zyxwvutsrqponm Copy right0 EuropeanAssociation Journal of Hepatology1996;25: 763- 768 Printed in Denmark All rights reserved M unksgaard Copenhagen for the Studv of the Liver 1996 Journal of Hepatology ISSN0168.8278 Rapid Publication zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH Responsiveness to interferon alpha treatment in patients with chronic hepatitis C coinfected with hepatitis G virus Thomas Berg’, Ullrike Dirla’, Uta Naumann’, Hans-Gerd Heuft’, Stefan Ktither’, Hartmut Lobeck’, Eckart Schreiers and Uwe Hopf’ ‘Virchow-Klinikum, Departments of ‘Internal M edicine and 2Pathology , ‘~2Humboldt- University ,‘Robert Koch-Institute, Berlin, Germany Background/Aims: Patients with chronic hepatitis C are often coinfected with the new identified Flaviviridae-like agent, termed hepatitis G virus (HGV). The aim of the study was to investigate the responsiveness of hepatitis G virus to interferon alpha and to evaluate whether a hepatitis G virus coinfection negatively influences the outcome of treatment in chronic hepatitis C. Methods: One hundred and fifteen patients with histologically proven chronic hepatitis C were treated with interferon alpha and investigated for the presence of hepatitis G virus coinfection by nested polymerase chain reaction with primers from the helicase region of hepatitis G virus. All patients received at least 3 MU (range 3-6) interferon alpha thrice weekly for at least 6 months (mean 8, range 612). Polymerase chain reaction products of seven pre- and post-treatment hepatitis G virus positive patients were directly sequenced for identification of sequence variability during the follow-up. Results: Eighteen (16%) patients were coinfected with hepatitis G virus. Although nine (50%) of these patients became HGV RNA negative during interferon alpha therapy, only three patients (17%) remained HGV RNA negative at the end of follow-up (mean 24 months). The rate of sustained response of chronic hepatitis C was not signiilcantly different between patients with hepatitis C virus infection and HCV/HGV coinfection (19% vs 28%). Severity of liver disease as determined by alanine aminotransferase levels, histology and hepatitis C virus viremia was not significantly different in patients with hepatitis C virus or HCV/ HGV coinfection. Sequence analysis of the helicase region revealed that our isolates all belonged to the hepatitis G virus aud not to the GBV-C like genotype. No amino acid exchanges during the observation period of up to 48 months were observed, indicating that this region is highly conserved. Conclusions: The responsiveness of hepatitis G virus to interferon alpha in chronic HCV/HGV coinfected patients is similar to that observed in chronic hepatitis C. Hepatitis G virus coinfection seems not to interfere with the efftcacy of interferon alpha treatment in patients with chronic hepatitis C. Key words: GB virus C; Helicase region; Hepatitis C virus; Hepatitis G virus; Interferon alpha. two new Flaviviridae-like agents were zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG the same virus, and both are only distantly related to independently discovered, tentatively named hepatitis C virus (HCV). GBV-C-like genotype origiGB virus C (GBV-C) and hepatitis G virus (HGV) nated from West Africa (reported as genotype l), (l-3). Comparison of full-length sequences revealed whereas HGV-like types were prevalent in the that GBV-C and HGV represent different strains of United States and Europe (reported as genotype 2). HGV is transmitted by blood and blood products, intravenous drug use and other behavior associated Received 28 M ay: accepted 19 July 1996 with a high risk of parenteral exposure to blood, and Correspondence: Prof. Dr. U. Hopf, Abt. Innere Medizin could be demonstrated as the possible causative und Poliklinik, m.S. HSm%ologie/Onkologie, Virchowagent in patients with acute and chronic non-A-E Klinikum, Humboldt Universit& Augustenburger Platz, hepatitis (1,3-5). However, many of the infected pa13353 Berlin, Germany. Tel. ++49-30-450-53-7 1. Fax. ++49-30-450-53903. tients had normal alanine aminotransferase (ALT) R ECENTLY, 763 T. