[Comparative study of MDCK and CaCo-2 cell lines for influenza virus isolation].

Study of effectiveness of CaCo-2 cell line for influenza virus isolation was carried out. It was shown that the properties of this cell line strongly depended on the source of its origin and cultivation conditions. The infectious activity of the influenza viruses on CaCo-2 cell line was virtually the same as in the MDCK cell line. The rate of the viral isolation was virtually identical for both cell lines tested, but viruses from post-mortem materials were isolated only in CaCo-2 cell line. In general, the CaCo-2 line is believed to be a valuable cell line for virological research, particularly for influenza virus isolation.

[Effect of different water-soluble forms of the fullerene C60 on the metabolic activity and ultra-structure of cells in culture].

In view of contradictory data on the toxicity of fullerenes for live organisms we studied the effect of water-soluble complexes of C60 with N-polyvivyl-pirrolidone (C60/PVP) and gamma-cyclodextrine (C60/gamma-CD) on MA-104 cells in culture. Both complexes proved to be non-toxic for cultured cells in the dark in wide range of concentrations. Both complexes provoke changes of cellular ultra-structure which reflect the enhancement of metabolic activity. At the same time only the exposition with the complex C60/PVP leads to the essential growth of number and size of mitochondria. However, the effect of two studied water-soluble forms of C60 under intensive UV-irradiation of cells proved to be opposite: C60/PVP had a cyto-protective action while C60/gamma-CD caused a significant growth of photo-toxicity. Possible reasons of the differences in the action of different forms of C60 on living organisms are discussed.

[Evolutionary variability of influenza B viruses in Russian Federation in 2005-2012].

Specific traits of influenza B viruses circulation in Russia and worldwide in 2005-2012 were studied and the amount of influenza B viruses in the whole population of influenza viruses isolated in Russia was estimated. The trend toward antigenic drift for both Victoria and Yamagata lineages was characterized. The genetic analysis revealed amino acid changes that influenced the antigenic properties of the viruses. The match of the epidemic isolates and vaccine strains was corroborated.

[Development of influenza surveillance in Russia in the system of the WHO national influenza center].

Analysis of development influenza activity season 2010-2011 is presented. Significant participation of influenza A(H1N1)pdm09 virus and influenza B of Victoria lineage virus in the epidemic morbidity structure with minor participation ofA(H3N2) virus was revealed. The influenza viruses isolated in Russia according to antigenic properties were similar to the strains included in the vaccine composition. Drift variants of influenza A(H1N1)pdm09 viruses isolated in Astrakhan and St.-Petersburg were recognized using WHO CC in London as representatives of three new genetic groups.

[H1N1V influenza epidemic of 2009 in Russia].

The paper describes dynamics, distribution and morbidity rate during the 2009 A(H1N1)v influenza epidemic in Russia. The epidemic appears to have been especially severe in the cities of the Far-East and Siberian Federal Districts where the average morbidity rate ranged from 6.4% to 19.2% (mean 10.3%) and the epidemic duration from 7.8 to 8 weeks. In less affected Southern and Central Federal Districts A(H1N1)v influenza occurred in 5.7% of the population. Schoolchildren aged 7-4 years showed the highest morbidity rate of 28.8%. The age group of 18-53 years accounted for 79.4% of the total lethality. Viral isolates were genetically stable and exhibited 98.9% hemagglutnin (HA) homology with reference viruses. None of the strains had an amino acid substitution at position 275 of neuraminidase (NA) responsible for resistance to oseltamivir. Towards the end of the epidemic, the viral population displayed a significant rise in the number of strains containing mutations in 4 genes (4 HA, 2 NA, 2 PB2 and 1 PA mutations respectively). 26.7% of the viral isolates obtained in the end of the epidemic had D222G substitution responsible for tropism of viruses to lung tissues. Epidemiologically, the 2009 A(H1NI)v influenza epidemic is described as moderate based on the absence of pathogenicity determinants typical of both A(H1N1) influenza virus of 1918 and A(H5N1) virus. The paper compares the 2009 epidemic with those caused by A/Honkong/68 and A/USSR/ 90/77 viruses. The necessity of classification for the discrimination between A(H1N1) subtype viruses is emphasized.

