Biomedical subjects

T Miyamura

Publications and source records attributed to T Miyamura.

At least 19 recordsLinked to original sources

PubMed2006

Genetic diversity of noroviruses in Taiwan between November 2004 and March 2005.

Noroviruses are a major health burden and are responsible for the majority of outbreaks of gastroenteritis in the world. Human noroviruses can be genetically divided into two main genogroups (GI and GII) and subdivided into many genotypes. In this study, stool specimens collected from 12 outbreaks of gastroenteritis in Taiwan were screened for viral agents between the 23rd of November 2004 and 9th of March 2005. Noroviruses were detected in all outbreaks. We detected six different norovirus genotypes: GI/11, GI/14, GII/3, GII/4, GII/6, and GII/18. Noroviruses belonging to GII/4 were dominant, 50 of 60 (83%) sequences, and were detected in 10 of 12 outbreaks. Furthermore, the norovirus GII/4 strains were detected throughout Taiwan, demonstrating their widespread distribution. We also found that three outbreaks had noroviruses from multiple genotypes. Our results have shown for the first time that noroviruses are an important cause of gastroenteritis in Taiwan.

Caliciviridae Infections↗

PubMed2005

Expression of sapovirus virus-like particles in mammalian cells.

Sapovirus (SaV) is an etiological agent of acute gastroenteritis in human and swine. SaV can be divided into five genogroups, GI to GV. Virus-like particles (VLPs) morphologically similar to native SaV have been expressed for GI, GII, GIII and GV strains in insect cells, although only low expression levels were observed for GII strains. In this study, we report the successful expression of SaV GII VLPs using cultured mammalian COS-7 and 293T cells. Our results demonstrated that this mammalian expression system was able to express and form SaV VLPs.

Animals↗

PubMed2005

Cleavage activity of the sapovirus 3C-like protease in Escherichia coli.

We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 x His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E(1055) and A(1056). In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.

Amino Acid Sequence↗

PubMed2005

Unrelated donor bone marrow transplantation for 100 pediatric patients: a single institute's experience.

In all, 100 unrelated donor bone marrow transplantations (UD-BMT) were performed in our institute between October 1993 and January 2003. Of 93 evaluable patients, 73 patients had hematological malignancy, 13 had nonmalignancy and seven had lymphoproliferative disease. The estimated 9-year event-free survival (EFS) rate was 57.1+/-5.5% in all patients. In the following analyses of the patients with hematological malignancy, the standard group had significantly better EFS than the high-risk group (61.5+/-7.0 vs 35.6+/-9.7%, P=0.02), and the EFS rate of the tacrolimus (FK-506)+methotrexate (MTX)+/-methylprednisolone prophylactic group for graft-versus-host disease was superior to that of the FK-506 without MTX group (75.7+/-8.0 vs 55.8+/-7.6%, P=0.02). When we compared the EFS rates of the FK506+MTX+/-methylprednisolone (mPSL) group and the HLA-matched related donor BMT group in our institute, these were almost similar (75.7+/-8.1 vs 68.4+/-9.3%). Therefore, UD-BMT using FK-506+MTX+/-mPSL is a safe and useful method for children with hematological malignancy who require allogeneic BMT.

Adolescent↗

PubMed2005

Age-specific antibody to hepatitis E virus has remained constant during the past 20 years in Japan.

We investigated the presence of antibodies to hepatitis E virus (anti-HEV) and hepatitis A virus (anti-HAV) by enzyme immunoassays in sera from 1015 individuals collected in 1974, 1984 and 1994. Age-specific profiles of anti-HEV remained unchanged with a peak at 40-49 years, while those of anti-HAV started to increase in individuals aged 20-29 years in 1974, 30-39 years in 1984 and 40-49 years in 1994. These results suggest that a silent HEV infection has been taking place in the last 20 years or so in Japan, while HAV infection has been terminated at least since 1974.

Adolescent↗

PubMed2004

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

AIDS Vaccines↗

PubMed2003

Cell cycle perturbation in a human hepatoblastoma cell line constitutively expressing Hepatitis C virus core protein.

Hepatitis C virus (HCV) is one of the major causes of chronic liver disease with the potential for development of hepatocellular carcinoma (HCC). The core protein of HCV has been shown to modulate expression of various cellular genes and to influence a number of cellular functions. We investigated the effect of constitutively expressed HCV core protein on cell cycle progression in HepG2 cell line, which is derived from a differentiated human hepatoblastoma and shows biosynthetic features similar to human hepatocytes. The results indicated that stable expression of the core protein in unsynchronized HepG2 cells induced a perturbation of the cell cycle with reduced cell doubling meantime and increased S phase fraction. Increase of c-myc protein above the basal expression level was demonstrated with a significant increase of c-myc stability, as revealed by its prolonged intracellular half-life, in HepG2 expressing HCV core protein. In contrast, p53 and p21 levels were unchanged. These results suggest that HCV core protein may promote cell cycle progression in HepG2 cells possibly through increasing stability of c-myc oncoprotein. These results are in support of important role played by HCV core protein in virus-mediated pathogenesis in persistently infected hosts and in hepatocarcinogenesis.

