Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo

A hepatitis A virus deletion mutant which lacks the first pyrimidine-rich tract of the 5' nontranslated RNA remains virulent in primates after direct intrahepatic nucleic acid transfection.

Cell culture-adapted variants of hepatitis A virus (HAV) in which the first pyrimidine-rich tract (pY1; nucleotides 99 to 138) of the 5' nontranslated region has been deleted (delta 96-137 or delta 96-139) replicate as well as parental virus in cultured cells (D.R. Shaffer, E.A. Brown, and S.M. Lemon, J. Virol. 68:5568-5578, 1994). To determine whether viruses with such large deletion mutations are able to replicate and to produce acute hepatitis in primates, we reconstructed the delta 96-137 deletion in the genetic background of a virulent virus which differs from the wild type by only one mutation in the 2B-coding region (HM175/8Y). Full-length synthetic delta 96-137/8Y RNA was injected into the livers of two HAV-seronegative marmosets (Saguinus mystax). Both animals developed serum liver enzyme elevations and inflammatory changes in serial liver biopsies within 3 to 4 weeks of inoculation which were comparable in magnitude to those observed previously following intrahepatic inoculation of marmosets with HM175/8Y RNA. Sequencing of RNA from virus shed in feces demonstrated the presence of the delta 96-137 deletion. These results indicate that the pY1 sequence of HAV is not required for efficient viral replication in hepatocytes in situ or for production of acute hepatic injury following intrahepatic RNA transfection in primates

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol

Prognostic value of Ishak fibrosis stage: Findings from the hepatitis C antiviral long-term treatment against cirrhosis trial

Studies of the prognostic value of Ishak fibrosis stage are lacking. We used multi-year follow-up of the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) Trial to determine whether individual Ishak fibrosis stages predicted clinical outcomes in patients with chronic hepatitis C. Baseline liver biopsy specimens from 1050 patients with compensated chronic hepatitis C who had failed combination peginterferon and ribavirin were reviewed by a panel of expert hepatopathologists. Fibrosis was staged with the Ishak scale (ranging from 0 = no fibrosis to 6 = cirrhosis). Biopsy fragmentation and length as well as number of portal tracts were recorded. We compared rates of prespecified clinical outcomes of hepatic decompensation and hepatocellular carcinoma across individual Ishak fibrosis stages. Of 1050 biopsy specimens, 25% were fragmented, 63% longer than 1.5 cm, 69% larger than 10 mm 2 , and 75% had 10 or more portal tracts. Baseline laboratory markers of liver disease severity were worse and the frequency of esophageal varices higher with increasing Ishak stage ( P < 0.0001). The 6-year cumulative incidence of first clinical outcome was 5.6% for stage 2, 16.1% for stage 3, 19.3% for stage 4, 37.8% for stage 5, and 49.3% for stage 6. Among nonfragmented biopsy specimens, the predictive ability of Ishak staging was enhanced; however, no association was observed between Ishak stage and outcomes for fragmented biopsy specimens because of high rates of outcomes for patients with noncirrhotic stages. Similar results were observed with liver transplantation or liver-related death as the outcome. Conclusion : Ishak fibrosis stage predicts clinical outcomes, need for liver transplantation, and liver-related death in patients with chronic hepatitis C. Patients with fragmented biopsy specimens with low Ishak stage may be understaged histologically. (H EPATOLOGY 2010;51:585–594.)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64929/1/23315_ftp.pd

Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus - associated hepatocellular carcinoma

Abstract Background The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. Methods We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence. Results Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. Conclusions Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets