scholarly journals Hepatitis C virus infection of primary tupaia hepatocytes leads to selection of quasispecies variants, induction of interferon-stimulated genes and NF-κB nuclear translocation

2005 ◽  
Vol 86 (11) ◽  
pp. 3065-3074 ◽  
Author(s):  
Anunciata Guitart ◽  
José-Ignacio Riezu-Boj ◽  
Edurne Elizalde ◽  
Esther Larrea ◽  
Carmen Berasain ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Semliki Forest Virus ◽  
Nuclear Translocation ◽  
Natural Infection ◽  
Hcv Infection ◽  
Interferon Response ◽  
Cell Phenotype

Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus–cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-κB in primary hepatocytes and upregulated NF-κB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell.

scholarly journals Expanding the Host Range of Hepatitis C Virus through Viral Adaptation

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Markus von Schaewen ◽  
Marcus Dorner ◽  
Kathrin Hueging ◽  
Lander Foquet ◽  
Sherif Gerges ◽  
...  
Keyword(s):  
Animal Model ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Envelope ◽  
Hcv Infection ◽  
Type I ◽  
Viral Adaptation ◽  
Mouse Hepatocytes

ABSTRACTHepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytesin vivoandin vitro. Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytesin vivoin the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV’s ability to enter murine hepatocytesin vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C.IMPORTANCEAt least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.

scholarly journals Hepatitis C Virus Enhances the Invasiveness of Hepatocellular Carcinoma via EGFR-Mediated Invadopodia Formation and Activation

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1395 ◽  
Author(s):  
Ninio ◽  
Nissani ◽  
Meirson ◽  
Domovitz ◽  
Genna ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tyrosine Phosphatase ◽  
Hcv Infection ◽  
Invasive Cancer ◽  
Cell Protein ◽  
Invadopodia Formation

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer.

scholarly journals Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

2005 ◽  
Vol 79 (9) ◽  
pp. 5774-5785 ◽  
Author(s):  
Heidi Barth ◽  
Raffaele Cerino ◽  
Mirko Arcuri ◽  
Marco Hoffmann ◽  
Peter Schürmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Scavenger Receptor ◽  
Hcv Infection ◽  
Type I ◽  
Scavenger Receptor Class ◽  
Class B

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.

scholarly journals Natural Killer Cell Function and Dysfunction in Hepatitis C Virus Infection

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Kayla A. Holder ◽  
Rodney S. Russell ◽  
Michael D. Grant
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Natural Killer ◽  
Nk Cell ◽  
Hcv Infection ◽  
Host Immune System ◽  
Chronic Hcv Infection ◽  
Chronic Hcv

Viruses must continually adapt against dynamic innate and adaptive responses of the host immune system to establish chronic infection. Only a small minority (~20%) of those exposed to hepatitis C virus (HCV) spontaneously clear infection, leaving approximately 200 million people worldwide chronically infected with HCV. A number of recent research studies suggest that establishment and maintenance of chronic HCV infection involve natural killer (NK) cell dysfunction. This relationship is illustratedin vitroby disruption of typical NK cell responses including both cell-mediated cytotoxicity and cytokine production. Expression of a number of activating NK cell receptorsin vivois also affected in chronic HCV infection. Thus, directin vivoandin vitroevidence of compromised NK function in chronic HCV infection in conjunction with significant epidemiological associations between the outcome of HCV infection and certain combinations of NK cell regulatory receptor and class I human histocompatibility linked antigen (HLA) genotypes indicate that NK cells are important in the immune response against HCV infection. In this review, we highlight evidence suggesting that selective impairment of NK cell activity is related to establishment of chronic HCV infection.

scholarly journals Differential reactivity of putative genotype 2 hepatitis C virus F protein between chronic and recovered infections

2008 ◽  
Vol 89 (8) ◽  
pp. 1890-1900 ◽  
Author(s):  
Wing Chia-Ming Chuang ◽  
Jean-Pierre Allain
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Cross Reactivity ◽  
Hcv Infection ◽  
Genotype 3 ◽  
F Protein ◽  
Genotype 2

To date, all studies regarding hepatitis C virus (HCV) F protein have been based on expression in vitro/in vivo of recombinant protein or monoclonal antibodies derived from genotype 1a or 1b sequences, but not from other genotypes. The objective of this study was to prepare a putative genotype 2 recombinant F protein and evaluate its reactivity in plasma from individuals with chronic HCV infection or who had recovered from infection. One genotype 2 strain was selected for F protein (F-2) and core expression in bacterial culture. An ELISA was developed and applied to samples from patients with chronic infection or recovered infection of various genotypes. The anti-F-2 response in 117 samples showed a significantly higher reactivity in chronic than in recovered HCV-infected blood donors (P<0.001), but no difference was found among genotypes. However, the correlation between anti-F and anti-core was more significant in genotypes 1 and 2 than in genotype 3. Anti-F-2 titres were also significantly higher in chronic than in recovered individuals (P<0.0001). Antibody titres to recombinant genotype 2 core protein or to genotype 1 multiple proteins used in commercial anti-HCV assays paralleled the anti-F-2 end-point antibody titre. This study thus demonstrated the antigenicity of genotype 2 HCV F protein, although the exact location of the natural frameshift position remains unknown. The difference in anti-F-2 response between chronic and recovered infection, the cross-reactivity irrespective of genotype and the correlation of antibody response with structural and non-structural antigens suggest that the immune response to F protein is an integral part of the natural HCV infection.

