Showgo Ohkoshi

Marked sequence diversity in the putative envelope proteins of hepatitis C viruses

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus

We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.

Detection of Hepatitis C Virus RNA in Sera and Liver Tissues of Non-A, Non-B Hepatitis Patients Using the Polymerase Chain Reaction

Sera obtained from patients with non‐A, non‐B hepatitis were examined for the presence of hepatitis C virus (HCV) genome by using the reverse transcription‐polymerase chain reaction assay, as well as for antibody to HCV (anti‐HCV) by using an enzyme‐linked immunosorbent assay (ELISA). We also examined the presence of HCV RNA in liver tissue obtained by surgical resection of hepatocellular carcinoma. Among 33 patients, HCV RNA was detectable in 21 (64%), and the antibody was also positive in 21 (64%). Eighteen (55%) patients were positive for both assays. The two assays gave inconsistent results in 3 patients who were positive for HCV RNA but negative for anti‐HCV, and in 3 other patients who were negative for HCV RNA and positive for anti‐HCV. HCV RNA was also detected in 6 out of 10 non‐cancerous liver tissue specimens and in 3 out of 7 tumor tissue specimens. Using the polymerase chain reaction, the HCV genome was detected directly in many specimens obtained from patients with non‐A, non‐B hepatitis, suggesting the presence of replicating virus in patients positive for anti‐HCV. In addition, the differing results of the two assay systems suggest that the application of both is important for evaluation of the status of HCV infection.

A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus

A putative core protein derived from hepatitis C virus was expressed in E. coli. More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein. Western blotting with this E. coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.

Distribution of plural HCV types in Japan

A detection system was developed to distinguish the four different HCV genomes [HCV-J, HCV-US, HCV-K2 and group II HCV (HCV-GII)], involving reverse transcription followed by a nested polymerase chain reaction using specific primers for each HCV type. The putative non-structural (NS) 5 regions of HCV-J, HCV-US and HCV-K2 and the putative NS3 region of HCV-GII were amplified. Of 95 specimens from patients with acute hepatitis, chronic hepatitis, liver cirrhosis or hepatocellular carcinoma, 67 specimens were positive for HCV-J, 2 for HCV-US, 23 for HCV-K2 and 11 for HCV-GII. About half the specimens that were positive for HCV-K2 or HCV-GII were coinfected with HCV-J and all those that were positive for HCV-GII were also positive for HCV-K2. Nucleotide sequence analysis of several amplified cDNA products revealed that HCV-K2 and HCV-GII could each be classified into two groups, and the pattern of classification of HCV-K2 was identical with that of HCV-GII. Therefore, our results strongly suggest that HCV-K2 is the same as HCV-GII.

Risk factors and the effect of interferon therapy in the development of hepatocellular carcinoma: A multivariate analysis in 343 patients

The aims of the present study were to clarify the risk factors for the development of hepatocellular carcinoma (HCC) in chronic hepatitis C virus (HCV) infection and to investigate the effectiveness of interferon (IFN) therapy. We retrospectively studied 343 patients who had been admitted to our hospital; 161 with chronic hepatitis, 49 with liver cirrhosis, 42 with chronic hepatitis bearing HCC and 91 with liver cirrhosis bearing HCC. The mean (±SD) observation period was 41.6 ± 31.1 months. The mean age of HCC and non‐HCC patients was 63.5 ± 7.6 and 56.9 ±12.5 years, respectively (P< 0.001). The HCV genotype II (1b) was the most prevalent genotype (92.5%) in HCC patients and the mean age was highest among patients with this genotype (63.6 ± 7.7 years). Multivariate analysis identified age (P< 0.001), the male gender (P<0.01), HCV genotype II (1b) (P<0.05) and excessive alcohol intake (P<0.05) as independent factors associated with the development of HCC. There was no relationship between the development of HCC and serum HCV levels as quantified by branched DNA assay or competitive reverse transcription polymerase chain reaction. The incidence of HCC in patients who had not received IFN therapy was 10.4/100 person‐year, while that of patients who had received IFN therapy was 1.2/100 person‐year (P<0.01) by the person‐year method. The low incidence of HCC in patients treated with IFN suggests that IFN may prevent the development of HCC.

