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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

This study, involving 20 laboratories and using currently available assays for hepatitis C virus RNA quantification, demonstrated that differences in viral load values are due not to interlaboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multicenter studies or when monitoring antiviral therapy.

The addition of ribavirin to alpha interferon therapy significantly increases response rates for patients with chronic hepatitis C virus (HCV) infection, but ribavirin's antiviral mechanisms are unknown. Ribavirin has been suggested to have mutagenic potential in vitro that would lead to “error catastrophe,” i.e., the generation of nonviable viral quasispecies due to the increment in the number of mutant genomes, which prevents the transmission of meaningful genetic information. We used extensive sequence-based analysis of two independent genomic regions in order to test in vivo the hypothesis that ribavirin administration accelerates the accumulation of mutations in the viral genome and that this acceleration occurs only when HCV replication is profoundly inhibited by coadministered alpha interferon. The rate of variation of the consensus sequence, the frequency of mutation, the error generation rate, and the between-sample genetic distance were measured for patients receiving ribavirin monotherapy, a combination of alpha interferon three times per week plus ribavirin, or a combination of alpha interferon daily plus ribavirin. Ribavirin monotherapy did not increase the rate of variation of the consensus sequence, the mutation frequency, the error generation rate, or the between-sample genetic distance. The accumulation of nucleotide substitutions did not accelerate, relative to the pretreatment period, during combination therapy with ribavirin and alpha interferon, even when viral replication was profoundly inhibited by alpha interferon. This study strongly undermines the hypothesis whereby ribavirin acts as an HCV mutagen in vivo.

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5′-untranslated region (5′UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5′UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5′UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5′UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log10 copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log10 copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

The hepatitis C virus (HCV) genome shows remarkable sequence variability, leading to the classification of at least six major genotypes, numerous subtypes and a myriad of quasispecies within a given host. A database allowing researchers to investigate the genetic and structural variability of all available HCV sequences is an essential tool for studies on the molecular virology and pathogenesis of hepatitis C as well as drug design and vaccine development. We describe here the European Hepatitis C Virus Database (euHCVdb, ), a collection of computer-annotated sequences based on reference genomes. The annotations include genome mapping of sequences, use of recommended nomenclature, subtyping as well as three-dimensional (3D) molecular models of proteins. A WWW interface has been developed to facilitate database searches and the export of data for sequence and structure analyses. As part of an international collaborative effort with the US and Japanese databases, the European HCV Database (euHCVdb) is mainly dedicated to HCV protein sequences, 3D structures and functional analyses.

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.