Clinical and therapeutic implications of hepatitis C virus compartmentalization

BACKGROUND & AIMS: Blood mononuclear cells (BMCs) frequently are infected by hepatitis C virus (HCV) variants that are not found in plasma. The influence of this compartmentalization on the natural and therapeutic outcome of hepatitis C is unknown. METHODS: We studied 119 patients with previously untreated chronic HCV infection. Sixty-five of these patients started first-line treatment with pegylated interferon-alfa and ribavirin after enrollment in the study. The internal ribosomal entry site (IRES) of HCV RNA was amplified and compared between plasma and BMCs by means of single-strand conformational polymorphism (SSCP) analysis, line-probe assay, and cloning sequencing. RESULTS: The IRES SSCP patterns differed between plasma and BMCs in 54 (48%) of 113 assessable patients. Twenty-seven (24%) of these patients were co-infected by 2 HCV types or subtypes, only 1 of which was detectable in BMCs (n = 25) or in plasma (n = 2). SSCP-defined compartmentalization was more frequent in former drug users than in others (35/56 [60%] vs 19/56 [34%]; P < .01), and less frequent in patients with genotype 1 HCV in plasma (26/73 [24%] vs 28/40 [65%]; P < .01). The only variables that were independently predictive of a sustained virologic response were SSCP-defined compartmentalization (25/31 vs 10/32; P = .0001) and genotype 2 or 3 infection of BMCs (22/31 vs 8/34; P = .002). CONCLUSIONS: A significant proportion of patients with hepatitis C are co-infected by 2 or more HCV variants with distinct IRES sequences and distinct cellular tropism. This compartmentalization is a strong independent predictor of treatment efficacy.     

重型丙型肝炎患者肝外脏器病毒感染和复制状态

目的 探讨丙型肝炎患者肝外脏器丙型肝炎病毒（HCV）感染和复制状态。方法 采用逆转录-聚合酶链反应（RT-PCR）、原位杂交（ISH）和免疫组织化学法,对9例重型丙型肝炎患者肝外8种脏器内HCV基因、HCV复制中间体和HCV抗原表达进行了研究。结果 采用RT-PCR,9例患者肝外脏器均可检出HCV基因,6例（66.7%）检出HCV复制中间体及HCV抗原;采用ISH和免疫组化法,5例（55.6%）检出HCV基因。除脾脏外,心脏、肾脏、胰腺、肾上腺、肠道、胆囊和淋巴结等脏器细胞内有HCV基因和HCV抗原表达。结论 肝外多种脏器细胞可能支持HCV复制;肝外HCV感染程度低,复制水平低。     

Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion

BACKGROUND & AIMS: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. METHODS: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2-3 weeks for 6 months, at 10 months, and at 11-16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5' untranslated region (5'UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). RESULTS: Seven patients cleared infection within 7-24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8-22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5'UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. CONCLUSIONS: Changes in E2/HVR1 quasispecies 8-22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host.     

Detection of hepatitis C virus RNA in the liver by in situ hybridization

ABSTRACT— To examine HCV infection histologically, we attempted nonradioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.     

Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in vivo

BACKGROUND & AIMS: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein-specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness. METHODS: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies. RESULTS: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region. CONCLUSIONS: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.     

The hepatitis C virus life cycle as a target for new antiviral therapies

The burden of disease consequent to hepatitis C virus (HCV) infection has been well described and is expected to increase dramatically over the next decade. Current approved antiviral therapies are effective in eradicating the virus in approximately 50% of infected patients. However, pegylated interferon and ribavirin-based therapy is costly, prolonged, associated with significant adverse effects, and not deemed suitable for many HCV-infected patients. As such, there is a clear and pressing need for the development of additional agents that act through alternate or different mechanisms, in the hope that such regimens could lead to enhanced response rates more broadly applicable to patients with hepatitis C infection. Recent basic science enhancements in HCV cell culture systems and replication assays have led to a broadening of our understanding of many of the mechanisms of HCV replication and, therefore, potential novel antiviral targets. In this article, we have attempted to highlight important new information as it relates to our understanding of the HCV life cycle. These steps broadly encompass viral attachment, entry, and fusion; viral RNA translation; posttranslational processing; HCV replication; and viral assembly and release. In each of these areas, we present up-to-date knowledge of the relevant aspects of that component of the viral life cycle and then describe the preclinical and clinical development targets and pathways being explored in the translational and clinical settings.     

Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes

BACKGROUND & AIMS: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. METHODS: We transfected antagomir of miR-122, 2'-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). RESULTS: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2'-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 mumol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. CONCLUSIONS: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.     

