Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy

Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.     

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.     

Hepatitis C virus--T-cell responses and viral escape mutations

Hepatitis C virus (HCV) is a small, enveloped RNA virus and the number of HCV-infected individuals worldwide is estimated to be approximately 170 million. Most HCV infections persist, with up to 80% of all cases leading to chronic hepatitis associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV-host interactions have a crucial role in viral survival, persistence, pathogenicity of infection, and disease progression. Maintenance of a vigorous, sustained cellular immune response recognizing multiple epitopes is essential for viral clearance. To escape immune surveillance, HCV alters its epitopes so that they are no-longer recognized by T cells and neutralizing antibodies, in addition to interfering with host cell cellular components and signaling pathways. The generation of escape variants is one of the most potent immune evasion strategies utilized by HCV. A large body of evidence suggests that single or multiple mutations within HLA-restricted epitopes contribute to viral immune escape and establishment of viral persistence. Further elucidation of the molecular mechanisms underlying immune escape will aid in the design of novel vaccines and therapeutics for the disease.     

Evasion of host immune surveillance by hepatitis C virus: potential roles in viral persistence

Hepatitis C virus (HCV) is a major human pathogen that causes mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. In order for a noncytopathic virus such as HCV to persist, the virus must escape immune recognition or evade host immune surveillance. Immune escape via the hypervariable region of the E2 envelope protein has been postulated as one mechanism for HCV persistent infection. Such hypervariability within the E2 protein may be under selective pressure from protective B cell or T cell responses and be able to escape immune recognition by rapid mutation of antigenic site. In addition to antigenic variation, HCV may also suppress immune response, leading to dampening of cellular immunity. This is supported by recent studies in our laboratory demonstrating that the HCV core protein can suppress host immune responses to vaccinia virus by downregulating viral specific cytotoxic T lymphocyte (CTL) responses and cytokine production. An understanding of the mechanisms behind HCV persistence will provide a basis for the rational design of vaccines and novel therapeutic agents targeting human HCV infection.     

The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.

CD8(+) cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.     

Quasispecies heterogeneity of the carboxy-terminal part of the E2 gene including the PePHD and sensitivity of hepatitis C virus 1b isolates to antiviral therapy

Two regions within the HCV genome, hypervariable region 1 (HVR1) within the envelope (E)2 region and the PKR-binding domain (PKRbD) comprising the interferon sensitivity determining region (ISDR) within the nonstructural (NS)5A protein, have been reported to correlate with the outcome of antiviral treatment. Recently, a PKR/eIF2alpha phosphorylation homology domain (PePHD) within the E2 protein of HCV-1 isolates was described to inhibit PKR in vitro. PePHD deleted HCV-1 mutants remain capable of binding PKR to some extent while inhibition of PKR was found to be abolished by carboxy-terminal truncated E2 protein. The importance of mutations and quasispecies heterogeneity within the carboxy-terminal part of the E2 protein comprising the PePHD of HCV-1b is unknown. Therefore, the carboxy-terminal part of the HCV E2 gene (codons 618-746) including the PePHD was analyzed by sequencing of PCR products and individual clones of 41 HCV-1b-infected patients with sustained (SR, n = 12), end-of-treatment (ETR, n = 10), or no virological (NR, n = 19) response to antiviral therapy. Two highly conserved regions (codons 658-673 comprising the PePHD and codons 675-704) and one variable region (codons 705-720) were detected within the carboxy-terminal part of E2. No significant correlation of specific mutations or number of mutations with treatment response was observed for the PePHD and the carboxy-terminal part of the E2 protein. Phylogenetic and conformational analyses showed no specific clusters related to treatment outcome. Calculation of genetic complexity and diversity based on nucleotide sequence analyses of 20 individual clones per patient showed no differences between matched SR, ETR, and NR patients. However, calculation of genetic complexity and diversity on the basis of amino acid sequences showed significantly lower normalized Shannon entropy as well as mean Hamming distances for SR patients than in ETR and NR patients (P = 0.029 and P = 0.027, respectively). This indicates that patients achieving a sustained virological response to interferon-alpha-based antiviral therapy may elicit more effective immunological pressure, resulting in continuous clearing of individual variants of HCV quasispecies.     

