Hepatitis C virus genetic variability in patients undergoing antiviral therapy

Hepatitis C virus (HCV) has been the subject of intense research and clinical investigations due to its worldwide prevalence and major role in chronic liver disease. Like most RNA viruses, HCV circulates in vivo as a complex population of different but closely related viral variants, commonly referred to as a quasispecies. Recent studies suggest that ribavirin might exert an antiviral effect against HCV through both mutagenic effect and an impairment of RNA replication. The introduction of alpha interferon (IFN-alpha) plus ribavirin combination therapy was an important breakthrough in the treatment of chronic HCV infection. However, the rate of sustained virological response is still unsatisfactory, particularly in patients infected with HCV genotype 1. Viral persistence, a hallmark of HCV, may result from a dynamic control of the host response by the virus. In children with chronic HCV infection, the viral population is initially highly homogeneous, but diversifies during prolonged infection which seems to be a common event during chronic hepatitis C in childhood. Coinfection of human immunodeficiency virus 1 (HIV-1) patients by HCV can complicate the treatment of these patients with highly active antiretroviral therapy (HAART). HIV coinfection is associated with a decrease of HCV quasispecies variability, which appears to be reversed by effective HAART.     

Characterization and evolution of NS5A quasispecies of hepatitis C virus genotype 1b in patients with different stages of liver disease

The quasispecies nature of hepatitis C virus (HCV) is thought to play a central role in modulating viral functions. Recent work has linked NS5A protein with viral replication, resistance to interferon (IFN), and control of cellular growth, probably through the interaction of its protein kinase R (double stranded RNA-activated protein kinase, PKR) binding domain (PKR-bd) with cellular PKR, but knowledge of how PKR-bd viral population evolves during disease progression is limited. Since we have previously described an association between amino acid composition of the PKR-bd and the presence of HCC, in this report we further investigated the dynamic behavior of viral population parameters by sequencing an average of 20 clones per sample in 27 samples from 19 untreated patients with different degrees of liver disease, 8 of whom were followed over time. Viral population parameters varied widely from patient to patient, but no differences were observed in the complexity, diversity, types of nucleotide changes, or evolutionary pattern of the quasispecies according to the stage of liver disease. In five samples, we detected "quasispecies-tails"; that is, clones whose minimum genetic distance to the remaining clones of their own quasispecies were higher than the maximum genetic distance found between any other two clones of the same sample. In summary, independent of the degree of liver disease, or the mutations detected within the consensus sequence of the PKR-bd, the NS5A of HCV presents a flexible and variable quasispecies structure that remains largely stable during the natural course of an HCV infection, highlighting the central role of NS5A protein in viral life cycle.     

Recurrence of hepatitis C virus (HCV) infection after liver transplantation for HCV-related disease: host factors and viral factors implicated in the occurrence and the severity of HCV recurrence

Cirrhosis due to chronic infection by hepatitis C virus (HCV), associated or not to a primary hepatocarcinoma, has become the first indication of liver transplantation. Graft reinfection by HCV is considered to be systematic while its prognosis is variable from one patient to another. A better knowledge of factors implicated in the occurrence and severity of hepatitis C recurrence is crucial in order to make optimal patients' monitoring. This article aims to present available data in this field, clarifying the role of viral factors (viral load, genotype, evolution of viral quasispecies) and host-related factors (immune response) which could take part in the development of hepatitis C recurrence.     

HCV quasispecies evolution during treatment with interferon alfa-2b and ribavirin in two children coinfected with HCV and HIV-1

Two children who acquired hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection by mother-to-child transmission were monitored during interferon alfa-2b and ribavirin treatment. In Patient C1, CD4(+) T cell counts were within normal range and HIV-1 viral load was undetectable. HCV viral load declined slightly following treatment initiation while novel variants rapidly emerged, indicative of quasispecies diversification. In Patient C2, CD4(+) T cell counts were low and HIV-1 replication was not fully controlled by antiretroviral therapy. HCV viral load rose during treatment and a striking conservation of the variant spectrum was observed. In both cases, there was no decline in quasispecies complexity following treatment initiation and sustained virological response was not achieved. These results suggest that reduction in quasispecies complexity, which is observed in adult responders following interferon treatment, may be mechanistically unrelated with evolution of the variant profile and/or selective pressure exerted on HCV.     

