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Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein β (C/EBP-β) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-β negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

Arbidol (ARB) is a broad-spectrum antiviral displaying activity against a number of enveloped and non-enveloped viruses. It was described as a viral entry inhibitor and shown to interact at the molecular level with lipid membranes and viral fusion glycoproteins to impede viral entry and fusion. However its mechanism of action at the cellular level remains unknown. Here, by using live-cell confocal imaging and the hepatitis C virus as a model virus, we show that ARB affects clathrin-mediated endocytosis by impeding dynamin-2-induced membrane scission. Moreover it induces the intracellular accumulation of clathrin-coated structures where viral particles are trapped. Collectively, our results shed light on the mechanistic aspects of ARB antiviral activity and suggest that ARB could prevent cell infection by viruses that enter through clathrin-mediated endocytosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Silymarin, an extract of milk thistle seeds, and silymarin-derived compounds have been considered hepatoprotective since the plant was first described in ancient times. Hepatoprotection is defined as several non-mutually exclusive biological activities including antiviral, antioxidant, anti-inflammatory and immunomodulatory functions. Despite clear evidence for silymarin-induced hepatoprotection in cell culture and animal models, evidence for beneficial effects in humans has been equivocal. This review will summarize the current state of knowledge on silymarin in the context of HCV infection. The information was collated from a recent workshop on silibinin in Germany.

Background: The hepatitis C virus (HCV) core protein is implicated in diverse aspects of HCV-induced pathogenesis. There is a paucity of information on core in acute hepatitis C infection., Methods: We analyzed core gene sequences and protein functions from 13 patients acutely infected with HCV genotype 1., Results: Although core isolates differed slightly between patients, core quasispecies were relatively homogeneous within each patient. In 2 of 4 patients studied temporally, core quasispecies did not change over time. Comparison with more than 2700 published core isolates indicated that amino acid changes from a prototype reference strain found in acute core isolates were present in chronically infected persons at low frequency (6.4%; range, 0%-32%). Core isolates associated with lipid droplets to similar degrees in Huh7 cells. Core diffusion in cells was not affected by nonconservative changes F130L and G161S in the lipid targeting domain of core. Core isolates inhibited interferon-stimulated response element- and nuclear factor kappaB-dependent transcription and tumor necrosis factor alpha-induced nuclear translocation of nuclear factor kappaB and were also secreted from Huh7 cells., Conclusions: The data suggest that upon transmission, core quasispecies undergo genetic homogenization associated with amino acid changes that are rarely found in chronic infection and that, despite genetic variation, acute core isolates retain similar functions in vitro.

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Background & Aims: Silymarin, an extract from the seeds of the milk thistle plant Silybum marianum, has been used for centuries for the treatment of chronic liver diseases. Despite common use by patients with hepatitis C in the United States, its clinical efficacy remains uncertain. The goal of this study was to determine whether silymarin has in vitro effects on immune function that might have implications for its potential effect on hepatitis C virus (HCV)-induced liver disease., Methods: Freshly isolated peripheral blood mononuclear cells (PBMC) and T cells from HCV-infected and uninfected subjects were tested in vitro for responses to nonspecific and antigenic stimulation in the presence and absence of a standardized preparation of silymarin (MK001)., Results: Minimal MK001 toxicity on PBMC was found at concentrations between 5 and 40 microg/mL. MK001 dose dependently inhibited the proliferation and secretion of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-2 by PBMC stimulated with anti-CD3. In addition, MK001 inhibited proliferation by CD4(+) T cells to HCV, Candida, and tetanus protein antigens and by HLA-A2/HCV 1406-1415-specific CD8(+) T cells to allogeneic stimulation. MK001 inhibited T-cell TNF-alpha and IFN-gamma cytokine secretion to tetanus and Candida protein antigens. Finally, MK001 inhibited nuclear factor-kappaB transcriptional activation after T-cell receptor-mediated stimulation of Jurkat T cells, consistent with its ability to inhibit Jurkat T-cell proliferation and secretion of IL-2., Conclusions: Silymarin's ability to inhibit the proliferation and proinflammatory cytokine secretion of T cells, combined with its previously described antiviral effect, suggests a possible mechanism of action that could lead to clinical benefit during HCV infection.

Unlabelled: In the majority of cases, infection with hepatitis C virus (HCV) becomes chronic and is often associated with impaired innate and adaptive immune responses. The mechanisms underlying viral persistence and lack of protective immunity are poorly understood. Considering that dendritic cells (DCs) play critical roles in initiating and modulating immune responses, we explored the effect of HCV proteins on DC gene and protein expression, phenotype, and function. Human DCs were generated following plastic adherence of monocytes and culture with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) from normal subjects. Autologous nonadherent peripheral blood mononuclear cells were infected with vaccinia constructs expressing various HCV proteins (core-E1, NS5A, NS5B) or an irrelevant protein beta-galactosidase (beta-gal) as the control, induced to undergo apoptosis, then co-cultured with DCs. Between 2% and 10% of the genes probed in a cDNA nylon array were differentially regulated within DCs that had engulfed HCV proteins. In particular, the presence of intracellular NS5A led to increased transcriptional and protein expression of IL-8 (CXCL-8), a chemokine with proinflammatory and anti-interferon properties, and impaired interferon induction of signal transducers and activators of transcription 1 (STAT1) serine and tyrosine and STAT2 tyrosine phosphorylation., Conclusion: These data provide novel mechanisms by which HCV subverts antiviral host immunity.

