Region Of Hepatitis Research Articles

Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin.

Nonstructural 5A (NS5A) and the second envelope (E2) proteins of hepatitis C virus (HCV) have the potential to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and may therefore interfere with the response to IFN therapy, but controversy still exists regarding the relevance of this. This study aimed to assess whether mutations in these regions correlated with the response to combination therapy, IFN and ribavirin. Pretreatment parameters were analysed in 57 HCV-1b patients who had received IFN-alpha2b (3 or 5 MU three times weekly) and ribavirin (800-1200 mg per day) for 24 weeks. The amino acid sequences of the NS5A and PKR-eIF2alpha phosphorylation homology domain (E2-PePHD) were deduced from the corresponding coding sequence, which were determinated by direct sequencing of the HCV genome amplified by the polymerase chain reaction. Twenty (36%) patients achieved a sustained virological response (SVR). The mean number of amino acid substitutions in the NS5A-PKR binding domain (2209-2274), interferon sensitivity-determining region (ISDR) (2209-2248), and E2-PePHD sequence (659-670) in patients with and without SVR were 4.53 +/- 3.31 vs 2.83 +/- 1.78 (P = 0.094), 2.45 +/- 2.74 vs 1.03 +/- 1.32 (P = 0.042) and 0.25 +/- 0.70 vs 0.03 +/- 0.17 (P = 0.109), respectively. Patients with a mutant-type (>/= 4) NS5A-ISDR had a higher rate of SVR (six of nine, 67%) than those with wild-type (five of 22, 23%) (P = 0.038). Stepwise multiple logistic regression analysis of the factors (age, gender, viral load, cirrhosis rate, IFN dosage and amino acid substitutions) revealed that the mutation in NS5A-ISDR (>/= 4 vs < 4) was the only independent variable of treatment outcome. Our study showed that NS5A-ISDR mutations were correlated with the SVR to combination therapy in chronic HCV-1b patients in Taiwan.

Two distinct types of hepatitis B virus core promoter variants in Yemeni blood donors.

Genetic variations in the basic core promoter (BCP) region of hepatitis B virus (HBV) occur during the natural history of chronic HBV infection. This study investigates the presence of basic core promoter variations in 28 asymptomatic Yemeni blood donors, correlating variations with HBeAg phenotype and viral load. The core promoter/precore and surface gene region of HBV DNA were amplified using nested PCRs and the PCR products were sequenced either directly or after cloning. HBeAg and viral load were measured when HBV DNA was detectable. Sequencing of 18 surface PCR products indicated that all were of genotype D. Two distinct types of variants were identified in the basic core promoter: substitution only (N = 14) and major deletion (N = 9). The commonest substitutions were located at nucleotide positions 1753, 1762, and 1764; 10/14 (71.4%) were associated with the precore 1896A substitution resulting in the premature stop of the precore reading frame and 6/14 (42.9%) had viral loads above 400 copies/ml. Two forms of deletion variants were found: 8 bp deletion (1763-1770) (N = 2) and a novel 12 (1746-1757) + 8 bp (1763-1770) deletion (N = 7). The deletion sequences were never associated with the precore 1896A substitution and all had viral load below 400 copies/ml with negative HBeAg phenotype. The polymorphism 1773C was found in 9/14 (64.3%) substitution sequences whereas all deletion sequences had 1773T. Two donors had mixed sequences of basic core promoter substitution and major deletions (both 8 bp and 12 + 8 bp). While the deletion variants in these two donors were similar to others found in isolation, the substitutions were of a different pattern. Further studies are required to understand the selection process behind these variants.

Nucleotide mutation profiles of core promoter and precore regions of hepatitis B virus genome in chronic carrier children

Nucleotide mutation profiles of core promoter and precore regions of hepatitis B virus genome in chronic carrier children

Single nucleotide insertion in the 5′-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfection

Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world-wide. Antiviral therapy-related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV-related liver cirrhosis, but the role of the 5'-untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5'-UTR in an HCV-infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5'-UTR of HCV showed a nucleotide A insertion at position 193 of the IRES.

DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV.

Both Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are major causative agents of transfusion-associated and community-acquired hepatitis worldwide. Development of a HCV vaccine as well as more effective HBV vaccines is an urgent task. DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against viral infection. The aim of this study is to achieve immune responses against both HCV and HBV by DNA immunization with fusion constructs comprising various HCV E2 gene fragments fused to HBsAg gene of HBV. C57BL/6 mice were immunized with plasmid DNA expressing five fragments of HCV E2 fused to the gene for HBsAg respectively. After one primary and one boosting immunizations, antibodies against HCV E2 and HBsAg were tested and subtyped in ELISA. Splenic cytokine expression of IFN-gamma and IL-10 was analyzed using an RT-PCR assay. Post-immune mouse antisera also were tested for their ability to capture HCV viruses in the serum of a hepatitis C patient in vitro. After immunization, antibodies against both HBsAg and HCV E2 were detected in mouse sera, with IgG2a being the dominant immunoglobulin sub-class. High-level expression of INF-gamma was detected in cultured splenic cells. Mouse antisera against three of the five fusion constructs were able to capture HCV viruses in an in vitro assay. The results indicate that these fusion constructs could efficiently elicit humoral and Th1 dominant cellular immune responses against both HBV S and HCV E2 antigens in DNA-immunized mice. They thus could serve as candidates for a bivalent vaccine against HBV and HCV infection. In addition, the capacity of mouse antisera against three of the five fusion constructs to capture HCV viruses in vitro suggested that neutralizing epitopes may be present in other regions of E2 besides the hypervariable region 1.

LIPOSOME (LIPODINE™)-MEDIATED DNA VACCINATION BY THE ORAL ROUTE

ABSTRACTPlasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen (HBsAg) was incorporated by the dehydration–rehydration method into Lipodine™ liposomes composed of 16 µmoles phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC), 8 µmoles of (dioleoyl phosphatidylethanolamine (DOPE) or cholesterol and 4 µmoles of the cationic lipid 1,2-dioleoyl-3-(trimethylammonium propane (DOTAP) (molar ratios 1 : 0.5 : 0.25). Incorporation efficiency was high (89–93% of the amount of DNA used) in all four formulations tested and incorporated DNA was shown to be resistant to displacement in the presence of the competing anionic sodium dodecyl sulphate molecules. This is consistent with the notion that most of the DNA is incorporated within the multilamellar vesicles structure rather than being vesicle surface-complexed. Stability studies performed in simulated intestinal media also demonstrated that dehydration–rehydration vesicles (DRV) incorporating DNA (DRV(DNA)) were able to retain significantly more of their DNA content compared to DNA complexed with preformed small unilamellar vesicles (SUV–DNA) of the same composition. Moreover, after 4h incubation in the media, DNA loss for DSPC DRV(DNA) was only minimal, suggesting this to be the most stable formulation. Oral (intragastric) liposome-mediated DNA immunisation studies employing a variety of DRV(DNA) formulations as well as naked DNA revealed that secreted IgA responses against the encoded HBsAg were (as early as three weeks after the first dose) substantially higher after dosing with 100 µg liposome-entrapped DNA compared to naked DNA. Throughout the fourteen week investigation, IgA responses in mice were consistently higher with the DSPC DRV(DNA) liposomes compared to naked DNA and correlated well with their improved DNA retention when exposed to model intestinal fluids. To investigate gene expression after oral (intragastric) administration, mice were given 100 µg of naked or DSPC DRV liposome-entrapped plasmid DNA expressing the enhanced green fluorescent protein (pCMV.EGFP). Expression of the gene, in terms of fluorescence intensity in the draining mesenteric lymph nodes, was much greater in mice dosed with liposomal DNA than in animals dosed with the naked DNA. These results suggest that DSPC DRV liposomes containing DNA (Lipodine™) may be a useful system for the oral delivery of DNA vaccines.

Biochemical response to interferon therapy correlates with interferon sensitivity-determining region in hepatitis C virus genotype 1b infection.

