Auricular chondritis in young ear-tagged Crj:CD(SD)IGS rats.

Gross and histopathological features of auricular chondritis in young Crj:CD(SD)IGS rats were examined. Although the rats were identified with metallic ear tags on the right pinnae, auricular chondritis was also observed on the contralateral (left) ear in some animals. Histopathologically, the lesions were characterized by granulomatous inflammation with destruction of the normal cartilaginous plate, formation of new cartilaginous nodules and osseous metaplasia. Proliferative cell nuclear antigen (PCNA) positive cells were present predominantly in chondrocytes found in the centre of the newly-formed cartilaginous nodules. The results suggest that the newly-formed cartilaginous nodules were due to interstitial proliferation of chondrocytes.

Transfection of single-stranded hepatitis A virus RNA activates MHC class I pathway.

Although infection of single-stranded RNA viruses can enhance expression of major histocompatibility complex (MHC) class I genes, the mechanism underlying this process remains unclear. Recent studies have indicated that exposure of non-immune cells to double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of viral origin can directly increase the expression of MHC class I and related molecules without immune cell interaction. In this report, we show that transfection of single-stranded hepatitis A virus RNA into cultured hepatocytes results in the induction of genes for MHC class I, LMP2 and transporter for antigen processing (TAP1), in addition to the generation of viral proteins. We suggest that this stimulatory effect is due to the double-stranded RNA formed during replication of single-stranded viral RNA, and involves both double-stranded, RNA-dependent protein kinase PKR and the secretion of IFNbeta.

Solitary fibroleiomyomatous hamartoma of the lung in a patient without a pre-existing smooth-muscle tumor.

A solitary well-demarcated tumor was found in the left lung of a 53-year-old man. It was located in the posterior region of the lower lobe just adjacent to, but apart from, the pleura. It was resected by video-associated thoracic surgery. Macroscopically, the tumor was a whitish solid nodule without hemorrhage or necrosis, and it was 1.5 cm in diameter. Histologically, the tumor consisted of a proliferation of fibromuscular tissue in interlacing fascicles in which many tubular or cleft-like epithelial inclusions were involved. The epithelial inclusions showed cystic changes with goblet cell metaplasia in part, but no atypical changes. Other mesenchymal components such as cartilaginous, myxomatous or adipose tissues were not seen. The patient had no history of neoplasm, including smooth-muscle tumor. Thus, we diagnosed this tumor as a "true" fibroleiomyomatous hamartoma, as distinct from so-called fibroleiomyomatous hamartoma or benign metastasizing leiomyoma, which are usually found in the lungs of women who have had hysterectomies, as multiple fibromuscular nodules. We report here this rare case and we review and discuss published reports of fibromuscular tumors of the lung.

N-Acetylglucosaminyltransferase V as a possible aid for the evaluation of tumor invasiveness in patients with hepatocellular carcinoma.

BACKGROUND: A close relationship has been shown to exist between the metastatic potential and beta1-6 branched oligosaccharides in human and rodent cells. N-acetylglucosaminyltransferase V (GnT-V) catalyzes this process. Although this phenomenon has been reported, little is known about the clinical usefulness of the determination of GnT-V in the evaluations of tumor invasiveness in hepatocellular carcinoma (HCC). In this study, we measured the GnT-V activity in serum of patients with HCC, together with its activity and gene expression in HCC tissues, and elucidated the clinical usefulness of the GnT-V level in evaluating tumor invasiveness. METHODS: Seventy-three serum samples from 38 patients with HCC, 11 with chronic hepatitis, eight with hepatic cirrhosis and 16 healthy controls were used. Twenty-one liver tissues were obtained by surgical resection from 17 patients with HCC, three with colorectal cancers and one with gallbladder cancer metastatic to the liver. The GnT-V activity was determined by using high performance liquid chromatography. The GnT-V mRNA was quantified by using competitive RT-PCR. RESULTS: There were statistically significant correlations between GnT-V activity in sera of HCC, and GnT-V activity and GnT-V mRNA expression in tumor tissue. The mean GnT-V activity in the sera of patients with HCC increased in accordance with the degree of tumor invasion. The HCC group with intrahepatic and extrahepatic metastases showed the highest serum GnT-V-value. CONCLUSIONS: The present study demonstrated that there was a close association between tumor invasiveness and GnT-V activity in sera, and that the measurement of GnT-V may improve prognostic estimates and therapeutic outcomes for patients with HCC.