Distribution of hepatitis C virus RNA in the liver and its relation to histopathological changes

Abstract: To investigate a cellular mode of HCV-infection in the liver and its pathological implications in relation to histopathological changes or clinical data, we studied the distribution of HCV-RNA in the livers of 21 patients with HCV-related chronic liver disease (chronic active hepatitis, 14 cases; cirrhosis, 7 cases) using the in situ hybridization technique. In situ hybridization was performed on 4% paraformaldehyde-fixed frozen sections with digoxigenin-labeled DNA probe deduced from the core region of HC–J4. In situ hybridization showed positive signals in the liver specimens of 20/21 cases. The signals were localized in the cytoplasm of hepatocytes. The distribution pattern of positive cells was individually different, whereas the pattern was identical in the right and left lobes. There were no correlations of the HCV-positive cell number with serum aminotransferase levels at biopsy or with genotypes of HCV. The positive hepatocytes were occasionally associated with infiltrating mononuclear cells, and they were sparsely distributed in the area of piecemeal necrosis. These findings suggest that factors such as host immunoreaction to the virus may be more important than its direct cytopathy in the pathogenesis of chronic hepatitis C virus infection.     

In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue

In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)‐labelled sense and anti‐sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig‐labelled riboprobes in detecting genomic and replicative‐intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome‐length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti‐sense probe detected HCV RNA in 20/21 specimens from HCV‐infected liver tissues obtained from patients and in 0/9 samples from patients with non‐HCV‐related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients.     

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.     

Molecular epidemiology of hepatitis C virus (HCV) in Greece: temporal trends in HCV genotype-specific incidence and molecular characterization of genotype 4 isolates

This study aimed to estimate the overall HCV genotype distribution and to reconstruct the HCV genotype-specific incidence in Greece during the recent decades. It also focused at the identification of genotype 4 subtype variability in Greek isolates. A total of 1686 chronically infected HCV patients with detectable serum HCV RNA by RT-PCR, belonging to different risk groups were studied. Amplified products from the 5'-noncoding region were typed using a commercially available assay based on the reverse hybridization principle. The HCV genotype-specific incidence was estimated using a previously described back calculation method. HCV genotype 1 was the most prevalent (46.9%) followed by genotype 3 (28.1%), 4 (13.2%), 2 (6.9%) and 5 (0.4%). A high prevalence of genotype 1 (66.3%) in haemophilia patients was recorded whereas HCV genotype 3 was found mainly among patients infected by I.V. drug use (58.2%). Data on the temporal patterns of HCV genotype-specific incidence in Greece revealed a moderate increase (1.3-1.6 times) for genotypes 1 and 4, and a decrease (1.5 times) for genotype 2 from 1970 to 1990, whereas there was a sharp (13-fold) increase for genotype 3. The molecular characterization of 41 genotype 4 HCV isolates belonging to various risk groups revealed that, subtype 4a was the most frequently detected (78%). Phylogenetic comparison of the Greek 4a isolates with all HCV-4a isolates reported worldwide so far revealed a topology which does not discriminate Greek isolates from the others. HCV-4 does not represent a recent introduction in Greece.     

Genetic diversity of the hepatitis C virus： Impact and issues in the antiviral therapy

The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.     

Relationship between histological prognosis of chronic hepatitis C and amount of hepatitis C virus core protein in serum

BACKGROUND: Hepatitis C virus (HCV) viraemia is one of the factors for histological prognosis of chronic hepatitis C. METHODS: One hundred and thirty-five patients who received hepatic biopsies twice at intervals of 5 years or longer were followed up for a mean of 9.7 +/- 4.0 years were studied retrospectively. The amount of HCV viraemia present was measured as the concentration of HCV core protein by using the fluorescence enzyme immunoassay method. RESULTS: Multiple-regression analysis, using deterioration of the histological stage as a dependent variable, showed that greater age (P = 0.041), higher stage of hepatic histology at the start of follow up (P = 0.029), and higher serum concentration of core protein (P < 0.001) were independent factors affecting the deterioration of the liver's histological stage. At follow up, no significant difference in histological stage was seen between patients with serum HCV core protein > or = 100 pg/mL (n = 60) and those with serum core protein < 100 pg/mL (n = 75). The histological grade in patients with high serum core-protein levels tended to be significantly worse and the deterioration rate of the histological stage was significantly higher than in those with low HCV core protein levels (68 vs 35%, P < 0.001). The mutation rate of the HCV envelope-2/non-structural 1 (E2/NS1) nucleotide region was compared in two patients who had high serum concentrations of HCV core protein and whose histological stage had deteriorated with two patients who had low serum concentrations of the core protein and whose histological stages remained unchanged. No significant difference in E2/NS1 mutation was found. CONCLUSIONS: The amount of HCV viraemia was suggested to be a significant factor for determining histological outcome in patients with chronic hepatitis C. The mutation rate in the E2/NS1 region did not seem to be associated with the prognosis of chronic hepatitis C.