[Analysis of pandemic influenza in Russia as a part of the global process based on data of an influenza monitoring reference center].

AIM: Characterization of features of influenza pandemic development in Russia in relation to global process. MATERIALS AND METHODS: Pandemic monitoring was performed by using results of integrative analysis of laboratory diagnostic and population morbidity data from 49 supporting bases of Federal center of influenza from various cities in Russian Federation. Isolation of influenza virus was carried out in MDCK cells and chicken embryos under BSL-3 conditions. Reference virus A/California/07/09 obtained from CDC (Atlanta, USA) and antisera against this strain contained in WHO kit were used for antigenic analysis; rat antisera, new monoclonal antibodies against pandemic influenza virus developed by Research institute of influenza were also used. RESULTS: Based on PCR monitoring during epidemic peak, rate of pandemic influenza identification reached 45-49% of examined patients. About 53% of lethal cases of respiratory infections were caused by pandemic influenza virus, while predominately young people died from pneumonia and acute respiratory distress syndrome. Russian isolates generally were antigenically and genetically similar to the parent pandemic strain--influenza virusA/California/07/09, but contained S203T substitution in hemagglutinin. A number of strains contained D222G mutation that is responsible for the expansion of substrate specificity, as well as strain specific substitutions in hemagglutinin and neuraminidase molecules. The investigated isolates were resistant to remantadin, but sensitive to oseltamivir. CONCLUSION: Due to the formation of population immunity after the end of the first pandemic wave new drift variants of the virus capable of overcoming this formed immunity should be expected that apparently will require the correction of vaccine composition for the 2011 - 2012 season.

[Effect of polymer carrier origin and physical state on fullerene C60 phototoxicity in vitro].

Biological effects of water-soluble inclusion complexes of fullerene C60 with poly(vinyl pyrrolidone) (C60/PVP) and gamma-cyclodextrin (C60/g-CD) as well as solid C60 (C60-coated surface) on cell viability have been studied in vitro. It is established that both inclusion complexes (in a broad range of concentrations) and solid fullerene coatings are nontoxic in the dark for the cell of all lines tested. In contrast, under intense UV illumination, the C60/PVP complex reliably protected test cells from the UV radiation damage, whereas the C60/g-CD and fullerene-coated surface exhibited pronounced phototoxicity. Moreover, solid fullerene caused a photodynamic effect under irradiation with both UV and visible light. The radiation damage could be blocked by some antioxidants (e.g., hypoxen) and singlet-oxygen scavenger (sodium azide). This is evidence for the participation of 1O2 in phototoxicity manifestations. The results indicate that the biological properties of fullerene C60 in vitro depend on its aggregate state, form of solubilization, and, probably, the nature of solubilizing medium.

[The 2009 pandemic influenza in Russia. I. Diagnosis and molecular biological characteristics of the virus].

The analysis of 1558 clinical samples revealed influenza virus A(H1N1v) RNA in 339 patients with influenza and 163 fatal cases,which was made in May to December 2009. Data on the antigenic properties of more than 250 of pandemic virus strains isolated at the Research Institute of Influenza and the molecular genetic characteristics of 31 strains are presented. All the test isolates were found to have the S203 substitution in hemagglutinin, which was characteristic of one of 5 minor genome A(H1N1v) virus variants found in the United States and Mexico in 2009. All the test strains contain the S31N substitution in the M2 protein, which determines viral resistance to adamantine, and have no H275Y substitution in neuraminidase, which determines oseltamivir resistance. The substitution of amino acid residue of Asp to Gly at position 222 of HA was found in 8 (73%) of 11 isolates from postmortem lung and trachea samples and in 2 (10%) of 20 isolates from nasopharyngeal swabs. The determination of the pathogenic role of this substitution calls for further investigations.

[Influence of rimantadine, ribavirine and triazavirine on influenza A virus replication in human monolayer and lymphoblastoid cell lines].

