Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

AbstractHCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection

Hepatitis C Virus Infection Suppresses the Interferon Response in the Liver of the Human Hepatocyte Chimeric Mouse

BACKGROUND AND AIMS: Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. METHODS: Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. RESULTS: HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16∼3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10(-10)∼1.95×10(-2)). CONCLUSIONS: These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy

Long-term elimination of hepatitis C virus from human hepatocyte chimeric mice after interferon-γ gene transfer.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment

Thromboxane A2 synthase inhibitors prevent production of infectious hepatitis C virus in mice with humanized livers.

[Background & Aims]A 3-dimensional (3D) culture system for immortalized human hepatocytes (HuS-E/2 cells) recently was shown to support the lifecycle of blood-borne hepatitis C virus (HCV). We used this system to identify proteins that are active during the HCV lifecycle under 3D culture conditions. [Methods]We compared gene expression profiles of HuS-E/2 cells cultured under 2-dimensional and 3D conditions. We identified signaling pathways that were activated differentially in the cells, and analyzed their functions in the HCV lifecycle using a recombinant HCV-producing cell-culture system, with small interfering RNAs and chemical reagents. We investigated the effects of anti-HCV reagents that altered these signaling pathways in mice with humanized livers (carrying human hepatocytes). [Results]Microarray analysis showed that cells cultured under 2-dimensional vs 3D conditions expressed different levels of messenger RNAs encoding prostaglandin synthases. Small interfering RNA–mediated knockdown of thromboxane A2 synthase (TXAS) and incubation of hepatocytes with a TXAS inhibitor showed that this enzyme is required for production of infectious HCV, but does not affect replication of the HCV genome or particle release. The TXAS inhibitor and a prostaglandin I2 receptor agonist, which has effects that are opposite those of thromboxane A2, reduced serum levels of HCV and inhibited the infection of human hepatocytes by blood-borne HCV in mice. [Conclusions]An inhibitor of the prostaglandin synthase TXAS inhibits production of infectious HCV particles in cultured hepatocytes and HCV infection of hepatocytes in mice with humanized livers. It therefore might be therapeutic for HCV infection

Absence of viral interference and different susceptibility to interferon between hepatitis B virus and hepatitis C virus in human hepatocyte chimeric mice

Background/Aims: Both hepatitis B virus (HBV) and hepatitis C virus (HCV) replicate in the liver and show resistance against innate immunity and interferon (IFN) treatment. Whether there is interference between these two viruses is still controversial. We investigated the interference between these two viruses and the mode of resistance against IFN. Methods: We performed infection experiments with either or both of the two hepatitis viruses in human hepatocyte chimeric mice. Huh7 cell lines with stable production of HBV were also established and transfected with HCV JFH1 clone. Mice and cell lines were treated with IFN. The viral levels in mice sera and culture supernatants and messenger RNA levels of IFN-stimulated genes were measured. Results: No apparent interference between the two viruses was seen ill vivo. Only a small (0.3 log) reduction in serum HBV and a rapid reduction in HCV were observed after IFN treatment, regardless of infection with the other virus. In ill vitro studies, no interference between the two viruses was observed. The effect of IFN on each virus was not affected by the presence of the other virus. IFN-induced reductions of viruses in culture supernatants were similar to those in ill vivo study. Conclusions: No interference between the two hepatitis viruses exists in the liver in the absence of hepatitis. The mechanisms of IFN resistance of the two viruses target different areas of the IFN system

Amphipathic DNA Polymers Inhibit Hepatitis C Virus Infection by Blocking Viral Entry

International audienceBACKGROUND & AIMS: Hepatitis C virus (HCV) gains entry into susceptible cells by interacting with cell surface receptor(s). Viral entry is an attractive target for antiviral development because of the highly conserved mechanism.METHODS: HCV culture systems were used to study the effects of phosphorothioate oligonucleotides (PS-ONs), as amphipathic DNA polymers (APs), on HCV infection. The in vivo effects of APs were tested in urokinase plasminogen activator (uPA)/severe combined immunodeficient (SCID) mice engrafted with human hepatocytes.RESULTS: We show the sequence-independent inhibitory effects of APs on HCV infection. APs were shown to potently inhibit HCV infection at submicromolar concentrations. APs exhibited a size-dependent antiviral activity and were equally active against HCV pseudoparticles of various genotypes. Control phosphodiester oligonucleotide (PO-ON) polymer without the amphipathic structure was inactive. APs had no effect on viral replication in the HCV replicon system or binding of HCV to cells but inhibited viral internalization, indicating that the target of inhibition is at the postbinding, cell entry step. In uPA/SCID mice engrafted with human hepatocytes, APs efficiently blocked de novo HCV infection.CONCLUSIONS: Our results demonstrate that APs are a novel class of antiviral compounds that hold promise as a drug to inhibit HCV entry. Hepatitis C virus (HCV) infects approximately 200 million people worldwide. 1 The majority of HCV-infected patients fails to clear the virus, and many develop chronic liver disease including cirrhosis with a risk of hepatocellular carcinoma. Treatment of chronic hepatitis