Expression of thrombomodulin in squamous cell carcinoma of the lung: its relationship to lymph node metastasis and prognosis of the patients.

Thrombomodulin (TM) is a type of thrombin receptor that was identified originally on the endothelium and acts as a natural anticoagulant through converting thrombin from a procoagulant protease to an anticoagulant. We reported previously that TM was also expressed in the squamous epithelium mainly at the intercellular bridges. In this study, we examined TM expression in the primary lesions of 81 patients with squamous cell carcinomas (SCCs) of the lung and in the lymph node metastatic lesions of 39 patients using immunohistochemical methods. The carcinoma tissues expressed TM mainly at the cell-cell boundaries and also in the cytoplasm. When TM expression was compared between the primary and metastatic lesions in the 39 patients who had lymph node metastasis, 26 (67%) showed decreased TM expression, 13 (33%) showed no change, and none (0%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells that were positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P < 0.0001). The present study also showed that the patients with TM-negative expression in the primary tumors showed significantly poorer survival than those with TM-positive expression, mainly due to distant metastases of poorly-differentiated SCCs with negative TM expression in the primary tumors. These results indicate that the reduction of TM expression seems to play an important role in the metastatic process of lung SCCs.

Accelerative effect of olive oil on adrenal corticosterone secretion in rats loaded with single or repetitive immersion-restraint stress.

The present study was performed to clarify the effects of dietary oils on physiological and metabolic changes induced by a stress, using one-time or repetitive water-immersion of restrained rats (single or repetitive stress) as an experimental stress load. In rats fed any test diets containing 20%) of the mixture of tripalmitin, tristearin, and corn oil (PSC), olive oil (OLI). safflower oil (SAF), and linseed oil (LIS) with repetitive stress loading, body weight gains and food intakes were generally reduced. The weights of the thymus and spleen also declined, but the adrenal weights were enhanced. Particularly, the increase in the adrenal weight of rats given the OLI diet was greater than of rats supplied with other diets. When the rats were loaded with the single or repetitive stress, the concentrations of urea, lipid peroxide, and corticosterone in the plasma were increased in rats fed any of dietary oils. The rise of plasma corticosterone level was especially great in rats fed the OLI diet. The concentrations of total cholesterol (T-CHOL) and triglyceride (TG) in the plasma and liver generally tended to be higher in rats fed the OLI diet than in rats given the other diets with and without stress exposure. Plasma corticosterone concentration was correlated to the adrenal weight (r=0.87, p<0.05). This study showed that OLI especially enhanced the adrenal weight in rats exposed to the repetitive stress and further raised the increased secretion of adrenal corticosterone in rats loaded with the single or repetitive stress compared with the other oils. The mechanism explaining these actions of OLI was inferred to be related to the levels of T-CHOL and TG in the plasma and liver generally enhanced by stress.

Comparison of loss of heterozygosity and microsatellite instability in adenocarcinomas of the distal esophagus and proximal stomach.

Adenocarcinoma of the gastroesophageal junction is rapidly rising in incidence. It has been proposed that these tumors be classified as three different types: distal esophageal (AEG I), cardia (AEG II), and subcardia (AEG III). Using comparative genomic hybridization (CGH) analysis, one recent study reported that the 14q chromosomal arm showed a significantly higher rate of deletion in esophageal than in cardia adenocarcinoma. Using a microsatellite analysis technique, we analyzed this area and regions in the vicinity of the APC, DCC, and p53 genes. Tumor and normal tissues were microdissected from 54 cases (27 AEG I and 27 AEG III). DNA was extracted and then amplified using seven fluorescent-labeled microsatellite markers, one pair each on 5q, 18q, and 17p and four on 14q. The results were analyzed for loss of heterozygosity (LOH) and microsatellite instability (MSI). LOH varied from 20% to 30% at each locus except for the 17p locus, where it was slightly above 50% in both groups. No significant differences in LOH or MSI were found between the esophageal and gastric tumors, including the 14q chromosomal arm. These results fail to confirm the finding that abnormalities on the 14q chromosomal arm distinguish between distal esophageal and proximal gastric tumors.

Upregulation of type I interferon receptor by IFN-gamma.

