Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges.

Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability.

Lymphocytes degranulation in liver in hepatitis C virus carriers is associated with IFNL4 polymorphisms and ALT levels.

BACKGROUND: The polymorphisms of IFNL4 are strongly associated with both spontaneous hepatitis C virus (HCV) clearance and response to peg-IFN-α/ribavirin treatment. To further establish the biological effects of the IFNL4 and rs1297860 variations, we studied the activity of liver immune cells. METHODS: Fresh liver samples were collected from HCV-infected patients before any treatment and from NASH patients as controls. Degranulation activity of each lymphocyte type was assessed by the surface expression of CD107a. IFNL4 polymorphisms and HCV genotypes were determined. RESULTS: In the liver, frequency of CD107a(+) immune cells was significantly higher in HCV patients compared to NASH patients. Higher degranulation activity was observed in lymphocytes of HCV patients with favorable IFNL4 genotypes compared to patients with unfavorable genotypes. Multivariate regression analyses indicated that serum ALT levels were dependent on both Metavir activity score and frequency of CD107a positive NKT cells. The high level of degranulation activity observed before treatment was associated with a high HCV RNA decline at the early stage of peg-IFN-α/ribavirin treatment in patients with favorable genotypes. CONCLUSIONS: These data underline that intrahepatic lymphocyte degranulation activity in HCV-infected patients is associated with IFNL4 polymorphisms and contributes to the clearance of HCV in patients with favorable genotypes under antiviral therapy.

Characterization and role of intra-hepatic regulatory T cells in chronic hepatitis C pathogenesis.

BACKGROUND & AIMS: In chronic hepatitis C (CHC), HCV-specific T-cell responses are often dysfunctionnal. In vitro data point out that regulatory T cells (Treg) are able to suppress HCV-specific lymphocyte proliferation and cytokine secretion but their implication in this pathology is still debated. METHODS: Three complementary approaches were performed to investigate phenotype, frequency or localization of intra-hepatic Treg in treatment naïve CHC patients. Double immunohistochemical analysis was performed in 20 formalin-fixed biopsies with CD8/FoxP3 and CD4/FoxP3 antibodies. Cellular markers and cytokines were investigated by quantitative RT-PCR in 27 additional frozen biopsies. Eight other fresh liver biopsies were selected for complementary analysis of immunophenotyping and frequency of intra-hepatic Treg. RESULTS: Immunohistochemical analyses showed the presence of intra-hepatic CD4(+)FoxP3(+)T cells while CD8(+)FoxP3(+)T cells were very scarce. CD4(+)FoxP3(+)T cells were located in necro-inflammatory areas in contact with CD8(+)T cells, suggesting that Treg-mediated inhibition of CD8(+)T cell proliferation may occur by cell-cell contact. RT-PCR analyses showed strong correlations between CD8, FoxP3, and IL-10 with emergence of four distinct gene clusters, CD8-FoxP3, CD8-IL-10, TGF-beta-IL-10, and TNF-alpha-TGF-beta. No correlation was found between serum viral load and any immune markers. Interestingly, the FoxP3(+)/CD8(+) cells ratio significantly decreased in severe fibrosis (F>3) due to the dramatic decline of FoxP3 cells. CONCLUSIONS: This study provides new insights into the histological localization of Treg within HCV-infected liver, with a special accumulation of CD4(+)FoxP3(+)Treg cells in necro-inflammatory areas, in contact with CD8(+)T cells. Our results suggest a link between Treg, CD8, and IL-10 which altogether could balance immune responses against the virus to avoid immunopathogenesis.

Prevalence and impact of occult hepatitis B infection in chronic hepatitis C patients treated with pegylated interferon and ribavirin.

The prevalence of occult hepatitis B, defined by absence of HBsAg and HBV DNA, ranges widely in patients with hepatitis C. This may influence the treatment of hepatitis C and the severity of liver disease. Sensitive and specific real-time PCR techniques are available commercially and can detect more reliably low HBV DNA levels. The aim of this study was to determine the prevalence of occult hepatitis B virus infection using the COBAS Taqman assay (Roche Diagnostics, Meylan, France) in the serum and liver of HBsAg negative patients with chronic hepatitis C and to evaluate its clinical consequences on liver pathology and its impact on the response to treatment with peg-IFNalpha and Ribavirin. HBV DNA detection was assessed retrospectively on 140 sera and 113 liver biopsies of HCV positive/HBsAg negative patients before treatment. A 4.4% (5/113) prevalence of occult hepatitis B was recorded in liver samples and in none of the sera. Anti-HBc was not detected in one, three of whom were sustained virological responders to treatment, one was relapsed responder and one was non-responder. Furthermore, in this cohort composed of 12% anti-HBs negative/anti-HBc positive and 20% anti-HBs positive/anti-HBc positive patients, anti-HBc was not associated with pre-therapeutic viral load, ALT serum levels, and histological activity or fibrosis. Using a commercial real-time PCR assay, we observed a low prevalence of occult B hepatitis. This, just as anti-HBC status, had no clinical impact in a large cohort of hepatitis C patients. It therefore does not appear useful to screen for occult hepatitis B in these patients with this test before beginning HCV treatment.

Fine characterization of intrahepatic NK cells expressing natural killer receptors in chronic hepatitis B and C.

