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Biomedical subjects

T Miyamura

Publications and source records attributed to T Miyamura.

At least 109 records · Page 6Linked to original sources
PubMed1996

Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus.

Replication-deficient recombinant adenoviruses expressing structural proteins of hepatitis C virus (HCV) were constructed. Each recombinant lacks adenoviral E1A and E3 genes and bears expression units for HCV structural proteins. The expression units contain HCV cDNAs coding for either the protein or core, one of two envelopes (E1 and E2) or all of these structural proteins (core, E1 and E2) under the control of the SR alpha promoter. In HeLa or HepG2 cells, the recombinants can express efficiently HCV genes after infection without replication of the recombinants. We detected 22-kDa core, 35-kDa E1 and 58-kDa E2 proteins of HCV in these cells. The recombinant expressing all three HCV structural proteins was inoculated into mice. Antibodies to each of the three HCV proteins were detected in all of the ten mice tested. The results indicate that the recombinant adenoviruses efficiently express HCV genes and induce specific antibody against the expressed HCV proteins in animals.

Adenoviridae↗
PubMed1996

Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region.

BACKGROUND/AIMS: We have analyzed the molecular basis of mother-to-child transmission of hepatitis C virus. METHODS/RESULTS: Healthy pregnant women were screened for anti-HCV antibody and babies born to hepatitis C virus carrier mothers were prospectively investigated. Among the 35 pairs studied, the hepatitis C virus genome was detectable in only one baby, who did not show any significant symptoms of hepatitis. The viral load in the blood of the mother was one of the highest of the 35, and the population of the hepatitis C virus genome was heterogeneous. Furthermore, she was found to have a mixed infection with type 1a and type 1b hepatitis C virus. However, the hepatitis C virus genome obtained from the baby was only from type 1b, less heterogeneous and composed of the clones which were detected in the blood of the mother. The selected hepatitis C virus had a 12-nucleotide insertion in the amino-terminus of the E2 hypervariable region of the genome. CONCLUSIONS: The incidence of mother-to-child transmission of hepatitis C virus from carrier mothers was shown by this prospective study to be low. The presence of selection pressure during transmission was suggested. The biological significance of the virus with 12-nucleotide insertion has to be determined.

Amino Acid Sequence↗
PubMed1996

Risk factors for hepatitis C virus infection among blood donors in an HIV-epidemic area in Thailand.

OBJECTIVE: The role of sexual transmission in hepatitis C virus (HCV) infection has not yet been completely elucidated. This study aimed to compare the risk factors for HCV and human immunodeficiency virus (HIV) infection in an HIV epidemic area of Thailand where HIV is mainly transmitted heterosexually. DESIGN AND SUBJECTS: Sera from 3053 blood donors were collected and tested for HCV and HIV between January and March 1994. Altogether 1756 (57.5%) of the donors were interviewed about demographics and several risk factors. RESULTS: The prevalence rates of HIV and HCV infections determined by antibody assays were 2.3% and 2.2%, respectively. Sexual risk factors were clearly shown among anti-HIV positive donors. These clear associations were not found, however, among anti-HCV positive donors. In contrast, previous histories of injecting drug use and being tattooed were found in some anti-HCV positive donors but less frequently in anti-HIV positive donors. CONCLUSIONS: Sexual transmission may play a relatively minor role in HCV transmission compared with HIV, in this area.

Adult↗
PubMed1996

[Effect of measuring bone mineral density on calcium intake].

The diet in Japan has improved, but calcium intake has not increased for the past ten years, and it remains insufficient. To prevent osteoporosis, instruction in nutrition is directed at increasing calcium intake. We studied the effect of measuring bone mineral density on calcium intake in people receiving nutrition education. Intake of other nutrients was also measured. The subjects were 87 healthy women living in an agricultural region (Yamanashi Prefecture). They were members of a group formed to improve the diet of people in their area. For three days in October 1992 and in August 1994 food-weight records were obtained. A total of 76 of the 87 women chose to have their bone mineral density measured. The measurements before the first nutrition assessment in 1992. The intake of almost all nutrients tended to be greater in 1994 than in 1992. Calcium intake exceeded the minimum daily requirement (600mg). Calcium intake increased between 1992 and 1994 only in the subjects whose bone mineral density had been measured. Calcium intake decreased in the other subjects. Therefore, nutrition education programs aimed at preventing osteoporosis may be more effective if bone mineral density is measured. In addition, an appropriate balance of other nutrients can be maintained as the intake of calcium is increased.

