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Biomedical subjects

Michiko Miyamoto

Publications and source records attributed to Michiko Miyamoto.

10 recordsLinked to original sources
PubMed2006

Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1).

Although replicon systems for hepatitis C virus (HCV) recently developed have enabled the replication of HCV in cultured cells, limited genotypes are available for them. We have isolated HCV cDNA of genotype 2a (JFH-1 strain) from serum of a patient with fulminant hepatitis. A subgenomic replicon of JFH-1 was constructed and compared with the HCV replicon of genotype 1b (Con1 NK5.1) which possessed adaptive mutations. Huh7 cells transfected with replicon RNAs that had been transcribed in vitro were cultured in the presence of neomycin sulfate (G418), and selected colonies were isolated and expanded. Then, growth rates and replication of HCV RNA were evaluated on isolated cells hosting replicons. Saturation densities were lower for cells propagating JFH-1 than Con1 NK5.1 or untransfected Huh7 cells, and the mean doubling time was longer for JFH-1 than for Huh7 cells. Levels of HCV RNA replication in isolated clones were similar between JFH-1 and Con1 NK5.1 cells. Replication of RNA decreased reciprocally with cell densities in both JFH-1 and Con1 NK5.1 cells. The replication of HCV RNA was more resistant to interferon-alpha in JFH-1 than in Con1 NK5.1 cells based on the comparison of an inhibitory concentration of 50%. In conclusion, we found differences between HCV replicon clones of genotypes 1b and 2a. However, these differences may result from strain-specific characteristics, such as the source of HCV, rather than characteristics of distinct genotypes. Therefore, further investigation may be needed on more HCV isolates of diverse genotypes.

Cell Division↗
PubMed2005

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.

Animals↗
PubMed2005

Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone.

A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus-cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus-cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.

Animals↗
PubMed2005

Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system.

Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 microg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 microg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.

Antiviral Agents↗
PubMed2004

Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.

A hepatitis C virus genotype 2a subgenomic replicon, JFH-1 replicon, was previously established using the consensus sequence of clone JFH-1 from a patient with fulminant hepatitis and, in a previous report, was indicated to replicate efficiently in Huh7. Here the replication of JFH-1 replicon was tested in HepG2, a human hepatocyte-derived cell line, and in IMY-N9, a cell line developed by fusing human hepatocytes and HepG2 cells. Following transfection with in vitro transcribed replicon RNA and selection by cultivation with G418, colonies formed in both cell lines although at efficiencies substantially lower than those of Huh7. The H2476L mutation identified in the Huh7 replicon in our previous study increased the colony formation efficiencies of the JFH-1 replicon in HepG2 and IMY-N9 cells. Higher amounts of replicon RNA were detected in IMY-N9 clones than in HepG2 clones by real time detection reverse transcription-PCR, and replicon RNA replication and viral protein expression were confirmed by Northern and Western blotting in isolated clones. Sequencing of replicon RNAs revealed that mutations found in hepatitis C virus-derived regions were not identical and that two of nine HepG2 clones and three of nine IMY-N9 clones had no or one synonymous mutation. This system with the JFH-1 replicon and three cell lines is useful not only for estimating the cellular factors affecting viral activity but also for clarifying the common gene response of the host.

Blotting, Northern↗
PubMed2004

Differences in hepatitis C virus core protein processing among genotypes 1 and 2.

Two forms of hepatitis C virus (HCV) core protein, p23 and p21, are produced from a precursor polyprotein. Production of p21 by cleavage at the c-terminus of p23 is considered essential for viral assembly and replication. In the present experiment, an in vitro translation and transcription assays were used to examine cleavage of p21 from p23 among 19 clones isolated from patients with chronic hepatitis, including 10 infected with genotype 1, and nine infected with genotype 2. Significantly greater p21 to p23 ratios were observed among genotype 1 clones, compared to genotype 2 clones. A comparison of the amino acid sequences of these clones revealed greater production of p21 core protein among clones which contained alanine, rather than valine, at amino acid residue 189. An exploration of Hepatitis Virus Database revealed that efficient p21 production related alanine at amino acid position 189 was observed in most clones of genotype 1 and in rare clones of genotype 2. These data suggest that the efficiency of core protein production differs among genotypes depending on differences in the c-terminus amino acid sequences of their core region. This may explain differences in some of the clinical characteristics of various genotypes or clones.

Journal Article↗
PubMed2003

Processing of hepatitis C virus core protein is regulated by its C-terminal sequence.

Polyprotein processing of plus-strand RNA viruses is important in the regulation of gene production and replication. The core protein of hepatitis C virus (HCV), constructing the viral particle, is processed from its precursor polyprotein and observed as two forms, p23 and p21. Production of p21 by cleavage at the C-terminus of p23 is considered crucial to viral assembly and replication. In this study, this processing step was compared between clones isolated from two patients with fulminant hepatitis and from five patients with chronic hepatitis by an in vitro translation assay and cell transfection assay. The p21 core protein was predominant from the clone isolated from one of the fulminant hepatitis patient (p21 core protein production was 65.98%), while p23 was abundant with clones from five chronic hepatitis patients (p21 core protein production was 7.11+/-1.62%) and clone from another fulminant hepatitis patient (p21 core protein production was 13.36%). Investigations with chimeric and mutation-introduced constructs revealed that four amino acid residues in the C-terminus of the core region are responsible for this difference. The data suggest that core protein processing is regulated by C-terminus mutations.

Adult↗
PubMed2003

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.

BACKGROUND & AIMS: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis. METHODS: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency. RESULTS: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/microg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/microg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay. CONCLUSIONS: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

Cell Line↗
PubMed2003

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice.

Although hepatitis C virus (HCV) is a well-known causative agent of hepatocellular carcinoma (HCC), the mechanism by which HCV induces HCC remains obscure. To elucidate the role of HCV in hepatocarcinogenesis, a model of hepatocyte injury was established using HCV core transgenic mice, which were developed using C57BL/6 mice transfected with the HCV core gene under control of the serum amyloid P component promoter. After 18-24 months, neither steatosis nor hepatic tumors were found in transgenic mice. The extent of hepatocyte injury and tumorigenesis were then examined in transgenic mice following repeated administration of carbon tetrachloride (CCl(4)) using various protocols (20%, 1/week; 10%, 2/week and 20%, 2/week). Serum alanine aminotransferase (ALT) levels did not differ among HCV core transgenic mice and non-transgenic littermates; however, after 40 weeks, hepatic adenomas preferentially developed in transgenic mice receiving 20% CCl(4) once weekly. Moreover, HCC was observed in transgenic mice receiving 2 weekly injections of a 20% solution of CCl(4), and was not observed in the non-transgenic control mice. In conclusion, the HCV core protein did not promote hepatic steatosis or tumor development in the absence of hepatotoxicity. However, the HCV core protein promoted adenoma and HCC development in transgenic mice following repeated CCl(4) administration. These results suggest that hepatotoxicity resulting in an increased rate of hepatocyte regeneration enhances hepatocarcinogenesis in HCV-infected livers. Furthermore, this experimental mouse model provides a valuable method with which to investigate hepatocarcinogenesis.

Animals↗