HCV准种多样性及其与临床关系的研究

目的     

Genetic diversity of hepatitis C virus quasispecies in chronic renal failure patients in Midwest Brazil

Hepatitis C virus (HCV) quasispecies constitute a dynamic population in a continuous process of variation and selection. To investigate effect of the immune system on the genetic variability of HCV, we compared the hypervariable region 1 (HVR1) of immunosuppressed patients with chronic renal failure (CRF group) to immunocompetent patients with HCV chronic infection (control group). The HVR1 from ten samples of each group was amplified, cloned and sequenced. The HCV quasispecies from the control group had a higher frequency of variable sites in HVR1 (83.9 % vs 59.3 %, p < 0.05), as well as a greater diversity within (intra-patient) and between samples, compared to the CRF group. The clustering of the majority of the quasispecies of the CRF group in the phylogenetic tree also showed the limited diversity of the quasispecies in immunosuppressed patients. Moreover, a higher variability of amino acids at positions 384, 386, 391, 394, 397, 398, 400, 405 and 410 was observed in the control group than in the CRF group, which showed a greater variability only at position 388 (p < 0.05). These data corroborates the hypothesis that the major selective pressure factor is the immune system, which promotes a high degree of diversity in the viral progeny and contributes to a constant evolution of HCV.     

Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system

Hepatitis C virus (HCV) exists in infected individuals as quasispecies, usually consisting of a dominant viral isolate and a variable mixture of related, yet genetically distinct, variants. A prior HCV infection system was developed using human hepatocellular carcinoma cells cultured in the three-dimensional radial-flow bioreactor (RFB), in which the cells retain morphological appearance and their differentiated hepatocyte functions for an extended period of time. This report studies the selection and alteration of the viral quasispecies in the RFB system inoculated with pooled serum derived from HCV carriers. Monitoring the viral RNA and core protein in the culture supernatants, together with nucleotide sequencing of hypervariable region 1 of the HCV genome, demonstrated that (1) the virus production intermittently fluctuated in the cultures, (2) the viral genetic diversity was markedly reduced 3 days post-infection (p.i.), and (3) dominant species changed on days 19-33p.i., suggesting that the virus populations can be selected according to susceptibility to the viral infection and replication. A therapeutic effect of interferon-alpha also demonstrated the inhibition of HCV expression. Thus, this HCV infection model in the RFB system should be useful for investigating the dynamic behavior of HCV quasispecies in cultured cells and evaluating anti-HCV compounds.     

Accurate representation of the hepatitis C virus quasispecies in 5.2-kilobase amplicons

Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 10(5) IU/ml, 15 of 16 (94%) at 10(4) to 10(5) IU/ml, 6 of 7 (86%) at 10(3) to 10(4) IU/ml, and 2 of 3 (67%) at less than 10(3) IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated during amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.     

Dynamics of serum hepatitis C virus load and quasispecies complexity during antiviral therapy in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) infection is a dynamic process during which viral genetic variants continuously develop as a result of the virus adaptation to the host's immune system. The level of viremia and the complexity of the hypervariable region 1 (HVR 1) quasispecies of hepatitis C virus during antiviral therapy reflect the dynamic balance between the viral and host components in response to therapy. OBJECTIVE: The aim of the study was to evaluate the dynamics of HCV viremia and the complexity of the HVR 1 quasispecies during the induction phase of a triple combination therapy regimen in nonresponders to earlier anti-HCV treatment. STUDY DESIGN: Ten patients with chronic hepatitis C undergoing antiviral combination therapy with interferon-alpha, ribavirin, and amantadine were studied. The serum HCV RNA level was monitored by a quantitative RT-PCR assay up to 3 months after start of treatment. The HVR 1 quasispecies complexity was analysed by an "in house" nested RT-PCR mediated single-strand conformation polymorphism (SSCP) assay. RESULTS: Baseline serum HCV RNA levels ranged from 1.94x10(6) to 5.53x10(6) copies/ml. In all patients, HCV subtype 1b was found. At the start of therapy, the SSCP assay revealed a high complexity pattern (at least six SSCP bands) in all patients. None of the patients responded within 4 weeks of treatment, however, the serum HCV RNA level decreased by one to two logs in eight patients. At week 4 after start of treatment, there was a decrease of SSCP bands in five patients. In four patients, SSCP bands remained unchanged and in one patient SSCP bands increased. At month 3 after start of treatment, serum HCV RNA was not detectable in one patient. CONCLUSION: Because of the low number of patients involved in this study, prediction of therapeutical success based on the quasispecies complexity was not possible. Larger studies are urgently needed.     

