Cyclic ferrocenylnaphthalene diimides as a probe for electrochemical telomerase assay.

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G-quartet (G4) specific electrochemical ligands via the reaction of 1,1'-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 × 105 M-1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

A Novel Mechanism Underlying Antiviral Activity of an Influenza Virus M2-Specific Antibody.

Protective immunity against influenza A viruses (IAVs) generally depends on antibodies to the major envelope glycoprotein, hemagglutinin (HA), whose antigenicity is distinctive among IAV subtypes. On the other hand, the matrix 2 (M2) protein is antigenically highly conserved and has been studied as an attractive vaccine antigen to confer cross-protective immunity against multiple subtypes of IAVs. However, antiviral mechanisms of M2-specific antibodies are not fully understood. Here, we report the molecular basis of antiviral activity of an M2-specific monoclonal antibody (MAb), rM2ss23. We first found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. rM2ss23 altered the cell surface distribution of M2, likely by cross-linking the molecules, and interfered with the colocalization of HA and M2, resulting in reduced budding of progeny viruses. However, these effects were not observed for another strain, PR8, despite the binding capacity of rM2ss23 to PR8 M2. Interestingly, HA was also involved in the resistance of PR8 to rM2ss23. We also found that two amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were critical for the insensitivity of PR8 to rM2ss2. These findings suggest that the disruption of the M2-HA colocalization on infected cells and subsequent reduction of virus budding is one of the principal mechanisms of antiviral activity of M2-specific antibodies and that anti-M2 antibody-sensitive and -resistant IAVs have different properties in the interaction between M2 and HA.IMPORTANCE Although the IAV HA is the major target of neutralizing antibodies, most of the antibodies are HA subtype specific, restricting the potential of HA-based vaccines. On the contrary, the IAV M2 protein has been studied as a vaccine antigen to confer cross-protective immunity against IAVs with multiple HA subtypes, since M2 is antigenically conserved. Although a number of studies highlight the protective role of anti-HA neutralizing and nonneutralizing antibodies, precise information on the molecular mechanism of action of M2-specific antibodies is still obscure. In this study, we found that an anti-M2 antibody interfered with the HA-M2 association, which is important for efficient budding of progeny virus particles from infected cells. The antiviral activity was IAV strain dependent despite the similar binding capacity of the antibody to M2, and, interestingly, HA was involved in susceptibility to the antibody. Our data provide a novel mechanism underlying antiviral activity of M2-specific antibodies.

Comparative Analyses of the Antiviral Activities of IgG and IgA Antibodies to Influenza A Virus M2 Protein.

The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.

Pyrolysis of Iron-Containing Polyanilines under Micropore Generation Control: Electrocatalytic Performance in the Oxygen Reduction Reaction.

Pyrolyzed iron-containing polyaniline (C-Fe-PANI) is one of the most promising candidates as a non-precious metal based electrocatalyst for oxygen reduction reaction (ORR). Although the ORR activity depends on the surface area arisen from pyrolysis-generated micropores on C-Fe-PANI particles, the micropore generation is hindered by pyrolysis-formed iron nanoparticles (Fe NPs) embedded inside C-Fe-PANI particles. Here, we demonstrate the pyrolysis of iron-containing PANIs under suppression of micropore-generation hindrance by blocking the Fe NPs formation. The higher-molecular-weight (MW: 100,000) PANI was dispersed in an FeCl3 solution before pyrolysis for preventing FeCl3 penetration inside PANI particles. As a result, as compared to the case of lower-MW (5,000) PANI, the Fe NPs formation was more suppressed inside catalyst particles to give 1.9 (1.8) times micropore volume (specific surface area), leading to a 11 % higher current density in ORR electrocatalytic performance test in acidic media.

In vitro Anti-Influenza Virus Activity of Agaricus brasiliensis KA21.

　Agaricus is known to have immunostimulatory and anti-tumor effects. However, the antiviral effects of Agaricus have not yet been examined. In the present study, the antiviral effects of an extract of Agaricus brasiliensis KA21 (AE) on the H1N1 influenza virus (PR8 strain) were investigated.　The anti-influenza virus effects of AE were examined by using the plaque formation inhibition test. AE inhibited the plaque formation of PR8 in a dose-dependent manner: 98 and 50% (IC50) inhibition at 2.5 and 0.99 mg/mL, respectively. To elucidate the mechanisms of AE, the direct actions and adsorption and invasion inhibition of AE were examined, and were found to have no inhibitory effect on PR8 infection. Thus, in vitro antiviral effects may somehow inhibit PR8 after the viral invasion of cells. These results demonstrated that it is expected that AE can effectively prevent the spread of the influenza virus.

Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line.

Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated I. scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of I. scapularis-associated virus-1, which was reported in a recent metagenomic study of I. scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks.

A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains.

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.

Genetic and antigenic characterization of H5 and H7 influenza viruses isolated from migratory water birds in Hokkaido, Japan and Mongolia from 2010 to 2014.

Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.