3 4 2 A A A S L D A B S T R A C T S HEPATOLOGY Oc t obe r 1995

941 RAPID QUASISPECIES ANALYSIS OF THE HEPATITIS C VIRUS BY FLUORESCENT SINGLE STRAND C O N F O R M A T I O N POLYMORPHISM (f-SSCP) T. Peters. H.Schlaver. B. Hiller: B. R6sler. H.E. Blum. J. Rasenack. Department of Internal Medicine, Albert-Ludwigs-University, Freiburg, Germany

Hepatitis C virus infection frequently causes chronic liver disease. Interferon-c~ is the only established treatment. The effectivity of this therapy is about 20% according to literature. Different factors are thought to be responsible for the response to treatment including virus titer and genotype. The aim of this study was to establish a rapid and reliable methode for the diagnosis of a heterogenous virus population (so-called 'quasi-species') in a given patient, which may be another predictor to the response of HCV infection to interferon-c~. The nu- cleotide sequence of the N-terminus of the putative envelope protein (high variable region I, HVRI), is extremely variant. Cloning and sequencing of multiple HCV clones - is very time-consuming and laborious, however. The SSCP analysis identifies single base substitutions on a non-denaturing gels due to different migration of the strands because of different secondary structures. We tested this system for the analysis of the 'quasispecies' in HCV infection with fluorescent labelled nucleic acids on an automated sequencer (ALF). 30 clones of a chronically infected patient were sequenced and showed a considerable degree of variation. Using these recombinant clones the conditions for SSCP were optimized with respect to primers, gel matrix, running temperature and fragment length. A single base pair substitution was detectable in a fragment of 170 b. Independent and repeated RNA extractions and amplifications from the same specimen and SSCP analysis of the viral RNA produced identical results of viral heterogeneity. It is shown that SSCP is a reliable method to analyze virus heterogeneity in hepatitis C.

9 4 2 QUASISPECIES OF HEPATITIS C VIRUS AND ITS CLINICAL RELEVANCE, TO HIGH SERUM LEVEL OF IMMUNE-COMPLEX. M Zeniya, A Kuramoto, Y. Okuaki, Y. Ohkawa, H Fukata, M Hara. H Takahashi, F Watanabe, Y Aizawa, and G Toda.. Dept. of Int. Med.(I), The JIKEI Uinversity School of Medicine, Tokyo

Background: Quasispacies at hypervariable domains that are present in the N terminus regions of the E1 and E2/NS1 regions, has been observed in gcaoms amplified from serum of chronic hepatitis C (CHC) patients. It is also reported high senun level of C3D-binding circulatory immune-complex (CIC) was observed in CHC patients. Aim: To darify the relationship between quasispecies of hyperzariable region-l(HVR-l) and high serum level of CIC. Patients and Methods: One-hundred and twenty-eight CHC patients were studied. As controls, 33 chronic hepatitis B (CHB) patients were used. CIC were detected by ELISA using anti-C3d and and-Clq mouse monodonal antibodies. Quasispacies of HVR- 1 was studied by fluorescence single saand conformation polymorphism (FSSCP) method and direct sequence analysis. The number of peripheral blood immunoglobulin secreting cell stimulated by PWM was studied using plaque assay. Results: In CHC the serum level of C3d-CIC was significantly higher than that of CHB (22-t-9.1 ttg/ml vs 144-8.7, mean-/-SD, p<0.01) and positive rates of C3d-CIC (>13) were 40 and 80%, in CHB and CHC, respectively. High serum C3d-CIC group (>=13) showed significantly higher serum level of ALT and IgG than low C3d-CIC group (<13) (p<0.01) and HCV-RNA was found in C3d-CIC of high C3d-CIC group. The number of immunoglobulin secreting cell of high C3d-CIC group was siginficanfly higher than that of low C3d-CIC group (p<0.01) in IgG, IgA, and tgM. However, there was no differences in positive rates of autoantibedies between two groups, and there were only 5 cases who showed positive for cryoglobulinemia. Five patients who showed high serum level of C3d-CIC (>40) had 6 donas ( average, range 1-8), and at least 7 different bases in the sequences ( range 7-20), whereas, in 2 out of 5 patients who showed low serum C3d-CIC (<13) did not show polyroorphism and the average base-polymorphism was 4 bases (range 0-13) in 3 out of 5 low-C3d-CIC patients. Polymorphism of amino acid sequence was markedly observed in high C3d-CIC group and this pulyroo~phism decreased after successful IFN therapy. Conclusion: The serum level of C3d-CIC was significantly high in CHC and this elevation was as sociated to clinical activity and augmentation of immunogiobulin secreting cells. High serum C3d level may partly associated to quasispecies of HVR- 1 of hepatitis C virus, indicating that variability of HVR-1 may cause pOlycloasl B cell activation resulting in formation of C3d-CIC.