Mode of HCV infection examined by polymorphism of hypervariable region-1 in cases of acute hepatitis C after accidental exposure to blood-borne pathogens

Acute hepatitis C is known to respond better to interferon therapy than chronic hepatitis C. The reason for this difference remains unclear. The present study was undertaken to examine HCV quasispecies in blood from patients with acute hepatitis C caused by accidental exposure to blood-borne pathogens and in blood from the source patients. Three patients who developed hepatitis C (recipient patients; R-Pt.) and two patients who served as a source of HCV infection (source patients; S-Pt.) were the subjects of this study. The number of quasispecies and the genetic diversity in hypervariable region-1 (HVR-1) were examined on the basis of fluorescence single-strand conformation polymorphism and sequence analysis (FSSA). On the day of the accident, the number of quasispecies and genetic diversity were 13 and 36 in S-Pt.1 and 6 and 20 in S-Pt.3, respectively. At the time of diagnosis of acute hepatitis, the number of quasispecies and nucleotide diversity were 2 and 2 in R-Pt.1, 2 and 0 in R-Pt.2, and 4 and 0 in R-Pt.3, respectively. Immediately before the start of treatment, the number of quasispecies and genetic diversity were 4 and 4 in R-Pt.1, 2 and 0 in R-Pt.2, and 3 and 0 in R-Pt.3., respectively. In three R-Pts, interferon therapy resulted in eradication of HCV. These findings indicate that in the early stage of HCV infection, only a portion of HCV transmitted from S-Pts to R-Pts can proliferate. The low number of quasispecies of HCV appears to be one of the reasons why acute hepatitis responds well to interferon therapy.     

Dynamics of viral quasispecies during interferon therapy in non responder chronic hepatitis C patients.

BACKGROUND: the reference method to study the HCV complexity was cloning and sequence analysis of a sufficient number of clones. The evolution of the viral complexity in chronic non responder patients during treatment with standard doses of interferon was not very well investigate because this method was expensive and labour intensive when large series of patients were concerned. Meanwhile, with the alternative Single-Strand Conformation Polymorphism (SSCP) method, a rough estimation of the quasispecies present in a given sample could be obtained. OBJECTIVES: the aim of the study was to analyse the evolution of HCV heterogeneity, investigated by SSCP analysis targeted to the HVR-1, in 30 nonresponders chronic hepatitis C patients treated by Interferon-alpha 3MUI. RESULTS: genotype 1 was the main HCV type found in this population (77% of non responder patients). Before treatment, the SSCP assay revealed a high complexity pattern: the median of SSCP band number was 9. During IFN-alpha treatment, SSCP band number didn't change. However a significant decrease of the viral load was observed (P<0.01). Patients with variations in their SSCP patterns after therapy significantly decreased HCV RNA levels (P<0.002). In one third of patients the SSCP profile didn't change at all. CONCLUSIONS: we observed that viral heterogeneity didn't change in non responder chronic hepatitis C patients during IFN-alpha treatment. Nevertheless patients with a low number of pre-treatment quasispecies exhibited an improvement of the response (P<0.02). These phenomena were probably due to a selection of resistant variants present prior onset of therapy.     