两种HCV不同结构基因重组质粒DNA诱导免疫应答的比较研究

目的：探讨丙型肝炎病毒（HCV）包膜基因E1E2,对核心基因CDNA疫苗诱生的免疫应答有无增强作用。方法：构建包含HCVC或CE1E2基因片段的真有达载体pHCV-C和pHCV-CE1E2,分别接种于Balb/c小鼠股四头肌（以空载体pcDNA3作为对照）,每间隔2wk l次,用ELISA法检测免疫小鼠血清中抗HCVC特异性抗体的产生。以pHCV-C转染并表达HCcAg r S p2/0细胞为靶细胞,采用^51Cr翻译放试验检测特异性CTL的杀伤作用。结果,两个实验免疫的20只小鼠均产生抗HCV C特异性抗体,当前／靶细胞比例为100：1时,CTL的杀伤率均明显高于对照组（P＜0．01）;pHCV-CE1E2与pHCV-C组之间,无论是抗HCV C抗体的滴度还是CTL的杀伤率均无显著性差异（P＞0．05）。结论E1E2基因的加入,并没有增加HCV C基因DNA疫苗诱导的抗HCcAg特异性抗体的滴度和CTL的杀伤作用。     

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.     

Recombinant glycoprotein vaccines for human immunodeficiency virus-infected children and their effects on viral quasispecies.

In individuals infected with human immunodeficiency virus type 1 (HIV-1), specific immunity is associated with a more diverse viral repertoire and slower disease progression. Attempts to enhance antiviral immunity with therapeutic vaccination have shown that recombinant glycoprotein (RGP) vaccines are safe, well tolerated, and immunogenic, but the effect of RGP vaccines on the viral repertoire is unknown. We evaluated diversification of the viral envelope in 12 HIV-infected children who received placebo or RGP vaccines. At baseline, 11 of 12 patients had multiple viral variants. On follow-up 6 months later, children who had a strong vaccine-associated lymphoproliferative immune response showed less viral diversification than those in whom the immune response was weak or absent. These results suggest that the immune response elicited by RGP vaccines does not exert a significant selection pressure on the viral quasispecies and therefore may not be helpful in changing the course of the disease.     

Structural constraints on viral escape from HIV- and SIV-specific cytotoxic T-lymphocytes

Cytotoxic T lymphocytes (CTL) play a central role in controlling lentiviral infections in both humans and monkeys. While they contain the spread of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), CTL are not capable of fully eradicating virus following infection. Ongoing viral replication can therefore lead to the accumulation of viral mutations within CTL epitopes that can undermine cellular immune control of virus. Here we review the importance of CTL in controlling HIV/SIV infection and how immunologic pressure exerted by effector T cells selects for viral variants that escape CTL recognition. We review two examples of viral escape from CTL at highly conserved epitopes that illustrate the extraordinary capacity of lentiviruses to adapt to their immunologic environment despite structural constraints on the ability of the virus to accommodate mutations.     

Quantitative analysis of specific Th1/Th2 helper cell responses and IgG subtype antibodies in interferon‐α‐treated patients with chronic hepatitis C

This study aimed to characterise the immune mechanisms relevant to viral clearance in interferon (IFN)‐α‐treated chronic hepatitis C virus (HCV) infection. Proliferative responses of peripheral blood mononuclear cells from sustained complete IFN‐α therapy responders (n = 8), nonresponders (n = 13), untreated patients (n = 10), and healthy controls (n = 5) were measured retrospectively upon stimulation with recombinant HCV‐antigens (core, helicase, NS3, NS4, and NS5) and the secretion of IFN‐γ and interleukins (IL‐4, IL‐5, IL‐10, and IL‐12) were tested by ELISA. Furthermore, IFN‐γ as well as IL‐10 secreting CD4+ T cells were quantitated by intracellular cytokine staining. Anti‐HCV core and NS3‐specific IgG subclass antibodies were quantitated in the corresponding patient sera. Sustained therapy responders had more frequent and stronger NS3 and helicase‐specific cellular immune responses than nonresponders, untreated HCV patients and healthy controls. Independent from therapy outcome HCV‐stimulated T cells in IFN‐α treated patients secreted preferentially IFN‐γ The Th2 cytokines IL‐4 and IL‐10 were even decreased in nonresponders, while the IL‐12 secretion was not influenced. With respect to the humoral immune response sustained complete responders showed significantly reduced IFN‐γ independent anti‐HCV‐core and ‐NS3 IgG1 antibody synthesis. In conclusion, vigorous NS3‐specific T‐helper cell responses were associated with viral clearance in IFN‐α recipients; however, the cytokine and antibody analysis argues against a Th1/Th2 imbalance as a major factor that influence the therapy outcome. J. Med. Virol. 64:340–349, 2001. © 2001 Wiley‐Liss, Inc.     