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.

Arbidol (ARB) is an antiviral compound that was originally proven effective for treatment of influenza and several other respiratory viral infections. The broad spectrum of ARB anti-viral activity led us to evaluate its effect on hepatitis C virus (HCV) infection and replication in cell culture. Long-term ARB treatment of Huh7 cells chronically replicating a genomic length genotype 1b replicon resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Pre-treatment of human hepatoma Huh7.5.1 cells with 15 microM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold. The inhibitory effect of ARB on HCV was not due to generalized cytotoxicity, nor to augmentation of IFN antiviral signaling pathways, but involved impaired virus-mediated membrane fusion. ARB's affinity for membranes may inhibit several aspects of the HCV lifecycle that are membrane-dependent.

Pretreatment hepatitis C virus (HCV)-specific lymphoproliferative (LP) responses, neutralizing antibody (NA) responses, intrahepatic cytotoxic T lymphocyte (CTL) responses, and HCV quasi-species (QS) diversity and complexity were examined in patients with advanced hepatic fibrosis (Ishak fibrosis score of > or = 3) and prior nonresponse to interferon (IFN)- alpha therapy who were enrolled in the initial phase of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis Trial. Positive baseline HCV E1- and/or E2-specific NA responses (P = .01) and higher baseline HCV QS diversity (P = .01) were more commonly found in patients who did not become sustained virologic responders (SVRs) at week 72 (W72) than they were in those who did. No patients with positive results for both the LP and NA assays achieved a sustained virologic response. Multiple logistic regression analysis revealed that, when the presence of cirrhosis, prior ribavirin therapy, genotype 1 infection, log serum HCV RNA level, and receipt of >80% of the prescribed medication were controlled for, a sustained virologic response (W72) was negatively correlated with positive baseline LP assay results (P = .02) and with 1 or more positive assays (LP, NA, or CTL) (P = .02). No differences were noted in baseline intrahepatic CTL activity between SVRs and non-SVRs. Thus, in patients with advanced hepatic fibrosis due to HCV infection, pretreatment HCV-specific immune responses and increased QS variability appear to hinder viral clearance by pegylated IFN- alpha 2a and ribavirin combination therapy.

In summary, HCV-cell interactions include those directly involved with the HCV life cycle such as virus attachment, entry, and replication. Included within this broad area of research are the interactions of HCV proteins with the IFN system, cytokine and chemokine pathways such as IL-8, and various other cellular proteins and pathways. The plethora of contradictory and sometimes confusing accessory HCV-host interactions defies precise predictions of their role in HCV biology. It is clear that these virus-cell interactions affect HCV replication, antiviral resistance, persistence, and pathogenesis. Because HCV-host interactions are initiated immediately on infection, they are operative during acute HCV infection, whereby HCV interacts with innate cellular antiviral and immune systems. The magnitude and duration of these HCV-host interactions therefore may influence the development of acquired immunity. Because HCV exists as a quasispecies in all infected individuals, heterogeneity in biological responses to HCV-host interactions is predicted, revealing opportunities for the development of various genotypic and phenotypic prognostic indicators. With the model systems in place, these hypotheses can be tested. The challenge for the future is to determine if there is a hierarchical importance to these interactions, to delineate how these virus-cell interactions affect the patient infected with HCV, and to determine whether any of these interactions represents a target for therapeutic intervention.

We conducted a multicenter clinical evaluation of the second versions of the manual AMPLICOR and the semiautomated COBAS AMPLICOR tests for hepatitis C virus (HCV) RNA (Roche Molecular Systems, Inc., Pleasanton, Calif.). The performance characteristics of these HCV RNA tests for diagnosis of active viral infection were determined by comparison to anti-HCV serological test results, alanine aminotransferase levels, and liver biopsy histology results. A total of 878 patients with clinical or biochemical evidence of liver disease were enrolled at four hepatology clinics. A total of 1,089 specimens (901 serum and 188 plasma) were tested with the AMPLICOR test. Sensitivity compared to serology was 93.1% for serum and 90.6% for plasma. The specificity was 97% for serum and 93.1% for plasma. A total of 1,084 specimens (896 serum and 188 plasma) were tested with the COBAS test. Sensitivities for serum and plasma were the same as with the AMPLICOR test. The specificity was 97.8% for serum and 96.6% for plasma. Of the 69 specimens with false-positive and false-negative AMPLICOR test results relative to those of serology, alternative primer set (APS) reverse transcription (RT)-PCR analysis showed that the AMPLICOR test provided the correct result relative to the specimens containing HCV RNA in 64 (92.7%) specimens. Similarly, 66 of 67 (98.5%) false-positive and false-negative COBAS test results were determined to be correct by APS RT-PCR analysis. There were no substantive differences in clinical performances between study sites, patient groups, specimen types, storage conditions (-20 to -80 degrees C versus 2 to 8 degrees C), or anticoagulants (EDTA versus acid citrate dextrose) for either test. Both tests showed >99% reproducibility within runs, within sites, and overall. We conclude that these tests can reliably detect the presence of HCV RNA, as evidence of active infection, in patients with clinical or biochemical evidence of liver disease.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