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Berg et al. levels, suggesting the existence of a healthy carrier HCV RNA levels (copies/ml) state (3). In patients with chronic hepatitis C and B, HGV coinfections were observed in a significant number, . probably as the consequence of shared risk factors . (3-5). The implications of this coinfection for the natural course of chronic hepatitis C and B are still unclear. In the present study we investigated whether the HGV coinfection in patients with chronic hepatitis C treated with interferon alpha (IFNa) had any influence on the rate of response to treatment in chronic hepatitis C, and whether HGV is responsive to IFNa. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA .. Materials and Methods Patients and ZFNa therapy One hundred and fifteen patients (mean age 45k12.5 years, range 20-73; male/female: 69/46) with histologically proven chronic hepatitis C were investigated. All patients were treated with at least 3 MU IFNa (IFNa-2a [n=25]; IFNa-2b [n=31] and lymphoblastoid IFNa [n=59]) for 6 months (mean total IFNa dosage 337 MU, range 216-720; mean treatment duration 8 months, range 6-12). Patients were followed after stopping IFNa treatment for up to 36 months (mean 24 months, range 6-36). Histology For all patients pre-treatment liver histology was available. Chronic persistent hepatitis was present in 37 patients, chronic active hepatitis in 68 and liver cirrhosis in 10, respectively. The histological activity was further graded by the Knodell score (6). Mean activity score in our patients was 8.6k2.9 (range 2-15). Definitions of treatment response The response pattern to IFNa was based on the viro- logical status for either HCV or HGV. No response was defined as continuous positive polymerase chain reaction (PCR) results for either HCV RNA or HGV RNA during the treatment period (at least determined at the end of treatment). Relapse was defined as HCV or HGV becoming negative during therapy but becoming again detectable at the end of follow-up. Patients who remained HCV or HGV RNA negative at the end of follow-up were considered to have a sustained response. Detection of HCV and HGV RNA by PCR RNA was extracted from serum, employing the spin column technique (Qiagen@; Hilden, Germany). For all patients, at least three serum samples (before IFNa treatment, at the end of treatment, and in the 764 0. . .: . zyxwvutsrqponmlkjihgfedcbaZYXWV IO' HCV illfectlon (n = 72) HCWHGV coinfectim (n = 18) Fig. 1. HCV RNA levels (copies/ml) in patients with HCV infection or HCV/HGV coinfection before IFNcl treatment. long-term follow-up [mean 24 months after the end of therapy, range 6-361) were tested for the presence of HCV and HGV RNA. For the detection of HCV RNA we used the HCV AmplicorW kit (Roche, Diagnostic Systems Inc., Branchburg, NJ, USA). HCV RNA levels were quantified using the HCV Amplicor MonitorTM(Roche, Diagnostic Systems) according to the manufacturer’s instructions. HCV-AmplicorTM products were further employed in the Inno-LiPA assay (Innogenetics N.V., Belgium) for typing of HCV. HGV sequences from the helicase-like region were amplified by nested reverse transcription PCR, using three published primers G8, G9, Gil (7) and one primer of our own GBV-C.hl (5’-CCN TIT TAT GGG CAT GG-3’; N=G, A, T, or C) as described (5). The 140-base pair nested PCR products were run on an agarose gel after ethidium bromide staining, and visualized by ultraviolet light. Direct sequencing of the biotinylated amplicons was performed with the use of Dynabeads’ M-280 Streptavidin (Dynal, Oslo, Norway) and the Sequenase Version 2.0 Sequencing kit (USB, Cleveland, Ohio, USA). Statistics Statistical analysis was performed with the SPSS-PC program (SPSS, Chicago, USA) with Chi-square, the Response of HCWHGV coinfection to IFNa TABLE 1 TABLE 2 Presentation of clinical data and HCV-related response to interferon alpha in patients with chronic HCV infection and HCV/ HGV coinfection (n=l15) Effect of interferon alpha therapy in 18 patients with HCV/HGV coinfection as defined by ALT values and HCV/ HGV RNA in serum Presentation Patients with HCV infection (n=97) HCV/ HGV coinfection (n=18) 45k12.7 (20-73) 44k11.2 (28-68) Age in years’ Male/female 56141 1414 ALT level before IFNa 114?71(39454) 83+33 (43-167) Treatment’ (III/l) Source l Sporadic 45 5 l Post-transfusional 22 7 15 2 l Occupational l IVDU 15 4 8.Ok2.6 (3-12)3 Histological activ8.8k3.0 (2 -15)* ity score’ (Knodell) HCV genotypes 71 13 lIIlpel 7 2 *Type2 19 3 l5pe3 HCV related virological response* l Sustained response 18 (19%) 5 (28%) l Relapse 31(32%) 5 (28%) l No response 48 (49%) 8 (44%) Significance n.s ns n.s n.s n.s n.s n.s. ‘Mea&standard deviation (range). ‘Eight (8%) and 3two (11%) patients with liver cirrhosis. *For definition, see Materials and Methods. Abbreviations: ALT, alanine aminotransferase. IVDU, intravenous drug use. n.s, not statistically significant. Wilcoxon U test and the Fisher exact test. Values were expressed as meatistandard deviation of mean unless otherwise stated. P-values less than 0.05 were considered as statistically significant. Results Pre-treatment characteristics Eighteen (16%) of the 115 patients with chronic hepatitis C were coinfected with HGV, as demonstrated by positive PCR results for HGV RNA. Table 1 shows the characteristics of the HGV-coinfected patients in comparison to the patients with only HCV infection regarding the age, sex, ALT levels, histological activity, source of infection and genotype distribution. Male patients were more often found to be coinfected with HGV, and ALT levels (p=O.O5 1) and histological activity score (Knodell) were less pronounced than in patients with HCV infection. However, these differences were not statistically significant. Eleven (61%) of the HGV-coinfected patients demonstrated parenteral risk factors (i.e. intravenous drug use [IVDU] or blood transfusion) as compared to 38% of only HCV-infected patients, whereas in five patients (28%) the mode of transmission was unknown. HCV Pat. Total no. IFNa dosage (MU) ALT before IFNa treatment (IUn) ALT at the Virological end of response to follow-up HCV* (IUfl) Viiological response to HGV* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 51 61 94 167 122 68 80 141 107 54 51 71 94 82 70 63 68 82 19 56 87 87 27 65 58 146 114 9 7 80 77 112 66 58 46 109 sustained sustained sustained relapse relapse no response relapse no response relapse no response relapse no response no response no response relapse no response no response no response 720 432 360 432 216 360 360 432 216 216 216 216 216 216 288 540 240 300 sustained relapse relapse no response sustained no response relapse relapse no response sustained sustained no response no response no response no response sustained no response no response *For definition, see Materials and Methods. genotype distribution was nearly identical in both groups (Table 1). HCV RNA concentrations were measured in all coinfected and 72 HCV-infected patients. Mean HCV RNA levels were not significantly different in HCV-infected or HCV/HGV coinfectedpatients (4.8f5.0~10~ [range 0.02-29.3~10~1 vs. 6.7+7.3x105 [range 0.02-22.6~10~1; p=O.56) (Fig. 1). Treatment response zyxwvutsrqponmlkjihgfedcbaZYXWVU HGV RNA became negative during IFNa treatment in nine (50%) of the 18 patients with HCV/HGV coinfection. However, after the end of follow-up only three (17%) of these remained HGV RNA negative. The virological response pattern for HCV induced by IFNa in relation to the presence