (type A/H5N2) are presented. The vaccine was developed based on strain /17/Duck/Potsdam/86/92 H5N2 [17/H5] - reassortant of two viruses, /Leningrad/134/17/57 (H2N2) and /Duck/Potsdam/1402-86 (H5N2), obtained from the Virology Department, St. Petersburg Institute of Experimental Medicine.Two schemes of immunization (with revaccination on days 10 and 21) were used. Evaluation of vaccine immunogenicity included determination of local, cellular and humoral immunity. A significant rise in the level of secretory IgA in the nasal cavity of vaccinated volunteers (with revaccination on days 10 and 21) was documented after application of the vaccine. The postvaccination humoral immune response was estimated from the level of significant (4-fold and more) antibody seroconversions, geometric mean titers of antibodies to two strains of influenza virus /17/Duck/Potsdam/86/92 H5N2 [17/H5] and /Chicken/Suzdalka/Nov-11/2005 (H5N1), and their incremental rate. Results of measurement of antibody titers in hemagglutination-inhibition assay are presented, with two antigens being used to analyse all serum samples from volunteers twice vaccinated with influenza vaccine "Ultragrivac" at 10 and 21 day intervals. Result of phase II of this clinical study show that influenza allantoic intranasal live vaccine "Ultragrivac" is nonreactogenic and safe for both vaccinated and surrounding individuals. Moreover, it is sufficiently immunogenic with respect not only to homologous virus A(H5N2) but also to the A(H5N1) strain.