Anti-Influenza Activity of Cetraria islandica Lichen Extracts in In Vitro Experiments.

The toxicity and antiviral activity of extracts obtained by the methods of aqueous and ethanol extraction of bioactive substances from Cetraria islandica lichen as a raw material were studied. Aqueous and ethanol extracts of lichen were characterized by low toxicity with respect to the passaged MDCK cell culture and exhibited antiviral activity. The ethanol extract showed more potent in vitro antiviral activity against human A/H3N2 and avian A/H5N1 influenza viruses: in a concentration of 50 µg/ml, it suppressed replication of these viruses by 3.5 and 4 log10, respectively, while the aqueous extract inhibited replication of viruses by 2 and 6 log10, respectively, when taken in a concentration of 500 µg/ml that was 10-fold higher than the concentration of the ethanol extract.

Estimation of Absolute Bioavailability of the Chemical Substance of the Anti-Smallpox Preparation NIOCH-14 in Mice.

We compared absolute bioavailability of the chemical substance of the anti-smallpox preparation NIOCH-14 and chemical compound ST-246 active against orthopoxviruses after oral administration to mice in doses of 10 and 50 µg/g and intravenous administration to mice in a dose of 2 µg/g body weight. The absolute bioavailability of NIOCH-14 is comparable with the absolute bioavailability of ST-246.

Evaluation of the Effectiveness of Metabolites of Bacterial Strains Bacillus thuringiensis against Human Influenza Virus A/Aichi/2/68 (H3N2) In Vitro and In Vivo.

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.

Study of Antiviral Activity of Metabolites of a New Serratia species K-57 Strain.

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.

[Impact of Delivery Method on Antiviral Activity of Phosphodiester, Phosphorothioate, and Phosphoryl Guanidine Oligonucleotides in MDCK Cells Infected with H5N1 Bird Flu Virus].

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.

Toxicity and Antiviral Activity of the Extracts of Submerged Mycelium of Nematophagous Duddingtonia flagrans Fungus in Vero Cell Culture.

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.

[Composites of Peptide Nucleic Acids with Titanium Dioxide Nanoparticles. IV+. Antiviral Activity of Nanocomposites Containing DNA/PNA Duplexes].

Antiviral activity of TiO2 · PL · DNA/PNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). PNA fragment in nanocomposites as a DNA/PNA heteroduplex is electrostatically bound to titanium dioxide nanoparticles precovered with polylysine (TiO2 · PL). It was shown that TiO2 · PL · DNA1/PNA1 nanobiocomposit bearing PNA1 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%. It was determined that the 50% cytotoxic concentration (TC50) of the TiO2 · PL · DNA1/PNA1 nanocomposite is more than 1200 mg/mL. And 50% effective inhibitory concentration (IC50) is less than 0.003 mg/mL. Based on these data, the selectivity index (SI) for TiO2 · PL · DNA1/PNA1 nanobiocomposite defined as the ratio TC50/LC50, is more than 400. Thus TiO2 · PL · DNA/PNA nanobiocomposites can not only penatrate through cell membrane, but and are able to exhibit a high specific antisense activity, without causing toxic effects on the living cells.

[Eficient inhibition of human influenza A virus by oligonucleotides electrostatically fixed on polylysine-containing TiO2 nanoparticles].

Antiviral activity of 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 pre-covered 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% (or by factors of~400 and 1000) at a low concentration of DNA(v3') in nanocomposite (0.1 and 0.2 µM, respectively). The TiO2 * PL * DNA(r) nanocomposite containing oligonucleotide noncomplementary to viral RNA or the oligonucleotide unbound to the nanoparticles show very low antiviral activity (inhibition by factors of~3.5 and 1.3, respectively).

[Design of deoxyribozymes for inhibition of influenza A virus].

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).

Synthesis of nanoscale TiO2 and study of the effect of their crystal structure on single cell response.

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.

[Comparative immunogenicity studies of cultural and peptide influenza vaccines].

AIM: 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. MATERIALS AND METHODS: Anti-hemagglutinin and virus neutralizing activity of mice sera was determined in MN and HI reactions in accordance with the WHO recommendations. RESULTS: 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. CONCLUSION: 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.

[Efficacy of combined administration of ridostin and oseltamivir (tamiflu) for therapy and prophylaxis of experimental infection induced by influenza virus A (H5N1) in mice].

AIM: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: Treatment effect during combined administration of ridostin and tamiflu after influenza virus infection increased.

[Evaluation of immunogenicity and safety of 2 immunizations with allantoic intranasal live influenza vaccine Ultragrivac].

AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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).

[Immunogenicity of influenza virus H5N2 vaccine strain samples produced by roller cultivation in media with plant derived components].

AIM: 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. MATERIALS AND METHODS: 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. RESULT: 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. CONCLUSION: 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.

[Use of plant-origin components in roller cultivation of vaccine reassortant influenza virus strain H5N2].

AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[In vitro and in vivo efficacy of Ingavirin against strains of pandemic influenza virus A(H1N1/09)v].

AIM: To study efficacy of Ingavirin in vitro and in vivo against strains of pandemic influenza virus A(H1N1/09)v and influenza virus A(H5N1) and A(H3N2). MATERIALS AND METHODS: Changes in hemagglutinating and cytopathic activity of influenza virus strains A(H1N1/09)v, A(H5N1) and A(H3N2) during their incubation in the presence of Ingavirin or Remantadin on MDCK cell culture were studied. In mice infected by influenza strains A(H1N1/09)v and A(H3N2) and orally treated with Ingavirin, Tamiflu or Remantadin virus titers in lungs were measured. RESULTS: There was decrease in hemagglutinating and cytopathic activity of influenza virus strains after incubation with Ingavirin in vitro. Ingavirin effectively inhibited reproduction of influenza virus strains A(H1N1/09)v and A(H3N2) in lungs of infected mice. Titers of these strains in lung homogenates decreased when Ingavirin was orally administered to infected mice. CONCLUSION: Strains of influenza virus A(H1N1/09)v were susceptible to Ingavirin and Tamiflu but resistant to Remantadin. Reference strains of A(H5N1) and A(H3N2) were susceptible to Ingavirin, Tamiflu and Remantadin.

[The morphological and karyological characteristics of MDCK and vero (B) cells cultures on plant hydrolyzate-based nutrient media].

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.

[Study of antiviral activity of extracts obtained from basidial fungi against influenza viruses of different subtypes in experiments in vitro and in vivo].

AIM: To study antiviral activity of extracts obtained from basidial fungi against influenza viruses of different subtypes. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: High antiviral activity of extracts obtained from basidial fungi against influenza viruses opens perspectives for development of drugs with preventive and treatment effects.

[Development of new nutrient medium for MDCK and Vero cells based on soy hydrolysate obtained using bromeline and assessment of growth characteristics of influenza virus vaccine strains cultivated on them].

AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[In vitro efficacy of Ingavirin against the pandemic influenza virus A(H1N1/09)v].

Ingavirin was shown to be efficient in inhibition of the influenza virus strains A/California/04/2009 (H1N1)v, A/California/07/2009 (H1N1)v, A/Moscow/225/2009 (H1N1)v and A/Moscow/226/2009 (H1N1)v, as well as the strains A/Chicken/Kurgan/05/2005 (H5N1) and A/Aichi/2/68 (H3N2) in the MDCK cell culture. The hemagglutinin and cytopathic activity of the influenza virus strains decreased at entering Ingavirin in vitro.