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.     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.     

Effect of different water-soluble forms of fullerene C<Subscript>60</Subscript> on the metabolic activity and ultrastructure of cells in culture

In view of contradictory data on the toxicity of fullerenes for living organisms we studied the effect of water-soluble complexes of C₆₀ with N-polyvinylpyrrolidone (C₆₀/PVP) and γ-cyclodextrin (C₆₀/γ-CD) on MA-104 cells in culture. Both complexes proved to be nontoxic for cultured cells in the dark in a wide range of concentrations. Both complexes provoke changes of cell ultrastructure that reflect enhancement of metabolic activity. At the same time only the exposition with C₆₀/PVP leads to substantial growth of the number and size of mitochondria. However, the effect of two studied water-soluble forms of C₆₀ under intense UV irradiation of cells proved to be opposite: C₆₀/PVP had a cytoprotective action while C₆₀/γ-CD caused a significant growth of phototoxicity. Possible reasons of the differences in the action of different forms of C₆₀ on living organisms are discussed.     

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.     

Interrelationship between immunologic and neurologic memory: learning ability of rats during immunostimulation]

Studies have been made on the effect of an immunostimulator - the complete Freund's adjuvant - upon the learning ability in Wistar rats for visual discrimination using food-obtaining and avoidance of the electric shock techniques. Injection of the adjuvant significantly increases learning ability provided negative reinforcement technique is used, but inhibits the former under the conditions of positive reinforcement. Analysis of the extinction of the conditioned reflexes yielded similar results. Possible relation of immunogenesis to the formation of memory is discussed.     

Human lung carcinoma (A-549) continuing cell line and human endothelial (ECV-304) continuing cell line responses to the influenza virus at different multiplicities of infection

The course of infection upon virus entry into the cell depends not only on the biological characteristics of the cells and of the virus itself, but also on the intensity of the cell infection by the virus, i.e., on the multiplicity of infection. The purpose of our work was to perform a comparative study of the responses of two human cell lines, the lung carcinoma cell line A-549 and the endothelium cell line ECV-304, to the infection with the influenza virus A at different multiplicities of infection. At the first passage, both cell lines responded by enhancement of proliferation and apoptosis induction only to the low doses of influenza virus (ID 1–10). In A-49 cells, the stimulatory effect of the low virus doses was observed 1–2 days earlier than in ECV-304 cells. Enhanced proliferation was observed in both cell lines from the second to the fourth passages, when cells were infected with higher virus doses (ID 100 and 1000). In addition, the response of the A-549 cells to low doses of the H3N2 strain of the influenza virus A depended on the virus propagation conditions—namely, no enhancement of cell proliferation was observed in response to the infection with the virus propagating in chicken embryonated eggs, in contrast to infection with the virus that propagated in cell culture. Immunocytochemistry of A-549 cells has demonstrated that, on the third day after infection, there could be observed a change (in the dose-dependent manner) in the intracellular localization of p53 and cyclin A, proteins involved in the cell cycle progression. At the low virus dose, cyclin A was predominantly detected in the nuclei (63%), while at the high virus dose it was p53 (54%), which was predominantly detected in this cellular compartment, this observation confirming that stimulation of cell proliferation in the case of very low multiplicity of infection and cell division arrest takes place in the case of high multiplicity of influenza virus infection. The study of the influenza virus A reproduction in A-549 and ECV-304 cells using a whole number of virology techniques showed low sensitivity of these cells to the influenza virus, which manifested in the gradual decrease in the viral RNA expression and the impairment of mature viral particles assembly during several passages. Therefore, the decrease in the multiplicity of infection is associated in the A-549 and ECV-304 cells with impairment of production of mature virus particles or certain virus protein synthesis, which is accompanied by cell proliferation enhancement and apoptosis induction. As a result of the comparative study of the two cell lines (A-549 and ECV-304) upon infection with different doses of influenza virus A, we have revealed common principles and specific features indicating the effects of the biological properties of the viruses and cells, as well as of the multiplicity of infection on the course of virus infection.     

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).