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1.  Hepatitis C Virus Serologic and Virologic Tests and Clinical Diagnosis of HCV-Related Liver Disease 
The use of serological and virological tests has become essential in the management of hepatitis C virus (HCV) infection in order to diagnose infection, guide treatment decisions and assess the virological response to antiviral therapy. Virological tools include serological assays for anti-HCV antibody detection and serological determination of the HCV genotype, and molecular assays that detect and quantify HCV RNA and determine the HCV genotype. Anti-HCV antibody testing and HCV RNA testing are used to diagnose acute and chronic hepatitis C. Only patients with detectable HCV RNA should be considered for pegylated interferon alfa and ribavirin therapy and the HCV genotype should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure. HCV RNA monitoring during therapy is used to tailor treatment duration in HCV genotype 1 infection, and molecular assays are used to assess the end-of-treatment and, most importantly the sustained virological response, i.e. the endpoint of therapy.
PMCID: PMC1415842  PMID: 16614740
Hepatitis C virus; serological and virological tests; HCV RNA
2.  Ultradeep Pyrosequencing and Molecular Modeling Identify Key Structural Features of Hepatitis B Virus RNase H, a Putative Target for Antiviral Intervention 
Journal of Virology  2014;88(1):574-582.
Last-generation nucleoside/nucleotide analogues are potent against hepatitis B virus (HBV) and have a high barrier to resistance. However, delayed responses have been observed in patients previously exposed to other drugs of the same class, long-term resistance is possible, and cure of infection cannot be achieved with these therapies, emphasizing the need for alternative therapeutic approaches. The HBV RNase H represents an interesting target because its enzyme activity is essential to the HBV life cycle. The goal of our study was to characterize the structure of the HBV RNase H by computing a 3-dimensional molecular model derived from E. coli RNase H and analyzing 2,326 sequences of all HBV genotypes available in public databases and 958,000 sequences generated by means of ultradeep pyrosequencing of sequences from a homogenous population of 73 treatment-naive patients infected with HBV genotype D. Our data revealed that (i) the putative 4th catalytic residue displays unexpected variability that could be explained by the overlap of the HBx gene and has no apparent impact on HBV replicative capacity and that (ii) the C-helix-containing basic protrusion, which is required to guide the RNA/DNA heteroduplex into the catalytic site, is highly conserved and bears unique structural properties that can be used to target HBV-specific RNase H inhibitors without cross-species activity. The model shows substantial differences from other known RNases H and paves the way for functional and structural studies as a prerequisite to the development of new inhibitors of the HBV cell cycle specifically targeting RNase H activity.
doi:10.1128/JVI.03000-13
PMCID: PMC3911741  PMID: 24173223
3.  Silymarin for hepatitis C virus infection 
Antiviral therapy  2012;18(2):141-147.
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 hepatitis C virus infection. The information was collated from a recent workshop on silibinin in Germany.
doi:10.3851/IMP2402
PMCID: PMC4076489  PMID: 23011959
4.  New Antiviral Agents for Hepatitis C 
Approximately 120-130 million individuals are chronically infected with hepatitis C virus (HCV) worldwide, although it is curable by therapy. Until recently, treatment of chronic hepatitis C was based on the combination of pegylated interferon-α and ribavirin. A number of models have been developed to study the HCV lifecycle and screen for potential HCV inhibitors. They led to the development of antiviral agents that specifically target a viral function (direct acting antivirals), and host-targeted agents that inhibit HCV replication. Direct acting antivirals in clinical development include NS3-4A protease inhibitors (two of which, telaprevir and boceprevir, have recently been approved for treatment of HCV genotype 1 infection in combination with pegylated interferon-α and ribavirin), nucleoside/nucleotide analogue and non-nucleoside inhibitors of HCV RNA-dependent RNA polymerase, and NS5A inhibitors. Host-targeted agents include cyclophilin inhibitors. This article describes the direct acting antivirals and host-targeted agents that have recently been approved or have been tested in HCV-infected patients and discusses their two current paths of clinical development: with or without interferon-α.