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.

[Comparative study of the differential susceptibility of different cell lines to pandemic H1N1v influenza viruses and avian influenza, swine influenza, and human influenza viruses].

The proliferation characteristics of influenza viruses of different origin were tested in various human and animal cell cultures. Pandemic H1N1v influenza and swine influenza viruses were shown to have a low infectious activity in virtually all the test lines. In spite of this, the replication of this group of viruses may be detected by de novo NP synthesis. These viruses are able to activate programmed cell death. Moreover, a low inoculative virus dose exerts a stimulating effect on cell proliferation in both suspension and monolayer cell lines.

[Etiological characteristics of the influenza epidemics of 2006-2009 in the Russian Federation (according to the data of the Research Institute of Influenza, North-Western Branch, Russian Academy of Medical Sciences)].

The basic trends in the evolution of influenza A and B in the Russian Federation during the epidemic seasons of 2006-2009 were studied on the basis of an antigenic analysis of 1774 Influenza isolated at the Research Institute of Influenza (RII), North-Western Branch, Russian Academy of Medical Sciences, and sent from resting bases (the regional centers of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare, which collaborate with the RII). Although the trends in the substitution of representative strains generally coincide with the world patterns, the authors revealed some specific features of the antigenic drift of influenza viruses in the Russian Federation and regional varieties. Data on some biological properties and those of the antigenic analysis of the first pandemic influenza A(H1NI)v strains isolated at the RII from Saint Petersburg patients in July-August 2009 are also given in the paper.

[Study of the antiviral activity of Russian anti-influenza agents in cell culture and animal models].

The study of the antiviral activity of Russian anti-influenza agents in the cultured MDCK cells demonstrated that arbidol and ribavirin inhibited the reproduction of various influenza A virus strains, including rimantadine- and ozeltamivir-resistant variants, as well as influenza B viruses (IC50 2-8.5 microg/ml). Rimantadine at concentrations of 1-5 microg/ml completely inhibited the reproduction of reference and ozeltamivir-resistant influenza A virus strains, and it had no effect on the reproduction of influenza B viruses and rimantadine-resistant influenza A viruses. Arbidol and ribavirin also inhibited the reproduction of pandemic influenza A/California/04/2009(H1N1), A/California/07/2009(H1N1), and A/Moscow/01/2009(H1N1)swl viruses in the cultured MDCK cells (IC50 = 1.5-4.0 microg/ml) while rimantadine had no effect on their reproduction. The cultured cells showed no significant antiviral activity of ingavirin at nontoxic concentrations (up to 200 microg/ml) against all study strains of influenza A and B viruses, including pandemic A(H1N1) influenza virus strains. The activity of rimantadine, arbidol, and ingavirin was found on a model of Influenza pneumonia in mice infected with their adopted influenza A/Aichi/2/69(H3N2) virus. The preventive efficacy of the three test agents was similar and most pronounced when they were used 96 hours before infection, by preventing 40-50% death in the animals and their body weight loss and by increasing their survival by 1.3-1.5 times. Arbidol and rimantadine were more effective when used for treatment and prophylaxis in doses of 30 and 10 mg/kg/day, respectively, by protecting the infected animals from 60-80% death, increasing their survival by 1.7-2 times, and preventing their body weight loss as compared with the control. The same experiments with ingavirin showed that this agent was less effective than arbidol and rimantadine. Thus, arbidol and rimantadine have a pronounced antiviral infection in both cell culture and a model of influenza pneumonia. The found efficacy of ingavirin on an integral model of murine influenza pneumonia without its activity in the cultured cells is likely to be due to other pharmacological properties of the drug rather than its direct virus-specific action.

[Synthesis and biological activity of complexes of sulfonate-containing polyanions and gentamycin].

