First next-generation sequencing full-genome characterization of a hepatitis C virus genotype 7 divergent subtype.

We report the near-full-length genome sequence of a hepatitis C virus (HCV) isolate from a man originating from Democratic Republic of Congo, the genotype of which could not be determined by the routinely used sequencing technique. The near-complete genome sequence of this variant BAK1 was obtained by the association of two next-generation sequencing technologies. Evolutionary analysis indicates that this isolate, BAK1, could be the first reported strain belonging to a new HCV-7b subtype. This new subtype has been incorrectly identified as genotype 2 by the Versant HCV Genotype 2.0 assay (LiPA). The requirement of three independent isolates has been filled, and a new subtype can be assigned. More examples of HCV-7 are required to better understand its origin, its pathogenicity and its relationship with genotype 2.

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types.

Recent evidence of underestimated circulation of hepatitis C virus intergenotypic recombinant strain RF2k/1b in the Rhône-Alpes region, France, January to August 2014: implications for antiviral treatment.

Since the beginning of 2014, hepatitis C virus (HCV) recombinant forms RF2k/1b have been detected in the Rhône-Alpes French region in 10 patients originating from the Caucasus area. Circulation of this particular HCV strain is very likely to be underestimated. It is also prone to be misgenotyped when using genotyping methods based on the 5' region of the viral genome, which may lead to suboptimal treatment.

IRES complexity before IFN-alpha treatment and evolution of the viral load at the early stage of treatment in peripheral blood mononuclear cells from chronic hepatitis C patients.

At the early stage of treatment, IFN alpha-2a induces inhibition of HCV replication. The viral load reflects mainly the degradation rate of the viruses. However, differences in the behavior of the viral population depend on changes, which occurred in the HCV-IRES genome. In this study, cloning and sequencing strategies permitted the generation of a large number of IRES sequences from the PBMCs of 18 patients (5 women, 13 men) with chronic hepatitis C. The HCV IRES appeared to be highly conserved structurally. However, some variability was found between the different isolates obtained: 467 substitutions with a median of 7 variants/patients. No relationship was observed between pre-treatment IRES complexity and the viral load at the beginning. However, on review of the evolution of viral load in the PBMCs during the first 3 days of IFN alpha-2a treatment, patients could be classified into two groups: Group 1, in which the viral population continued to replicate and Group 2, in which the viral load decreased significantly (P = 0.01727). Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. At the early stage of treatment, IFN alpha-2a was efficient in reducing the viral replication in a significant number of patients; mechanisms of response might affect the virus directly. However, pre-treatment genomic variations observed in the 5'NCR of HCV were not a parameter of a later response to antiviral therapy in chronic hepatitis C patients. (244)

Influence of three successive antiviral treatments on viral heterogeneity in nonresponder chronic hepatitis C patients.

The purpose of the present study was to assess the viral diversity of hepatitis C virus (HCV) in six nonresponder patients during three unsuccessful treatments. These patients were treated successively with IFN-alpha2a (IFN-alpha) at a posology of 3.10(6) units (MIU) three times a week, 10 MIU three times a week, and a combination of IFN-alpha (3 MIU) plus ribavirin (1,000 mg/day). However, only two chronically infected patients could be included in the study due to the persistence of HCV RNA during the three successive treatments. The viral diversity was analysed by cloning and sequencing the HVR-1 region. The treatment of the two nonresponder patients was associated with the persistence of a wide diversity in the viral population and with the emergence of new or minor variants. Under the influence of standard doses of IFN-alpha, a rearrangement of the quasispecies present was observed at this time point. No significant change in viral load or in the complexity of the quasispecies was observed. A second treatment with a high dose of IFN-alpha induced a significant decrease in the associated viral load and, in one case, resulted in a radical change of the viral diversity. Administration of a combination of IFN-alpha and ribavirin did not affect the evolution of the variants but was followed by the emergence of various multiple variants. These results reinforce the hypothesis of the presence of preexisting quasispecies best adapted to the host environment, and therefore resistant to any current therapy.

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.

Mosquito cells bind and replicate hepatitis C virus.

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.

Hepatitis C virus RNA in plasma and blood mononuclear cells in patients with chronic hepatitis C treated with alpha-interferon.

