Mutations in the E2 and NS5A regions in patients infected with hepatitis C virus genotype 1a and their correlation with response to treatment

Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF‐2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated‐IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN. J. Med. Virol. 83:1332–1337, 2011. © 2011 Wiley‐Liss, Inc.     

Metabolic syndrome in patients with chronic hepatitis C virus genotype 1 infection who do not have obesity or type 2 diabetes

OBJECTIVE:

The individual components of metabolic syndrome may be independent predictors of mortality in patients with liver disease. We aimed to evaluate the prevalence of metabolic syndrome and its related components in hepatitis C virus–infected patients who are not obese and do not have type 2 diabetes.

METHODS:

This cross-sectional study included 125 patients infected with hepatitis C virus genotype 1. Metabolic syndrome was defined according to the International Diabetes Federation. Anthropometric data were measured according to standardized procedures. Bioimpedance analysis was performed on all patients.

RESULTS:

Metabolic syndrome was diagnosed in 21.6% of patients. Of the subjects with metabolic syndrome, 59.3% had hypertension, 77.8% had insulin resistance, 85.2% were overweight, 48.1% had a high waist circumference, 85.2% had an increased body fat percentage, and 92.3% had an elevated waist:hip ratio. In the bivariate analysis, female sex (OR 2.58; 95% CI: 1.09–6.25), elevated gamma-glutamyl transferase (γGT) (OR 2.63; 95% CI: 1.04–7.29), elevated fasting glucose (OR 8.05; 95% CI: 3.17-21.32), low HDL cholesterol (OR 2.80; 95% CI: 1.07–7.16), hypertriglyceridemia (OR 7.91; 95% CI: 2.88–22.71), elevated waist circumference (OR 10.33; 95% CI: 3.72–30.67), overweight (OR 11.33; 95% CI: 3.97–41.07), and increased body fat percentage (OR 8.34; 95% CI: 2.94–30.08) were independent determinants of metabolic syndrome. Using the final multivariate regression model, similar results were observed for abdominal fat (OR 9.98; 95% CI: 2.63–44.41) and total body fat percentage (OR 8.73; 95% CI: 2.33–42.34). However, metabolic syndrome risk was also high for those with blood glucose ≥5.55 mmol/L or HDL cholesterol <0.9 mmol/L (OR 16.69; 95% CI: 4.64–76.35; OR 7.23; 95% CI: 1.86–32.63, respectively).

CONCLUSION:

Metabolic syndrome is highly prevalent among hepatitis C virus–infected patients without type 2 diabetes or obesity. Metabolic syndrome was significantly associated with hypertension, insulin resistance, increased abdominal fat, and overweight.     

Variations within hepatitis C virus E2 protein and response to interferon treatment

To determine whether the hepatitis C virus (HCV) E2 PePHD sequence (aa 659-670; PKR-eIF2alpha phosphorylation homology domain) is the determinant for the response of interferon treatment, we have analyzed PePHD sequences in HCV-infected patients who had received interferon-alfa treatment. The PePHD sequence from all (6/6) of the patients, who are non- or partial responders to the interferon treatment, is the wild-type sequence (RSELSPLLL-TT, consensus sequence of HCV-1a and HCV-1b). However, there are sequence variations from more than half (5/9) of the patients, who are complete responders to the treatment. We have also analyzed the NS5A ISDR sequence (aa 2209-2248, interferon sensitivity-determining region) variation in HCV-1b-infected patients. No such correlation has been observed. Thus, our data suggest that HCV E2 should play a more important role than NS5A in determining the interferon responses.     

Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain

Infections with hepatitis C virus (HCV) genotype 3 exhibit differences in clinical phenotype including an increase in response to interferon therapy and development of steatosis. To initiate studies on genotype 3, we created a chimeric genotype 1b replicon containing a genotype 3a NS5A domain. The chimera was capable of efficient colony formation after the selection of a novel dominant adaptive mutation. Thus, domains from highly different strains can interact to form a functional replicase. A new genotype 1a replicon was constructed as well. Genotype specific influence on interferon sensitivity was examined using genotype 1a, 1b and chimeric 1b-3a replicons. The genotype 3a NS5A domain did not increase the sensitivity of the chimeric replicon to IFNalpha. The results suggest that NS5A is not sufficient to convey the increased IFNalpha response by genotype 3 or the replicon model is not capable of mimicking the events involved in increased sustained viral response.     

Significance of pretreatment analysis of hepatitis C virus genotype 1b hypervariable region 1 sequences to predict antiviral outcome

The heterogeneity of hypervariable region 1 (HVR1), located at the amino terminus of the E2 envelope, may be involved in resistance to alpha interferon (IFN-alpha) treatment. We investigated whether peculiar HVR1 domain profiles before treatment were associated with the maintenance of sensitivity or the appearance of resistance to treatment. Fifteen patients infected with hepatitis C virus genotype 1b and treated with IFN with or without ribavirin were selected. Ten responded to treatment (groups R1 and R2) and five did not (group NR). The amino acid sequences of 150 naturally occurring HVR1 variants present in the serum before therapy were compared in relation to treatment outcome. HVR1 variants from the NR group contained a constant nonantigenic amino acid segment that was not found in HVR1 variants from the R groups.     