doi:10.3410/B4-5
PMCID: PMC3292288  PMID: 22403588
6.  Hepatoprotective and Antiviral Functions of Silymarin Components in HCV Infection 
Hepatology (Baltimore, Md.)  2013;57(3):1262-1271.
doi:10.1002/hep.26179
PMCID: PMC3594650  PMID: 23213025
silymarin; silibini; hepatitis C; HCV; liver
7.  Therapeutic Implications of Hepatitis C virus Resistance to Antiviral Drugs 
Treatment of chronic hepatitis C is currently based on a combination of pegylated interferon-o! and ribavirin. Neither drug exerts direct selective pressure on viral functions, meaning that interferon-a/ribavirin treatment failure is not due to selection of interferon-a- or ribavirin-resistant viral variants. Several novel antiviral approaches are currently in preclinical or clinical development, and most target viral enzymes and functions, such as hepatitis C virus protease and polymerase. These new drugs all potentially select resistant viral variants both in vitro and in vivo, and resistance is therefore likely to become an important issue in clinical practice.
doi:10.1177/1756283X09336045
PMCID: PMC3002526  PMID: 21180544
hepatitis C virus; resistance; interferon-a; ribavirin; protease inhibitors; polymerase inhibitors
8.  Regulation of Hepatitis C Virus Replication by Nuclear Translocation of Nonstructural 5A Protein and Transcriptional Activation of Host Genes 
Journal of Virology  2013;87(10):5523-5539.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is involved in regulating viral replication through its direct interaction with the HCV RNA-dependent RNA polymerase. NS5A also alters infected cell metabolism through complex interactions with numerous host cell proteins. NS5A has furthermore been suggested to act as a transcriptional activator, although the impact on viral replication is unclear. To study this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected patient, and their abilities to activate gene transcription were analyzed in a single-hybrid yeast (Saccharomyces cerevisiae) model. Different variants isolated from the same patient displayed different transactivational activities. When these variants were inserted into the HCV subgenomic replicon system, they demonstrated various levels of RNA replication, which correlated with their transactivational activities. We showed that the C-terminal fragment of NS5A was localized to the nucleus and that a functional NS5A nuclear localization signal and cellular caspase activity were required for this process. Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, we demonstrate that nuclear NS5A binds to host cell promoters of several genes previously identified as important for efficient HCV RNA replication, inducing their transcription. Taken together, these results demonstrate a new mechanism by which HCV modulates its cellular environment, thereby enhancing viral replication.
doi:10.1128/JVI.00585-12
PMCID: PMC3648193  PMID: 23468497
9.  The Cobas AmpliPrep/Cobas TaqMan HCV Test, Version 2.0, Real-Time PCR Assay Accurately Quantifies Hepatitis C Virus Genotype 4 RNA 
Journal of Clinical Microbiology  2013;51(4):1078-1082.
Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. The first-generation Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV) underestimated HCV RNA levels by >1-log10 international units/ml in a number of patients infected with HCV genotype 4 and occasionally failed to detect it. The aim of this study was to evaluate the ability of the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), to accurately quantify HCV RNA in a large series of patients infected with different subtypes of HCV genotype 4. Group A comprised 122 patients with chronic HCV genotype 4 infection, and group B comprised 4 patients with HCV genotype 4 in whom HCV RNA was undetectable using the CAP/CTM HCV. Each specimen was tested with the third-generation branched DNA (bDNA) assay, CAP/CTM HCV, and CAP/CTM HCV v2.0. The HCV RNA level was lower in CAP/CTM HCV than in bDNA in 76.2% of cases, regardless of the HCV genotype 4 subtype. In contrast, the correlation between bDNA and CAP/CTM HCV v2.0 values was excellent. CAP/CTM HCV v2.0 accurately quantified HCV RNA levels in the presence of an A-to-T substitution at position 165 alone or combined with a G-to-A substitution at position 145 of the 5′ untranslated region of HCV genome. In conclusion, CAP/CTM HCV v2.0 accurately quantifies HCV RNA in genotype 4 clinical specimens, regardless of the subtype, and can be confidently used in clinical trials and clinical practice with this genotype.
doi:10.1128/JCM.02004-12
PMCID: PMC3666793  PMID: 23325825
10.  Discovery of naturally occurring aurones that are potent allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase 
Journal of Medicinal Chemistry  2011;54(15):5395-5402.
