Toshio Shikata

Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986–1988) of China

Thein Thein Aye, Toshikazu Uchida*, Xue-Zhung Ma, Fusae lida, Toshio Shikata, Hui Zhuang and Khin Maung Win Department of Pathology, Nihon University School of Medicine, Ooyaguchi-Kamimachi, Itabashi-ku, Tokyo 173, Japan, 1 Department of Epidemiology, School of Public Health, Beijing Medical University, Beijing 100093, China and department of Medical Research, No. 5 Ziwaka Road, Dangon PO, Yangon, Myanmar

Detection of Hepatitis C Virus Antibody in the Absence of Viral RNA in Patients with Autoimmune Hepatitis

OBJECTIVE To determine whether laboratory findings showing antibodies to hepatitis C virus (HCV) in patients with autoimmune hepatitis represent false-positive results and to identify possible explanations for true-positive results in these patients. DESIGN Cross-sectional. SETTING University-based hospital. PATIENTS Fifty-two patients with non-A, non-B chronic hepatitis as a control group and 26 patients with classic chronic active autoimmune hepatitis. MEASUREMENTS Comparison of the results of five kinds of assays of HCV antibodies and HCV RNA. MAIN RESULTS Of 52 patients with non-A, non-B chronic hepatitis, HCV antibodies (anti-HCV) were detected in 42 patients (81%; 95% CI, 67% to 90%) by a first-generation enzyme-linked immunosorbent assay (ELISA-I), in 39 patients (75%) by Sp42 ELISA, in 37 patients (71%) by RIA-I, in 49 patients (94%) by ELISA-II, and in 48 patients (92%) by RIBA-II. We found HCV RNA in 47 patients (90%; CI, 79% to 97%). Of the 26 patients with autoimmune hepatitis, anti-HCV were detected in 23 patients (88%; CI, 70% to 98%) by ELISA-I, in 12 (46%) by both RIA-I and Sp42 ELISA, in 20 (77%) by ELISA-II, and in 9 (35%) by RIBA-II. However, HCV RNA was found in only five of these patients (19%; CI, 7% to 39%). None of our patients, including controls, had antibodies to superoxide dismutase. Of the 21 patients who had autoimmune hepatitis that was completely responsive to steroid therapy, 18 had anti-HCV by ELISA-I, but 13 of these patients had negative results by RIBA-II, and only two patients had HCV RNA. Of the five patients who did not respond to steroid treatment, all had anti-HCV by ELISA-I, four had negative results by RIBA-II, and three had HCV RNA. CONCLUSIONS Testing for HCV antibodies in patients with autoimmune hepatitis frequently elicits positive results when the ELISA-I or ELISA-II tests are used. Most of these appear to represent false-positive results because HCV RNA is usually absent from the serum. Such false positivity may result from previous infection with HCV or from cross-reaction of an epitope of HCV. Other patients with apparent autoimmune hepatitis who fail to respond to corticosteroid therapy may actually have chronic hepatitis C (or other non-A, non-B hepatitis) infection.

Hepatitis E Virus: cDNA Cloning and Expression

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus‐specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Isolation and purification of a non-A, non-B hepatitis-associated microtubular aggregates protein

Blood-borne type non-A, non-B (NANB) hepatitis-associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB-infected chimpanzees and were found to have a buoyant density in sucrose solution of 1.21 to 1.23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44,000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.

Production of antibodies directed against microtubular aggregates in hepatocytes of chimpanzees with non-A, non-B hepatitis.

We have previously used Epstein-Barr virus transformation to established two clonal lymphoblastoid cell lines (48-1 and S-1) producing monoclonal antibodies against microtubular aggregates that appear in the hepatocytes of chimpanzees with non-A, non-B hepatitis (NANBH). To obtain additional antibodies directed against the same structure, the mouse hybridoma method was employed. Partially purified microtubular aggregates were prepared from liver homogenates of a chimpanzee with NANBH and used as the immunogen. Hybridoma cultures were first screened by radioimmunoassay against the partially purified antigen and secondly by immunofluorescence (IF) using liver sections from a chimpanzee with NANBH. Twenty-seven cultures exhibited positive IF reactions similar to those observed with the original antibodies, 48-1 and S-1, and were cloned by limiting dilution. The specificities of the monoclonal antibodies were tested by IF on liver biopsy specimens from chimpanzees with hepatitis A, B, D or NANBH and from normal chimpanzees. All the antibodies proved to be IgG. Immunoelectron microscopy revealed that all 27 antibodies bound to the same structure, the microtubular aggregates, in hepatocytes of chimpanzees with NANBH. To determine the size of the antigen polypeptide recognized by these antibodies, polyacrylamide gel electrophoresis and Western blot assays were performed. Nine of the 27 antibodies specifically reacted with a single polypeptide of Mr 44K (p44). The remaining 18 antibodies detected no antigen polypeptide on the filters. The anti-p44 antibodies were then tested using cross-competition assays with 125I-labelled antibodies, and were found to be classifiable into three groups. In addition, the results indicate that at least three distinct epitopes are located on p44: epitope A recognized by group 1, epitope B recognized by group 2 and epitope C recognized by group 3.

Expression of HCV core protein inEscherichia coli and the establishment of ELISA system

Abstract Protein encoded by the cDNA of the core region of hepatitis C virus (HCV) was expressed as a fusion protein with β-galactosidase (β-gal) inEscherichia coli, and an ELISA system was established using this fusion protein for diagnosis of HCV infection. Using this ELISA system, a comparative study with anti-C100 antibody ELISA was undertaken in 150 cases of NANB acute hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Anti-C100 antibody positivity rates with these diseases were 62.5, 71.1, 75.0 and 75.0%, respectively, while anti-JCC antibody positivity rates were as high as 75.0, 85.6, 92.5 and 91.7%, respectively. On average, the positivity rate of anti-C100 antibody was 72.0%, while that of anti-JCC antibody was 87.3%. In the long-term analyses of two clinical cases regarding the antibody responses to these different proteins, anti-JCC antibody had a tendency to be detected earlier than anti-C100 antibody, although the time of detection in relation to ALT peaks was dependent on the case.