In vivo analysis at the cellular level reveals similar steatosis induction in both hepatitis C virus genotype 1 and 3 infections.

Steatosis is a frequent histological feature of hepatitis C virus (HCV) infection. Cohort studies of patients with chronic hepatitis C identified HCV genotype 3 (HCV GT3) as the prevalent steatotic genotype. Moreover, Huh-7 cells over-expressing HCV GT3 core protein accumulate more triglyceride in larger lipid droplets than cells expressing core proteins of other HCV genotypes. However, little is known about the relationship of steatosis and HCV infection at the cellular level in vivo. In this study, we used highly sensitive multiplex in situ hybridization methodology together with lipid staining to investigate HCV-induced lipid droplet accumulation at the cellular level in liver biopsies. Consistent with previous reports, histological steatosis grades were significantly higher in GT3 compared to GT1 infected livers, but independent of viral load. Using nile red lipid stainings, we observed that the frequency of lipid droplet containing cells was similar in HCV GT1- and HCV GT3-infected livers. Lipid droplet formation preferentially occurred in HCV-infected cells irrespective of the genotype, but was also observed in noninfected cells. These findings demonstrate that the main difference between GT1- and GT3-induced steatosis is the size of lipid droplets, but not the number or relative distribution of lipid droplets in infected vs uninfected hepatocytes.

Hepatitis B virus covalently closed circular DNA homeostasis is independent of the lymphotoxin pathway during chronic HBV infection.

Current treatment options for patients with chronic hepatitis B virus (HBV) infection are not curative as they are not effective in eliminating covalently closed circular DNA (cccDNA). cccDNA is a stable template for HBV transcription in the nucleus of hepatocytes and is thought to be one of the main factors responsible for HBV persistence. Recently, activation of the lymphotoxin beta receptor (LTßR) has been shown to trigger degradation of cccDNA through induction of cytidine deaminases of the APOBEC3 family in HBV cell culture model systems. To assess the presence and relevance of such mechanisms in the liver of chronically HBV-infected patients, we compared intrahepatic cccDNA levels with the expression levels of lymphotoxins and some of their target genes (eg APOBEC deaminases) in liver biopsy tissue. Our results confirm elevated gene expression levels of components of the lymphotoxin pathway including lymphotoxin alpha (LTα), lymphotoxin beta (LTß), APOBEC3B (A3B) and APOBEC3G (A3G) in the chronically HBV-infected liver compared to uninfected liver. Furthermore, expression levels of the genes of the APOBEC deaminase family were correlated with those of LTα and LTß gene expression, consistent with lymphotoxin-mediated upregulation of APOBEC gene expression. However, intrahepatic cccDNA and HBV replication levels were not correlated with LTα, LTß and APOBEC gene expression. In conclusion, these results suggest that although the lymphotoxin pathway is activated in the chronically HBV-infected liver, it has no major impact on HBV cccDNA metabolism in chronic HBV infection.

Limited hepatitis B virus replication space in the chronically hepatitis C virus-infected liver.

We compared the kinetics and magnitude of hepatitis B virus (HBV) infection in hepatitis C virus (HCV)-naive and chronically HCV-infected chimpanzees in whose livers type I interferon-stimulated gene (ISG) expression is strongly induced. HBV infection was delayed and attenuated in the HCV-infected animals, and the number of HBV-infected hepatocytes was drastically reduced. These results suggest that establishment of HBV infection and its replication space is limited by the antiviral effects of type I interferon in the chronically HCV-infected liver.

Human plasmacytoid dendritic cells sense lymphocytic choriomeningitis virus-infected cells in vitro.

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/ß) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).

Pathogenesis of hepatitis B virus infection.

The adaptive immune response is thought to be responsible for viral clearance and disease pathogenesis during hepatitis B virus infection. It is generally acknowledged that the humoral antibody response contributes to the clearance of circulating virus particles and the prevention of viral spread within the host while the cellular immune response eliminates infected cells. The T cell response to the hepatitis B virus (HBV) is vigorous, polyclonal and multispecific in acutely infected patients who successfully clear the virus and relatively weak and narrowly focussed in chronically infected patients, suggesting that clearance of HBV is T cell dependent. The pathogenetic and antiviral potential of the cytotoxic T lymphocyte (CTL) response to HBV has been proven by the induction of a severe necroinflammatory liver disease following the adoptive transfer of HBsAg specific CTL into HBV transgenic mice. Remarkably, the CTLs also purge HBV replicative intermediates from the liver by secreting type 1 inflammatory cytokines thereby limiting virus spread to uninfected cells and reducing the degree of immunopathology required to terminate the infection. Persistent HBV infection is characterized by a weak adaptive immune response, thought to be due to inefficient CD4+ T cell priming early in the infection and subsequent development of a quantitatively and qualitatively ineffective CD8+ T cell response. Other factors that could contribute to viral persistence are immunological tolerance, mutational epitope inactivation, T cell receptor antagonism, incomplete down-regulation of viral replication and infection of immunologically privileged tissues. However, these pathways become apparent only in the setting of an ineffective immune response, which is, therefore, the fundamental underlying cause. Persistent infection is characterized by chronic liver cell injury, regeneration, inflammation, widespread DNA damage and insertional deregulation of cellular growth control genes, which, collectively, lead to cirrhosis of the liver and hepatocellular carcinoma.

Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice.

We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-alpha/beta) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-alpha/beta inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA-containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-alpha/beta triggers intracellular events that either inhibit the assembly of pgRNA-containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte.