Natural non-homologous recombination led to the emergence of a duplicated V3-NS5A region in HCV-1b strains associated with hepatocellular carcinoma.

BACKGROUND: The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies. METHODS: Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplication-like event. RESULTS: Quasispecies were entirely composed of exclusively mutant or wild-type strains respectively. Mutant quasispecies were found to have been present since contamination and had persisted for at least 10 years. This V3 duplication-like event appears to have resulted from non-homologous recombination between HCV-1b wild-type strains around 100 years ago. The association between increased liver disease severity and these HCV-1b mutants may explain their persistence in chronically infected patients. CONCLUSIONS: These results emphasize the possible consequences of non-homologous recombination in the emergence and severity of new viral diseases.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin.

The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.

Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study.

The epidemic history of HCV genotype 5a is poorly documented in France, where its prevalence is very low, except in a small central area, where it accounts for 14.2% of chronic hepatitis C cases. A Bayesian coalescent phylogenetic investigation based on the E1 envelope gene and a non-structural genomic segment (NS3/4) was carried out to trace the origin of this epidemic using a large sample of genotype 5a isolates collected throughout France. The dates of documented transmissions by blood transfusion were used to calibrate five nodes in the phylogeny. The results of the E1 gene analysis showed that the best-fitting population dynamic model was the expansion growth model under a relaxed molecular clock. The rate of nucleotide substitutions and time to the most recent common ancestors (tMRCA) of genotype 5a isolates were estimated. The divergence of all the French HCV genotype 5a strains included in this study was dated to 1939 [95% HPD: 1921-1956], and the tMRCA of isolates from central France was dated to 1954 [1942-1967], which is in agreement with epidemiological data. NS3/4 analysis provided similar estimates with strongly overlapping HPD values. Phylodynamic analyses give a plausible reconstruction of the evolutionary history of HCV genotype 5a in France, suggesting the concomitant roles of transfusion, iatrogenic route and intra-familial transmission in viral diffusion.

Interpretation of real-time PCR results for hepatitis C virus RNA when viral load is below quantification limits.

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.

Influence of the HCV subtype on the virological response to pegylated interferon and ribavirin therapy.

The hepatitis C virus genotype is considered to be the most important baseline predictor of a sustained virological response in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. The influence of the subtype on the sustained virological response was investigated in patients infected with genotypes 1, 4, 5, or 6. This study was done on 597 patients with chronic hepatitis C who were given pegylated interferon and ribavirin for 48 weeks. The overall rate of sustained virological response in the 597 patients was 37.8%. Univariate analysis indicated that the sustained virological response of patients infected with subtype 1b (39%) tended to be higher than that of patients infected with subtype 1a (30.6%; P = 0.06) and it was similar to those patients infected with subtypes 4a (51.3%; P = 0.12) or 4d (51.7%; P = 0.16). Multivariate analysis indicated that five factors were independently associated with sustained virological response: the age (OR 0.97; 95% CI = 0.95-0.99), absence of cirrhosis (OR: 2.92; 95% CI = 1.7-5.0; P < 0.01), absence of HIV co-infection (OR: 2.08; 95% CI = 1.2-3.5; P < 0.01), low baseline plasma HCV RNA concentration (OR: 1.74; 95% CI = 1.2-2.6; P < 0.01), and the subtype 1b (OR: 1.61; 95% CI = 1.0-2.5; P = 0.04) or subtypes 4a and 4d (OR: 2.03; 95% CI = 1.1-3.8; P = 0.03). In conclusion, among difficult-to-treat genotypes, the subtype 1a is associated with a lower response to anti-HCV therapy than subtypes 1b, 4a, and 4d.