Biochemical responders maintain normal alanine aminotransferase levels after interferon (IFN) therapy despite persistent presence of hepatitis C virus (HCV) RNA in their sera. There have been few reports on predictive factors for biochemical response. A region associated with sensitivity to IFN was identified in the nonstructural protein 5 A of genotype 1b [aa 2209-2248; IFN sensitivity-determining region (ISDR)]. The substitutions in ISDR correlate with sustained response to IFN. In this report, we assessed the association of ISDR with biochemical response. The sequences of ISDR were determined in 62 patients with HCV genotype 1b treated by IFN in two randomized controlled trials. 30 patients had wild ISDR (identical to HCV-J), 20 intermediate ISDR (1-3 amino acid substitutions compared with HCV-J), and 12 mutant ISDR (four or more amino acid substitutions). All 12 patients with mutant ISDR had a sustained response, while only one of those with wild or intermediate ISDR had a sustained response (P < 0.0001). In the 49 patients other than sustained responders, the patients with intermediate ISDR obtained biochemical response significantly more frequently (52.6%, 10/19) than those with wild-type ISDR (20.0%, 6/30) (P < 0.05). Multivariate analysis indicated the number of substitutions in ISDR as the most important predictor for biochemical response (discriminant coefficient=1.08, P < 0.05) and sustained response (discriminant coefficient=6.13, P < 0.0001). In phylogenetic analysis, clustering of sustained responders and biochemical responders was observed. These results demonstrate that the substitutions in ISDR are the most important predictor for biochemical response to IFN in patients infected with genotype 1b as well as for sustained response.

Genetic variability of hepatitis A virus in South America reveals heterogeneity and co-circulation during epidemic outbreaks.

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. In order to gain insight into the genetic variability and mode of evolution of HAV in South America, an analysis was performed of sequence data obtained from the VP1 amino terminus and the VP1/2A region of HAV strains isolated over a short period of time in Uruguay, Argentina and Chile. Sequences obtained from 22 distinct HAV isolates were compared with published sequences from 21 different strains isolated all over the world. Phylogenetic analysis revealed that all strains isolated belong to a unique sub-genotype (IA). Strains isolated during an outbreak period showed a higher degree of heterogeneity than anticipated previously and the co-circulation of different isolates. The genetic variability among strains isolated in this region seems to be higher in comparison with strains isolated in other regions of the world.

Genetic evolution of the hypervariable region in hepatitis C virus patients with persistently normal aminotransferase levels

Genetic evolution of the hypervariable region in hepatitis C virus patients with persistently normal aminotransferase levels

Genetic evolution of the hypervariable region in hepatitis C virus patients with persistently normal aminotransferase levels

Genetic evolution of the hypervariable region in hepatitis C virus patients with persistently normal aminotransferase levels

Liposome-mediated DNA vaccination: the effect of vesicle composition

Liposome-entrapped DNA has been shown to enhance the potency of DNA vaccines, possibly by facilitating uptake of the plasmid by antigen-presenting cells (APC). In this paper, we have investigated the influence of the liposomal composition and surface charge on such potency. Plasmid DNA pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was entrapped within cationic liposomes of various compositions and surface charges with high efficiency (88–97% of the amount used) by the dehydration–rehydration method that generates dehydration–rehydration vesicles (DRV). Cryo-electron microscopy revealed that DNA-containing DRV (DRV(DNA)) were multilamellar. In immunisation studies, female Balb/c mice were given two to four intramuscular injections of 10 μg naked or liposome-entrapped pRc/CMV HBS and bled at time intervals. Results indicate that the lipid composition of the DRV(DNA) influences the strength of the humoural response (immunoglobulin (Ig)G subclasses) with inclusion of dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylethanolamine (PE) in the liposomal structure contributing to greater responses. DRV(DNA) in which the DOPE or PE were omitted or substituted with cholesterol led to significant reduction of humoural responses against the encoded antigen. Replacing phosphatidylcholine (PC) in the DRV(DNA) with the high-melting distearoyl phosphatidylcholine also contributed to lower responses. In other experiments, IgG responses were monitored in mice immunised with pRc/CMV HBS entrapped in DRV composed of PC and DOPE as before but incorporating increasing amounts of DOTAP (1–16 μmol). Maximal IgG responses were observed at 10 weeks after the first of four injections and suggested a trend of higher responses when 4 or 8 μmol DOTAP was present in the DRV(DNA) formulation. Cell-mediated immunity (measured in terms of endogenous antigen-specific splenic interferon-γ) in mice immunised with pRc/CMV HBS entrapped in liposomes composed of PC, DOPE and DOTAP (16:8:4 molar ratio) was much greater than in animals treated with naked plasmid. These results indicate that liposome-mediated DNA immunisation is more effective than the use of naked DNA, and also suggest that the presence of fusogenic phosphatidylethanolamine in DRV in conjunction with a low-melting phosphatidylcholine and an appropriate content of cationic lipid might contribute to more effective liposomal DNA vaccines. The notion that liposomes improve immune responses to the plasmid-encoded vaccine by facilitating the latter's uptake by APC was supported by the observation that in Balb/c mice injected intramuscularly with liposome-entrapped pCMV. Enhanced green fluorescent protein, expression of the gene in terms of fluorescence intensity in the draining lymph nodes, was much greater than in animals treated with the naked plasmid.

Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3' end of the hepatitis G virus/GB-virus C genome.

Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787-5794, 1999 ). In this study we have carried out a detailed comparison of the structure of the 3'untranslated region (3'UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1-4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem-loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.

Integrity of the NS5A (amino acid 2209 to 2248) region in hepatitis C virus 1b patients non-responsive to interferon therapy

In hepatitis C virus-1b, it has been suggested that an amino acid stretch (aa 2209-2248) of the carboxy terminal half of the non-structural 5A (NS5A) region participates in the response to interferon treatment. We tested the hypothesis that absence of mutations in the NS5A (aa 2209-2248) sequence is required for interferon resistance. We also investigated the importance of different HCV-1b isolates in interferon response in France. We determined the NS5A sequences of 70 patients with chronic hepatitis C before IFN therapy and then compared them with HCV-J prototype sequence. The isolates were determined by NS5B sequencing, the "gold standard" method for genotyping and subtyping. Pre-therapeutic viral load was also measured. No sustained virological response was observed in the patients without amino acid substitutions in the NS5A (aa 2209-2248) sequence, and in the patients with HCV-J isolates. Viral load was significantly higher in the patients with no amino acid substitutions in the NS5A (aa 2209-2248) sequence. In HCV-lb infected patients, an HCV-J strain with no amino acid substitution in the NS5A (aa 2209 2248) region indicates a poor prognosis for response to IFN therapy. The low interferon response rate in HCV-lb infection in Europe is probably not due to a difference between isolates.

Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS-TTEGDA support for the investigation of HCV infection in human blood.

Four peptides were designed and synthesized on a highly solvating copolymer of tetraethyleneglycol diacrylate cross-linked polystyrene (PS-TTEGDA) support with very high purity and yield. The polymer was synthesized in various cross-linking densities (1, 2, 3, 4, 5 and 10%) using radical aqueous suspension polymerization. Four per cent PS-TTEGDA resin showed rigidity and mechanical characteristics comparable with those of divinylbenzene cross-linked polystyrene (PS-DVB) support. Swelling and solvation characteristics of PS-TTEGDA were much higher than PS-DVB support in all solvents used in solid-phase peptide synthesis. Forty-eight hour treatment of the support with neat trifluoroacetic acid did not show any change in its infrared spectra. PS-TTEGDA could be functionalized with chloromethyl, aminomethyl and hydroxymethyl functional groups under various controlled conditions. Synthetic utility of the support was demonstrated by the synthesis of four peptides selected from the envelope and nonstructural protein region of the prototype hepatitis C virus (HCV). These peptides were later used successfully to develop a peptide-based immunoassay (PBEIA) for the detection of HCV immunity. Peptides designed from the NS1 and NS4 protein regions were found to be very promising for the development of a new diagnostic kit to detect HCV infection in human blood. Peptide purity was tested by RP-FPLC and the peptide identity was confirmed by amino acid analysis.

