[The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses].

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

[Immunologic aspects of renal involvement in patients with type II diabetes mellitus]. / Immunologicheskie aspekty porazheniia pochek u bol'nykh sakharnym diabetom II tipa.

Besides the known factors contributing to the pathogenesis of diabetic nephropathy, the role of immune mechanisms in types I and II diabetes is discussed of late; the contribution of autoimmune mechanisms to pathogenesis of noninsulin-dependent diabetes (NIDDM) is virtually unknown. Seventy-six patients with NIDDM and 48 with insulin-dependent condition were examined. Under study were levels of antibodies to FxIA and renal glomerular basal membrane antigens in the blood sera of donors and patients with types I and II diabetes, as well as concentrations, size, and pathogenicity of immune complexes. Antibodies to FxIA antigen were detected in patients with both types of diabetes with diabetic nephropathy. Detection of circulating antibodies to FxIA antigen in more than 70% of diabetics in the absence of protein in the urine may be used as a test system for the laboratory diagnosis of diabetic nephropathy prestage and as a criterion for prescription as early as at the initial stages of nephroprotective agents.

[Effectiveness of pulse-therapy in seronegative spondyloarthritis (preliminary report)]. / Effektivnost' pul's-terapii pri seronegativnykh spondiloartritakh (predvaritel'noe soobshchenie).

Pulse-therapy with 6-methylprednisolone was provided to 12 patients (4 with Bekhterev's disease--BD resistant to indomethacin, voltaren and phenylbutazone therapy, 8 with psoriatic arthritis--PA). The drug was injected i.v. at a dose of 1000 mg for 3 days running. A rapid analgetic and antiinflammatory effect was noted in both groups. Ten patients (3 with BD and 8 with PA) demonstrated considerable improvement, 2 improvement. A positive trend in all indices of therapeutic efficacy was noted. In BD change in 33% of indices was statistically significant, in PA in 76%. A decrease in initially elevated serum IgG concentrations in both groups was statistically insignificant. The content of IgM and IgA before treatment was within normal, and the change resulting from pulse-therapy was statistically insignificant. A decrease in initially elevated levels of blood serum circulating immune complexes was statistically significant in both groups. Pulse-therapy was well tolerated.

[Immunological aspects of pulse therapy in systemic lupus erythematosus]. / Immunologicheskie aspekty pul's-terapii pri sistemnoi krasnoi volchanke.

Twenty SLE patients were examined against a background of pulse-therapy with methylprednisolone. The analysis showed that there was a decrease in the CIC concentration, antibodies to native DNA and an increase in the level of C3c and C4 components of the complement against a background of pulse-therapy. A more rapid time course of the CIC level was noted shortly after pulse-therapy as compared to changes in other immunological indices. A dynamic study of immunological indices in SLE against a background of pulse-therapy was appropriate for a clinical assessment and a study of the mechanisms responsible for the therapeutic efficacy of this method.