Adult↗
PubMed1996

Circulation of type 1 wild poliovirus in northern Vietnam during 1991-1994.

From 1991 to 1994, 143 polioviruses were isolated from patients with acute flaccid paralysis in northern Vietnam. Of these 143 isolates, 133 were type 1 and five of each were type 2 and type 3. These isolates were intratypically differentiated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Of the 133 type 1 isolates, 113 were wild strains and only 20 isolates were of Sabin vaccine-like strains. These 113 isolates were divided into seven groups by PCR-RFLP patterns and were also classified into three genomic groups by nucleotide sequencing and phylogenetic analysis. One of the three genomic groups accounted for the majority (75) of these isolates. The isolates belonging to the predominant group were found during 1991-1993. The second group, which included 17 isolates, were detected only in 1993. These isolates had genome sequence similar to isolates in southern Vietnam in the same year. The third group of isolates were detected only in 1991 and were considered to be Mahoney-like wild strains. All isolates examined were different from those obtained in other countries. In 1994, however, no wild-type polioviruses were isolated in our study. These results reveal that three unique strains were circulating in northern Vietnam in recent years, and indicate that the incidence of poliomyelitis due to wild poliovirus is decreasing.

DNA, Viral↗
PubMed1996

Detection of hepatitis C virus core protein in the glomeruli of patients with membranous glomerulonephritis.

Two Japanese patients suffered from membranous glomerulonephritis associated with hepatitis C virus (HCV) infection. Renal histologic changes were characterized by granular deposits of IgG and C3 along the capillary wall and numerous subepithelial deposits in glomeruli. Hypocomplementemia was present in one patient, but both cryoglobulins and rheumatoid factors were absent. HCV RNA was detected in both their sera by RT-PCR, both free and in the form of circulating immune complexes. The HCV core protein was found in the glomeruli from both patients by indirect immunofluorescence. These results suggest that in some patients chronic HCV infection causes membranous glomerulonephritis through immune complex deposition involving HCV proteins.

Aged↗
PubMed1996

[Primary macroglobulinemia with t(11;18) (q21;q21)].

This is the first case of primary macroglobulinemia with t(11;18) (q21;q21) reported in the literature. A 77-year-old man was admitted to a hospital in December, 1994, with acute renal failure and pleural effusion. He was treated with prednisolone pulse therapy and his symptoms improved. He was referred to our hospital for further examination. Analysis of blood chemistry revealed macroglobulinemia (IgM-kappa). There were no other findings that would indicate a diagnosis of malignant lymphoma. A complete blood count revealed a hemoglobin level of 8.7 g/dl and a white blood cell count of 5,300/microliters with 11% abnormal lymphoid cells. Immunologic and karyotype analyses revealed that these abnormal cells were positive for IgM-kappa, CD19, and CD20, negative for CD5, and CD10, and had t(11;18) (q21;q21). The bone marrow had also been infiltrated by 8.6% abnormal lymphoid cells. Six other cases with t(11;18) (q21;q21) have been reported including 5 of small lymphocytic lymphoma and 1 of mucosa-associated lymphoid tissue-type lymphoma. The tumor cells in these cases were the same as in our case. Therefore, our report is in agreement with the finding that t(11;18) (q21;q21) might be one of the characteristic chromosomal abnormalities in mature B-lymphoid neoplasms.

Aged↗
PubMed1996

Variations in the hypervariable region 1 of the envelope region E2 of hepatitis C virus RNA appear associated with virus persistence independently of liver disease.