A series of copolymers of acrylamide (Am) with 2-acrylamido-2-methylpropanesulfonic acid (AmMPSA) and N-2-hydroxypropyl-methacrylamide (HPMA) with acrylic acid (AA) have been synthesized. Complexes of gentamycin in the base form with these polymers and dextran sulfate were obtained. It is established that both poly(AmMPCA) and its complex with gentamycin exhibit an antiviral activity in vitro against human A(H3N2) influenza virus and herpes simplex virus of type 1 (HSV-1). The HPMA-AA copolymer and its complex with gentamycin are only active against HSV-1. Complexes of gentamycin with the polyanions based on the Am derivatives and dextran sulfate showed a high level of antibacterial activity against four bacterial strains. All the polymers studied as well as their complexes with gentamycin demonstrated a low toxicity in vitro, which makes them promising for the creation of preparations with combined antiviral and antimicrobial properties.

[Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005].

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.

[Antiviral action of some antioxidants/antihypoxants and their combinations with remantadine against human influenza A(H3N2) virus studied in in vitro models].

The possible antiviral activity of preparations with antioxidant and/or antihypoxant properties was studied on two in vitro models of influenza infection: (i) in cultures of chorio-allantoic membranes of chicken embryos and (ii) in MDCK cells. Preparations under study were hypoxene, reduced glutathione, dihydroquercetin, trolox, coenzyme Q10, and the enzymatic preparation of superoxide-dismutase (recsod). Preparations possessing combined antioxidant/antihypoxic and detoxicating properties (reduced glutathione and hypoxene) produced a significant antiviral effect and enhanced the antiviral effect of rimantadine. The antiviral effect of these preparations was manifested by a decrease in the production of viral particles and, to a more pronounced degree, by the inhibition of cytopathogenic action of virus on cultured cells, which was revealed in the tests for the activity of respiratory enzymes. In contrast to the compounds containing thio or sulfo groups, the antioxidants of "direct action" (free radical scavengers) - coenzyme Q 10, trolox, quercetin and the enzymatic preparation recsod did not show any pronounced protective effect and in some cases even enhanced the production of viral particles and decreased the antiviral action of rimantadine.

[Studying the effect of antioxidants and/or antihypoxants on cell cultures under conditions of cytotoxic action of rimantadine].

The effect of some preparations with antioxidant and/or antihypoxic properties was studied under the conditions of cytotoxic action of the antiviral drug rimantadine in an MDCK cell culture. The preparations under study were hypoxene, reduced and oxidized glutathione, metadoxil, trolox (water-soluble analog of vitamin E), dihydroquercetin, coenzyme Q, and recsod (superoxide dismutase enzyme preparation). The protective drug action on the model of cytotoxicity in vitro was observed for the preparations possessing a complex of antioxidant/antihypoxic and detoxicant properties: hypoxene, metadoxil, and reduced glutathione. The protective effect of preparations did not correlate with their direct antiradical activity with respect to the stable free radical DPPH. Some compounds of phenolic nature (trolox, coenzyme Q) and recsod enhanced the harmful effect of rimantadine on the culture cells under the conditions studied. A possible explanation of this fact could be the conversion, under certain conditions, of the effect of phenolic compounds from antioxidant to pro-oxidant, which is confirmed by some literature data. The results do not allow antioxidant preparations to be recommended for routine application as cytoprotectors during toxic stresses of different nature. The protective activity of such preparations should be estimated separately for each particular xenobiotic.

[Evaluation of metabolic parameters in vitro as a model for testing the cytotoxicity of antiviral drugs].

A new test system based on in vitro assessment of the cellular viability and/or metabolism is proposed, which offers an informative approach to the screening of antiviral drugs. Cytotoxic effects of the antiviral drugs rimantadine and polyrem were studied on the cultures of some mammalian cells. The results of short-term (2 h) exposures with the drugs tested were close to their LD50 values in mammals, which makes the proposed test system promising for the assessment of drug toxicity in vivo for the whole organism. A study of the state of cell metabolism after a long-term (48 h) exposure showed that the system of endocytosis is more sensitive than other indices with respect to antiviral drugs. Is was demonstrated on the cell level that the binding of drugs into polymeric complexes can decrease the degree of its cytotoxicity: the toxicity of polyrem (a polymeric complex of rimantadine) was lower as compared to that of the equimolar concentration of rimantadine or a mixture of rimantadine with a polymeric carrier.