Evolution of viral quasispecies in four dominant HlA-A2 restricted T cell epitopes is not a major reason for viral persistence in interferon-treated patients with chronic hepatitis C

In most patients, chronic hepatitis C virus (HCV) infection persists despite antiviral treatment with interferon-alpha (IFN-alpha) and ribavirin. The aim of the study was to determine whether HCV could evade cellular immune responses through mutations within T cell epitopes. Viral sequences flanking four major CTL epitopes within the HCV core and envelope regions were analyzed by PCR amplification, cloning and sequencing in seven HLA-A2 positive HCV patients before, during and after antiviral therapy. In addition, cytotoxic T lymphocyte precursor (CTLp) frequencies specific to these epitopes were quantitated by ELISPOT. A total of 13 coding mutations were observed among 650 cloned and sequenced PCR products under or post IFN treatment but no clear selection of viral variants. In detail, the diversity of quasispecies in the two core epitopes remained fairly stable over time despite variable CTLp induction in some individuals. The overall mutation rate in the two envelope epitopes was higher but there was no correlation with specific CTLp frequencies. In conclusion, although evolution of the viral quasispecies during and after antiviral therapy was demonstrated, immune evasion by epitope specific mutations seemed to be not common in interferon nonresponders because the viral complexity did not increase.     

Trends for genetic variation of Hepatitis C Virus quasispecies in Human Immunodeficiency virus-1 coinfected patients

Chronic infection by Hepatitis C Virus (HCV) causes liver fibrosis, which is accelerated by unknown mechanisms in patients with HIV-1 coinfection. The evolution of HCV quasispecies in this setting of coinfection is not fully understood. To compare HCV quasispecies between HIV–HCV coinfection and HCV monoinfection, we sequenced 340 HCV clones from the HVR-1 and NS3 regions at two different time points in two groups of treatment-naïve patients with HCV-1a infection: (1) HIV–HCV positive (n = 6); and (2) HIV negative–HCV positive (n = 3). In HCV/HIV coinfection, we found a trend for reduced HCV genetic complexity and diversity, and a trend towards reduced dN/dS ratios in the HVR-1 region, especially in those patients with CD4 < 200 cells/mm³, who lost positive selective immune pressure in the HVR-1 region. Differences in immune regulation of HCV quasispecies in HIV coinfected individuals deserve further exploration to clarify the different outcomes of chronic hepatitis C noted between the immunocompromised and the immunocompetent host.     

Replicative homeostasis: a mechanism of viral persistence

Acute viral infection is characterised by high-level replication before prompt decline of viraemia and, commonly, viral clearance. This kinetic pattern is generally held to be due to immune control. However, infection with some viruses, notably hepatitis C (HCV), hepatitis B (HBV) and the human immunodeficiency virus (HIV), often results in chronic stable low-level spontaneously fluctuating viraemia, kinetics that are difficult to rationalize on this basis. The persistence of HCV, an RNA virus, is especially problematic and its stability, occurring despite rapid, genomic mutation is highly paradoxical. This paper outlines the hypothesis, and evidence, that viruses autoregulate replication and mutation and describes a mechanism--replicative homeostasis--explaining viral stability. Replicative homeostasis results in stable, but reactive, replicative equilibria that drive quasispecies expansion and immune escape and explain all observed viral behaviours and host responses. This paradigm implies new approaches to antiviral therapy and is broadly relevant to modulation of gene expression.     

Clinical implications of viral quasispecies heterogeneity in chronic hepatitis C

To determine the clinical significance of viral quasispecies heterogeneity, 59 patients with chronic hepatitis C were studied using single-stranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVR1); of these, 48 were subsequently treated with interferon-α. The SSCP method was validated using clones of known nucleotide sequence. HVR1 was amplified in 54 of 59 (92%) patients. The median number of SSCP bands per sample was 6 (range: 2–12). Increased quasispecies heterogeneity correlated with the estimated duration of HCV infection (P < 0.05), parenteral-acquired HCV infection (vs. sporadic, P < 0.05), serum HCV RNA levels (P < 0.05), and HCV genotype 1 infection (P < 0.05), but not with age, serum AST, ALT, or Knodell score. Patients who had complete and sustained response to interferon-α (n = 11) had lower pre-treatment quasispecies heterogeneity compared to patients who had complete response with relapse (n = 18, P < 0.05) or no complete response (n = 16, P < 0.01). However, multivariate analysis revealed that HCV viremia was a stronger predictor of response to interferon-α. These findings indicate that the estimated duration of HCV carriage, serum HCV RNA levels, and HCV type 1 are important determinants for the evolution of HCV quasispecies heterogeneity; and that increased HCV quasispecies heterogeneity is another marker associated with a poor subsequent response to interferon-α. © 1996 Wiley-Liss, Inc.     