We have identified naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) as new templates for non-nucleoside hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) inhibitors. The aurone target site, identified by site-directed mutagenesis, is located in Thumb Pocket I of HCV RdRp. The RdRp inhibitory activity of 42 aurones was rationally explored in an enzyme assay. Molecular docking studies were used to determine how aurones bind to HCV RdRp and to predict their range of inhibitory activity. Seven aurone derivatives were found to have potent inhibitory effects on HCV RdRp, with IC50s below 5 μM and excellent selectivity. The most active aurone analogue was (Z)-2-((1-butyl-1H-indol-3-yl)methylene)-4,6-dihydroxybenzofuran-3(2H)-one (compound 51), with an IC50 of 2.2 μM. Their potent RdRp inhibitory activity, together with their low toxicity, make these molecules attractive candidate direct-acting anti-HCV agents.
doi:10.1021/jm200242p
PMCID: PMC3579765  PMID: 21699179
Antiviral Agents; chemical synthesis; pharmacology; Benzofurans; chemical synthesis; pharmacology; Hepacivirus; enzymology; Models, Molecular; RNA Replicase; antagonists & inhibitors; metabolism
11.  New virologic tools for management of chronic hepatitis B and C 
Gastroenterology  2012;142(6):1303-1313.e1.
Molecular biology techniques are routinely used to diagnose and monitor treatment of patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. These tools can detect and quantify viral genomes, and analyze their sequence, in order to determine their genotype or subtype and to identify nucleotide or amino acid substitutions associated with resistance to antiviral drugs. They include real-time target amplification methods, which have been standardized and are widely used in clinical practice to diagnose and monitor HBV and HCV infections, and next-generation sequencing techniques, which are still restricted to research laboratories. In addition, new enzyme immunoassays can quantify hepatitis B surface and hepatitis C core antigens, and point-of-care tests and alternatives to biologic tests that require whole-blood samples obtained by venipuncture have been developed. We review these new virologic methods and their clinical and research applications to HBV and HCV infections.
doi:10.1053/j.gastro.2012.02.027
PMCID: PMC3477068  PMID: 22537437
12.  Hepatitis B Virus Kinetics under Therapy Sheds Light on Differences Between e-antigen Positive and Negative Infection 
The Journal of infectious diseases  2010;202(9):1309-1318.
BACKGROUND
Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B has a divergent presentation and clinical course from that of HBeAg-positive infection. The former usually presents with lower viral levels, but faster progression to liver disease. We sought to understand better the balance between replication and the immune response against hepatitis B virus (HBV).
METHODS
Viral kinetics in 50 HBeAg-negative patients under various treatment protocols with interferon-α and/or nucleos(t)ide analogues was analyzed. HBV DNA level was measured frequently and the data fitted to a viral dynamic model. A meta-analysis of all published studies of viral kinetics in HBeAg-positive and negative infection was also conducted.
RESULTS
We found that the clearance of both HBV virions and infected cells was significantly faster in HBeAg-negative than -positive infection. In HBeAg-negative infection, there was also a negative correlation between baseline HBV DNA levels and infected cell half-life, suggesting that the higher the viral load the faster the turnover of infected cells.
CONCLUSIONS
These results reveal the dual role of the immune response in maintaining lower viral levels and inducing faster turnover of infected cells, the latter of which may be responsible for the more aggressive nature of HBeAg-negative infection.
doi:10.1086/656528
PMCID: PMC3058754  PMID: 20874517
HBeAg; mathematical model; e antigen; HBV; dynamics
13.  Interpretation of Real-Time PCR Results for Hepatitis C Virus RNA When Viral Load Is Below Quantification Limits▿ 
Journal of Clinical Microbiology  2011;49(3):1113-1115.
Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with 10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation of their results, leading to ambiguous conclusions.
doi:10.1128/JCM.01511-10
PMCID: PMC3067753  PMID: 21227989
14.  Downregulation of Gadd45beta expression by hepatitis C virus leads to defective cell cycle arrest 
Cancer Research  2010;70(12):4901-4911.
Members of the Gadd45 family play central roles in the cellular response to genotoxic stress, and have been implicated in several human cancers including hepatocellular carcinomas. Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of primary hepatocellular tumors, although the underlying mechanisms are unclear. Here, we demonstrate a novel link between diminished Gadd45β expression and HCV infection. Inhibited Gadd45β expression was observed in both non-tumoral and tumoral tissues from infected individuals, and in cell lines harboring an HCV replicon and the infectious HCV strain JFH1. Decreased Gadd45β expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame. Mechanistically, hypermethylation of the Gadd45β promoter in the presence of HCV is responsible for this defect. Diminished Gadd45β expression leads to aberrant cell cycle arrest and diminished DNA excision repair. Together, these results provide a novel insight into the mechanisms involved in HCV-associated hepatocellular carcinomas, showing that reduced Gadd45β expression may play a contributory role to this process, and providing evidence that HCV may interfere with epigenetic gene expression by altering promoter methylation.
doi:10.1158/0008-5472.CAN-09-4554
PMCID: PMC3125696  PMID: 20530689
hepatocellular carcinoma; HCV; Gadd45β; Adult; Aged; Animals; Antigens, Differentiation; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cells, Cultured; DNA Methylation; DNA Repair; Down-Regulation; Female; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Liver ; Liver Neoplasms; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; pharmacology; Reverse Transcriptase Polymerase Chain Reaction; Virus Replication
15.  Performance of Version 2.0 of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification ▿  
Journal of Clinical Microbiology  2010;48(10):3641-3647.
The detection and quantification of hepatitis B virus (HBV) DNA are essential for the diagnosis and treatment of chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of version 2.0 (v2.0) of the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/ml and a claimed upper limit of quantification of 1.7 × 108 IU/ml. The specificity of the assay was 99% (95% confidence interval, 94.7 to 100%). Intra-assay and interassay coefficients of variation ranged from 0.21% to 2.67% and from 0.65% to 2.25%, respectively. The calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed good correspondence between HBV DNA levels measured by the CAP/CTM v2.0 assay, version 1.0 of the same assay, and the third-generation “branched DNA” assay. The CAP/CTM v2.0 assay quantified HBV DNA levels in serum or plasma from the same patients equally. In conclusion, the new version of the CAP/CTM assay is sensitive, specific, and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay, version 2.0, is well suited to monitoring clinical HBV DNA levels according to current clinical practice guidelines.
doi:10.1128/JCM.01306-10
PMCID: PMC2953086  PMID: 20720031
16.  Performance of version 2.0 of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification 
Journal of Clinical Microbiology  2010;48(10):3641-3647.
Hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose and treat chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performances of version 2.0 (v2.0) of the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/mL and a claimed upper limit of quantification of 1.7 × 108 IU/mL. The specificity of the assay was 99% (95% confidence interval: 94.7–100%). Intra-assay and inter-assay coefficients of variation ranged from 1.10% to 3.07%, and 0.82% to 2.95%, respectively. Calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed a good correspondence between HBV DNA levels measured with CAP/CTM v2.0, version 1.0 of the same assay and the third generation “branched DNA” assay. CAP/CTM v2.0 equally quantified HBV DNA levels in serum or plasma from the same patients. In conclusion, the new version of the CAP/CTM assay is sensitive, specific and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay version 2.0 is well suited to monitoring clinical HBV DNA levels according to current Clinical Practice Guidelines.
doi:10.1128/JCM.01306-10
PMCID: PMC2953086  PMID: 20720031
17.  Characterization of V36C, a Novel Amino Acid Substitution Conferring Hepatitis C Virus (HCV) Resistance to Telaprevir, a Potent Peptidomimetic Inhibitor of HCV Protease▿ †  
We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.
doi:10.1128/AAC.01796-09
PMCID: PMC2876365  PMID: 20368394
18.  Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice 
The Journal of Biological Chemistry  2009;284(48):33466-33474.