Hepatitis C virus (HCV), a positive stranded RNA virus, is the main causative agent of post-transfusion and sporadic non-A non-B hepatitis worldwide. Paired samples of plasma and peripheral blood mononuclear cells (PBMC) from 11 patients with chronic hepatitis C treated with alpha-interferon (IFN) were tested, using a single step polymerase chain reaction (PCR), for the presence of HCV RNA. Before treatment, the viral genome was detected in all the plasma samples and 81.8% of PBMC. After 3 months of treatment, HCV RNA was still present in 63.6% of plasma samples but in only 27.3% of PBMC. A good correlation was observed between serum alanine aminotransferase level normalisation and disappearance of the viral genome in plasma. Among the six responder patients, five relapsed shortly after IFN withdrawal; HCV RNA became detectable again, especially in PBMC. These results show the presence of HCV in PBMC from most patients infected chronically. IFN therapy had an inhibitory effect on viral replication in lymphoid cells, but frequent relapses observed after cessation of treatment with IFN suggested persistence of HCV in these cells.

Different prevalence of precore mutants in five members of a hepatitis-B-virus-infected family: evidence for a precore variant type in an asymptomatic anti-HBs patient.

Chronic active hepatitis B (CAH-B), anti-HBe (+) has been associated with a hepatitis B virus variant carrying a stop codon at the distal pre-C region that prevents HBeAg synthesis. We analyzed the HBV DNA pre-C region in five members of a Turkish family. The mother presented an anti-HBe (+) CAH-B and the four children different hepatitis B virus serological and clinical profiles. The pre-C region was analyzed by cloning after DNA amplification in sera and peripheral blood mononuclear cells. A method for rapid screening of a large number of cloned polymerase chain reaction products was developed for the presence of the most frequent pre-C mutations (G to A substitution at nucleotide position 1896 and 1899). At least 60 independent clones were tested for each patient by selective oligonucleotide hybridization using non-mutated (M0), one (M1) and two (M2) point-mutated probes. Results were confirmed by sequencing. The mutation 1896 was present in 91% of DNA clones from the mother. The same mutation was also found in 85% of the clones in the youngest child (D), but in less than 10% of the clones from children A and C. Only the pre-C wild-type strain was observed in child B. X gene deletions (3 to 20 nt) were also present in some clones from the mother and children A, B and C. No significant difference between serum and peripheral blood mononuclear cells concerning the viral population was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay.

A quantitative, non-radioactive hybrid capture HBV DNA assay (Digene Diagnostics), which uses an efficient solution hybridization procedure coupled to a sensitive chemiluminescent signal amplification system, was compared with the quantitative, radioactive solution hybridization assay (Genostics, Abbott Laboratories), in hepatitis B virus carriers, particularly in those undergoing antiviral therapy. The qualitative reproducibility of the chemiluminescent method, tested on 30 sera, was acceptable, with a reproducibility rate of 93.3%. A comparison of this hybrid capture HBV DNA assay with the radioactive test on 113 sera obtained from 48 patients (39 HBsAg-positive patients) gave a sensitivity of 87.2%, a specificity of 100% and an agreement between the two tests of 89.4% (101 sera including 82 HBV DNA positive and 19 negative samples). Changes in HBV DNA levels measured by the two assays showed a good correlation with each other during interferon therapy. However, the hybrid capture values were higher than the radioactive assay values, with the ratio of the two values being variable in the same patient during the course of treatment. The Genostics assay therefore seems to be a more accurate procedure for evaluating changes in viral replication, particularly at high HBV DNA levels. However, the hybrid capture method is faster and has the advantage of being a non-radioactive procedure. This chemiluminescent assay is easy to perform as a routine diagnostic procedure and may be a useful alternative to the radioactive solution hybridization method.

c-Ha-ras polymorphism in patients with hepatocellular carcinoma. Comparison of healthy subjects and alcoholic patients with cirrhosis.

It has been observed that certain rare alleles of the c-Ha-ras gene occur more frequently in patients with certain malignant tumors than in healthy individuals, suggesting that these alleles may serve as markers for particular types of cancer. In this study, we compared the restriction fragment length polymorphism at the c-Ha-ras gene locus in 40 patients with cirrhosis and hepatocellular carcinoma with that in 39 patients with cirrhosis and in 42 normal subjects, all of Caucasian origin. Southern blotting of leukocyte DNA from the above patients, after digestion with either BamH1 or AvaII, revealed the presence of allele fragments of different sizes, corresponding to 4 common alleles and 3 rare alleles. The occurrence of the 3 rare alleles was not significantly different in the 3 populations studied. On the other hand, 2 of the common alleles, a3 (P < 0.01) and a4 (P < 0.03), were found at a significantly higher frequency in patients with cancer than in the 2 other groups. These results suggest that, in hepatocellular carcinoma, there is no increase in the frequency of occurrence of the rare alleles of the c-Ha-ras, but that the distribution of the common alleles may be modified.

Interest of the detection of hepatitis C virus RNA in patients with alcoholic liver disease. Comparison with the HBV status.

HCV-RNA detection was investigated in 66 chronic alcoholic patients divided into 3 groups according to the severity of liver injury: group 1 included 22 chronic alcoholics without cirrhosis, group 2, 20 patients with alcoholic cirrhosis and group 3, 24 patients with alcoholic cirrhosis and hepatocellular carcinoma. The 'nested' polymerase chain reaction (PCR) technique amplifying the 5' non-coding region was used to detect HCV-RNA. For comparison, ELISA1, ELISA2 and RIBA2 tests (Ortho Diagnostics System) were also used to detect anti-HCV antibodies. Finally HBV markers (HBsAg, anti-HBc and anti-HBs antibodies) were detected in all patients as well as HBV-DNA by PCR. In group 1, only 1 patient (4.5%) showed an HCV-RNA-positive PCR, while 3 patients (13.6%) were found to have anti-HCV antibodies detected by RIBA2. In group 2, 3 patients (15%) showed positive PCRs, whereas 4 patients (20%) had anti-HCV antibodies. Finally, in group 3, the PCR was positive in 3 patients (12.5%), while 9 (37.5%) had anti-HCV antibodies. All patients with positive PCRs showed positive anti-HCV antibodies detected by second-generation assays. On the other hand, these patients often had past HBV infection markers but rarely had HBV-DNA detected by PCR. These results suggest that in chronic alcoholic patients, regardless of the severity of liver injury, HCV replication is rarely observed by PCR. Indeed, replication is only observed when anti-HCV antibody detection is positive in second-generation assays, particularly with strong reactivity against C33-C and C22-3 antigens. The relatively high prevalence of anti-HCV antibodies in this population compared to the usual rates could be explained by the age, geographic and perhaps even socioeconomic origin of the patients.

Advantage of PCR for detecting low amounts of HBV DNA in patients' sera.

Serum hepatitis B virus DNA (HBV DNA) is now the most important and reliable marker for monitoring viral replication. Quantitative detection of HBV DNA in serum is based on a commercial standardized solution hybridization assay (Genostics). In this work, we studied the sensitivity and specificity of this method, in comparison with the polymerase chain reaction (PCR) technique, for low-value HBV DNA serum samples. Fifty-four patients with or without HBV serological markers were divided into 4 groups according to their HBV DNA values. Genomic amplification was found to affect 2 conserved regions of the viral genome, the S and C regions. Samples with an HBV DNA concentration equal to or greater than 1.5 pg/ml were considered positive in the "Genostics" test. A total of 38% of patients considered negative in the quantitative assay (less than 1.5 pg/ml) were found to be positive for HBV DNA in serum after PCR. Only 26% of patients with an HBV DNA concentration of between 1.5 and 10 pg/ml in the Genostics test had PCR-detectable viral DNA in serum. Some 56% of patients with HBV DNA values between 10 and 20 pg/ml were found to be positive after amplification. All patients whose HBV DNA values were above 20 pg/ml had PCR-detectable viral DNA in serum. Our PCR results suggest that the positive limit level of the Genostics test has to be re-evaluated. Indeed, for low values of HBV DNA (under 20 pg/ml and especially under 10 pg/ml), it is not possible to conclude about the positivity from the quantitative assay, and results have to be estimated according to the clinical and serological status of the patients. Moreover, PCR can be falsely negative because of methodological problems. Nevertheless, this study confirms that PCR does enable detection of the viral genome in HBV-seronegative patients and in "old" and "cured" HBV-infection marker carriers.