Frequencies of epitope-specific cytotoxic T lymphocytes in active chronic viral hepatitis B infection by using MHC class I peptide tetramers

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are thought to play key roles in viral control and liver damage. We have used HLA-A*02 tetramer complex to human HBV core 18-27 (Tc 18-27), envelope 183-191 (Te 183-191), envelope 335-343 (Te 335-343), and polymerase 575-583 (Tp 575-583) epitopes to characterize HLA class I-restricted CD8+ T cells in active chronic HBV infection. The frequencies of specific epitopes circulating tetramer+ cells were determined in whole-blood samples by analysis of flow cytometry. The correlation of HBV epitope-specific CTL, between viral replication and liver damage, was analyzed by multiple regression. Our data shown that HBV-specific CD8+ T cells can be easily detected in peripheral blood of active chronic HBV infections. No significant correlation was found between either the frequency of HBV-specific CD8+ T cells and the viral load, or the frequency of HBV-specific CD8+ T cells and the levels of alanine transaminase. These results suggest that the existence of epitope-specific HBV CTLs are not directly correlated to hepatocyte injury, and the frequencies of HBV-specific T cells are not determinant of immune-mediated protection in chronic HBV infection.     

Molecular therapies for viral hepatitis

Current treatment modalities available for hepatitis B virus (HBV) or hepatitis C virus (HCV) infections are not efficient. The enormous disease burden caused by these two infections makes the development of novel therapies critical. For HCV, the development of an effective vaccine is urgent in view of the escalating number of infected individuals. Molecular therapies for HBV and HCV infection can be directed at reducing viral load by interfering with the life cycle of the viruses or at generating immune response against viral epitopes. The antiviral approaches consist of the delivery or expression of antisense RNAs, ribozymes or dominant negative proteins. Viral biology can be interrupted by attacking various potential targets within the two viruses. DNA-based vaccination strategies are being explored for both prevention and treatment of these diseases. Both non-viral and recombinant viral vectors are being developed for safe, effective and long-term gene transfer to the liver. Although no "ideal" vector is available at this time, the ingenuity of numerous investigators is leading to the improvement of the vector systems, promising successful application of gene therapy to the prevention and treatment of viral hepatitis in the foreseeable future.     

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.     

Chemiluminescence enzyme immunoassay for monitoring hepatitis C virus core protein during interferon-alpha2b and ribavirin therapy in patients with genotype 1 and high viral loads

This study evaluated an updated chemiluminescence enzyme immunoassay (CLEIA) for hepatitis C virus (HCV) core protein for monitoring viral kinetics during treatment with interferon (IFN)-alpha and ribavirin. Using the CLEIA, serum levels of HCV core protein were measured in 17 patients with genotype 1 and high baseline viral loads during the first 4 weeks of combination therapy. HCV RNA was measured by the Amplicor Monitor test for comparison. At the start of therapy, the median HCV level (interquartile range) was 700 (540-940) kIU/ml of viral RNA and 11,310 (5,528-14,238) fmol/L of core protein. HCV RNA was above the upper limit of the linear range of the Amplicor Monitor test in 13 of the 17 patients, while the core protein level was within the linear range of the CLEIA in all patients. During therapy, the proportion of patients with HCV levels below the cutoff values at each time point was less with the Amplicor Monitor test than with CLEIA. Serum HCV core protein level decreased rapidly during the first 24 hr of therapy and more slowly thereafter, with median exponential decays of 1.08 and 0.046 log10/day, respectively. In the second phase, between day 1 and 28, the median decrease in HCV core protein level was higher in four patients with sustained virologic response (0.13 log10/day) than in 13 patients with no response (0.028 log10/day, P = 0.042). The wide linear range of the HCV core protein assay is appropriate for measuring viral loads during therapy with IFN-alpha and ribavirin.     