scholarly journals Griffithsin Has Antiviral Activity against Hepatitis C Virus

2011 ◽  
Vol 55 (11) ◽  
pp. 5159-5167 ◽  
Author(s):  
Philip Meuleman ◽  
Anna Albecka ◽  
Sandrine Belouzard ◽  
Koen Vercauteren ◽  
Lieven Verhoye ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Envelope ◽  
Hcv Infection ◽  
Liver Graft ◽  
Chimeric Mice ◽  
Content Type

ABSTRACTHepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluatedin vitrousing the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infectionin vivowas evaluated using uPA+/+-SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infectionin vitroand mitigate HCV infectionin vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.

scholarly journals Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

2008 ◽  
Vol 89 (9) ◽  
pp. 2108-2113 ◽  
Author(s):  
Takashi Kimura ◽  
Michio Imamura ◽  
Nobuhiko Hiraga ◽  
Tsuyoshi Hatakeyama ◽  
Daiki Miki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Small Animal ◽  
Genetically Engineered ◽  
Human Hepatocyte ◽  
Hcv Infection ◽  
Chimeric Mice ◽  
Genotype 1B

The establishment of clonal infection of hepatitis C virus (HCV) in a small-animal model is important for the analysis of HCV virology. A previous study developed models of molecularly cloned genotype 1a and 2a HCV infection using human hepatocyte-transplanted chimeric mice. This study developed a new model of molecularly cloned genotype 1b HCV infection. A full-length genotype 1b HCV genome, HCV-KT9, was cloned from a serum sample from a patient with severe acute hepatitis. The chimeric mice were inoculated intrahepatically with in vitro-transcribed HCV-KT9 RNA. Inoculated mice developed viraemia at 2 weeks post-infection, and this persisted for more than 6 weeks. Passage experiments indicated that the sera of these mice contained infectious HCV. Interestingly, a similar clone, HCV-KT1, in which the poly(U/UC) tract was 29 nt shorter than in HCV-KT9, showed poorer in vivo infectivity and replication ability. An in vitro study showed that no virus was produced in the culture medium from HCV-KT9-transfected cells. In conclusion, this study developed a genetically engineered genotype 1b HCV-infected mouse. This mouse model will be useful for the study of HCV virology, particularly the mechanism underlying the variable resistance of HCV genotypes to interferon therapy.

Funktion von CEACAM-1 im Rahmen der Therapie der Hepatitis C-Virus Infektion mit Interferon-alpha in vivo und in vitro

2006 ◽  
Vol 44 (08) ◽  
Author(s):  
P Hilgard ◽  
R Bröring ◽  
M Trippler ◽  
S Viazov ◽  
G Gerken ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Interferon Alpha

scholarly journals Mass Balance, Metabolite Profile, andIn Vitro-In VivoComparison of Clearance Pathways of Deleobuvir, a Hepatitis C Virus Polymerase Inhibitor

2014 ◽  
Vol 59 (1) ◽  
pp. 25-37 ◽  
Author(s):  
Lin-Zhi Chen ◽  
John P. Sabo ◽  
Elsy Philip ◽  
Lois Rowland ◽  
Yan Mao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mass Balance ◽  
Metabolite Profile ◽  
Fecal Samples ◽  
Acyl Glucuronide ◽  
Polymerase Inhibitor ◽  
Main Components

ABSTRACTThe pharmacokinetics, mass balance, and metabolism of deleobuvir, a hepatitis C virus (HCV) polymerase inhibitor, were assessed in healthy subjects following a single oral dose of 800 mg of [14C]deleobuvir (100 μCi). The overall recovery of radioactivity was 95.2%, with 95.1% recovered from feces. Deleobuvir had moderate to high clearance, and the half-life of deleobuvir and radioactivity in plasma were ∼3 h, indicating that there were no metabolites with half-lives significantly longer than that of the parent. The most frequently reported adverse events (in 6 of 12 subjects) were gastrointestinal disorders. Two major metabolites of deleobuvir were identified in plasma: an acyl glucuronide and an alkene reduction metabolite formed in the gastrointestinal (GI) tract by gut bacteria (CD 6168), representing ∼20% and 15% of the total drug-related material, respectively. Deleobuvir and CD 6168 were the main components in the fecal samples, each representing ∼30 to 35% of the dose. The majority of the remaining radioactivity found in the fecal samples (∼21% of the dose) was accounted for by three metabolites in which deleobuvir underwent both alkene reduction and monohydroxylation. In fresh human hepatocytes that form biliary canaliculi in sandwich cultures, the biliary excretion for these excretory metabolites was markedly higher than that for deleobuvir and CD 6168, implying that rapid biliary elimination upon hepatic formation may underlie the absence of these metabolites in circulation. The lowin vitroclearance was not predictive of the observedin vivoclearance, likely because major deleobuvir biotransformation occurred by non-CYP450-mediated enzymes that are not well represented in hepatocyte-basedin vitromodels.

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