Clinical examinations based on nucleic acids of hepatitis viruses

Viral hepatitis is the most common cause of acute and chronic hepatitis. The term, viral hepatitis, generally refers to infections resulting from one of the hepatotrophic viruses: hepatitis A, B, C, D and E. As hepatitis B virus (HBV) and hepatitis C virus (HCV) mainly cause chronic hepatitis and hepatocellular carcinoma, early diagnosis, prevention and viral elimination are necessary. Eight genotypes of HBV have been detected by sequence divergence of >8% in the entire HBV genome, and designated by capital letters from A to H in the order of documentation. They have distinct geographical distribution and influence the severity of liver disease as well as the response to antiviral therapies. Similarly, HCV has at least six genotypes, of which HCV-1 and -2 are the major genotypes worldwide. Only interferon therapy can eradicate HCV at present, and the sustained virological response depends on HCV genotypes and viral loads. Here, we summarize examinations based on viral nucleic acids and the clinical manifestations of HBV and HCV genotypes.     

HCV准种多样性及其与临床关系的研究

目的     

Twenty-four hour kinetics of hepatitis C virus and antiviral effect of alpha-interferon

Numerous studies reported that patients infected by the genotype 1 of hepatitis C virus (HCV) and/or with a high baseline viral load responded poorly to antiviral therapy. Study of viral kinetics has provided clues to the understanding of non-response to alpha-interferon (IFN-alpha)-based therapy. The objective of this study was to clarify the influence of viral factors such as the genotype and baseline viral load on HCV resistance to treatment through the study of their impact on the first phase of viral decline. HCV RNA levels were determined frequently following the administration of 3 million units of IFN-alpha in 22 chronic HCV carriers. The evolution of HCV RNA level over 24 hr was different in genotype 1-infected patients, compared to that in patients infected by other genotypes. The viral load decline at 24 hr was lower in patients with genotype 1. Patients with a high baseline viral load exhibited a viral dynamics different from patients with a lower level of viremia; the extent of the first phase was also lower in these patients. Non-responder patients had a slower viral decay on day 1 of therapy than patients who cleared the virus under treatment. In conclusion, 24 hr HCV dynamics is regulated by genotype and baseline viral load. Genotype 1 strains and those that produce high viral loads are the most resistant to the antiviral action of IFN-alpha. Resistant HCV strains could be distinguished from sensitive viruses as early as a few hours after the beginning of the treatment.     

Rapid viral response and treatment outcome in genotype 2 and 3 chronic hepatitis C: comparison between two HCV RNA quantitation methods

Fifty consecutive patients with genotype 2 or 3 chronic hepatitis C were treated with peg-IFN alfa-2a 135 microg weekly and ribavirin (11 mg/kg body weight) daily during 24 weeks. Rapid viral response treatment week 4, end-of-treatment response, and sustained viral response were analyzed by two different HCV RNA quantitation methods, the Cobas Amplicor Monitor test and the TaqMan test with a sensitivity of 600 and 15 IU/ml, respectively. The TaqMan test differentiated patients with rapid viral response finally achieving sustained viral response better. Hence, patients with and without rapid viral response as tested by the TaqMan test finally achieved sustained viral response in 97% (32/33) versus in 75% (12/16), P < 0.017. The corresponding figures for the Cobas Amplicor test was 91% (41/45) versus (80%) 4/5 a non-significant difference. In conclusion, the more sensitive TaqMan test yielded a lower number of patients with rapid viral response than the less sensitive Amplicor Monitor test, but predicted sustained viral response in a higher percentage of patients with rapid viral response than the Amplicor Monitor test. A rapid viral response meaning HCV RNA levels <15 IU/ml predicted a sustained viral response in 97% of patients with genotype 2 or 3.     

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.     

Transmission of hepatitis C virus in a gynecological surgery setting

A cluster of hepatitis C virus (HCV) infections among gynecological patients who underwent surgical intervention in the same setting is described. An epidemiological investigation was conducted to identify the cases, the likely source of infection, and the route of transmission. Four recent HCV infections were identified. Based on molecular fingerprinting analysis and epidemiological investigation, transmission between the putative source patient (an HCV-positive woman who was the first patient of the surgical session) and outbreak patients was highly suggestive. All patients, including the source patient, were infected with HCV type 1b. Molecular characterization of HCV clones by sequence analysis of both structural envelope regions (20 clones from the source patient and 58 from the outbreak patients) and the nonstructural NS5 region of the viral genome (12 clones from the source patient and 32 from the outbreak patients) showed close homology between the viral isolates from the source and those from the outbreak patients that was higher than that observed between the viral isolates from the source and those from four unrelated, HCV type 1b-infected patients from the same geographical area (in the latter case, 33 clones were sequenced for the envelope regions and 30 were sequenced for the NS5 region). The mean percent divergence between clones was 4.69 for the envelope and 3.71 for the NS5 region in the source patient and the outbreak patients compared with 6.76 (P = 0.001) and 5.22 (P = 0.01) in the source patient and control patients, respectively. Among the risk factors investigated, only that of having undergone surgery in the morning session of the same day reached statistical significance (P = 0.003). The investigation showed that the source patient and outbreak patients shared only the administration of propofol in multidose vials. The study documents the risk of nosocomial transmission of HCV and the importance of infection control procedures in the operating room and highlights the crucial role of molecular strategies, especially sequence-based phylogenetic analysis of cloned viral isolates, in the investigation of HCV outbreaks.     