Sequence elements correlating with circulating viral load in genotype 1b hepatitis C virus infection

The correlation between hepatitis C virus (HCV) genomic sequences and circulating HCV RNA levels was assessed to investigate the genetic elements affecting viral load. The interferon sensitivity-determining region (ISDR) sequence and the serum viral load were strongly correlated in 226 patients examined. Analysis of the entire HCV genome from six patients (three with a high and the others with a low viral load) with similar ISDR sequences identified several candidate residues associated with viral load. The amino acid (aa) sequences of these candidate residues and flanking regions in 67 additional patients revealed that only the residue at aa 962 varied significantly between the HCV patients with low and high serum loads (P=0.042). At this position, alanine was observed more frequently in the patients with a high viral load. In conclusion, our results strongly suggest that serum HCV RNA loads are inversely correlated with amino acid substitutions in the ISDR, and aa 962 was identified as a possible second determinant of serum HCV RNA load.     

Prediction of response to peginterferon-alfa-2b plus ribavirin therapy in Japanese patients infected with hepatitis C virus genotype 1b

Variation at the IL‐28B locus was recently reported to be a significant predictive factor of viral response to pegylated‐interferon plus ribavirin combination therapy against chronic hepatitis C. Predictive factors for the effect of therapy, including IL‐28B polymorphism rs8099917 and viral and clinical factors were investigated. A total of 288 patients were enrolled who were chronically infected with hepatitis C virus (HCV) genotype 1b and treated with combination therapy. Among them, 87 patients completed 48 weeks of therapy without dose reduction or discontinuation. In multivariate regression analysis, the rs8099917 TT genotype was the only independent factor significantly associated with sustained viral response (P = 0.016, OR 61.5), whereas substitutions at amino acid 70 (aa 70) of the HCV core protein (P = 0.038, OR 5.9) and non‐TT genotypes (P = 0.002, OR 17.2) were associated with nonvirological response. Both factors were also associated with viral dynamics during the initial stage of the therapy. Correlation analysis revealed that rs8099917 genotype was correlated with γ‐glutamyl transpeptidase, hyaluronic acid, and HCV core aa 70. In conclusion, host (IL‐28B polymorphism) and viral (aa 70) factors independently affect response to combination therapy. J. Med. Virol. 83:981–988, 2011. © 2011 Wiley‐Liss, Inc.     

2a型HCV E1、E2/NS1区基因cDNA的克隆及序列分析

目的：了解丙型肝炎病毒（HCV）2a型基因组E1 E2／NS1区序列，为进一步研究HCV包膜蛋白的生物学功能奠定基础。方法：用逆转录套式PCR（RT－nPCR)从江苏省1例丙型肝炎患者血清中扩增出两条分别约770bp、1100bp的片段，分别以限制性内切酶EcoR I、BamH I和EcoR I、Hand Ⅲ双酶切后连入pUC19载体中，转化感受态细胞，经限制性酶切长度多态性分析（RFLP）和PCR法证实为阳性克隆后，用全自动序列分析仪测序。结果：分别测得约770bp、1100bp的核苷酸序列，拼接后得到的完整核苷酸序列及其编码的氨基酸序列分别与HCV－1、HC－C2、HCV－BK、HC－J6、HC－J8相应序列作比较，显示分离株HCV在核苷酸水平上与以上分离株的同源性分别为60．5％、60．1％、59．7％、87．8％、67．5％；在氨基酸水平上的同源性分别为67．3％、66．4％、65．0％、87．8％、79．0％。结论：分离株（HCV－JS）与HC－J6同属2a型，但同源性有一定差异。     

基因1b型HCV核心蛋白不同功能区域对HepG2细胞生长的影响

目的 研究基因1b型丙型肝炎病毒(HCV)不同病毒株编码的核心蛋白(CORE):癌中心株(T)、癌旁株(NT)、C191(HCV-J6)及同一病毒株(T)编码的不同功能区域(1～172、1～126、1～58、59～126、127～172 AA)在HCV致病机制中的作用,为治疗HCV感染提供靶位.方法 将含有3个不同病毒株(T、NT及C191)及T不同功能区域的CORE真核表达质粒,转染到HepG2细胞,用流式细胞仪检测Annexin Ⅴ/PI,并用细胞生长实时观察仪观察细胞生长曲线.结果 基因1b型T、NT、C191和T不同功能区域的CORE均能诱导HepG2细胞凋亡和坏死且抑制了细胞的生长,其中,T诱导细胞凋亡和坏死及抑制细胞生长强于NT和C191, T的CORE的N端1～58 AA诱导细胞凋亡和坏死及抑制细胞生长均高于其他功能区域.结论 HCV基因1b型不同病毒株及同一病毒株编码的CORE不同功能区域的分子致病机制存在一定的差异,N端的1～58 AA在CORE的致病机制中起重要作用,可能是基因治疗的重要靶位之一.     

