[Antiviral and virucidal activity of sodium deoxyribonucleate and its complex with iron against viruses of different kingdoms and families].

INTRODUCTION: The urgent problem of modern medicine is the fight against acute respiratory viral infections (ARVI). To combat ARVI, drugs of wide antiviral potency are needed, as well as immunomodulating drugs. Such antiviral and immunomodulatory effects has sodium deoxyribonucleate (DNA-Na) and its complex with iron (DNA-Na-Fe) developed on the basis of double-stranded DNA of natural origin. AIM OF THE STUDY: To assess antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against viruses of different kingdoms and families. MATERIALS AND METHODS: Antiviral and virucidal activity of DNA-Na and DNA-Na-Fe was assessed in cell cultures infected with viruses. RESULTS AND DISCUSSION: DNA-Na and DNA-Na-Fe had antiviral activity against adenovirus at concentrations of 2501000 mcg/ml. Antiviral effect of both drugs was not detected in case of poliovirus. DNA-Na and DNA-Na-Fe had antiviral activity against coronavirus in all administration schemes. EC50 for DNA-Na ~ 2500 mcg/ml, for DNA-Na-Fe ~ 1000 mcg/ml. In cells treated with DNA-Na-Fe, secretion of following proinflammatory cytokines was detected: Interleukin (IL) 1, IL-2, IL-6, IL-18, interferon- (IFN-), IFN-, as well as anti-inflammatory cytokines: IL-4, IL-10, antagonist of IL-1 receptor. Evidently, DNA-Na and DNA-Na-Fe have antiviral effect, but mechanism of action does not seem to be associated with specific effect on viral replication. Presence of virucidal activity of drugs against representatives of Coronaviridae, Adenoviridae, Picornaviridae, Retroviridae, Herpesviridae in vitro test in range of 1.03.0 lg TCID50 was identified. CONCLUSION: Presence of simultaneous antiviral and virucidal activity of DNA-Na and DNA-Na-Fe against adeno- and coronaviruses shows their prospects for prevention and treatment of ARVI.

Determination of Bactericidal Activity Spectrum of Recombinant Endolysins of ECD7, Am24, Ap22, Si3, and St11 Bacteriophages.

The bactericidal activity of recombinant endolysins LysECD7, LysAm24, LysAp22, LysSi3 and LysSt11 was assayed in multidrug resistant strains (n=120) of Salmonella enterica, E. coli, Acinetobacter baumannii, Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Campylobacter jejuni. The assay showed that the recombinant endolysins had a wide spectrum of bactericidal activity compared to endolysins of their progenitor phages. Among examined endolysins, we selected the active pharmaceutical substances with broad spectrum of bactericidal activity. Most strains were sensitive to LysECD7 (70.7%), LysAm24 (65%), and LysAp22 (58.6%), which seems to be promising causative agents for the development of finished dosage form.

Analysis of the Pharmacokinetics of Suppository Forms of Bacteriophages.

Pharmacokinetics of suppository forms of bacteriophages was studied on male Chinchilla rabbits. Suppositories with various composition of bacteriophages were administered once per rectum to rabbits, and the presence of phage particles was estimated in the blood, urine, and feces over 24 h. Pharmacokinetic study showed that the phages were detected in the blood, urine, and feces at various terms of the experiment irrespective of the size of viral particles, which confirmed the possibility of their systemic effects after rectal administration. Thus, the use of suppository form of bacteriophages can ensure the presence of phage particles even in infection foci that cannot directly contact with the preparation.

Genetic Basis of Attenuation of Cold-Adapted Influenza Strain B/Leningrad/14/17/55 - Backup Master Donor Virus for Influenza Type B Live Attenuated Vaccines.

The reassortant vaccine strain of live attenuated influenza vaccine inherits temperature sensitivity and areactogenicity from cold-adapted attenuated master donor virus. In Russia, B/ USSR/60/69 master donor virus (B60) is currently in use for the preparation of live attenuated type B influenza vaccine candidates. Trivalent live attenuated influenza vaccine based on A/ Leningrad/134/17/57 and B60 are licensed for the use in Russia for single dose vaccination of adults and children over 3 years. B/Leningrad/14/17/55 (B14) cold-adapted virus is a backup master donor virus for live attenuated type B influenza vaccine. According to our preliminary estimates, it is more attenuated than B60, which can allow expanding applicability of this vaccine for children under 3 years of age. In this paper, the role of B14 genes in its attenuation was assessed. Representative collection of reassortants of B14 with epidemic influenza B viruses was obtained, a phenotypic analysis of reassortants was performed, and their pathogenicity for animals was assessed. The leading role of PB2 and PA genes in attenuation of B14 master donor virus was proven.