Separation of near full-length hepatitis C virus quasispecies variants from a complex population

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163-1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.     

Comparative rates of nucleotide sequence variation in the hypervariable region of E1/E2 and the NS5b region of hepatitis C virus in patients with a spectrum of liver disease resulting from a common source of infection

The association of the severity of liver disease and the molecular evolution of hepatitis C virus (HCV) during chronic infection remains unclear and controversial. To address this we have studied the interpatient variability in the nucleotide sequence of two regions of the HCV genome, E1/E2, which contain the hypervariable region 1 and the nonstructural NS5b region, in a cohort of Irish female patients who were all recipients of a single source of HCV genotype 1b-contaminated anti-D immunoglobulin in 1977 and 1978 and who over the subsequent 20 years developed a spectrum of liver disease. In addition, quasispecies analysis was used to evaluate intrapatient variability in the E1/E2 region in four patients with mild and four with severe disease. Phylogenetic and evolutionary rate analyses of the nucleotide sequences demonstrated that there was no significant difference between those who developed mild disease and those who had progressed to severe disease or cirrhosis. These findings suggest that other factors, either additional viral or host, may be important in the pathogenesis and clinical outcome of chronic hepatitis C virus infection.     

Influence of the dynamics of the hypervariable region 1 of hepatitis C virus (HCV) on the histological severity of HCV recurrence after liver transplantation

Recurrence of hepatitis C virus (HCV) infection after liver transplantation is almost universal and usually leads to chronic hepatitis with different degrees of severity. The pathogenic mechanisms underlying the variable outcome of HCV infection recurrence are not well defined, but recent data suggest that the dynamics of HCV quasispecies may be involved. HCV quasispecies evolution was traced by longitudinal single strand conformation polymorphism, direct sequencing, and cloning analyses of pre- and post-transplant HCV-1b isolates from patients with histologically severe (seven cases) or mild or moderate (nine cases) HCV infection recurrence. Differences between the two groups of patients that concerned the level of viremia or the degree of HCV quasispecies complexity and diversity were not observed at any of the three time points analyzed. However, emergence of nucleotide and amino acid changes during the 12 months follow-up was significantly more frequent in patients with mild or moderate than in those with severe HCV infection recurrence. The ratio of non-synonymous to synonymous nucleotide substitutions 12 months after transplantation was also greater in the former, suggesting that the HVR1 of HCV is under stronger selective pressure in these subjects. These findings suggest that the degree of amino acid diversification in the HVR1 of HCV, which probably reflects the strength of immune pressure on HCV, is inversely related to the histological severity of HCV infection recurrence.     

Evolution of hepatitis C virus hypervariable region 1 in chronically infected children

Hepatitis C virus (HCV) quasispecies diversification plays an essential role in the establishment of chronic infections. Our earlier analysis of HCV population structure in children subjected to interferon-ribavirin treatment demonstrated that viral quasispecies is homogenous in patients who failed to respond to the therapy and heterogeneous in sustained responders. We also showed that certain variants of HCV hypervariable region 1 (HVR1) are conserved in non-responders. To better elucidate the pathways of HCV evolution, here we examined the changes of HVR1 in viral populations isolated from sera of eight treatment-naive pediatric patients. We found that HCV evolution in untreated chronically infected children occurs according to two pathways and results in the formation of either genetically homogenous or variable quasispecies. Variable populations are prone to quasispecies shifts. In contrast, homogenous populations are composed of closely related variants that undergo only minor changes. In addition, we observed that a phenomenon of inter-quasispecies conservation of HVR1 is associated with some of the homogenous HCV populations. The collected data suggest that there exist HVR1 variants with superior fitness, capable of persisting in different hosts.     

Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5' noncoding (5'NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5'NC quasispecies genetically. Similar to coding regions, the HCV 5'NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5'NC quasispecies were observed after interferon-alpha treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.