Tsuyoshi Yamashiro

Synergistic Inhibition of Intracellular Hepatitis C Virus Replication by Combination of Ribavirin and Interferon-α

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.

Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1

ABSTRACT Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.

Quantitation of the Level of Hepatitis Delta Virus RNA in Serum, by Real-Time Polymerase Chain Reaction—and Its Possible Correlation with the Clinical Stage of Liver Disease

Some hepatitis B virus (HBV) carriers with chronic hepatitis delta virus (HDV) superinfection show progressive chronic hepatitis, whereas others show no apparent signs of liver disease. In the present study, we established a sensitive method for the quantitation of the level of HDV RNA in serum on the basis of real-time reverse-transcription polymerase chain reaction (RT-PCR), to clarify the role that the level of HDV RNA in serum plays in the diverse natural course of clinical manifestation. In 48 subjects who were positive for hepatitis B surface antigen and for anti-hepatitis delta antibody, the levels of HDV RNA in serum were quantitated by RT-PCR. The levels of HBV DNA in serum were determined by a transcription-mediated amplification assay. The levels of HDV RNA in serum of subjects with chronic hepatitis and of subjects with liver cirrhosis were significantly higher than those in asymptomatic carrier subjects. The levels of HBV DNA in serum did not differ significantly among these 3 groups. In conclusion, HDV RNA quantification by real-time RT-PCR is possibly a useful tool for understanding the pathophysiology of HDV infection.

Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication.

Summary.  The number of amino acid substitutions in the interferon sensitivity‐determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon‐based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

Correlation between serum transaminase activity and virus load among patients with chronic liver disease type B

Newly developed hepatitis B virus (HBV)-DNA quantitative assays, transcription-mediated amplification and hybridization protection assay (TMA-HPA) and branched-DNA assay were clinically evaluated. The subjects consisted of 160 chronic HBV carriers; 48 were hepatitis Be antigen (HBeAg)-positive, whereas 109 were anti-HBe-positive (three were both negative). All subjects with HBeAg, except one, showed high HBV-DNA replication levels (>/=10(5.8) copies/ml). In HBeAg negative subjects, there was a strong correlation between the serum HBV-DNA and alanine aminotransferase (ALT) levels; ALT level was usually normal if the samples tested showed an HBV-DNA level less than 10(5)/ml, whereas, the majority of the sera with an HBV-DNA concentration greater than 10(7)copies/ml showed elevation in serum ALT level. An intermediate range of HBV-DNA level (10(5)-10(7) copies/ml) was associated with variable ALT activity. In conclusion, a serum HBV-DNA level associated with ALT elevation was lower in patients with type B chronic liver disease negative for HBeAg compared with their HBeAg-positive counterparts. There was usually no or mild liver disease activity when patients with chronic HBV infection have serum HBV-DNA levels less than 10(5)copies/ml.

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells.

Summary.  Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh‐7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC‐J4, one of chimpanzee‐infectious HCV clones. An HCV replicon derived from HC‐J4 (RpJ4) consists of HCV‐5′ untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV‐3′ untranslated region. The adaptive mutations known to be required for HCV‐Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC‐J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4‐S2197P, RpJ4‐S22001del, and RpJ4‐S2204I). RpJ4/ISDR mutant and RpJ4‐S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh‐7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418‐resistant cells were transduced efficiently by RpJ4‐S2197P, RpJ4‐S2204I, RpJ4‐S2201del and RpJ4‐S2201del/ISDR mutant, with the RpJ4‐S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC‐J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications.

Alcoholic liver cirrhosis complicated with torsade de pointes during plasma exchange and hemodiafiltration

A 36-year-old man with severe alcoholic hepatitis was treated with plasma exchange combined with hemodiafiltration to remove endotoxins and inflammatory cytokines. During the treatment, he had critical arrhythmia (torsade de pointes [TdP]). His laboratory data showed hypomagnesemia, which was suspected to be responsible for the development of TdP. Patients with alcoholic liver disease tend to have hypomagnesemia and Q-T interval prolongation. Furthermore, hemodiafiltration may cause hypomagnesemia. Careful observation for electrolytic imbalance is necessary when clinicians treat patients with alcoholic liver failure with a liver support system.

A patient with primary biliary cirrhosis associated with autoimmune hemolytic anemia.

Abstract: Primary biliary cirrhosis is often associated with autoimmune conditions, such as thyroid disease, sicca complex, and rheumatoid arthritis. However, an association with autoimmune hemolytic anemia has rarely been reported. We present a case of primary biliary cirrhosis associated with warm type autoimmune hemolytic anemia, and we review prior reports.

Negative regulation of intracellular hepatitis C virus replication by interferon regulatory factor 3

BackgroundInterferon regulatory factor (IRF)-3 plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. In the present study, we evaluated the effects of IRF-3 expression and activation on intracellular hepatitis C virus (HCV) replication using an HCV replicon system.MethodsAn HCV replicon was constructed that expressed a neomycin-selectable chimeric firefly luciferase reporter protein. A small interfering (si) RNA oligonucleotide directed against IRF-3 mRNA was designed and synthesized. A eukaryote expression plasmid vector was constructed that expressed IRF-3 mRNA under control of the cytomegalovirus early promoter/enhancer. To evaluate transcriptional activity of the interferon-stimulated genes, a reporter vector was used that expressed firefly luciferase under control of the interferon-stimulated response element (ISRE).ResultsThe baseline expression of IRF-3 did not significantly differ between cells with and without expression of the replicon. Transfection of an IRF-3 expression plasmid into the cells raised the ISRE-luciferase activities. The increase of ISRE activity was significantly more potent in the replicon-expressing cells than in cells without replicon expression. Concomitantly, the overexpression of IRF-3 suppressed HCV replication levels. In contrast, siRNA knockdown of IRF-3 suppressed ISRE activity by 38% ± 2%. Interestingly, the suppression of IRF-3 resulted in a significant increase of HCV replication, by up to twofold, depending on the IRF-3 suppression levels.ConclusionsIRF-3 negatively regulated intracellular HCV replication, and was partially activated in cells that expressed the HCV replicon. Thus, IRF-3 is a key molecule controlling HCV replication through modulation of host interferon gene responses.

Monitoring low level hepatitis B virus by a newly developed sensitive test

We have recently developed a sensitive quantitative test for hepatitis B virus (HBV) DNA using a real-time polymerase chain reaction (HBV RTD DIRECT), which can detect HBV DNA levels as low as 10(0.7) copies/ml. The aim of this study was to explore the significance of viremia changes below the detection limit of the other currently developed sensitive assays, transcription-mediated amplification and hybridization protection assay (TMA-HPA) and Amplicor HBV Monitor. The subjects consisted of 11 patients with chronic liver disease type B who showed undetectable test results of HBV DNA by TMA-HPA or Amplicor HBV Monitor during the observation period. A total of 150 serial serum samples were examined for viremia level by HBV RTD DIRECT: 139 were positive and 11 were negative. HBV RTD DIRECT could detect viremia in 72 of 78 serum samples negative for HBV DNA by TMA-HPA, or in 38 of 43 serum samples negative for HBV DNA by Amplicor HBV Monitor. The HBV DNA level was gradually increased from its lowest level before the spontaneous reactivation of hepatitis or the emergence of YMDD mutant during lamivudine treatment. However, such a phenomenon was not revealed by either TMA-HPA or the Amplicor HBV Monitor test.