Chronic hepatitis C virus (HCV) infection is associated with altered lipid metabolism and hepatocellular steatosis. Virusinduced steatosis is a cytopathic effect of HCV replication. The goal of this study was to examine the mechanisms underlying HCV-induced lipid metabolic defects in a transgenic mouse model expressing the full HCV protein repertoire at levels corresponding to the human infection. In this model, expression of the HCV full-length open reading frame was associated with hepatocellular steatosis and reduced plasma triglyceride levels. Triglyceride secretion was impaired while lipogenesis was activated. Increased lipogenic enzyme transcription resulted from activation at the maturation step and nuclear translocation of sterol regulatory element binding protein 1c (SREBP1c). No ER-stress marker was expressed at significantly higher levels in HCV transgenic mice than in their wild-type counterparts, suggesting that SREBP1c proteolytic cleavage was independent of ER stress. In conclusion, transgenic mice expressing the HCV full-length polyprotein at low, physiological levels, have decreased plasma triglyceride levels and develop hepatocellular steatosis in the same way as HCV-infected patients. In these mice, de novo triglyceride synthesis is induced by direct SREBP1c activation by one or several HCV proteins through induction of the lipogenic pathway, independently of ER stress, while triglyceride secretion is simultaneously reduced.
doi:10.1074/jbc.M109.019810
PMCID: PMC2785191  PMID: 19808675
Animals; Blotting, Western; Disease Models, Animal; Endoplasmic Reticulum; metabolism; Fatty Liver; blood; etiology; metabolism; Hepacivirus; genetics; metabolism; Hepatitis C; blood; complications; metabolism; Humans; Lipogenesis; physiology; Liver; metabolism; pathology; virology; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Reverse Transcriptase Polymerase Chain Reaction; Sterol Regulatory Element Binding Protein 1; genetics; metabolism; Triglycerides; blood; secretion; Viral Proteins; genetics; metabolism
19.  Seroconversion to hepatitis C virus alternate reading frame protein during acute infection 
Hepatology (Baltimore, Md.)  2009;49(5):1449-1459.
The existence of hepatitis C virus proteins encoded by alternate reading frames overlapping the core-encoding region has been suggested. Several mechanisms of production have been postulated and the function(s) of these proteins in the HCV life cycle remain(s) unknown. We analyzed cases of seroconversion to an alternate reading frame protein in a group of 17 patients infected by the one of two hepatitis C virus genotype 1b strains during an outbreak in a hemodialysis unit. Three patients seroconverted and antibodies were transiently detected in another patient. Three of these patients were infected by one of the two HCV strains, whereas the strain infecting the remaining patient could not be identified. Quasispecies sequence analysis of the core-coding region showed no differences in the core or +1 reading frame sequences that could explain alternate reading frame protein seroconversion in some but not all of the patients infected by one of the HCV strains, and no such difference was found between the two strains. As differences in the structure of RNA elements could play a role in frameshift events, we conducted a predictive analysis of RNA folding by using RNAfold software. No difference was found between the patients who did and did not seroconvert to alternate reading frame protein.
Conclusion
our findings prove that alternate reading frame proteins can be produced during acute HCV infection. However, seroconversion does not occur in all patients for unknown reasons. Alternate reading frame protein could be generated by minority quasispecies variants or variants that occur transiently.
doi:10.1002/hep.22821
PMCID: PMC2956746  PMID: 19350656
Alternative Splicing; Disease Outbreaks; Enzyme-Linked Immunosorbent Assay; Female; Genotype; Hemodialysis Units, Hospital; Hepacivirus; genetics; immunology; Hepatitis Antibodies; blood; Hepatitis C; virology; Humans; Male; Middle Aged; Protein Structure, Secondary; RNA, Viral; blood; genetics; Reading Frames; Sensitivity and Specificity; Sequence Analysis, RNA; Viral Proteins; genetics; immunology; ARFP; antibodies; acute hepatitis C; seroconversion
20.  Complex dynamics of hepatitis B virus resistance to adefovir 
Hepatology (Baltimore, Md.)  2009;49(1):50-59.
