Fuyoko Sasao

Isolation and characterization of conglutinin as an influenza A virus inhibitor.

Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.

Differential susceptibility of resting CD4+ T lymphocytes to a T-tropic and a macrophage (M)-tropic human immunodeficiency virus type 1 is associated with their surface expression of CD38 molecules

Recent evidence has accumulated which definitively shows that chemokine receptors CCR5 and CXCR4 play an essential role as coreceptors for human immunodeficiency virus type 1 (HIV-1) infection. Flow cytometric analysis permitted us to detect CD38, a surface marker of early differentiation, as well as activation of T cells, on about half of healthy donor-derived CD4(+) T cells. In this study, we focused on the susceptibility of CD38(+) and CD38(-) subsets of CD4(+) T cells to HIV-1 infection with different coreceptor tropisms. About 20% of peripheral blood mononuclear cell-derived resting CD4(+) T cells were recovered into the CD38(+) subset fraction by panning with a monoclonal antibody to CD38. Most of the cells in this CD38(high) fraction also expressed CD45RA and CD62L at higher intensities compared with those of CD38(low) fraction. CCR5(+) T cells predominated in the CD38(-) subset, although cell surface expression of CD4 and CXCR4 was almost similar between both subsets. This difference was consistent with a significantly higher susceptibility of the CD38(-) subset to a macrophage (M)-tropic HIV-1 strain. In contrast, it was shown that a T-tropic strain of HIV-1 could replicate more efficiently in the CD38(+) subset, although viral adsorption rates were similar between both subsets. Thus, the differential susceptibility of CD4(+) T cells to M(-) and T-tropic HIV-1 was associated with their surface expression of CD38.

Detection by Radioligand Assay of Antibodies against Borna Disease Virus in Patients with Various Psychiatric Disorders

ABSTRACT Using a radioligand assay, which preserves the natural form of the antigen, antibodies against Borna disease virus nucleoprotein and phosphoprotein were detected in 11 and 19 sera of 171 psychiatric patients, respectively. Compared with results by Western blotting, three and nine sera were concordantly positive, respectively. The four sera showing the highest levels of antibodies by radioligand assay were all negative by Western blotting; however, dilution and inhibition tests supported the positive results. Our results suggest the importance of conformational structure to detect human anti-Borna disease virus antibodies.

Cell-mediated lysis of heat-inactivated influenza virus-coated murine targets

The involvement of inoculated virus antigens in the induction of target susceptibility to cytotoxic T lymphocyte (CTL)-mediated lysis was investigated using heat-inactivated influenza virus, PR8 strain, and various inhibitors in comparison to the cases for live or ultraviolet (u.v.)-irradiated influenza and Sendai viruses. Induction of target susceptibility with heated PR8 was not inhibited by cycloheximide and actinomycin D as in the case of u.v.-irradiated Sendai virus, whereas live virus and u.v.-irradiated PR8 were inhibited under conditions which suppress protein synthesis. Induction of target susceptibility with the live and inactivated PR8 tested was suppressed in the presence of chloroquine, contrary to the case of Sendai virus, and was dependent on the cleavage type of influenza virus haemagglutinin. These findings suggest that the viral target antigens recognized by CTL in heated PR8-coated targets came from inoculated virus proteins, whereas those in PR8-infected or u.v.-irradiated PR8-coated targets involved newly synthesized viral proteins. The former viral target antigens seem to be transferred or processed from the endosome, depending on low pH fusion in the endosomes into which they were engulfed. In this point, the induction of viral target antigens with heated PR8 was different from that induced by u.v.-inactivated Sendai virus. Targets made with heated PR8 were recognized by cross-reactive CTL over the HA subtype.

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.

Mutants of Getah and Japanese encephalitis viruses isolated from field-caught Culex tritaeniorhynchus using Aedes albopictus clone C6/36 cells.

Summary Attempts to isolate mosquito-borne arboviruses from field-caught Culex tritaeniorhynchus using Aedes albopictus clone C6/36 cells resulted in the detection of some mutant Getah (GET) and Japanese encephalitis (JE) viruses. These mutants were isolated by plaque formation on C6/36 cells at 28°C and showed more than 1,000-fold higher plating efficiency (EOP) on C6/36 at 28°C than on BHK21 cells at 37°C. Examination on EOP at various temperatures on these 2 cell lines indicated that they appear to have the characteristics of host-dependent temperaturesensitive (ts) mutant.