Sofía Pérez-del-Pulgar

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny, an Alternative to Current Methods

ABSTRACT Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Virology and Pathogenesis of Hepatitis C Virus Recurrence

1 In hepatitis C virus (HCV)–infected patients undergoing liver transplantation (LT), the virus infects the liver graft immediately after transplantation. The main source of HCV infection is circulating virions. Nevertheless, some data suggest that HCV present in extrahepatic compartments may contribute to HCV infection in some cases of hepatitis C recurrence. 2 Studies on early kinetics have shown that HCV replication starts a few hours after transplantation and that HCV‐RNA concentrations increase a few hours or days after the procedure, suggesting that HCV has an enormous ability to adapt to the new environment. 3 The quasispecies population may change significantly after transplantation, most likely because of the need to adapt to a new environment. There are no conclusive data supporting the role of HCV quasispecies composition and disease outcomes. 4 Persistence of HCV infection is the rule after transplantation. This is due to immunosuppression and to the immune exhaustion of the previously exposed immune system. 5 In general, HCV is not thought to be directly cytopathic. Thus, it is believed that the immune response against HCV causes liver damage. However, understanding the mechanisms of liver damage in HCV‐infected LT recipients is extremely complex because of the existence of a human leukocyte antigen–mismatched organ, the preexisting virus‐specific T cells that may be dysfunctional and/or tolerized, and the immunosuppression. 6 Despite the possible effect of immune‐mediated liver damage, it is clear that strong immunosuppression is associated with severe forms of hepatitis C recurrence (cholestatic hepatitis, fibrosing cholestatic hepatitis, and accelerated fibrosis progression). Thus, in the absence of a strong anti‐HCV immune response, HCV is able to directly (HCV proteins) or indirectly (cytokines) produce liver damage. 7 The activation of stellate cells and accelerated deposition of fibrosis are the final consequences of HCV infection in the graft. There are several mechanisms that may act synergistically to activate and perpetuate stellate cell activation in the setting of LT: ischemia‐reperfusion damage, old donor age, HCV proteins, cholestasis, rejection, infection with other viruses (cytomegalovirus), and immune‐mediated injury.

Hepatitis C virus receptors claudin-1 and occludin after liver transplantation and influence on early viral kinetics

Liver transplantation (LT) is a unique model to study hepatitis C virus (HCV) entry into hepatocytes. Recent in vitro studies suggest significant changes in the expression of the HCV receptors claudin‐1 and occludin after HCV infection. Our aims were: (1) to characterize claudin‐1 and occludin expression in grafts from LT recipients and (2) to explore their potential influence on early HCV kinetics and their changes after HCV infection. We included 42 HCV‐infected LT recipients and 19 uninfected controls. Claudin‐1 and occludin were detected in paraffin‐embedded liver biopsies obtained during reperfusion and 3 and 12 months after LT. HCV receptors were characterized by confocal immunofluorescence microscopy; quantification and colocalization studies were performed with dedicated software. Claudin‐1 and occludin expression were restricted to the apical pole of hepatocytes. There was a significant correlation between the amount of scavenger receptor B1 at the time of reperfusion and the HCV‐RNA decay during the first 24 hours following LT (r = 0.55, P = 0.007). Similarly, there was a significant correlation between the levels of claudin and occludin and the slope of HCV‐RNA increase during the first week after LT (r = 0.63, P = 0.005). Occludin and claudin‐1 levels increased significantly 12 months after LT (P = 0.03 and P = 0.007, respectively). The expression pattern of both proteins, however, remained unchanged, colocalizing strongly (60%‐94%) at the apical membrane of hepatocytes. Conclusions. HCV receptor levels at the time of LT seem to modulate early HCV kinetics. Hepatitis C recurrence after LT was associated with increased levels of claudin‐1 and occludin in the hepatocyte cell membrane, although it did not alter their localization within the tight junctions. (HEPATOLOGY 2011;.)

Concanavalin-A-induced liver injury is severely impaired in mice deficient in P-selectin

P‐selectin (CD62P) is an adhesion molecule that mediates the initial attachment of leukocytes to activated platelets and endothelial cells in damaged tissues. We evaluated the role of P‐selectin in concanavalin A (Con A)‐induced hepatitis, a model characterized by CD4+ T cell activation and infiltration of the liver. Con A injection induced transient P‐selectin expression on hepatic venules and platelets. Mice lacking P‐selectin showed impaired lymphocyte adhesion to liver venules and sinusoids, a striking reduction in intrasinusoidal occlusion, and decreased lymphocyte infiltration of liver parenchyma. The reduction in transaminase levels and the almost complete abolition of necrotic injury demonstrated that liver damage was lower in P‐selectin‐deficient mice. In wild‐type mice, pretreatment with the P‐selectin‐blocking monoclonal antibody attenuated the sinusoidal occlusion and reduced the rise in transaminases after Con A treatment. These results implicate P‐selectin in the development of Con A‐induced liver injury and reveal the protective effect of blocking P‐selectin in this hepatitis.