Phylogenetic and structural analysis of HCV nonstructural protein 4A from Pakistani patients

Hepatitis C virus nonstructural protein, NS4A, is a small protein comprising of about 54 amino acids. Despite its small size, it plays key role in many viral and cellular functions. The most important of which is its role as the co-factor of viral serine protease and helicase (NS3). Our study examines the phylogenetic and structural analysis of this coding region after isolation from Pakistani HCV patient samples. Phylogenetic analysis of the gene revealed that Pakistani 3a HCV strains do not show significant divergence from those reported from the rest of the world. The findings of this study also depict that NS4A sequence is conserved within genotypes, whereas it shows variations among different genotypes. While predicting the tertiary structure of the protein two important mutations (H28Y & E32G) were observed when comparing the Pakistani sequences with that of a reference HCV (genotype 3a) strain NZL (D17763). These mutations were observed in the central domain of NS4A which is responsible for interaction with NS3. Taken together, these mutations within the NS4A coding region can play an important role in the binding capacity of NS4A with HCV serine protease NS3.     

Simultaneous runs of the Bayer VERSANT HIV-1 version 3.0 and HCV bDNA version 3.0 quantitative assays on the system 340 platform provide reliable quantitation and improved work flow

Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63 degrees C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround.     

Predictors of viral kinetics to peginterferon plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus genotype 1b

For chronic hepatitis C virus (HCV) infection, evaluation of response to peginterferon (PEG-IFN) plus ribavirin (RBV) therapy based on viral kinetics is useful as an early predictor of treatment efficacy, but the underlying mechanisms of the different viral kinetics to treatment are still unclear. The response to 48-week PEG-IFN-RBV combination therapy was evaluated in 160 Japanese adult patients infected with HCV genotype 1b and determined the rapid virological response (at 4 weeks), early virological response (at 12 weeks), end-of treatment response, and sustained virological response (6 months after end of treatment). The proportion of patients who showed rapid, early and sustained virological, and end-of treatment responses were 50%, 73%, 47%, and 71%, respectively. Furthermore, 66% of patients who achieved early virological response also showed sustained virological response. Multivariate analysis identified substitutions of amino acid (aa) 70 and 91 in the HCV core region (double-wild-type) as a predictor of early HCV-RNA negativity, rapid, early, and sustained virological responses and end-of treatment response, and lipid metabolic factors (high levels of LDL cholesterol and total cholesterol) as predictors of early and rapid virological responses and end-of treatment response. Male sex and low levels of alpha-fetoprotein were other predictors of sustained virological response. Furthermore, female sex and severity of liver fibrosis were determinants of lack of sustained virological response in spite of early virological response. This study identified predictors of efficacy of PEG-IFN-RBV therapy based on viral kinetics in Japanese patients infected with HCV genotype 1b.     

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.     

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 in liver transplantation

HCV-related cirrhosis represents the leading indication for liver transplantation in the Western countries. HCV reinfection after liver transplantation occurs in virtually all patients transplanted for HCV-related liver disease Histological evidence of chronic HCV infection develops in 50 to 90 % of patients by 12 months after liver transplantation, and cirrhosis occurs in about 20 % of patients within 5 years after transplant. Several studies have evaluated host, viral, and transplant-related factors that might be associated with the severity of HCV recurrence. Among host factors, immunosuppression is one of the major factors that accounts for accelerated HCV recurrence and it has been an area of extensive research and controversy. Donor age, steatosis, and immunogenetic factors are also relevant in determining the outcome in patients transplanted for HCV-related cirrhosis. A major step to prevent complications of HCV recurrence related to the rapid fibrosis is the posttransplant antiviral treatment. Two strategies have been tried: pre-emptive or other strategies as soon as possible after liver transplantation or elective therapy once there is histological evidence of recurrent hepatitis C. Retransplantation due to graft failure from recurrent hepatitis C is rarely an option in the era of organ shortage as it is associated with poor outcome, but many case needs to be considered early in the evolution of disease. New antivirals may change the outcome dramatically of patients transplanted for HCV cirrhosis.     

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.     