Liposome-entrapped plasmid DNA: characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 μg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmol dioleoylphosphatidylcholine (DOPE) and 1,2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95–97 and 48–54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 μg plasmid DNA also led to high complexation values of 73–97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA–SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors

Background/Aims: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV, cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV.Methods: Active NS3 protease domains of genotype 1–3 (representing five subtypes: 1a, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B.Results: Our data revealed that NS3 proteases crossinteracted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner.Conclusion: Our study suggests that the NS4A cofactor function is well conserved among HCV. It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.

Different regions of hepatitis B virus X protein are required for enhancement of bZip-mediated transactivation versus transrepression.

The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIgamma and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIgamma interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX(49-115) is as effective as the full-length pX in enhancing the ATF3- and ICERIIgamma-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX(49-140) are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.

Detection of mutations in hepatitis B virus enhancer 2/core promoter and X protein regions in patients with fatal hepatitis B virus infection.

The enhancer 2/core promoter and the X protein regions located upstream of the precore and core regions in hepatitis B virus regulate expression of core/e antigen peptides. Mutations in the precore and core regions have been reported to be associated closely with the severity of type B hepatitis, and regions regulating expression of these peptides may also be involved in severe liver damage. Mutations were examined in regions that may be related to fatal liver diseases. Nucleotide sequences and deduced amino acid sequences from 20 patients with fatal type B hepatitis (12 with fulminant hepatitis and 8 with severe exacerbation) and 10 patients with self-limited acute hepatitis were analyzed. There were 50 nucleotide alterations in the enhancer 2/core promoter region of virus from 12 patients with fulminant hepatitis (average 4.1/case), 37 alterations in 8 patients with severe exacerbation (4.6/case), and 10 mutations in 10 cases of acute hepatitis (1.0/case). The numbers of amino acid mutations in X protein were 53 in 12 cases of fulminant hepatitis (4.4/case), 27 in 8 cases of severe exacerbation (3.3/case), and 9 in 10 cases of acute hepatitis (0.9/case). In fatal cases, approximately 50% of the nucleotide mutations were located within the region spanning nucleotides 1741-1777 (14.2% of the enhancer 2/core promoter region) and 30% of the amino acid mutations in X protein were located within the region containing codons 122-132 (7.1% of X protein). In addition to mutations in the precore and core regions, mutations in the enhancer 2/core promoter and the X protein regions may be associated with the pathogenesis of fatal B hepatitis infection.

Genetic changes in the interferon sensitivity determining region of hepatitis C virus during the natural course of chronic hepatitis C.

Amino acid mutations in the interferon sensitivity determining region (ISDR) are closely associated with the response to interferon in patients with hepatitis C virus genotype 1b (HCV-1b) infection. In this study, 36 patients chronically infected with HCV-1b, with no history of interferon therapy with respect to ISDR changes in HCV were studied. Two serum samples were obtained from each patient, with an average interval of 3.5 years, and predominant nucleotide and amino acid sequences of the ISDR at initial and subsequent time points were compared for each patient. Three of 12 patients with the wild type ISDR (no amino acid mutation in the ISDR compared to the consensus sequence) changed to the intermediate type (1 to 3 mutations) at later time points, whereas the other 9 still had the wild type. Similarly, 2 of 18 patients with the intermediate type changed to the wild type, whereas the other 16 patients continued to have the intermediate type. One of 6 patients with the mutant type (4 or more mutations) changed to the intermediate type, and the other 5 continued to have the mutant type. Although ISDR nucleotide changes/site/year were not significantly different among the 3 groups of patients, percentages of non-synonymous nucleotide changes were greater in the mutant type (63%) than the wild (9%) or the intermediate type (20%) (P < 0.05). These results show that mutations in the ISDR do not occur frequently, suggesting that interferon sensitivity does not change greatly during the natural course of the disease in each patient.

Diversity of hepatitis G virus within a single infected individual.

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5' non-coding region (5'NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5'NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23-0.84% nucleotide position and at 0.42-0.61% amino acid position.