Type I interferon (IFN) receptor has a multichain structure composed of at least two distinct subunits, IFNAR-1 and IFNAR-2. In the present study, we demonstrated that IFN-gamma induced the expression of mRNA for IFNAR-1 and IFNAR-2 in a human hepatoma cell line, HepG2 cells. The induction was dose and time dependent. Because of this result, we examined the effect of combined treatment with type I IFN and IFN-gamma. The intracellular 2-5A-synthetase activity induced by combined treatment was significantly higher than that by type I IFN alone. This study suggests that combined treatment with type I IFN and IFN-gamma may be more effective than that of type I IFN alone and that the upregulation of type I IFN receptor may be one of the reasons. Our findings may have some relevance to the clinical use of IFN.

Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.

Of the six major genotypes of hepatitis C virus (HCV), infectious cDNA clones of only genotype 1 have been reported. Here, we report the construction of an infectious cDNA clone representing a second major HCV genotype, genotype 2. This infectious clone (pJ6CF) encodes the consensus polyprotein of strain HC-J6(CH), genotype 2a. Its encoded polyprotein differs from those of the infectious clones of genotypes 1a and 1b by approximately 30%. Intertypic chimeric cDNA clones constructed from infectious clones of genotypes 1a and 2a of HCV were not infectious. RNA transcripts of four chimeras containing the 2a structural genes (C, E1, and E2) in the backbone of an infectious genotype 1a clone (pCV-H77C) were not viable in a chimpanzee regardless of whether p7 was from the 1a or 2a clone. However, the chimpanzee was subsequently infected with RNA transcripts of each of the two infectious parent clones, indicating that the inability of the chimeras to replicate was intrinsic to the clones and not the result of preexisting protective immune responses.

Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.

Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development.

Simple and reliably sensitive diagnosis and monitoring of Philadelphia chromosome-positive cells in chronic myeloid leukemia by interphase fluorescence in situ hybridization of peripheral blood cells.

Philadelphia (Ph) chromosome or the bcr/abl fusion gene is the hallmark of chronic myeloid leukemia (CML) and serves as a prognostic marker during its treatment. Its detection has been primarily done by karyotype analysis of bone marrow cells. The major limitation of the karyotypic technique is an absolute need for metaphases, often difficult to obtain in an appropriate number in patients under therapy. Fluorescence in situ hybridization (FISH) is a sensitive and quantitative method to detect the bcr/abl fusion gene in cells in both metaphase and interphase. Using M-bcr and abl probes, we performed the interphase FISH in the peripheral blood of 30 healthy volunteers and in 20 hematologically normal bone marrow samples. False-positive cells were detected in 2.7 +/- 0.7% (mean +/- standard deviation) and 2.3 +/- 0.7% among 500 cells, respectively. Then we tested 31 patients with CML at various stages of disease on 50 occasions. Although there was a good correlation between the percentage of FISH-positive cells in the peripheral blood and that in the bone marrow (r = 0.977), between the percentage of FISH-positive cells in the peripheral blood and that of Ph chromosome in the bone marrow (r = 0.841), and between the percentage of FISH-positive cells and that of Ph chromosome in the bone marrow (r = 0.933), the limits of agreement in each group were not small, and thus the peripheral blood FISH test can not be interpreted as the same method with conventional karyotyping. Additionally, we could easily rule out CML in 15 individuals with leukocytosis without performing bone marrow aspiration. The present study indicates that FISH analysis in the peripheral blood is a simple and reliably sensitive test for the detection and quantitative monitoring of the M-bcr/abl fusion gene in CML in routine clinical practice, although this can not entirely replace karyotype analysis of bone marrow cells.

The reduced expression of e-cadherin, alpha-catenin and gamma-catenin but not beta-catenin in human lung cancer.

Cadherins are Ca2+-dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the histo-architecture. Using a combination of biochemical and immunohistochemical methods, we analyzed the expression of cadherin-catenin complexes in 37 non-small cell lung carcinomas. In 19 cases, decreased expression of E-cadherin protein was observed. In 12 of them, decreased expression of alpha-catenin protein was also observed. Thus, decreased expression of alpha-catenin was apparently preceded by decreased expression of E-cadherin. In no cases was decreased expression of beta-catenin observed. In the 12 cases in which mRNA expression was analyzed by Northern blot analysis, decreased expression of mRNAs for E-cadherin and alpha-catenin was observed in 11 and 9 cases, respectively. In cases with reduced E-cadherin and alpha-catenin expression, immunohistochemistry revealed two types of staining pattern for the proteins. In the first type, almost all the cells in a tumor were stained weakly (homogeneous pattern). In the second type, different percentages of cells were stained strongly, the rest being almost negative for the staining (heterogeneous pattern).