or absence of an additional HGV infection is also shown in Table 1. Sustained HCV RNA negativity by PCR was achieved in 19% of the patients only infected with HCV and in 28% of the coinfected patients (p=n.s). A sustained response of HCV was independent of sustained clearance of HGV or vice versa. No sustained response of HGV could be observed in four (no. 5, 10, 11, 16) of the five patients with sustained clearance of HCV. But ALT levels remained elevated in only one of these four patients (no. 16) (Table 2). HGV sequence analysis In seven patients with pre- and post-treatment HGV viremia the amplicons of the helicase-like region 765 T. Berg et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA al. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA A HCV 1 GBV-C 4551 HGV GTA --C -A- GGG --G A-A --T _-_ A-C _-_ __- -__ --A -__ AA- A-- __- --C GA- --C --T --- A-G e-T --T C-C --- --T --A _-_ __- --_ -_- --_ --G --- -_- _-_ A-A T-G CGG ATG CGA ACC GGA AGG CAC CTC GTG TTC TGC CAT TCT AAG GCT GAG TGC GAG CGC CTT 4-b 4-(48 months) --_ --T --A --_ --_ _-- --- --- --C -aC ___ v-G ___ ___ ___ -_T ___ _-_ -_- --A --- --_ __- --- _-- --C --C ___ w-G ___ ___ ___ --T --C 5-b 5-(45 months) ___ __T C_- ___ ___ -_A ___ ___ __- _-A _-- --- _-- ___ _-- -__ _-_-- --G -_- --- --- --A _-_ -__ ___ --A ___ _-_ -__ _-_ -__ _-_ -__ 6-b 6-(48 months) --_ -_G --C --T _-- e-T _-- --- ___ --A ___ e-G -__ _-_ -__ _-G -aG -_- __- --G --_ --T --- _-T _-- --_ _-_ -_A ___ __G __- ___ -_- _-G -_G 7-b 7-(47 months) --- --- --- -_T _-- --A _-_ -_- _-C -_A -__ e-G -__ _-- --A _-- T-G --_ --- C-e --T _-- --A ___ --_ --C --A ___ _-G -_- _-_ -_A __- -_G 8-b 8-(41 months) --- -_T --- -_- _-_ -_- _-_ -__ ___ e-C -_A e-G --- _-- --- --- --_ ___ e-T -__ e-T -__ --R -_y _-- -_C e-G --R --G --- -_- --- --S --G A-- ___ --G ___ --T --_ e-T --_ --A -__ _-- --C --A --- --- --_ -_- --- --- -_ G 10-b lo-(29 months) A-- --- --G --- --T --- --T --- _-A --- --- --C --A ___ ___ -__ _-_ -_- --_ --G 12-b 12-(28 months) --T --T ___ ___ _-_ -__ e-C -__ --G --- --- -__ -_- --G -__ _-- --G -__ _-T --_ --T --T -__ ___ ___ ___ _-_ ___ S-G __- _-_ -_- _-- --G HCVl GBV-C HGV 4611 --- -a A-- C-G GT- --A TT- e-C A-C -_- --- G-G e-C --- -_- ___ ___ _-G C-- --_ _-T ___ ___ __C GCT GGC CAG TTC TCC GCT AGG GGG GTC AAT GCC ATT 4-b 4-(48 months) _-C --_ -_- _-_ --T --C ___ __- e-T --_ --- -__ --C -_- --- --T _-T _-C --- -_- _-T -_C __- ___ 5-b S-(45 months) e-C --_ -__ --T __T _-A --A --_ ___ -_C ___ ___ _-C --- _-- --T --T _-A --A __- --_ --y _-- -__ 6-b 6-(48 months) --C --T ___ __T --_ T-G -_- -_A ___ --- --- --__C __T _-- --T ___ T-A ___ --A _-_ --- _-- -_- 7-b 7-(47 months) -_G __- --A ___ --A _-_ ___ _-- --_ --_ --T ___ v-G __- --A -__ --A ___ ___ --- -__ _-_ _-T ___ 8-b 8-(41 months) --C e-T _-_ w-T --T --C ___ _-- --- --- --_ -__ --- -- y _-- --T --y --C ___ ___ -_S _-_ _-- ___ 10-b lo-(29 months) v-G __- _-_ --- --T T-Y ___ __- --- -_C ___ ___ --G _-_ ___ ___ --T T-Y __- ___ ___ --C --_ --- 12-b 12-(28 months) _-C --- ___ _-T _-_ --- ___ __- --_ -_- --T -__ v-C __- --_ --T _-_ ___ -__ _-- --S -_- _-T ___ were directly sequenced. The sequences showed a mean homology on the nucleotide level of 88% (range 84-91%) to the reported HGV sequence (3) and of 80% (77-85%) to the GBV-C sequence (1,2). On the amino acid level the identity was 97-100% to both the published HGV and GBV-C sequence. Individual pre- and post-treatment sequences (mean interval 41 months [28+8]) differed in a mean of four nucleotides (range O-13, including positions in which two bases, the previous and a new one, were readable), but none of these mutations led to an amino acid exchange (Fig. 2). 