Cell Cycle↗

PubMed2003

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in Yokohama City between 1982 and 2000.

Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980's and 1990's. Six EV71 isolates in the early 1980's in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970's. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan.

Amino Acid Sequence↗

PubMed2003

Inhibition of attachment of virions of Norwalk virus to mammalian cells by soluble histone molecules.

Viral infection is usually initiated by the binding of virus particles to specific receptor molecule(s) on the host cell surface. Blocking of this step prevents the following step, penetration into the cell. In the present study, we investigated the virus-cell interactions of virions of Norwalk virus (NV), a major etiological agent for viral diarrhea. We found that histone was an extremely strong NV-binding protein. Histone H1, a heterologous histone molecule, appeared to be able to interact not only with NV particles, but also with the cell surface. Histone H1 appeared capable of effectively preventing the attachment of NV to intestinal cells, but not of other viruses. No cytotoxic effects of histone were observed under the assay conditions. These results indicate that nonsecretory histone molecules may inhibit the attachment of viruses to cells like lactoferrins. Our results suggest that by searching virus-binding molecules, we might find antiviral agents, even for new viruses.

Animals↗

PubMed2001

Characterization of pseudotype VSV possessing HCV envelope proteins.

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.

Animals↗

PubMed2001

Oral administration of hepatitis E virus-like particles induces a systemic and mucosal immune response in mice.

We evaluated the potential of recombinant hepatitis E virus (rHEV) virus-like particles (VLPs) as an oral immunogen by analyzing the response of serum IgM, IgG, and IgA and fecal IgA in mice after oral administration. The capsid proteins of HEV with its N-terminal 111 amino acids truncated were expressed with a recombinant baculovirus in insect cells, where the capsid proteins self-assembled into VLPs. Mice were orally inoculated four times with purified rHEV VLPs in concentrations ranging from 10 to 100 microg without adjuvant. Serum IgM response was obtained with as little as 10 microg of the VLPs, and the level reached its maximum in all mice groups within 2 weeks after the first administration. Serum IgG was detected by 4 weeks post-immunization (p.i.) in the majority of mice given doses of 50 and 100 microg and continuously increased at least until the 10 week mark. Serum IgA was also detected by 4 weeks p.i. in the majority of mice given doses of 50 and 100 microg, and the level reached the maximum at 8 weeks p.i. Furthermore, the maximum level of intestinal IgA responses was detected in the groups of mice receiving 50 and 100 microg rHEV VLPs at 8 weeks p.i. All these antibody responses were obtained without a mucosal adjuvant. We therefore concluded that oral immunization of rHEV VLPs is capable of inducing systemic as well as intestinal antibody responses. Furthermore, serum IgG and fecal IgA thus induced were reactive to the native HEV antigen, as determined by Western blot assays and antigen-capture ELISA.

Administration, Oral↗

PubMed2001

Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus.

Hepatitis C virus core protein, in addition to being a component of the viral capsid, has a number of regulatory functions. Here we showed two bodies of evidence indicating that a fraction of the core protein species is a substrate of the ubiquitin (Ub)-proteasome pathway of targeted proteolysis. First, the core protein processing the C-terminal hydrophobic region is metabolically unstable, and incubation with a proteasome inhibitor led to a significant accumulation of the protein. Second, an in vivo ubiquitylation assay indicates conjugation of multi-Ub chain to the unstable core protein. In contrast, a stable form of core protein, p21, is also able to be ubiquitylated, but it links to a single or only a few Ub moiety. Therefore, processing event(s) at the C-terminal hydrophobic domain of HCV core protein may affect the ubiquitylation pathway, particularly the efficiency of the multi-Ub chain assembly, resulting in stable, matured core proteins.

Binding Sites↗

PubMed2001

Characterization of cell-surface determinants important for baculovirus infection.

Baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. To investigate the cell-surface molecules important for infection of baculovirus into mammalian cells, we constructed a recombinant baculovirus, Ac64-CAluc, which has gp64 and luciferase genes under the polyhedrin and the CAG promoter, respectively. For controls, we constructed recombinant viruses possessing vesicular stomatitis virus (VSV) G protein, mouse hepatitis virus (MHV) S protein, or green fluorescent protein (GFP) gene under the polyhedrin promoter and the luciferase gene under the CAG promoter (AcVSVG-CAluc, AcMHVS-CAluc, and AcGFP-CAluc). Treatment of HepG2 cells with phospholipase C markedly reduced the reporter gene expression by Ac64-CAluc or AcVSVG-CAluc in a dose-dependent manner, whereas AcMHVS-CAluc was shown to be resistant to the treatment. Inhibition with purified lipids and susceptibility to the mutant CHO hamster cell lines deficient in phospholipids synthesis suggest that the interaction of gp64 and phospholipids on the cell surface might play an important role in baculovirus infection into mammalian cells.

Animals↗

PubMed2001

Increase in the concentration of carbon 18 monounsaturated fatty acids in the liver with hepatitis C: analysis in transgenic mice and humans.

Steatosis is one of the histologic characteristics of chronic hepatitis C and is well reproduced in a transgenic mouse model for hepatocellular carcinoma (HCC) in which the core protein of hepatitis C virus (HCV) plays a pivotal role in inducing steatosis and HCC. In the present study, the lipid composition in the liver of the HCV core gene transgenic mice as well as in those of chronic hepatitis C patients was determined. The concentration of carbon 18 monounsaturated (C18:1) fatty acids, such as oleic and vaccenic acids, which are known to increase membrane fluidity leading to higher cell division rates, significantly increased in the livers of transgenic mice compared to nontransgenic control mice. The concentration of C18:1 fatty acids also significantly increased in the livers of chronic hepatitis C patients compared to subjects without HCV infection. These results suggest that HCV may affect a specific pathway in the lipid metabolism and cause steatosis in the liver.

Animals↗

PubMed2001

Seroepidemiological study of hepatitis E virus infection in Japan using a newly developed antibody assay.

PURPOSE: A seroepidemiological study of hepatitis E virus (HEV) infection was conducted in Japan, where HEV infection is not considered endemic. METHODS: IgG and IgM class antibodies to HEV were measured with a newly developed enzyme-linked immunosorbent assay in which recombinant virus-like particles were used as an antigen. A total of 1253 individuals (401 males and 852 females; age range, 6-89 years) were enrolled from two different areas: area 1 (n = 478), in which hepatitis C was endemic; and area 2 (n = 775), in which it was not endemic. RESULTS: The HEV antibody (IgG class) positive rate was 6.7% in area 1 and 4.6% in area 2. Similarly, the HAV antibody (IgG class) positive rates were 65.3% and 72.3%. The age- and sex-specific prevalence of both HAV and HEV antibodies was quite similar in the two areas, and the HAV antibody positive rate clearly increased with age in both males and females. On the other hand, the HEV antibody positive rate showed a slight tendency to increase with age in males, but not in females. None of the 32 individuals with the HEV antibody who were interviewed had a history of visiting countries in which hepatitis E was endemic. In both areas, the mean age, percentage of males, and HAV antibody positive rate were significantly higher in the group of individuals with the HEV antibody than in the group of those without it, according to conventional statistical analyses. Of the three factors age, male sex, presence of HAV antibody, and the area factor, only male sex was statistically significant (P < 0.001) on multivariate logistic regression analysis. Two (0.2%) of the total of 1253 individuals were positive for the IgM class antibody to HEV. CONCLUSIONS: Our results suggest the possibility that HEV infection is circulating in Japan at a low level. HEV infection was associated with male sex, but not with HAV infection.

Adolescent↗

PubMed2001

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens.

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.

Adenoviridae↗

PubMed2001

Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.

Cell Line↗

PubMed2001

[Structural quantification of bone from radiographs using power spectral analysis].

Bone mineral density (BMD) and bone structure are very important indices for prevention of fracture. However, it is very difficult to quantify bone structure, and only a few indices for structural quantification of bone have been reported. The purpose of this research was to investigate a new index for bone structure. The subjects were 52 women aged from 20 to 85 years. Directivity index (DI) is a new index of bone structure calculated by directivity of power spectrum from radiographs of metacarpal bone using fast Fourier transform (FFT). DI was obtained by subtracting the integral power value at 0 and 90 degree directions on the x-y plane of the two-dimensional power spectrum of bone from the integral power value at a direction of 45 degrees. A significant relationship between BMD and DI was indicated by correlation coefficient. However, no significant relationship between BMD and the first moment of the Fourier power spectrum or the fractal dimension was found. There is a possibility that DI estimates a slight deformation of bone structure. In the future, we will apply DI to the prevention of fractures and osteoarthritis.

Adult↗