In patients with hepatitis B e antigen-negative chronic hepatitis B, adefovir dipivoxil administration selects variants bearing reverse transcriptase rtN236T and/or rtA181V/T substitutions in 29% of cases after 5 years. The aim of this work was to characterize the dynamics of adefovir-resistant variant populations during adefovir monotherapy in order to better understand the molecular mechanisms underlying hepatitis B virus resistance to this class of nucleotide analogues. Patients included in a 240-week clinical trial of adefovir monotherapy who developed adefovir resistance-associated substitutions were studied. The dynamics of hepatitis B virus populations were analyzed over time, after generating nearly 4000 full-length reverse transcriptase sequences, and compared with the replication kinetics of the virus during therapy. Whatever the viral kinetics pattern, adefovir resistance was characterized by exclusive detection of a dominant wild-type, adefovir-sensitive variant population at baseline and late and gradual selection by adefovir of several coexisting resistant viral populations, defined by the presence of amino acid substitutions at position rt236, position rt181, or both. The gain in fitness of one or other of these resistant populations during adefovir administration was never associated with the selection of additional amino acid substitutions in the reverse transcriptase.
Conclusions
Our results suggest that adefovir administration selects poorly fit pre-existing or emerging viral populations with low-level adefovir resistance, which subsequently compete to fill the replication space. Viral kinetics depends on the initial virological response to adefovir. Lamivudine add-on restores some antiviral efficacy but adefovir-resistant variants remain predominant. Whether these adefovir resistance-associated substitutions may confer cross-resistance to tenofovir in vivo will need to be determined.
doi:10.1002/hep.22634
PMCID: PMC2956748  PMID: 19065672
Adenine; analogs & derivatives; pharmacology; therapeutic use; Adult; Antiviral Agents; pharmacology; therapeutic use; DNA, Viral; chemistry; Drug Resistance, Viral; Female; Hepatitis B virus; drug effects; Hepatitis B, Chronic; drug therapy; Humans; Male; Middle Aged; Molecular Sequence Data; Phosphonic Acids; pharmacology; therapeutic use; Reverse Transcriptase Polymerase Chain Reaction; Virus Replication; drug effects; quasispecies; amino acid substitutions; nucleotide analogues; treatment failure; viral populations
21.  Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice 
PLoS ONE  2009;4(12):e8209.
Background
With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.
Methodology/Principal Findings
A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.
Conclusions/Significance
In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.
doi:10.1371/journal.pone.0008209
PMCID: PMC2785465  PMID: 19997618
22.  Performance of the Abbott Real-Time PCR Assay Using m2000sp and m2000rt for Hepatitis C Virus RNA Quantification▿  
Journal of Clinical Microbiology  2009;47(6):1726-1732.
Quantification of hepatitis C virus (HCV) RNA is essential for the everyday management of chronic hepatitis C therapy. “Real-time” PCR techniques are potentially more sensitive than classical PCR techniques, are not prone to carryover contamination, and have a consistently wider dynamic range of quantification. Thus, they are rapidly replacing other technologies for routine quantification of HCV RNA. We extensively evaluated the intrinsic characteristics and clinical performance of the m2000sp-m2000rt Abbott real-time PCR platform for HCV RNA quantification. The study shows that the m2000sp-m2000rt platform is sensitive, specific, and precise; that the results are reproducible; and that the platform has a broad dynamic range of quantification. When comparing HCV RNA levels measured in the same individuals with the m2000sp-m2000rt platform and the third-generation branched-DNA assay, a trend toward a modest overestimation of HCV RNA levels was observed in the m2000sp-m2000rt platform in all genotypes except genotype 5. The differences, however, were unlikely to have any impact in clinical practice. In conclusion, our study shows that the Abbott m2000 real-time PCR system for HCV RNA quantification is sensitive, specific, and precise; that the results are reproducible; and that the platform's broad dynamic range of quantification is well suited to HCV RNA monitoring in the clinical setting.
