High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease.

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.

Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation.

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a scavenger receptor class B type I antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV-infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received 150 mg of ITX5061 immediately before and after transplant and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing (UDPS). ITX5061 was well tolerated with measurable plasma concentrations during therapy. Although the median HCV RNA reduction was greater in ITX-treated patients at all time points in the first week after transplantation, there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype (GT) 1-infected patients, treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, P = 0.004). UDPS revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group. In conclusion, ITX5061 reduces plasma HCV RNA after transplant notably in GT 1-infected patients and slows viral evolution. Following liver transplantation, the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies.

CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism.

INTRODUCTION: CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. OBJECTIVE: To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. DESIGN: CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. RESULTS: Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-ß at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. CONCLUSIONS: Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.