766 Discussion This study has confirmed that in Germany a significant number of chronic HCV-infected patients were also chronically coinfected with HGV (3,4). Most of these patients had parenteral risk factors but in some patients the mode of transmission could not be clarified. The severity of the liver disease, as determined by ALT levels and liver histology, was not significantly different in patients with or without HGV coinfection. Rather, coinfected patients showed slightly lower disease activity, as demonstrated by lower mean ALT levels and Knodell score. Measure- Response of HCVZIGV coinfection to IFNa B HCV-1 GBV-C HGV VI -RM KG----I- - - - - - - - RTGRHLVF - - - -KK-DE---------CHSKAECERL -AKLV-L-I---------AGQFSARGVN - V A 4-b 4-(48 months) 5-b 5-(45 months) 6-b 6-(48 months) ------- _ - - - - - - 7-b 7-(47 months) 8-b 8-(41 months) 10-b lo-(29 months) - - ------__ - - - - - - - - - - 12-b 12-(28 months) - - -------- - Fig. 2. Alignment of the nucleotide (A) and deduced amino acid (B) sequences within the HGV helicase-like region from seven patients with HCV/HGV coinfection before (b) and after IFNcx treatment (mean interval 41 months). The nucleotide sequences were described using the standard IUPAC ambiguity code where Y=C or r R=A or G; S=C or G. Lines indicate the same nucleotide/amino acid as in the above HGV sequence. Nucleotide position number according to GBV-C, Genebank accession number U 25538. HGV sequence according to Kim et al. (PCT/US95/06169) and HCV-I sequence according to prototype sequence (8). ment of HCV RNA levels in serum revealed no significant differences between HCV and HCV/HGV coinfected patients, indicating that HGV coinfection does not seem to interfere with HCV replication. This suggests the conclusion that HGV coinfection has no influence on the activity of chronic hepatitis C. The rate of sustained response in our 18 HCV/ HGV-coinfected patients did not differ significantly from that observed in patients with chronic hepatitis C (9). The high rate (50%) of response of HGV to IFNcx treatment in patients with HCVMGV coinfection indicates that HGV is an interferon-sensitive agent. However, the rate of sustained remission was only 17%, and this response rate is similar to that found in patients with hepatitis C (9). KekulC et al. (10) described two sustained responses of HGV in eight HCVEIGV-coinfected patients treated with IFNa but without elimination of HCV. We also observed a dissociation between HCV and HGV response to IFNa. But normalization of the ALT levels corresponded with the response to HCV. Interestingly, three of the four HCV/HGV-coinfected patients with sustained response to HCV and normal ALT levels showed persistent HGV viremia, implying that HGV infection per se may not always induce a pro- nounced inflammatory response. A healthy carrier state may therefore exist in patients with chronic HGV infection. Sequence analysis of the amplicons from the helicase-like region revealed that our isolates all belonged to the HGV-like type, reported as genotype 2. During the observation period of up to 4 years, only O-13 (mean 4) nucleotide exchanges were observed, and none of these led to an amino acid exchange. The high conservation of the helicase-like region on the amino acid level might be indicative that this region is functionally important and not under strong immunological pressure. In conclusion, we have shown that HGV in chronic HCV/HGV coinfected patients is responsive to IFNa and that HGV coinfection did not negatively influence the response of chronic hepatitis C. It still remains to be seen whether patients with only HGV infection respond similarly to those with HCV/I-IGV coinfection, and whether the different HGV/GBV-C genotypes have any influence on the responsiveness to IFNa treatment. Acknowledgements We thank Heidrun assistance. Roeske for excellent technical 767 T. Berg zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA References 1. Simons JN, Leary TP, Dawson, Pilot-Matias TJ, Muerhoff AS, Schlauder GG, Desai SM, Mushahwar D, Mushahwar IK. 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