The high genetic variability of the 5' end of the envelope protein-coding region E2 (HVR1 E2) of Hepatitis C Virus (HCV) RNA has been suggested by many authors to play an important role in both virus persistence and outcome of liver disease. We studied the relations between HVR1 E2 variability and HCV genotypes, HCV-RNA levels and liver disease in 8 chronic HCV carriers (5 males and 3 females, median age 41 years, followed-up for a mean period of 3 years). Four were healthy HCV carriers with persistently normal ALT levels and normal liver histology and 4 patients with chronic liver disease. In each patient, the HVR1 E2 variability of 2 serum HCV-RNA isolates obtained at least 12 months apart were evaluated by direct sequencing. Nucleotide and amino acid homologies ranged between 97.6%-57.1% and 92.8%-25% in healthy carriers and 95.2%-55.9% and 89.3%-32.1% in patients, respectively. We did not observe any correlation between HVR1 E2 heterogeneity and HCV genotypes, viraemia levels, presence and extent of liver necroinflammation. Our findings suggest that HVR1 E2 heterogeneity has no direct implications in hepatitis, pathogenesis but it could play a major role in virus persistence.

Adult↗
PubMed1995

Genotyping of hepatitis C virus by a simple ELISA method.

BACKGROUND: Hepatitis C virus (HCV) has been classified into five distinct types by nucleotide sequence analysis of the genome. The correlation between genotypes of HCV and sensitivity to treatment or prognosis is still controversial. OBJECTIVES: We tried to establish a simple antibody assay to determine the HCV serotype instead of genotype determined by PCR or nucleotide sequence. STUDY DESIGN: We made an enzyme-linked immunosorbent assay (ELISA) system by using synthetic oligopeptides of NS4 regions of types 1 (CH) and 2 (KN) HCV, and examined sera of various stages of hepatitis C patients. RESULTS: Among 13 HCV RNA-positive sera, serotyping was consistent with genotyping. Four sera of type 1 and 7 sera of type 2 were positive to anti-CH and anti-KN antibodies, respectively. Two sera of patients mixedly infected with type 1 and 2 HCV were positive to both antibodies. The number of type 1 and 2 in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma was 27 and 18, 28 and 15, and 46 and 12 respectively. CONCLUSIONS: Our result suggests that this simple ELISA method is useful for typing of HCV, and there is no significant relationship between HCV type and liver failure.

Journal Article↗
PubMed1995

Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted.

The core protein of hepatitis C virus (HCV) is considered to be cleaved from the N terminus of the large precursor polyprotein by cellular signalase. The HCV cDNA encoding the core protein was expressed (i) in monkey COS cells by a plasmid expression vector driven by the SR alpha promoter, and (ii) in insect cells by a recombinant baculovirus. The expressed product had an M(r) of 22,000 and was located in the cytoplasm. When the C-terminal hydrophobic domains were deleted, however, the truncated core proteins were translocated into the nucleus. The truncated core proteins were located in the nucleus even when they were expressed as a fusion protein with E. coli beta-galactosidase, which is essentially localized in the cytoplasm. Plasmids containing HCV cDNAs with a deletion in one of the regions encoding clusters of basic amino acids were expressed in COS cells and the localization of the core protein was examined. The residues PRRGPR were suggested to play an important role in nuclear localization. HCV is an RNA virus and its life cycle was originally considered to be confined to the cytoplasm; the present study, however, suggests that the HCV core protein can translocate into the nucleus under certain circumstances.

Amino Acid Sequence↗
PubMed1995

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses.

By the use of recombinant baculoviruses, the trans-cleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein. Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also found that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained wil provide basic knowledge on processing of the HCV polyprotein.

Amino Acid Sequence↗
PubMed1995

Expression of hepatitis C virus envelope proteins in transgenic mice.