943 QUASISPECIES OF THE HEPATITIS C VIRUS IN ACUTE AND CHRONIC INFECTION. T. Peters. O. Czerwinski. H . L Sehlayer, B, Hiller. H.E. Blum & J. Rasenack. University of Freiburg, Dept. of Internal Medicine

Various types and subtypes of HCV have been isolated all over the world. Even within: the same patient closely related viridiae with sequence variations of the genome can be observed, which led to the term "quasispecies". The degree of the heterogeneity may be a predictor for the responsiveness of the disease to a-interferon. The spontaneous evolution of HCV quasispecies in chronic infection is unknown, however. Therefore we analyzed the quasispecies of prospectively followed patients with chronic and with resolving hepatitis C contracted by blood transfusion during open heart surgery. Blood samples of six patients were analyzed over a period of time of six years. HCV-RNA was isolated and amplified by rT/PCR with primers specific for the envelope protein I (El) and hypervariable region I (HVRI) and the fragments cloned. From each patient twenty to thirty clones of the first and latest specimen were sequenced. HVRI was also tested by single strand conformation polymorphism analysis (SSCP). Point mutations were found in the El region with an average frequency of 0.73% compared to 5.5% in the HVRI. The HVRI region showed a highly variable degree o f mutations during the follow-up in the individual patient. The predominant virus species changed in all patients during the course of chronic infection. A patient with spontaneous resolution of the HCV infection had a very mild degree of variation. In summary we found a variable number of circulating HCV quasispecies, varying from patient to patient as well as during the course of the disease. In conclusion it may be beneficial to sequentially analyze heterogeneity of the HCV population by SSCP to determine the optimal timing for interferon-c~ therapy.

9 4 4 THE SEVERITY OF LIVER DISEASE INFLUENCES HCV REPLICATION IN PATIENTS WITH CHRONIC HEPATITIS C. C. Duvoux 1. JM. Pawlotskv 2. A. Bustle-l-, D. Chamui.l-.. JM. Mttreau 1. J. DuvalZi D. Dhumaaqx-t-.'lLiver Transplant Unit and 2Department of Virology, Htpital Henri M0ndo~', Universit6 Paris XlI, Crtteit, France.

Recent data, based on short-term repeated measurements of hepatitis C virus (HCV) RNA levels, have suggested that HCV replication is stable in chronically-infected patients. However, chronic HCV infection is a long-lasting disease, which can lead to chronic active hepatitis of various severity and cirrhosis. The aim of this study was to determine whether the severity of liver disease might influence the level of serum HCV RNA, supposed to reflect the level of HCV replication in the liver.

Methods. 148 patients with chronic hepatitis C were studied (96M, 52F, mean age 48 yr), They were divided into three groups according to the severity of liver disease, as assessed by biochemical evaluation and liver histology : (i) group 1 included 92 patients with chronic active hepatitis (CAH) without cirrhosis ; (ii) group 2 included 25 patients with CAll and cirrhosis, without signs of liver failure ; (iii) group 3 included 28 patients with end-stage liver cirrhosis, referred for liver transplantation. In all these patients, serum HCV RNA was sought for by a highly sensitive "nested"-PCR procedure and quantified by means of the "branched-DNA" (bDNA)-based signal amplification assay (Quantiplex TM HCV RNA, Chiron Diagnostics) prior to any treatment or transplantation.

Results. Mean age (years) Positive PCR Mean HCV RNA

n (%) level (xl 05 Eq/ml I Group 1 (n=92) 44-.t:13 92/92 (100%) 36.4~48.0 Group 2 (n=28) 53.+.13 28•28 (100%) 51.3+66.5 Group 3 (n=28) 54!-_6 25•28 (89%) 8.8+_.11.7

HCV RNA level was significantly lower in group 3 (end-stage liver cirrhosis) than in the two other groups (p<0.0003). HCV RNA was belowthe cutoff of bDNA assay (i.e. <3.5 x 105 Ecl/ml) in 14 patients in group 1 (15"/o, all PCR (+)), in 9 patients in group 2 (32%, all PCR (+)) and in 16 patients in group 3 (57%, 13/16 PCR (+)) (p<0.001). In group 3, HCV RNA levels were inversely related to the severity of the disease, as assessed by the prothrombin time (p < 0.05). In all of the patients of group 3 with follow-up, including those with negative HCV RNA detection in serum pratransplant, HCV infection recurred after liver transplantation. In 14 patients serially tested poet-transplantation, HCV RNA levels were markedly increased compared to pretransplant values and fluctuated.

Conclusions. (i) HCV RNA levels were lower in patients with end-stage cirrhosis compared to patients with less severe forms of HCV infections. (ii) In patients with end- stage liver cirrhosis, RNA levels were inversely correlated to the severity of the disease. (iii) In these patients, high levels of replication were restored when cirrhotic livers were replaced by normal livers after transplantation, suggesting that lower replication was not due to intrinsic characteristics of viral strains. Altogether, these results suggest that lower HCV replication in end-stage cirrhosis could be related to the severity of liver disease. Tha roles of the lower hepatocytio mass in end-stage cirrhotic livers and/or of fibrosis, that might impair transmission of the virus from cell to cell, can be evoked.