The hepatitis C virus (HCV) envelope glycoprotein-2 inhibits the interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) via the PKR eukaryotic initiation factor-2alpha phosphorylation homology domain (PePHD). The present study examined the genetic variability of the PePHD in patients receiving IFN therapy. The PePHD from 12 HCV genotype 1 (HCV-1)-infected patients receiving daily IFN therapy was amplified by reverse-transcriptase polymerase chain reaction and analyzed by direct and clonal sequencing. The PePHD was highly conserved in 38 HCV GenBank isolates. There was no difference in pretreatment PePHD sequences isolated from IFN responders versus nonresponders. The major PePHD quasi-species variant did not change after 6 weeks of daily IFN therapy, and in 1 patient the major quasi-species variant did not change during 9 months of observation. Sequencing of 25 pretreatment PePHD clones from 3 patients confirmed that there was extremely low sequence variability surrounding the PePHD. The PePHD is highly conserved in HCV-1-infected IFN responders and nonresponders and does not appear to evolve in response to IFN therapy.

Hepatitis C virus (HCV) infection is an important clinical problem, with a world-wide prevalence of approximately 1-2%. HCV infection is associated with an increased risk for the development of severe liver disease. HCV is inherently resistant to anti-viral therapy with interferon (IFN). The virus circulates in infected individuals as a mixture of related, yet genetically distinct variants, or quasispecies. Many studies have implicated HCV quasispecies in IFN responsiveness. Effective containment of HCV quasispecies mutation and selection through more aggressive therapy (e.g. daily induction), combination therapy (e.g. IFN plus ribavirin), or longer lasting therapy (e.g. pegylated IFN) is required for IFN responsiveness. Recently, several HCV proteins including the non-structural 5A and envelope gene 2-glycoprotein have been implicated in HCV anti-viral resistance. It is likely that multiple HCV genes disrupt IFN-induced anti-viral responses at many levels and that these virus-host cell interactions are associated with IFN resistance. Characterisation of HCV-encoded mechanisms of anti-viral resistance has important implications for the development of new anti-virals.

Resistance of the hepatitis C virus (HCV) to interferon-alpha (IFN-alpha) therapy in patients with hepatitis C may be genetically controlled by an IFN sensitivity-determining region (ISDR) within the non-structural 5A (NS5A) gene. To assess whether HCV 1b strains carrying a 'resistant' type of ISDR are selected during unsuccessful IFN therapy, we analysed the evolution of the NS5A quasispecies, as detected by the clonal frequency analysis technique, and of the ISDR sequence by nucleotide sequence determination, in 11 patients showing no virological response during two consecutive cycles of IFN-alpha therapy. IFN-resistant patients had a homogeneous ISDR quasispecies with sequences identical to those described as 'resistant-' or 'intermediate-' type ISDR. After retreatment with IFN, further selection towards a homogeneous viral population was observed and 10 out of 11 patients had only one variant of HCV with no or just one single amino acid mutation within the ISDR sequence. Treatment and retreatment with IFN was associated in our non-responder patients with evolution of the ISDR quasispecies towards a rather homogeneous viral population carrying a conserved or minimally mutated ISDR motif, supporting the idea that this motif may be relevant for IFN resistance in HCV 1b-infected individuals.

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.

Hepatitis C virus (HCV) quasispecies heterogeneity was assessed in pretreatment sera of 22 patients treated with interferon (IFN). Quasispecies heterogeneity was quantitated by analysis of 490 hypervariable region 1 (HVR1) clones using gel shift analysis (GSA), which allowed determination of two components of HCV quasispecies heterogeneity: genetic complexity (number of variants) and genetic diversity (mean genetic distance between variants). HCV genotype and pretreatment RNA titer were similar between responders (n = 12) and nonresponders (n = 10). GSA correlated well with nucleotide sequencing for estimating HCV genetic diversity (R = .952; P < .001). A positive correlation of .666 (P < .001) was observed between genetic diversity and complexity, suggesting that these components of quasispecies heterogeneity were related. However, while there were no significant differences in pretreatment complexity or genetic diversity among response groups, higher pretreatment genetic diversity was predictive of response failure (P = .041). Assessment of HVR1 heterogeneity by GSA revealed important relationships between quasispecies genetic diversity, complexity, and response to IFN therapy and suggested a need for analysis of other HCV genes.

Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.