[Antiviral activity of extracts of basidiomycetes and humic compounds substances against Human Immunodeficiency Virus (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus 1) and Herpes Simplex Virus (Herpesviridae: Simplexvirus: Human alphaherpesvirus 1)].

INTRODUCTION: One of the most urgent problem of modern medicine is the fight against the disease caused by the Human Immunodeficiency Virus (HIV) - HIV infection. The chemical compounds have improved the situation for infected people, but they are toxic, disrupt the metabolism and cannot eliminate the integrated virus from the body. The emergence of resistant HIV strains makes these treatments ineffective. Often, the death of HIV-infected people occurs as a result of the development of opportunistic infections caused by viruses of the Herpesviridae family. Therefore, the search for new therapeutic and preventive drugs that are less toxic and active against several viruses at the same time is relevant. Basidiomycetes, higher fungi, are a source of medicinal compounds that have antimicrobial properties, as well as antiviral ones. Humic compounds (HS) of various nature also have antiviral activity.The aim of the study was to obtain nontoxic compounds from the basidiomycete Inonotus obliquus and humic compounds from brown coals and to test their activity against viruses that are pathogenic to humans: HIV and Herpes Simplex Virus (HSV). MATERIAL AND METHODS: The antiviral activity of melanin extracts obtained from the culture of the chaga fungus Inonotus obliquus and HS from the brown coal of the Kansko-Achinsk Deposit was studied using a model of MT-4 lymphoblastoid cells infected with HIV type 1 (HIV-1) strains and a monolayer culture of Vero cells infected with HSV type 1 (HSV-1) using virological and statistical research methods. RESULTS AND DISCUSSION: It was found that all the studied compounds did not have a cytotoxic effect on cells at a concentration of 100 mcg/ml. It was shown that extracts of basidiomycetes and HS have antiviral activity against HIV-1 and HSV-1. EC 50 (50%-effective concentration) for HIV-1 was 3.7-5.0 mcg/ml, selectivity index 28-35. Antiherpetic activity was detected at a dose of 50-100 mcg/ml. The antiviral effectiveness of melanin compounds was established both in the «preventive¼ (2 hours before cell infection) and in the «therapeutic¼ regimen of drug administration, both for HIV-1 and HSV-1. The presence of antiviral activity of melanin and HS in relation to the RNA-containing HIV-1 virus and DNA-containing HSV-1 virus in our study coincides with the results of a number of authors in relation to influenza viruses, herpes virus, HIV, hepatitis B virus, Coxsackievirus, smallpox vaccine virus, which suggests that the type of nucleic acid in the virus does not play a fundamental role in the antiviral action of these drugs. It is also clear that HS is effective against both enveloped and non-enveloped viruses. CONCLUSION: In general, it can be concluded that melanin and humic compounds are characterized by low toxicity in the presence of both virucidal and antiviral activity. This allows us to consider the studied compounds as the basis for creating safe medicines that are effective against pathogens of various viral infections.

Bacteriophage Cocktail Effectively Prolonging the Shelf-Life of Chilled Fish.

We developed a technological accessory bacteriophage-based preparation and a method for phage-mediated bioprocessing for elimination of pathogenic microorganisms from the surface of fresh fish and for prolongation of the shelf-life of chilled hydrobionts. Specimens of rainbow trout (Salmo irideus) served as the objects of the study carried out at a fish-processing plant in the Republic of Karelia. The specimens were decontaminated by a bacteriophage cocktail containing six original virulent phage strains characterized by their pheno- and genotypical properties. A new method of biodecontamination (plunging the rainbow trout for 30 sec into a solution of bacteriophage cocktail (bacteriophage titers ≥108 PFU/ml) delayed bacterial degradation of hydrobionts by 3 days. The use of the new method for decontamination of food half-products - phage-mediated bioprocessing - promoted preservation of the initial ecological purity, nutritive value, and taste of the products and prolonged their shelf-life in comparison with the actual standards.

Development of a Bacteriophage Complex with Superhydrophilic and Superhydrophobic Nanotextured Surfaces of Metals Preventing Healthcare-Associated Infections (HAI).

We studied antibacterial properties of organo-inorganic hybrid coatings on the AMg2 aluminum alloy including superhydrophilic and superhydrophobic nanotextured metal substrates with applied bacteriophage particles. Bactericidal activity of surfaces after artificial contamination with a bacterial suspension was evaluated. To increase bactericidal effect of the plates, bacteriophage was sorbed on their surface. In the experiments simulating possible spreading of HAI pathogens, higher bactericidal activity of superhydrophilic surfaces in comparison with superhydrophobic ones. Application of bacteriophage particles did not prevent primary colonization of textured metal surfaces by strains used in the experiment, but in some cases increases their bactericidal activity.