Mutations in the interferon sensitivity‐determining region of hepatitis C virus genotype 2a correlate with response to pegylated‐interferon‐alpha 2a monotherapy

The interferon sensitivity‐determining region (ISDR) is thought to be inhibited by the double‐stranded RNA‐dependent protein kinase (PKR). Several studies have reported a relationship between the ISDR and interferon (IFN) responsiveness. However, this relationship is controversial. The aim of this study was to investigate whether genomic heterogeneity of the ISDR among patients with hepatitis C virus (HCV) genotype 2a affects the response to pegylated‐IFN‐alpha 2a monotherapy. Eighty patients (47 men, 33 women; mean age: 54.2 ± 12.9 years) infected with HCV genotype 2a were evaluated. HCV viral loads were determined by real‐time PCR. The ISDR (amino acids 2193–2228) was examined by direct sequencing. Thirty‐one patients received subcutaneous injections of pegylated‐IFN‐alpha 2a (180 µg) once weekly for 24 weeks, and 35 patients received injections for 48 weeks. Fourteen patients withdrew from treatment. Of the remaining 66 patients, 51 (77.3%) showed a sustained virologic response. Factors related to sustained virologic response on multivariate analysis were rapid virologic response (negative HCV at 4 weeks; odds ratio: 0.033; 95% confidence interval (95% CI) 0.003–0.363; P = 0.0052) and the number of mutations in the ISDR (odds ratio: 0.025; 95% CI 0.001–0.476; P = 0.0141). There were no significant differences in other factors, including sex, age, aspartate aminotransferase, alanine aminotransferase, platelet count, duration of treatment, and HCV viral load. Rapid virologic response and the ISDR sequence variations are significantly associated with response to pegylated‐IFN‐alpha 2a monotherapy in Japanese patients with HCV genotype 2a. J. Med. Virol. 81:459–466, 2009. © 2009 Wiley‐Liss, Inc.     

The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death

The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.     

Response prediction and treatment tailoring for chronic hepatitis C virus genotype 1 infection

We monitored early viral response during the treatment of hepatitis C virus (HCV) infection with the aim of identifying predictors of treatment outcome. We studied 53 patients with genotype 1 infection who received 180 microg/week pegylated interferon alfa-2a and 1,000 or 1,200 mg/day ribavirin depending on body weight and serially assessed HCV RNA in serum, using the Cobas TaqMan assay. Thirty-one patients (58%) achieved sustained viral response (SVR). SVR was obtained in 100% (10/10) of patients with pretreatment viremia concentrations below 400,000 IU/ml, in 100% (14/14) of patients with more than 1.5 log reduction of HCV RNA after 4 days of treatment, and in 95% (22/23) of patients with a rate of decline in viremia higher than 0.70 log units/week during the second phase. Non-SVR was seen in all patients with a second-phase decline rate lower than 0.35 log units/week. Patients with slopes between 0.50 and 0.80 log units/week achieved SVR (4/4) unless the treatment dose was modified (3/3). We conclude that the second-phase slope appears to be an accurate and useful predictor of treatment response. On the basis of these findings, we propose a model of tailored treatment which takes into account the second-phase slope and the amount of HCV RNA after 21 days of treatment.     

Detection of hepatitis E virus-specific immunoglobulin a in patients infected with hepatitis E virus genotype 1 or 3

Currently, diagnosis of acute hepatitis E virus (HEV) in patients is primarily based on anti-HEV immunoglobulin M (IgM) detection. However, several investigations suggest the use of HEV-specific IgA for diagnosing acute HEV infections. We evaluated two commercially available assays, an IgA enzyme-linked immunosorbent assay (ELISA) (Diacheck) and an adapted immunoblot protocol (Mikrogen) for IgA detection and compared the performance in genotype 1- and 3-infected patients. The specificity of the IgA assays was high, with no positive reactions in a control group of 18 acute hepatitis patients who were negative for HEV. The sensitivity calculated in nine PCR-positive type 1-infected patients was 100% in both assays but was clearly lower in genotype 3-infected patients (n = 14), with sensitivities of only 67% and 57% for the ELISA and immunoblot assay, respectively. The lower IgA responses detected in genotype 3-infected patients could be caused by the use of only the genotype 1 and 2 antigens in the serological assays. Interestingly in two patients with possible infection through blood transfusion no response or intermediate IgA responses were detected, and this might confirm the parenteral route of transmission. In both the type 1- and type 3-infected patients both the IgA and IgM responses disappeared simultaneously. We conclude that IgA detection is of limited value for the serodiagnosis of acute HEV cases, particularly with genotype 3.     

Molecular epidemiology of hepatitis C virus genotype 4 isolates in Egypt and analysis of the variability of envelope proteins E1 and E2 in patients with chronic hepatitis

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.