Molecular structure of the Japanese hepatitis C viral genome.

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive‐stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin‐like serine proteinase. NTPase and RNA‐dependent RNA polymerase

A retrospective study of hepatitis C virus carriers in a local endemic town in Japan. A possible presence of asymptomatic carrier.

Chronic hepatitis, cirrhosis, and hepatocellular carcinoma are the accepted sequelae of chronic hepatitis C virus (HCV) infection. However, the real natural history of HCV infection is not still well understood. To approach this problem, we investigated 91 individuals positive for antibodies against HCV (anti-HCV), who have received annual liver function examination in a local town known to have had high carrier rates of hepatitis B virus (HBV) and HCV. Among the 91 anti-HCV-positive individuals, 63 had undertaken the annual examination more than five times in the past 14 years. We analyzed retrospectively the past liver function test results of these 63 subjects and evaluated their present virological status by determining HCV genotypes and estimating quantity of HCV RNA in the sera. Among the 63 subjects, 50 (79.4%) had HCV RNA in the serum and 40 (80%) of the 50 subjects with HCV RNA had abnormal alanine aminotransferase or aspartate aminotransferase level more than once in their records. However, the other 10 (20%) had no abnormal levels during the period examined. Six of 50 (12%) had ultrasonographic findings suggestive of cirrhosis. Thus, HCV-infected individuals in this area did not seem to have progressive liver diseases. Considering the advanced ages of the individuals examined (mean 64 years old), we may have observed a stage in the natural history of HCV infection in which viremia persists in most individuals and the tendency to progress to serious chronic liver disease is mild.

Detection of HBV DNA in non-A, non-B hepatic tissues using the polymerase chain reaction assay

SummaryThe polymerase chain reaction (PCR) followed by Southern blotting was used to examine the presence of hepatitis B virus (HBV) DNA in non-cancerous liver tissue specimens from 22 Japanese hepatocellular carcinoma (HCC) patients, who were negative for HBV surface antigen (HBsAg). By Southern blot analysis, HBV DNA was negative in all 22 patients, but it was detected by the PCR in 8 of the 15 patients who were positive for antibodies against HBsAg or HBV core antigen. Seven patients who were negative for those antibodies were also negative for HBV DNA by the PCR. These results suggest that HBV may be involved in the etiology of the liver disease of some patients with what is presently classified as non-A, non-B hepatitis, if they are positive for HBV antibodies.

Sequence analysis of the proximal promoter region of the human α-fetoprotein gene in hepatocellular carcinoma

We have examined whether or not mutations exist in the proximal promoter region of the human alpha-fetoprotein (AFP) gene in the hepatocellular carcinoma (HCC) tissue. Genomic DNA was extracted from four patients: one HCC tissue, one HCC and its corresponding non-cancerous (cirrhosis) tissues, one liver cirrhosis (LC) tissue without HCC and one matching HCC tissue and peripheral blood leukocytes. Serum concentrations of AFP in the patients ranged from less than 5 to 10,138 ng/ml. Nucleotide sequence was determined by direct sequencing using a single-stranded DNA template that was produced first through the polymerase chain reaction (PCR) amplification and then asymmetric PCR. In one HCC tissue taken from the patient with a high concentration of serum AFP, nucleotides different from published ones were detected at -120 and -113. These changes, however, probably reflect a DNA polymorphism, because peripheral blood leukocytes of the same patient had the same changes. Including this patient, no mutations in the region from -160 to -10 were detected in the HCC specimens we have examined. These results suggest that the extremely proximal promoter region of the AFP gene where glucocorticoid-responsive element and HNF-1 binding sites exist is not responsible for the re-expression of AFP in HCC.