Experimental Study of Genetic Constellation of Cold-Adapted Master Donor Viruses for Live Attenuated Influenza Vaccine Type B.

We studied the constellation of genes encoding polymerase complex proteins of master donor viruses for Russian live attenuated influenza vaccine type B. Reassortants of the reserve attenuation donor B/Leningrad/14/17/55 with B/USSR/60/69 master donor virus currently used for manufacturing seasonal influenza vaccine were prepared and examined. Most reassortants obtained by the classical reassortment method inherited all genes from the B/Leningrad/14/17/55 virus except the gene encoding PB1 subunit of the polymerase complex. One reassortant was selected for further evaluation of the role of PB1 gene. Greater attenuation of the strain for experimental animals (mice) in comparison with the original strains was demonstrated. This indicates high degree of constellation of genes of cold-adapted master donor viruses and the important compensating role of amino acid substitutions in the PB1 protein of B/Leningrad/14/17/55 donor virus in preventing viral hyperattenuation.

[Diagnostic bacteriophage V32 as a tool for the rapid identification of Escherichia coli serogroup O157.]

Bacteriophage V32, a representative of bacterial viruses of the Myoviridae family Ounavirinae subfamily, is proposed for search and identification of E. coli O157 serogroup, including Shiga-toxin producing E. coli O157:H7 (STEC O157:H7), among cultures of enterobacteria from the primary seeding of the material studied. Phage genome containes a linear double-stranded DNA of 87875 base pairs with G/C-content of 38.9% and includes 132 open reading frames (ORF). In the genome, there are no determinants of antibiotic resistance, virulence genes of STEC and other well-known pathogroups of E. coli. It has been established that phage V32 has lytic activity against all studied cultures of E. coli O157 serogroup (n=183) isolated from people and farm animals in various regions of the Russian Federation, as well as in Japan and Italy. At the same time, the phage lyses only 6 of 182 strains (3.3%) of E. coli not belonging to the O157 serogroup and is not active against strains of other enterobacteria. That is, the phage has a high specificity. The use of bacteriophage V32 as a diagnostic tool is a highly efficient, fast, cheap and simple method for identifying E. coli serogroup O157, including the serotype E. coli O157: H7, in any bacteriological laboratory without special equipment and special training of performers.

Experimental Application of Organic-Inorganic Hybrid Coatings with Adsorbed Bacteriophages for Reducing the Risk of STEC Infections.

A new approach to prevention of STEC infection is based on the synergic effect of combined bactericidal activity of bacteriophages and organic-inorganic hybrid coating of metals. The coatings are characterized by superhydrophilic or superhydrophobic properties and multimodal granularity with the texture incorporating metal oxide nanoparticles and bacteriophages. Superhydrophilic surfaces are characterized by significantly higher bactericidal activity than superhydrophobic ones. The cytotoxicity of superhydrophobic surfaces can be increased due to antibacterial activity of bacteriophages combined with the superhydrophobic characteristics of the materials.

Development of Pyrosequencing Assay for Evaluation of Genetic Stability of Vaccine Strains of Live Attenuated Influenza Type B Vaccine.

We developed a pyrosequencing protocol for monitoring of stability of attenuating mutations in the genome of vaccine reassortants based on master donor virus of Russian live attenuated influenza vaccine B/USSR/60/69. The developed protocol allows rapid and accurate assessment of mutations and can be used for analysis of genetic stability of reassortants during vaccine strain development and manufacturing, as well as genetic stability of vaccine isolates of influenza B virus during pre-clinical and clinical trials.

Composition of the Stabilizer and Conditions of Lyophilization for Preserving Infectious Activity of Influenza Virus.

For stabilization of vaccine preparations, they are lyophilized. The composition of the protective medium is an important parameter affecting the quality of the vaccine after drying. In view of the risk of spreading prion diseases, the use of media containing animal proteins is not recommended. In this study, protective media containing no animal proteins and lyophilization regimen were determined. The optimum lyophilization regimen consisted of three stages: freezing at -70°C, main stage at -35°C, and drying at 24°C. Protective medium containing 4% trehalose or protective medium with 10% sucrose and 5% soy peptone ensured highest stability of the lyophilized vaccine preparation in temperature range of 4-24°C. This can help to overcome possible break in the cold chain, which is important during transporting or storage of vaccine preparations.

[Morphological Features of the Transgenic Tobacco Plant Shoot Expressing the 3-Hydroxy-3-Methylglutagyl-CoA Reductase (HMG1) Gene in the Direct and Reverse Orientations Towards the Promoter].

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.

[Syncope in Young Elite Athletes].

