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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.

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.

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.

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.