Assessment of rat polyclonal antisera's suitability in hemagglutination inhibition assay for influenza surveillance and antigenic mapping.

This paper presents comparative hemagglutination inhibition (HI) assay data obtained using ferret or rat antisera to analyze influenza viruses. The results indicate that rat antisera can be successfully applied both for identification and for antigenic analysis of human influenza A and B viruses. Data gained with rat antisera were comparable to those obtained with ferret antisera. In-depth statistical analysis, based on Confusion Matrix analysis and Receiver Operating Characteristic (ROC) analysis, confirmed good coincidence between ferret antisera-based and rat antisera-based results. Two-dimensional antigenic mapping, based on HI assays using rat and ferret antisera, supported these findings. Both antisera types yielded identical antigenic attributions for the viruses analyzed, and both permitted visualization of contemporary human influenza virus evolutionary trends.

The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

PROBLEMS OF ISOLATION, IDENTIFICATION AND ANTIGENIC CHARACTERIZATION OF RECENT HUMAN A(H3N2) INFLUENZA VIRUSES.

Human A (H3N2) influenza viruses are distinguished by a high rate of evolution and regularly cause epidemics around the world. Their ability to adapt and to escape from the host's immune response and to change their receptor specificity is very high. Over the past 20 years, these viruses have lost the ability to agglutinate red blood cells of chickens and turkeys and have practically ceased to propagate in chicken embryos - the main source of influenza vaccines. Isolation of viruses in the MDCK cell culture led to the selection of strains that lose one of the potential glycosylation sites. Many of the A (H3N2) strains have acquired mutations in neuraminidase, which distort the results of antigenic analysis in the hemagglutination inhibition test - the cornerstone method for the analysis of the match between viral isolates circulating in human population to strains selected for the influenza vaccines. In this regard, the characteristics of the antigenic properties of influenza A (H3N2) viruses by traditional methods become poorly informative, and the selection of vaccine strains of this subtype is erroneous, which is reflected in the discrepancy between vaccine and circulating A (H3N2) viruses in recent years (2013-2014, 2014 -2015, 2015-2016). The search, development and implementation of new algorithms for the isolation and antigen analysis of influenza A (H3N2) viruses are extremely urgent.

Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters.

Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N'-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10-29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30).

[IMPACT OF VARIOUS MULTIPLICITY OF INFECTION OF INFLUENZA A VIRUS ON PROLIFERATION AND APOPTOSIS INDUCTION IN CULTURED CELL LINES OF LYMPHOCYTIC AND MONOCYTIC ORIGIN (JURKAT, NC-37, THP-1 AND U-937)].

The severity of disease caused by influenza A infection depends not only on biological characteristics of the virus but also on the number of viral particles than penetrate the body. T- and B-lymphocytes as well as monocytes (macrophages) play a key role in the development of cell-based and humoral immunity as well as influenza virus elimination from the body. The present study describes the effect of influenza A virus infection on cell proliferation and induction of apoptosis in human cultured cell lines of T-, B-lymphocytic and monocytic origin infected with various multiplicity of infection (moi). Low moi of the virus stimulated cell proliferation; maximal effect has been registered 3-4 days after infection. But the fate of T-cells, B-cells and monocytes after initial infection was different: Jurkat cells continued intense proliferation while proliferation of NC-37, THP-1 and U-937 cells lowered. Prolonged (for 3 passages) cultivation of Jurkat, NC-37 and U-937 cell lines has shown that infection of these cell lines not only with low but also with medium and high moi also leads to stimulation of proliferation. Using a variety of methods for the detection of viral reproduction has clearly shown that infection of non-permissive human T-, B-cells and monocytes with influenza A virus leads to latent infection. So, low moi interferes with normal formation of viral particles, which in turn might stimulate cell proliferation and then be followed by induction of apoptosis. Antiviral drags rimantadine and ribavirin suppressed virus-induced cell proliferation; at the same time, induction of apoptosis was suppressed only by rimantadine and was enhanced by ribavirin. The data obtained provide strong support for the role of influenza A virus in the observed effects.

[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.

The European Virus Archive: a new resource for virology research.

The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7-EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

[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.

[Pandemic influenza 2009 in Russia. Characteristics of the isolation and biological properties of viruses].

Research Institute of Influenza, Ministry of Health and Social Development of Russia, Saint Petersburg The characteristics of the isolation of pandemic influenza A(H1N1)v viruses were studied on chick embryos (CE) and MDCK cell culture. The materials (nasal swabs and autopsies) were collected in different regions Russia in the period from 20 July to 30 December 2009. The paper gives the data of the antigenic analysis of isolates, their capacity to multiply in different species-specific and tissue cell cultures. The viruses isolated on CE were shown to have higher hemagglutination titers and to be more stable. Isolation from the autopsies was effective only on CE. All the test cell lines other than MDCK were insensitive to the isolated pandemic influenza strains. The antigenic analysis showed no significant antigenic drift of the viruses isolated during the first wave of the pandemic in the Russian Federation.

[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.