Syncope - one of the most common syndrome in the general population - may be a manifestation of diseases associated with risk of sudden death. OBJECTIVE: To evaluate the prevalence, pathogenesis particular syncope, near syncope in children involved in elite sport. MATERIAL AND METHODS: The study involved 500 young elite athletes aged 9-18 years (mean age 16.1+/-2.2 years), member of Russian national teams.

A small-scale experiment of using phage-based probiotic dietary supplement for prevention of E. coli traveler's diarrhea.

Traveler's diarrhea (TD) is caused by Escherichia coli in 30% of cases. We have developed a phage cocktail for prophylaxis of TD caused by E.coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes or Staphylococcus aureus, and investigated its effectiveness against infection caused by the non-pathogenic Lac (-) strain of E.coli K12 C600 in animal and human trials. On the 6th day of both animal and human trials E. coli K12 C600 strain was detected in titer of 104 CFU/g of mice feces and 106 CFU/g of human feces in the control (untreated) groups, while it was not detected in the samples of either of the study (phage-treated) groups. These results have great significance because the original coliphages included in the cocktail have a broad host-range including ETEC, EAEC and EHEC strains which cause severe cases of TD.

[Microvolt T-wave alternans in adolescent elite athletes].

UNLABELLED: Microvolt T-wave alternans (MTA) is one of markers of electrical myocardial instability. STUDY AIM: To elucidate normal range of MTA parameters during bicycle exercise test (ET) in adolescent elite athletes. MATERIAL AND METHODS: We examined 500 athletes aged 9-18 (mean 16.1 ± 2.2) years members of national teams of 27 kinds of sport. There were 333 females with mean age 16.0 ± 1.01 years and 167 males with mean age 16.3 ± 1.4. All athletes underwent ECG registration in clino- and ortho- positions and bicycle ET with automatic analysis of MTAT-wave alternans. RESULTS: Mean MTA value was 26 ± 13 (9-51) mV. MTA above 65 mV was registered in 10 persons (2.1%) 5 of whom had signs of overtraining. Sensitivity (Se) of MTA for detection of overtraining state was 21%, specificity (Sp)--99%, positive and negative predictive value--50 and 96%, respectively. CONCLUSION: Normal MTA range during ET is 9-51 mV. MTA values during ET do not depend on age and sex but are determined by duration of participation in sports and myocardial mass. MTA value is related to length of QT-interval. Increase of maximal MTA values above 65 mV is insensitive (Se 21%) but highly specific (Sp 99%) sign of overtraining in adolescent elite athletes.

[A panel of the drug-resistance HIV-1 clinical isolates].

A panel of 16 HIV-1 isolates was designed. Those isolates were isolated from patients undergoing HAART and developing resistance to the antiretroviral drugs. It was shown that the isolates were resistant to nucleoside RT inhibitors (retrovir, epivir) and non-nucleoside inhibitors (viramun). Isolates had stable replication activity. Average rate of cells expressing viral Ag was 14-20%. The infectious titer was 2.4 Ig TCID50. The sequencing showed that all isolates were of the subtype A dominating in the major part of Russian Federation. This panel could be used as the biotechnological base for studying antiretroviral drugs of new generation and for the design of experimental vaccines.

[Microvolt T-Wave Alternans in Adolescent Elite Athletes].

Microvolt T-wave alternans (MTA) is one of markers of electrical myocardial instability. STUDY AIM: to elucidate normal range of MTA parameters during bicycle exercise test (ET) in adolescent elite athletes. MATERIAL AND METHODS: We examined 500 athletes aged 9-18 (mean 16.1+/-2.2) years members of national teams of 27 kinds of sport. There were 333 females with mean age 16.0+/-1.01 years and 167 males with mean age 16.3+/-1.4. All athletes underwent ECG registration in clino- and ortho- positions and bicycle ET with automatic analysis.

[Contribution of neuraminidase of influenza viruses to the sensitivity to the serum inhibitors and reassortment efficiency].

The live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental and the 6 internal protein-encoding gene segments from the cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles to developing LAIV vaccine strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. The genomic composition analysis of 849 reassortants revealed that over 80% of the reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of the vaccine genotype reassortants was achieved when WT parental viruses were resistant to the non-specific serum inhibitors. We demonstrate that NA may play a role in the influenza virus sensitivity to the non-specific serum inhibitors. Replacing NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to the non-specific inhibitors.

[Codon optimization of hemagglutinin as a promising tool for improving of the influenza vaccines immunogenicity].

Modification of the codon bias of sequences is a promising tool of the gene expression control. The theoretical basis of the codon optimization is reviewed, data on experiments in changing the viral gene codon bias for purposes of vaccine development are discussed. Research into the field of the influenza vaccine immunogenicity improvement with codon optimization method is reviewed. Prospects of the use of the codon optimization technique for influenza vaccine development are considered.