In an attempt to establish a model for hepatitis C virus (HCV) infection, we produced mice transgenic for the HCV envelope genes, E1 and E2, under the control of a regulatory element from hepatitis B virus. F1-generation mice from the established founders demonstrated expression of both E1 and E2 proteins as glycosylated forms in their organs including the liver. Immunostaining revealed the localization of envelope proteins principally in the cytoplasm of hepatocytes around the hepatic central veins. Furthermore, E1 and E2 proteins were shown by immunoprecipitation to be associated with each other in the mouse liver. There was no evidence of tissue pathology in the mouse liver up to 16 months of age, suggesting that E1 and E2 proteins per se may not have direct pathogenic effects. Our results demonstrate the first successful expression of HCV gene products in the mouse liver and the association of in vivo expressed HCV envelope proteins. This transgenic mouse would be a good model to study the virus-cell interactions of HCV such as intracellular transport and assembly of envelope proteins. Also it may be a good model system to determine the role of these proteins in the pathogenesis of hepatitis or extra-hepatic manifestations in HCV infection by the introduction of cytotoxic T lymphocytes specific for the envelope proteins.

Animals↗
PubMed1995

Identification and characterization of the thymidine kinase gene of Yaba virus.

DNA of Yaba virus, a member of the poxviruses, was mapped by cross-hybridization between fragments of various restriction enzymes. The genome was approximately 135 kb in length and possessed two characteristic features of poxviruses: cross-links and inverted terminal repeats at both termini. Hybridization of fragments of Yaba virus DNA to known vaccinia virus DNA fragments indicated that the thymidine kinase (TK) gene mapped within the 0.9 kb XhoI-HincII fragment between 52.5 and 53.5 kb from the left end of the genome. The fragment could rescue the TK+ phenotype in TK- cells preinfected with a TK vaccinia virus mutant. Nucleotide sequencing of the fragment revealed an ORF capable of encoding 181 amino acids. The sequence TAAAAATGAAAAATTA upstream of the ORF was considered to be the promoter and the downstream sequence TTTTTAT to be the early transcription termination signal. These sequences are in good accord with the consensus regulatory sequences for the expression of early genes of other known poxviruses. The amino acid sequence similarity among the poxvirus TK genes suggests that Yaba virus is most closely related to swinepox virus and less similar to fowlpox virus.

Amino Acid Sequence↗
PubMed1995

Characterization of an established human hepatoma cell line constitutively expressing non-structural proteins of hepatitis C virus by transfection of viral cDNA.

A human hepatoma cell line constitutively expressing proteins of hepatitis C virus (HCV) was established by transfection with cDNA encoding part of the virus nonstructural (NS) genome region. Proteins consistent with authentic processing at NS3/NS4A, NS4A/NS4B and NS4B/NS5A were identified. Pulse-chase experiments indicated that the cleavage between NS3 and NS4A occurred first and cleavage at other sites followed. Expression of specific surface antigens in response to the presence of HCV proteins was analysed by flow cytometry. A significant increase in CD26 expression was observed in cells expressing the HCV proteins. CD26 plays an important role in cellular signal transduction. Its upregulation in response to the presence of HCV proteins may play a role in viral pathology.

Base Sequence↗
PubMed1995

Establishment of a cell line constitutively expressing E2 glycoprotein of hepatitis C virus and humoral response of hepatitis C patients to the expressed protein.

A Chinese hamster ovary cell line was established which abundantly expresses the second envelope protein (E2) of hepatitis C virus under the control of an exogenous promoter. The expressed E2 protein was found to be a glycoprotein of 58 kDa by immunoprecipitation with sera from patients that had chronic hepatitis C. Using this cell line as antigen in immunofluorescence tests, as high as 93% of patients with non-A non-B hepatitis had antibodies against E2 protein. In Western blots using SDS-denatured E2 protein, however, the detectability of the antibody was drastically reduced to 30%. Immunoprecipitation assays and ELISA, using both native and denatured E2 protein, revealed that antibodies to E2 protein were present in most of the chronic hepatitis C patients and that they reacted only to the native forms.

Animals↗