[Epizootics of "avian" influenza as the manifestation of pandemic].

In this review of literature modern notions on the role of birds in the evolution of the pathogenicity signs and immune system of the main and intermediate hosts of influenza viruses, as well as on the mechanisms of overcoming interspecific barriers, are analyzed. The chronology of the spread of "avian" influenza among humans, starting from 1997, the properties of the natural reservoir of this infection, and in particular influenza viruses A, the ways of their variability and evolution are presented. The conclusion has been made that the mixing, joint evolution, recombination and reassortment of viral genomes may be caused by global events in individual geographical regions.

[Effect of the bone cement on human fibroblast culture and possible ways of its correction by cytoprotector preparations]. / Osobennosti deistviia kostnogo tsementa na fibroblasty cheloveka v kul'ture i vozmozhnye puti ego korrektsii s pomoshch'iu tsitoprotektornykh preparatov.

The results of in vitro investigation are used to assess the effect of poly(methyl methacrylate) based bone cements (BCs) upon human fibroblast culture. The dose dependent cytotoxic effect of BCs (Polacris) was manifested by the development of oxidative stress and hypoxia (on the cell level) and by a decrease in the cell ability to multiplication. The drugs possessing antihypoxant (mafusol) and antioxidant (erysod) activity, introduced into the incubation medium in clinically adequate doses, significantly reduced the toxic effect of BCs. The maximum positive effect was observed upon a combined administration of both drugs.

[The study of the Ca2+ role in cytotoxic response of human cells in culture to the action of xenobiotics]. / Issledovanie uchastiia ionov kal'tsiia v toksicheskom deistvii ksenobiotikov na kletki cheloveka v kul'ture.

The role of Ca2+ in mechanisms of cell death, necrosis and apoptosis is diverse and generally recognized. The purpose of this work was to study Ca2+ participation in a cytotoxic response of human cultured cells in the presence of toxic concentrations of cationic antiseptic substance poly(hexamethylene guanidine), anionic surfactant SDS and monomeric methyl methacrylate (a component of bone cement applied in surgery). Human cell line U-937 grown in suspension was used for this study. A fluorescent probe chlortetracycline was used, as an indicator of Ca2+ transport through biologic membranes. Our results show that weakly toxic concentrations of xenobiotics under study, close to the minimum toxic doses, nearly always provoke a fair but statistically significant drop in Ca2+ binding by cells. At the same time, higher toxic doses lead to significant increase in Ca2+ influx. The latter event well compares with the majority of literary data, while the mentioned decrease in Ca2+ influx at low toxic concentrations of xenobiotics presumably correlates with the initial stage of acute cytotoxic response, accompanied by a metabolic activation and enhanced resistance of cells to injuring stimuli, demonstrated by the authors elsewhere. In parallel, a possible effect of Ca(2+)-channel antagonist nifedipine was explored under conditions of cytotoxic response of cell lines U-937, A-549 and human embryonic lung fibroblasts to poly(hexamethylene guanidine). Nifedipine (10 microM) was introduced in the incubation medium simultaneously with the toxic agent, and the cells were further maintained for 5 or 24 h in culture; their viability was monitored with the microtetrasolium test or by assessment of LDH leakage into the incubation medium. The effect of nifedipine proved to be dual, depending on the applied concentration of toxic agent: at low toxic concentrations the improvement of viability could be noticed, while at more pronounced toxic doses aggravation of viability was evident. From our point of view the explanation of this result could be the following. In weakly toxic conditions, as in intact cells, Ca2+ influx is brought about by specific mechanisms, mainly through Ca(2+)-channels, that is why nifedipine partly abolishes Ca(2+)-dependent cytotoxic response. At high concentrations, cell plasma membrane is directly damaged by toxic agent, Ca2+ enters cells mainly non-specifically, so that Ca2+ antagonist cannot protect cell injury. The reason of toxic effect aggravation by nifedipine in these conditions is still waiting for its explanation.