Determination of IL28B polymorphisms in liver biopsies obtained after liver transplantation

BACKGROUND & AIMS Recipient and donor IL28B polymorphisms seem to play an important role in the response to hepatitis C treatment after liver transplantation (LT). Since donor peripheral blood mononuclear cells (PBMC) are not always available, the aim of our study was to assess whether follow-up biopsies obtained after LT could be used to determine donor IL28B genotype. METHODS Genotyping of IL28B rs12979860 was performed by TaqMan real-time PCR and direct sequencing in 56 HCV-infected LT recipients and their donors. Liver biopsies were obtained at the moment of LT (reperfusion) and at any time when clinically indicated (follow-up). Direct sequencing always confirmed the real-time PCR results. RESULTS Genotyping of donor IL28B rs12979860 polymorphisms showed a 100% match both in PBMC and reperfusion biopsies. The frequency of IL28B rs12979860 polymorphisms differed significantly between donors and follow-up biopsies (p=0.024). We found an enrichment of the IL28B rs12979860 CT genotype (72%) in follow-up biopsies compared to donor samples (46%). Recipient alleles were clearly detected in 14 heterozygous follow-up samples: 10 CT/CC, 1 CT/TT, and 3 TT/CC (recipient/donor), thus reflecting a mixture of both donor and recipient genotypes. CONCLUSIONS Our results support that follow-up liver biopsies from LT recipients are not suitable for determining donor IL28B rs12979860 genotype by TaqMan real-time PCR or direct sequencing and that PBMC or reperfusion biopsies should be used instead. Thus, it is very important to obtain adequate samples in order to accurately determine the relative contributions of both donor and recipient.

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H386 and R408), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.

Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV.

Hepatitis C virus infection inhibits P-body granule formation in human livers

BACKGROUND & AIMS Decoding the myriad of interactions that hepatitis C virus (HCV) establishes with infected cells is mandatory to obtain a complete understanding of HCV biology and its associated pathogenesis. We and others have previously found that HCV infection disrupts the formation of P-bodies in cell culture. These are cytoplasmic RNA granules with key roles in post-transcriptional regulation of gene expression. Therefore, P-body disruption might have consequences beyond viral propagation. However, whether P-body disruption occurs also in vivo is unknown. Aim of this study was to address this important issue. METHODS Formalin-fixed paraffin-embedded liver biopsies from four groups of patients (healthy donors, patients with non-virus related liver inflammation, HCV- and HBV-infected patients) were immunostained to detect DDX6 and Dcp1, two core P-body components. Changes in the localization of these proteins were assessed by confocal microscopy. RESULTS HCV specifically inhibited P-body formation in hepatocytes from human livers regardless of viral genotype, inflammation grade or whether the infection was recent or long established. Importantly, this alteration was reversed once HCV was eliminated by therapy. Furthermore, we observed in vivo an unexpected heterogeneity in P-body composition, which might reflect functional specializations. CONCLUSIONS This is the first comprehensive in vivo P-body analysis that links a pathogenic condition to P-body alterations. Because of their role in gene expression, the alteration of P-bodies should be further studied to understand fully complex HCV-associated pathologies.

HEV infection in two referral centers in Spain: epidemiology and clinical outcomes.

BACKGROUND AND OBJECTIVES Hepatitis E virus (HEV) is one of the major causes of icteric hepatitis worldwide. In industrialized countries it is considered an emerging disease, as a growing number of autochthonous cases have been reported in recent years. Occasional extrahepatic manifestations have been described in the setting of HEV infection. STUDY DESIGN To characterize the epidemiological pattern and clinical outcomes of new cases of HEV infection diagnosed in two referral centers during the period 2011-2013. RESULTS During the study period, four cases of self-limited acute hepatitis E after travel to endemic areas were recorded, as well as five cases of HEV infection after solid organ transplantation. Four patients failed to spontaneously clear the virus and received ribavirin monotherapy; all of them had HEV genotype-3. Ribavirin was effective in inhibiting HEV replication, although in one patient a virological relapse occurred after the end of therapy. Finally, we report a case of HEV-genotype-3 related agranulocytosis in an immunocompetent patient, resulting in a fatal outcome; this is the first case reported of its kind. CONCLUSION Diagnosis of HEV infection needs to be taken into consideration in patients with acute or chronic hepatitis in whom other etiologies have been excluded. Although hematological complications related to acute HEV infection are infrequent, these may affect any of the bone marrow series, even after viral clearance.

Selective Inhibition of Hepatitis C Virus Infection by Hydroxyzine and Benztropine

ABSTRACT Hepatitis C virus (HCV) infection is a major biomedical problem worldwide as it causes severe liver disease in millions of humans around the world. Despite the recent approval of specific drugs targeting HCV replication to be used in combination with alpha interferon (IFN-α) and ribavirin, there is still an urgent need for pangenotypic, interferon-free therapies to fight this genetically diverse group of viruses. In this study, we used an unbiased screening cell culture assay to interrogate a chemical library of compounds approved for clinical use in humans. This system enables identifying nontoxic antiviral compounds targeting every aspect of the viral life cycle, be the target viral or cellular. The aim of this study was to identify drugs approved for other therapeutic applications in humans that could be effective components of combination therapies against HCV. As a result of this analysis, we identified 12 compounds with antiviral activity in cell culture, some of which had previously been identified as HCV inhibitors with antiviral activity in cell culture and had been shown to be effective in patients. We selected two novel HCV antivirals, hydroxyzine and benztropine, to characterize them by determining their specificity and genotype spectrum as well as by defining the step of the replication cycle targeted by these compounds. We found that both compounds effectively inhibited viral entry at a postbinding step of genotypes 1, 2, 3, and 4 without affecting entry of other viruses.