How chronic viral infections impact on antigen‐specific T‐cell responses

Persistent viral infections are, by definition, associated with ineffective antiviral immunity, in particular those infections caused by viruses that are highly productive and replicative (including HIV, HBV and HCV). The reasons for ineffective antiviral immunity in these types of infections are complex and manifold, and only recently a more comprehensive picture of the parameters responsible for attenuation of immune function is emerging. One reason for poor viral control in these types of infections is the functional deterioration of antiviral T‐cell responses and understanding the underlying mechanisms is of key importance. This review summarizes our current knowledge of cell‐intrinsic and cell‐extrinsic parameters that contribute to T‐cell exhaustion during chronic viral infections and discusses related implications for host survival, immunopathology, and control of infection.     

Predictors of spontaneous viral clearance and outcomes of acute hepatitis C infection

Background/Aims

This study evaluated the predictors of spontaneous viral clearance (SVC), as defined by two consecutive undetectable hepatitis C virus (HCV) RNA tests performed ≥12 weeks apart, and the outcomes of acute hepatitis C (AHC) demonstrating SVC or treatment-induced viral clearance.

Methods

Thirty-two patients with AHC were followed for 12-16 weeks without administering antiviral therapy.

Results

HCV RNA was undetectable at least once in 14 of the 32 patients. SVC occurred in 12 patients (37.5%), among whom relapse occurred in 4. SVC was exhibited in 8 of the 11 patients exhibiting undetectable HCV RNA within 12 weeks. HCV RNA reappeared in three patients (including two patients with SVC) exhibiting undetectable HCV RNA after 12 weeks. SVC was more frequent in patients with low viremia than in those with high viremia (55.6% vs. 14.3%; P=0.02), and in patients with HCV genotype non-1b than in those with HCV genotype 1b (57.1% vs. 22.2%; P=0.04). SVC was more common in patients with a ≥2 log reduction of HCV RNA at 4 weeks than in those with a smaller reduction (90% vs. 9.1%, P<0.001). A sustained viral response was achieved in all patients (n=18) receiving antiviral therapy.

Conclusions

Baseline levels of HCV RNA and genotype non-1b were independent predictors for SVC. A ≥2 log reduction of HCV RNA at 4 weeks was a follow-up predictor for SVC. Undetectable HCV RNA occurring after 12 weeks was not sustained. All patients receiving antiviral therapy achieved a sustained viral response. Antiviral therapy should be initiated in patients with detectable HCV RNA at 12 weeks after the diagnosis.     

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.     

Influence of CD4+ T cell counts on viral evolution in HIV-infected individuals undergoing suppressive HAART

We analyzed the viral C2-V4 envelope diversity, glycosylation patterns, and dS/dN ratios of plasma HIV-1 in an attempt to better understand the complex interaction between viral quasispecies and the host-selective pressures pre- and post-HAART. Phylogenetic analysis of the envelope gene of five patients revealed monophyletic clustering in patients with higher CD4+ T cell counts and sequence intermingling in those with lower CD4+ T cells in relation to the stage of HAART. Our analyses also showed clear shifts in N-linked glycosylation patterns in patients with higher CD4+ T cells, suggesting possible distinct immunological pressures pre- and post-HAART. The relative preponderance of synonymous/nonsynonymous changes in the envelope region suggested a positive selection in patients with higher CD4+ T cells, whereas lack of evidence for positive selection was found in the patients with lower CD4+ T cells. An exception to the last analysis occurred in the only patient who reached complete viral suppression, maybe due to drug pressure exerted over the pol gene that may obscure the immune pressure/selection at the envelope in this analysis. All these indications may suggest that even when HAART generates viral suppression, quasispecies evolve in the envelope gene probably resulting from host-selective pressure.     

Ultrasensitive Amplification Refractory Mutation System Real-Time PCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis B Virus-Resistant Strains in the Era of Personalized Medicine

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.