doi:10.1128/JCM.01300-08
PMCID: PMC2691100  PMID: 19369435
23.  Virologic Monitoring of Hepatitis B Virus Therapy in Clinical Trials and Practice: Recommendations for a Standardized Approach 
Gastroenterology  2007;134(2):405-415.
Treatment of chronic hepatitis B virus (HBV) infection is aimed at suppressing viral replication to the lowest possible level, and thereby to halt the progression of liver disease and prevent the onset of complications. Two categories of drugs are used in HBV therapy: the interferons, including standard interferon alfa or pegylated interferon alfa, and specific nucleoside or nucleotide HBV inhibitors that target the reverse-transcriptase function of HBV-DNA polymerase. The reported results of clinical trials have used varying definitions of efficacy, failure, and resistance based on different measures of virologic responses. This article discusses HBV virologic markers and tests, and their optimal use both for planning and reporting clinical trials and in clinical practice.
doi:10.1053/j.gastro.2007.11.036
PMCID: PMC2676233  PMID: 18242209
24.  Performance of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification▿  
Journal of Clinical Microbiology  2008;46(5):1716-1723.
Hepatitis B virus (HBV) DNA quantification is used to establish the prognosis of chronic HBV-related liver disease, to identify those patients who need to be treated, and to monitor the virologic response and resistance to antiviral therapies. Real-time PCR-based assays are gradually replacing other technologies for routine quantification of HBV DNA in clinical practice. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the real-time PCR Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform for HBV DNA quantification. Specificity was satisfactory (95% confidence interval, 98.1 to 100%). Intra-assay coefficients of variation ranged from 0.22% to 2.68%, and interassay coefficients of variation ranged from 1.31% to 4.13%. Quantification was linear over the full dynamic range of quantification of the assay (1.7 to 8.0 log10 IU/ml) and was not affected by dilution. The assay was accurate regardless of the HBV genotype. Samples containing HBV DNA levels above 4.5 log10 IU/ml were slightly underestimated relative to another accurate assay based on branched-DNA technology, but this is unlikely to have noteworthy clinical implications. Thus, the CAP/CTM HBV DNA assay is sensitive, specific, and reproducible, and it accurately quantifies HBV DNA levels in patients chronically infected by HBV genotypes A to F. Samples with HBV DNA concentrations above the upper limit of quantification need to be diluted and then retested. Broad use of fully automated real-time PCR assays should improve the management of patients with chronic HBV infection.
doi:10.1128/JCM.01248-07
PMCID: PMC2395079  PMID: 18287319
25.  Spontaneous Recovery in Acute Human Hepatitis C Virus Infection: Functional T-Cell Thresholds and Relative Importance of CD4 Help▿  
Journal of Virology  2007;82(4):1827-1837.
The mechanisms mediating protective immunity to hepatitis C virus (HCV) infection are incompletely understood because early infection in humans is rarely identified, particularly in those individuals who subsequently demonstrate spontaneous virus eradication. We have established a large national network of patients with acute HCV infection. Here, we comprehensively examined total HCV-specific CD4+ and CD8+ T-cell responses and identified functional T-cell thresholds that predict recovery. Interestingly, we found that the presence of HCV-specific cytotoxic T lymphocytes (CTLs) that can proliferate, exhibit cytotoxicity, and produce gamma interferon does not ensure recovery, but whether these CTLs were primed in the presence or absence of CD4+ T-cell help (HCV-specific interleukin-2 production) is a critical determinant. These results have important implications for early prediction of the virologic outcome following acute HCV and for the development of novel immunotherapeutic approaches.
doi:10.1128/JVI.01581-07
PMCID: PMC2258714  PMID: 18045940

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