Modeling suggests that virion production cycles within individual cells is key to understanding acute hepatitis B virus infection kinetics

Hepatitis B virus (HBV) infection kinetics in immunodeficient mice reconstituted with humanized livers from inoculation to steady state is highly dynamic despite the absence of an adaptive immune response. To recapitulate the multiphasic viral kinetic patterns, we developed an agent-based model that includes intracellular virion production cycles reflecting the cyclic nature of each individual virus lifecycle. The model fits the data well predicting an increase in production cycles initially starting with a long production cycle of 1 virion per 20 hours that gradually reaches 1 virion per hour after approximately 3–4 days before virion production increases dramatically to reach to a steady state rate of 4 virions per hour per cell. Together, modeling suggests that it is the cyclic nature of the virus lifecycle combined with an initial slow but increasing rate of HBV production from each cell that plays a role in generating the observed multiphasic HBV kinetic patterns in humanized mice

Common Variation of IL28 affects Gamma-GTP Levels and Inflammation of the Liver in Chronically Infected Hepatitis C Virus Patients

Background & Aims: A common genetic variation at the IL28 locus has been found to affect the response of peg-interferon and ribavirin combination therapy against chronic hepatitis C virus (HCV) infection. An allele associated with a favorable response (rs8099917 T), which is the major allele in the majority of Asian, American, and European populations, has also been found to be associated with spontaneous eradication of the virus. Methods: As no studies have yet analyzed the effect of the polymorphism on biochemical and inflammatory changes in chronic infection, we analyzed a cohort of patients with chronic hepatitis C (n = 364) for the effect of the IL28 polymorphism on viral, biochemical, and histological findings. Results: We found that the proportion of HCV wild type core amino acids 70 and 91 was significantly greater (p = 1.21 × 10-4 and 0.034) and levels of gamma-GTP significantly lower (p = 0.001) in patients homozygous for the IL28 major allele. We also found that inflammation activity and fibrosis of the liver were significantly more severe in patients homozygous for the IL28 major allele (p = 0.025 and 0.036, respectively). Although the higher gamma-GTP levels were also associated with higher inflammatory activity and fibrosis, multivariate analysis showed that only the IL28 allele polymorphism, sex, alcohol consumption, and liver fibrosis were independently associated with gamma-GTP levels (p = 0.001, 0.0003, 0.0013, and 0.0348, respectively). Conclusions: These results suggest that different cytokine profiles induced by the IL28 polymorphism resulted in different biochemical and inflammatory conditions during chronic HCV infection and contribute to the progression of liver diseases

Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients

A First Case of Hepatic Angiosarcoma Treated with Recombinant Interleukin-2

A 60 year-old woman was admitted to our hospital because of management of multiple liver tumors. According to image findings and liver biopsy, she was diagnosed as having epithelioid hemangioendothelioma of the liver accompanied by metastases in the spleen, lungs and bones. Based on the spread of the liver tumors and the extensive systemic metastases, she was considered inoperable. Instead, she received hepatic arterial infusion therapy using recombinant interleukin-2. However, she died due to liver failure about two months after admission. Autopsy revealed that the liver tumor was angiosarcoma. It is difficult to differentiate angiosarcoma from epithelioid hemangioendothelioma based on the image findings and pathological findings of percutaneous liver biopsy. Many cases are diagnosed as angiosarcoma at autopsy. There is no established effective treatment for hepatic angiosarcoma, because the tumor stage at the time of diagnosis is often progressive. To date, immunotherapy with recombinant interleukin-2 has been reported to be effective clinically for cutaneous angiosarcoma, such as of the scalp and facial skin. To our knowledge, there have been no reported cases of hepatic angiosarcoma treated with recombinant interleukin-2. Our case is important should recombinant interleukin-2 be considered effective for hepatic angiosarcoma in the future