Michinori Kohara

Real-Time Detection System for Quantification of Hepatitis C Virus Genome

BACKGROUND & AIMS For diagnosis of hepatitis C virus infection and monitoring of viral load in patients, a highly sensitive and accurate hepatitis C virus quantification system is essential. METHODS Hepatitis C virus genome was detected by real/time detection system using an ABI Prism 7700 sequence detector (Perkin Elmer Corp./Applied Biosystems, Foster City, CA). RESULTS As few as 10 copies of the genome were detected, and the quantification range was between 10(1) and 10(8) copies (r > 0.99). This system was 10-100-fold more sensitive than an Amplicor monitor (Roche Diagnostic Systems, Branchburg, NJ). The coefficient of variation values for both intra-assay precision and interassay reproducibility of identifying the genome quantification ranged from 0.37% to 2.00% and 0.88% to 4.66%, respectively. The system could detect the genome in 98% of patients with chronic hepatitis, 95.8% of patients with liver cirrhosis, and 100% of patients with hepatocellular carcinoma who had the antibody to hepatitis C virus, but could not detect the genome in patients without the antibody. CONCLUSIONS The establishment of a real-time detection system enables more accurate diagnosis of infection and monitoring of viral load in interferon-treated patients via quantification of viral genome.

Efficient Conditional Transgene Expression in Hepatitis C Virus cDNA Transgenic Mice Mediated by the Cre/loxP System

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294–3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.

Inhibition of Cytochrome c Release in Fas-mediated Signaling Pathway in Transgenic Mice Induced to Express Hepatitis C Viral Proteins

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochromec release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome cfrom mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA)

A protein-capture fluorescence enzyme immunoassay (FEIA) was developed using monoclonal antibodies (mAbs) against recombinant hepatitis C virus (HCV) core protein. Four hybridoma cell lines (5E3, 5F11, 515S, 1080S) were established and characterized. These monoclonal antibodies (mAbs) each had IgG1 and OgG2 isotypes, and recognized major B cell epitopes within the immunodominant nucleoprotein amino terminal subregion. Using mAb 5F11 as the first antibody to the solid phase and beta-D-galactosidase-conjugated mAb 5E3 as the second antibody to the protein, we established a specific HCV core protein capturing FEIA capable of detecting as little as 20 pg/ml of recombinant HCV core protein. HCV core protein in serum was detectable after treatment with 4.0% polyethyleneglycol, 0.5 NaOH, and 5% Triton X-100. The results of a peptide inhibition assay indicated that this FEIA is specific for HCV RNA positive sera. The quantity of HCV core protein detected in serum was significantly correlated to the level of HCV RNA. The detection limit for HCV core proteins was an HCV RNA per titer of approximately 10(4)/ml. Using this FEIA system, the detection ratio of HCV core protein in patients with chronic HCV infection was 92.3% (70/76).

Hepatitis C virus structural proteins induce liver cell injury in transgenic mice

To develop an animal model of hepatitis C virus (HCV) infection, transgenic mice carrying part of the HCV cDNA (C980) encoding HCV‐core and envelope proteins under control of the mouse class I major histocompatibility complex gene (H‐2K) regulatory region were produced. HCV‐C980 RNA and HCV‐core protein were present in livers from line H36 as determined by RNase protection assay and immunostaining, respectively. More than 40 animals from line H36 were examined histologically. Most of these H36 mice after 10 months of age developed spontaneous focal infiltration of lymphocytes, hepatocyte necrosis, degeneration, and altered foci with mitotic hepatocytes. These pathological lesions were absent in livers from the age‐matched control littermates. Liver cells from these H36 mice were sensitive to damage induced by intravenous administration of an anti‐Fas antibody. It is suggested that HCV‐C980 proteins by themselves may be one causative agent of liver cell injury in subjects with HCV infection. J. Med. Virol. 59:281–289, 1999.

Quantification of serum hepatitis C virus core protein level in patients chronically infected with different hepatitis C virus genotypes.

BACKGROUND/AIM: A novel fluorescent enzyme immunoassay (FEIA) for the detection and quantification of serum hepatitis C virus (HCV) core protein was developed. The aim of this study was to evaluate the relation among serum HCV core protein level, HCV RNA level, and HCV genotype in patients with chronic HCV infection. PATIENTS AND METHODS: Serum HCV core protein, HCV RNA, HCV genotype were determined in 175 patients using the FEIA, branched DNA assay (Quantiplex HCV RNA ver 1.0), and serologically defined genotyping assay, respectively. For the specificity, all 13 patients seronegative for anti-HCV were negative for serum core antigen and HCV RNA by FEIA and bDNA, respectively. RESULTS: FEIA assay seems to be more sensitive than bDNA for patients with HCV type 2 infection (detection: 83.4% v 63.4%, p < 0.01). There was a good overall correlation between the FEIA and bDNA results. However, when the patients were stratified into their HCV types, a correlation was observed in HCV type 1 but not in type 2 infection. Patients with HCV type 2 infection had a lower serum HCV core protein level (median 56 RFI) compared with type 1 infection (median 149 RFI, p < 0.01). Thirty seven patients subsequently received interferon alpha therapy, patients who showed a complete and sustained response had a lower pretreatment serum HCV core protein level compared with patients who had a relapse and nonresponders (36 v 338 RFI, p < 0.01). CONCLUSIONS: This study showed that FEIA (1) is a good assay for the detection and quantification of serum HCV core protein level, (2) is also very sensitive in detecting HCV core protein in patients with HCV type 2 infection, and (3) may have a role as a predictor of subsequent response to interferon therapy.

HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice

BACKGROUND/AIMS Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The aim of this study was to elucidate pathological effects of HCV-core protein on liver cells. METHODS We have generated transgenic mice carrying HCV-core cDNA (Px-core) and pathologically examined livers of Px-core mice. RESULTS HCV-core protein was detectable in livers from lines 5 (C5) and 8 (C8) of Px-core transgenic mice. Since chronic hepatitis and cirrhosis precede hepatocellular carcinoma in patients with HCV infection, we tried to examine the effect of repetitive injection of a small dose of anti-Fas antibody in the transgenic mice. Surprisingly, an initial injection of anti-Fas antibody induced resistance of liver cells to the second injection of anti-Fas antibody in both Px-core and littermate control mice. The insensitivity of liver cells induced in the control mice continued for more than 24 weeks after the first injection but was broken within 1 week after partial hepatectomy. However, the sensitivity was restored in the Px-core mice within 12 weeks after the injection. CONCLUSION HCV-core protein in liver cells may affect persistence of Fas-mediated liver cell injury.

Correlation of interferon treatment response with GBV-C/HGV genomic RNA and anti-envelope 2 protein antibody

The clinical significance of GB virus C/hepatitis G virus (GBV‐C/HGV) co‐infection was studied retrospectively in 100 consecutive patients with hepatitis C virus (HCV) infection. All 100 patients had been treated with interferon‐α (IFN‐α). Co‐infection with GBV‐C/HGV and HCV was detected in 10 of the 100 patients (10%) and anti‐envelope 2 region (anti‐E2) antibody was detected in 25 patients. None of the patients with GBV‐C/HGV RNA had anti‐E2 antibody. Co‐infected patients were younger (P < .005) and their serum transaminase levels were lower than HCV‐only infected patients (P < .01). In 7 of the 10 co‐infected patients, HCV RNA was eradicated from serum after IFN‐α treatment and normal alanine transaminase (ALT) levels continued in 6 of these 7 patients. In one patient who was negative for HCV RNA but positive for GBV‐C/HGV RNA, the ALT level relapsed transiently. The rate of clearance of HCV and normalization of the ALT level was significantly higher in co‐infected patients than in HCV‐only infected patients (P < .05). GBV‐C/HGV RNA disappeared from 6 of the 10 co‐infected patients (60%) upon cessation of IFN‐α treatment. However, continuous clearance of GBV‐C/HGV was observed in only two patients and anti‐E2 antibody could not be detected in the serum of these patients. These results indicate that co‐infected patients tend to be younger and more sensitive to IFN‐α treatment. However, long‐term clearance of GBV‐C/HGV after IFN‐α treatment may be difficult. Moreover, anti‐E2 antibody may act to neutralize GBV‐C/HGV. J. Med. Virol. 57:370–375, 1999.

Acute hepatitis caused by sexual or household transmission of GBV-C

BACKGROUND/AIMS Transfusion transmission is the only known transmission route of the recently identified GBV-C. To date, no evidence of household transmission of GBV-C has been reported. In the present study, a case of acute GBV-C hepatitis was investigated with respect to the mode of transmission. METHODS A 24-year-old woman with acute GBV-C hepatitis and her asymptomatic husband were examined. Two GBV-C RNA samples, isolated at different times from both the patient and her husband were amplified by reverse transcriptase-polymerase chain reaction using sequences from the NS3 region. Sequencing of this region was subsequently performed for all four samples. RESULTS/CONCLUSIONS The genomic RNA sequences of GBV-C isolated from each partner at two different time points were found to be 100% homologous. However, these genomic GBV-C RNA sequences differed in the NS3 (helicase) region from the genomic RNA isolated from the GBV-C prototype previously reported by Simons et al. Nonetheless, high homology (83%) was found between the GBV-C prototype and the GBV-C isolated from our patients. In addition, four nucleotides of the GBV-C RNA in the present study are unique and are not present in any other reported sequences of GBV-C. These results strongly suggest that GBV-C is one of the etiological agents involved in severe liver injury and was transmitted between this couple.

Hepatitis C virus replication and pathogenesis

The mechanism involved in the development of persistent hepatitis C virus (HCV) infection and the pathogenesis remain unclear. The present review is an accumulation of evidence gathered to date. In addition, it discusses the system developed to characterize HCV.