Expression of interferon alpha/beta receptor in the liver of chronic hepatitis C patients.

Interferon (IFN) demonstrates antiviral activity by binding to receptors on the cell surface. Expression of the IFN receptor in hepatocytes may be directly associated with a hepatitis C virus (HCV) infection and the response to IFN therapy. A competitive PCR method was developed to measure IFN alpha/beta (alphabeta) receptor mRNA in liver samples obtained by needle biopsy. Thirty-one patients with chronic hepatitis C (21 without cirrhosis, 10 with cirrhosis) and six normal subjects were used. Eighteen of the 21 patients without cirrhosis received the IFN therapy. Competitive PCR was carried out using IFN alphabeta receptor gene-specific primers and a specific competitor. Expression of the receptor was detected in all liver samples. There was no association between the expression level and serum alanine aminotransferase level, serum (2'-5') oligo (A) synthetase level, amount of serum HCV RNA, or HCV genotype. The expression level in patients with chronic hepatitis was significantly higher than that in normal livers (P < 0.05) and in cirrhotic livers (P< 0.01). Seven of the 18 patients treated with IFN demonstrated a sustained response to IFN (sustained responders), and the remaining 11 did not (nonsustained responders). The expression level of IFN alphabeta receptor mRNA in the sustained responders was significantly higher than that in the nonsustained responders (P< 0.01). Thus, the expression of IFN alphabeta receptor mRNA may be one of the host factors influencing the response to IFN therapy.

Serum N-acetylglucosaminyltransferase III activities in hepatocellular carcinoma.

N-Acetylglucosaminyltransferase III (GnT III) catalyses the addition of N-acetylglucosamine through a beta 1-4 linkage to the mannose of the trimannosyl core, resulting in conversion of the concanavalin A (Con A)-reactive glycan into a non-reactive state. In this study, we measured GnT III activity to evaluate its diagnostic efficacy and its therapeutic effect on hepatocellular carcinoma (HCC). Concanavalin A-non-reactive fraction of serum transferrin (Tf) was also determined since the sugar chains of Tf are one of the possible candidates for the product of GnT III. Serum samples (159) were used from patients with HCC (89), liver cirrhosis (30), chronic hepatitis (19), alpha-fetoprotein (AFP) producing gastric carcinoma metastatic to the liver (five) and healthy controls (16). N-Acetylglucosaminyltransferase III activity was determined by high performance liquid chromatography. The reactivity of serum Tf to Con A was also analysed in 21 paired HCC samples before and after treatment by crossed immuno-affinoelectrophoresis. N-Acetylglucosaminyltransferase III activity from the HCC group (153 +/- 72pmol/mL/h) was significantly higher than that from liver cirrhosis (99 +/- 67 pmol/mL per h), chronic hepatitis (84 +/- 39 pmol/mL per h) and the normal controls (62 +/- 16 pmol/mL per h). N-Acetylglucosaminyltransferase III activity of 21 patients with HCC was significantly reduced after treatment such as transcatheter arterial chemoembolization and/or percutaneous ethanol infection therapy, (123 +/- 77 to 100 +/- 60 pmol/mL per h). Commensurate decreases of AFP and des-gamma-carboxy prothrombin with GnT III activity were also observed after treatment. The Con A-non-reactive fraction (n = 21; 6.4 +/- 2.3%) in patients with HCC after treatment was significantly lower than before (8.2 +/- 2.4%). The present study suggests that GnT III activity is a possible aid in the diagnosis and evaluation of HCC, especially when other tumour markers are negative.

The usefulness of determining des-gamma-carboxy prothrombin by sensitive enzyme immunoassay in the early diagnosis of patients with hepatocellular carcinoma.