Suppression of NF-kappaB and AP-1 activation in monocytic cells persistently infected with measles virus

HIV-1 evolution in the envelope gene (env) was analyzed in four asymptomatic antiretroviral therapy na?ve patients with typical and slow disease progression rates. In typical progressors, viral populations were monophyletic and two distinct evolutionary patterns were observed. In one patient, HIV-1 evolution displayed a strong temporal structure similar to the consistent pattern previously described. In the other, viral evolution displayed a lack of temporal structure with no increase in genetic heterogeneity and divergence over time. In slow progressors, several clades were observed in viral populations. However, analysis within the major sub-population revealed the same two evolutionary patterns described for typical progressors. Synonymous and non-synonymous substitution rate analyses indicated that positive selection was the major force driving HIV-1 evolution in viral populations with temporal structure, while evolution in viral populations with an atemporal structure was dominated by genetic drift and purifying selection. These results support the existence of distinct patterns of env evolution in untreated HIV-1-infected patients.     

Patterns of viral load decline with telaprevir-based therapy in patients with genotype 1 chronic HCV infection

BackgroundTelaprevir-based therapy is associated with rapid decline in HCV RNA, enabling the application of early futility rules.ObjectivesTo familiarize physicians with this paradigm, a comprehensive analysis of the most frequent HCV viral load profiles observed during treatment with telaprevir/Peg-IFN/RBV in Phase III trials is provided.DesignHCV RNA profiles were analyzed from 320 HCV genotype 1 treatment-naïve patients enrolled in the ADVANCE study, and 225 prior Peg-IFN/RBV treatment-experienced patients enrolled in the REALIZE study. Patients received 12 weeks of telaprevir with either 24 or 48 weeks of Peg-IFN alfa-2a/RBV. Patients with missing SVR assessments during follow-up, detectable HCV RNA at end of treatment but who did not have viral breakthrough (vBT), or with early vBT who discontinued telaprevir before time of failure were excluded.ResultsAll analyzed patients experienced a rapid decline in HCV RNA (>2.0 log₁₀) by Day 14, irrespective of baseline characteristics and/or prior response to Peg-IFN/RBV (relapse, partial response and null response). Subsequently, HCV RNA continued to decline to undetectable levels in most patients. These patients went on to have one of the following outcomes: sustained virologic response, late vBT (after Week 12, i.e. during the Peg-IFN/RBV phase), or relapse. In the small subset of patients with early vBT or meeting a futility rule before Week 12, HCV RNA usually never became undetectable and/or increased rapidly after reaching the nadir.ConclusionsHCV RNA profiles with telaprevir/Peg-IFN/RBV are different from those with Peg-IFN/RBV alone. It is important that clinicians understand these HCV RNA profiles and monitor patient viral load in order to apply futility rules correctly.     

Rapid changes in hepatitis C virus quasispecies produced by a single dose of IFN-alpha in chronically infected patients.

The effects of a single dose of 3 international megaunits of interferon-alpha2b (IFN-alpha2b) on hepatitis C virus (HCV) load and quasispecies were examined 24 h after administration in 12 previously untreated, chronically infected patients. All patients had viremia loads appreciably reduced relative to baseline values, thus confirming that the viral load is rapidly affected by IFN-alpha2b. Five patients also exhibited changes in plasma HCV quasispecies composition that were clearly evident by single-strand conformation polymorphism, analysis, thus indicating that one dose of IFN-alpha2b may suffice to produce rapid perturbations in the genetic heterogeneity of circulating HCV. Prior to IFN-alpha2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. The latter finding raises the possibility that in these patients, the differences in quasispecies composition between pre-IFN and post-IFN plasma resulted from increased representation of lymphoid tissue-originated variants in the latter sample, possibly because of poor sensitivity to IFN-alpha2b of HCV replication in the lymphoid compartment.     

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.     

HIV/HCV Co-infection: Pathogenesis, Clinical Complications, Treatment, and New Therapeutic Technologies

World-wide, hepatitis C virus (HCV) accounts for approximately 130 million chronic infections, with an overall 3% prevalence. Four to 5 million persons are co-infected with HIV. It is well established that HIV has a negative impact on the natural history of HCV, including a higher rate of viral persistence, increased viral load, and more rapid progression to fibrosis, end-stage liver disease, and death. Whether HCV has a negative impact on HIV disease progression continues to be debated. However, following the introduction of effective combination antiretroviral therapy, the survival of coinfected individuals has significantly improved and HCV-associated diseases have emerged as the most important co-morbidities. In this review, we summarize the newest studies regarding the pathogenesis of HIV/HCV coinfection, including effects of coinfection on HIV disease progression, HCV-associated liver disease, the immune system, kidney and cardiovascular disease, and neurologic status; and effectiveness of current anti-HIV and HCV therapies and proposed new treatment strategies.     

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.