Monitoring of Avian Influenza Viruses and Paramyxoviruses in Ponds of Moscow and the Moscow Region.

The ponds of the Moscow region during the autumn migration of birds are a place with large concentrations of mallard ducks, which are the main hosts of avulaviruses (avian paramyxoviruses) and influenza A viruses (IAV). The purpose of this study was the determination of the biological diversity of IAV and avulaviruses isolated from mallards in Moscow's ponds. A phylogenetic analysis of IAV was performed based on complete genome sequencing, and virus genomic reassortment in nature was studied. Almost all IAV genome segments clustered with apathogenic duck viruses according to phylogenetic analysis. The origin of the genes of Moscow isolates were different; some of them belong to European evolutionary branches, some to Asian ones. The majority of closely related viruses have been isolated in the Western Eurasian region. Much less frequently, closely related viruses have been isolated in Siberia, China, and Korea. The quantity and diversity of isolated viruses varied considerably depending on the year and have decreased since 2014, perhaps due to the increasing proportion of nesting and wintering ducks in Moscow.

Studying the Effect of Amino Acid Substitutions in the M2 Ion Channel of the Influenza Virus on the Antiviral Activity of the Aminoadamantane Derivative In Vitro and In Silico.

Purpose: The aminoadamantane derivative of L-histidyl-1-adamantayl ethylamine hydrochloride (HCl*H-His-Rim) has showed a high inhibition level against influenza A virus strains in vitro. The aim of this work is to search and establish evidence of the direct effect of the drug on influenza A virus proton channel M2. Methods: The compound HCl*H-His-Rim was obtained by classical peptide synthesis methods. Influenza A virus mutants of A/PuertoRico/8/34(H1N1) strain were obtained by reverse genetics methods. The mutant samples of the virus were cultured on chicken embryos with a virus titer in the hemagglutination test. ELISA was carried out on Madin-Darby canine kidney (MDCK) monolayer cells when multiplying the virus 10-4-10-6. The binding stability of HCl*H-His-Rim was compared to those of M2 (S31N) and M2 (S31N_A30T) channels by molecular dynamic (MD) modeling. The calculation was performed taking into account the interaction with the model lipid bilayer (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) in the presence of water molecules in accordance with the three-center model. Results: It was found that HCl*H-His-Rim is a direct action drug against influenza A. The most likely conformation of drug binding to target protein has been shown. It has been found that the A30T mutation reduces the binding energy of the drug, and the results obtained in vitro have confirmed the data calculated in silico. Conclusion: The mechanism of action of HCl*H-His-Rim is directly related to the suppression of the function of the proton channel M2 of influenza A virus.

Chromosomal flhB1 gene of the alphaproteobacterium Azospirillum brasilense Sp245 is essential for correct assembly of both constitutive polar flagellum and inducible lateral flagella.

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327-HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1:: Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase-response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.

Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla- Laf- fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

Location and architecture of an antibody-binding site of influenza A virus nucleoprotein.

Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope. Ten amino acid residues involved in the reaction of NP with the MAb 3/1 and the MAb 469/6 were identified. Our data are the first to outline a compact site recognized by MAbs in the 3D structure of the influenza virus NP.

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses.

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.

Effective expression of recombinant cytotoxic protein via its attachment to a polyglutamine domain.

Inadvertent cytotoxicity may hinder the expression of many recombinant proteins that are of industrial or medicinal importance. Here, we show that covalent binding of the influenza A cytotoxic protein M2 to a polyglutamine domain (polyQ-M2; QM2) results in significant delay of its cytotoxic effects when compared to wild-type protein (M2wt). We also show that while expression of recombinant M2wt from A/WSN/1933 strain could not be attained in vaccinia virus (VV), polyQ-M2 was successfully expressed in this system. Moreover, we demonstrate that in cell culture, the polyQ domain is cleaved off following 48 h of expression, thus releasing free and active M2. Similarly, we show the spontaneous cleavage and polyQ release from fusion with another distinct polypeptide, green fluorescent protein (GFP). Expression of M2 from QM2 construct was more prolonged than one based on M2wt-expressing construct, markedly exceeding it at the later time points. Therefore, cell death caused by a toxic polypeptide may be suppressed via genetic fusion with polyQ, resulting in its enhanced expression, followed by slow release of the free polypeptide from the fusion. Collectively, covalent fusion with polyQ or other aggregate-forming domains presents a novel approach for industrial production of cytotoxic proteins and also holds promise for gene therapy applications.

Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule.

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

Mobile elements of an Azospirillum brasilense Sp245 85-MDa plasmid involved in replicon fusions.

Sequence analysis of approximately 25kb of an Azospirillum brasilense Sp245 85-MDa ( approximately 142kb) plasmid, p85, identified two novel IS elements mediating p85 fusions with a suicide plasmid vector, pJFF350. These IS elements, 1465-bp ISAzba1 and 1112-bp ISAzba3, belong to the IS256 family and to the IS5 family/IS903 group, respectively. Truncated ISAzba2 from the ISL3 family was found near one of the copies of ISAzba1 that flank pJFF350 in p85::pJFF350. As another factor potentially contributing to the known genetic plasticity of p85, a phage integrase gene was identified in this plasmid.

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components.

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.

Signal sequences modulate the immunogenic performance of human hepatitis C virus E2 gene.

Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472-586 of E2, and a novel epitope at aa 774-796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines.

Immunization with hepatitis C virus core gene triggers potent T-cell response, but affects CD4+ T-cells.

Numerous attempts to induce immunity against HCV core (HCV-C) by DNA immunization met serious difficulties in optimizing T-helper cell and antibody responses. Immunomodulatory properties of HCV-C could be blamed that seem to be dependent on the genotype of HCV source. Here, we characterized HCV-C gene from HCV 1b isolate 274933RU. Eukaryotic expression of HCV-C was effectively driven by CMVIE, while human elongation factor 1 alpha promoter directed low levels of HCV-C expression. C57BL/6 mice were immunized with CMVIE-driven HCV-C gene, and assessed for specific antibody production, T-cell proliferation and cytokine secretion. The number and proportion of CD19+, CD3+, CD3+/CD4+, and CD3+/CD8+ splenocytes in HCV-C gene recipients was evaluated by flow cytometry. A significant mounting drop in CD3+/CD4+ T-cell counts occurred in HCV-C gene-recipients as compared to the controls. Despite that, 75% of mice exhibited core-specific cellular reactivity revealed as high proliferative responses to HCV-C and HCV-C peptides. Stimulated T-cells secreted predominantly IFN-gamma and IL-2. A shift of epitope specificity was observed with the early response being broad, and the late limited to the HCV-C C-terminus. Thus, we demonstrate both T-cell immunogenicity and T-cell modulation by core of HCV 1b. Immune modulation by HCV core may affect host ability to mount long-lasting cellular and antibody response and should be dealt with in designing core-based HCV vaccines.

Sugar transport through maltoporin of Escherichia coli: role of the greasy slide.

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.