BACKGROUND: Measurements of serum concentrations of des-gamma-carboxy prothrombin (DCP) are widely used for diagnosing hepatocellular carcinoma (HCC). However, the DCP is not always sensitive enough to detect small HCCs. In the current study, the authors investigated the usefulness of DCP in the early diagnosis of HCC, using a more sensitive enzyme immunoassay than is conventionally employed. METHODS: The authors examined 148 serum samples with DCP concentrations from a conventional assay of less than 100 mAU (arbitrary unit)/mL from 91 patients with HCC and 57 with cirrhosis. DCP concentrations were determined by a more sensitive enzyme immunoassay (ED-036 kit, Eisai Laboratory, Tokyo, Japan) with a minimal detection level of 10 mAU/mL. Ninety-one HCC patients had 43 solitary small HCCs (with a greatest dimension of less than 2 cm). Of these 43 HCCs, 12 were well differentiated. RESULTS: The mean serum concentration of DCP in HCC (48.3 +/- 24.3, mean +/- standard deviation [SD]) was higher than in cirrhosis (20.3 +/- 10.3); this difference was statistically significant. When the tentative cutoff level of 40 mAU/mL (almost corresponding to the mean value + 2SD in patients with cirrhosis) was used as the level of discriminating HCC from cirrhosis, 62% of patients (56 of 91) with HCC had DCP values above this level (sensitivity). However, only three patients with cirrhosis had higher DCP levels. Thus, the specificity of this test was 95% (54 of 57 patients). The total accuracy was 74% (56 + 54/91 + 57). Twenty-three of 43 solitary small HCCs (53%) had DCP values above the cutoff level. Furthermore, 7 of 12 (58%) small, well-differentiated HCCs less than 2 cm in greatest dimension had higher DCP values. CONCLUSIONS: The results of this study indicate that DCP determination by sensitive enzyme immunoassay is useful in the early diagnosis of HCC because a high specificity is maintained.

Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo.

We constructed a chimeric cDNA clone of hepatitis C virus (HCV) that is infectious. The chimeric genome encodes the polyprotein of a genotype 1b strain (HC-J4) of HCV and replicates via 5' and 3' untranslated regions of a genotype 1a strain. The infectivity of three full-length cDNA clones was tested by direct injection of RNA transcripts into the liver of a chimpanzee. The chimpanzee became infected with HCV and the viral titer increased over time from 10(2) genome equivalents (GE)/ml at week 1 postinoculation (p.i.) to 10(4)-10(5) GE/ml during weeks 3-11 p.i. Antibodies to HCV were detected from week 18 p.i. However, the chimpanzee did not develop hepatitis. Sequence analysis of PCR products amplified from the serum of the chimpanzee demonstrated that only one of the three clones was infectious. Sequence comparisons with the cloning source, an acute-phase infectious plasma pool derived from an experimentally infected chimpanzee, showed that this infectious clone had three amino acids that differed from the consensus sequence of HC-J4, whereas the two noninfectious clones had seven and nine amino acid differences, respectively. Together, genotype 1b, represented by the infectious molecular clone described herein, and genotype 1a, represented by the two cDNA clones previously shown to be infectious for chimpanzees, account for the majority of HCV infections in the United States, Europe, and Japan.

Serum thrombopoietin levels in patients correlate inversely with platelet counts during chemotherapy-induced thrombocytopenia.

We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

Quantitation of telomerase activity in hepatocellular carcinoma: a possible aid for a prediction of recurrent diseases in the remnant liver.

Because telomerase activity is necessary for cell immortality and probably associated with tumor progression, we have evaluated a possible aid for quantitation of the activity to predict intrahepatic recurrences after surgery in patients with hepatocellular carcinoma (HCC). HCC tissues obtained by surgical resection from 20 patients were studied. Telomerase activity was expressed as peaks with a periodicity through a fluorescence-based telomeric repeat amplification protocol using an autosequencer, and the quantity of activity was calculated from peak areas. A ratio of fluorescence intensity depending on telomerase to that of an internal standard was used as a value of relative telomerase activity (RTA). RTA in serially diluted S100 extracts from HepG2 cells was well correlated with the amount of the extracts. The mean RTA value of 36.4 +/- 27.8 (mean +/- SD, 3.21 to 105) in 9 patients suffering from early recurrences after surgery was significantly higher than that (9.84 +/- 7.65; mean +/- SD, 3.00 to 29.0) in 11 patients without intrahepatic recurrences during the early period (P = .004). These results indicate that RTA value can be a useful predictor for intrahepatic recurrences during the early period after surgical resection of HCC.

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase-PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6-28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.