BACKGROUND/AIMS: The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood. METHODS: Blood and intrahepatic CD56(+) NK cell subsets (expressing NKG2A, CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies. RESULTS: A significant reduction in NK cell frequency and a quantitative imbalance between CD56(bright) and CD56(dim) subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56(+)CD3(-) cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56(+)CD3(-) cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3(-)CD56(bright)NKG2A(+) and CD3(-)CD56(dim)NKG2A(+) cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients. CONCLUSIONS: HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3(-)CD56(dim)NKG2A(+) and CD3(-)CD56(bright)NKG2A(+) cells, may be of interest for disease monitoring.

Baseline and post-treatment hepatitis C NS5A resistance in relapsed patients from a multicentric real-life cohort.

BACKGROUND: Recent data have suggested that failure to achieve sustained virological response with direct-acting antiviral therapy is usually due to relapse and is primarily associated with the emergence of resistance-associated substitutions. The aim of this study was to investigate the prevalence and characterization of non-structural-5A resistance-associated substitutions in patients infected with HCV genotypes 1, 3 and 4 treated by direct-acting antiviral therapy, including anti-non-structural-5A, and to characterize the pre-existing resistance-associated substitutions in subjects treated with anti-non-structural-5A inhibitors. METHODS: From January 2014 to March 2016, 2,995 patients infected with HCV genotypes 1, 3 and 4 were exposed to non-structural-5A inhibitors. Sequencing results at the time of virological failure were available for 61 patients; sequencing at baseline was available for 35 of these patients. RESULTS: Among the 35 patients with sequencing results available at baseline, 15 had no resistance-associated substitution, 16 had only one resistance-associated substitution, and 4 had more than one resistance-associated substitution. Resistance-associated substitutions were harbored in 57% of the sequences in the non-structural-5A region. Among the 61 patients sequenced at virological failure, 50 (82%) patients presented at least one resistance-associated substitutions inducing a high level of resistance to non-structural-5A inhibitors (>10-fold resistance). CONCLUSIONS: This pooled analysis suggests that non-structural-5A resistance-associated substitutions screening should be recommended when considering retreatment with a non-structural-5A inhibitor regimen in patients who have previously experienced failed non-structural-5A treatment.

Functions of liver natural killer cells are dependent on the severity of liver inflammation and fibrosis in chronic hepatitis C.

During chronic hepatitis C virus (HCV) infection, the role of intra-hepatic (IH) natural killer (NK) cells is still controversial. To clarify their functions, we investigated anti-viral and cytotoxic activity of NK cells in human fresh liver biopsies. We compared the functions of IH-NK cells in HCV-infected and NASH patients in physiological conditions as well as after stimulation using flow cytometric and immunohistochemical analyses. Interestingly, few IH-NK cells produced anti-viral cytokine IFN-γ in HCV-infected patients similarly as in non-infected individuals. Spontaneous degranulation activity was extremely low in peripheral NK cells compared to IH-NK cells, and was significantly higher in IH-NK cells from HCV-infected patients compared to non-infected individuals. Immunohistochemical analysis revealed that perforin granules were polarized at the apical pole of IH-NK cells. The presence of CD107a and perforin in IH-NK cells demonstrated that NK cells exerted a cytolytic activity at the site of infection. Importantly, IH-NK cell functions from HCV-infected patients were inducible by specific exogenous stimulations. Upon ex vivo K562 target cell stimulations, the number of degranulating NK cells was significantly increased in the pool of IH-NK cells compared to circulating NK cells. Interestingly, after stimulation, the frequency of IFN-γ-producing IH-NK cells in HCV-infected patients was significantly higher at early stage of inflammation whereas the spontaneous IH-NK cell degranulation activity was significantly impaired in patients with highest inflammation and fibrosis Metavir scores. Our study highlights that some IH-NK cells in HCV-infected patients are able to produce INF-γ and degranulate and that those two activities depend on liver environment including the severity of liver injury. Thus, we conclude that critical roles of IH-NK cells have to be taken into account in the course of the liver pathogenesis associated to chronic HCV infection.

Lack of clinical significance of variability in the internal ribosome entry site of hepatitis C virus.

The extreme 5'-proximal sequence of the hepatitis C virus (HCV) genome including the 5' non-coding region (5'NCR) of 341 nucleotide long and the first 30 nucleotides of the core region is highly conserved among different HCV genotypes. It contains a segment termed Internal Ribosome Entry Site (IRES) that regulates the cap-independent translation of HCV-RNA to polyprotein. Sequence variability in this region has important implications for structural organisation and function of the IRES element and could correlate with HCV RNA concentration or response to antiviral therapy. Fourteen patients (seven women, seven men) with chronic hepatitis C were separated into two groups according to their response to antiviral therapy. Seven of these were sustained responders to treatment by Interferon alpha 2b and Ribavirin and seven were non-responders. After cloning-sequencing, the IRES (nt 21 to 374) appears to be structurally highly conserved. However some variability was found between the different isolates obtained: 209 substitutions with a median of four variants/patients. Comparison of the number of variants present in the viral population of the sustained responders and non-responders patients do not show any difference. 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. The translation initiator AUG-4 codon, located in the stem-loop IV, is never modified. Variations observed in the IRES are not a parameter of response to antiviral therapy, but the integrity of this region is a necessary condition to maintain its activity.