Robust hepatitis C virus infection<i>in vitro</i>

Zhong, Jin; Gastaminza, Pablo; Cheng, Guofeng; Kapadia, Sharookh B.; Kato, Takanobu; Burton, Dennis R.; Wieland, Stefan; Uprichard, Susan L.; Wakita, Takaji; Chisari, Francis V.

doi:10.1073/pnas.0503596102

Cited by 1,614 publications

(1,739 citation statements)

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“…The lack of an efficient viral culture system and a small animal infection model has hampered HCV researches (2). However, the production of infectious HCV in tissue culture was established recently from a cloned JFH-1 viral genome and has been widely applied in many labs around the world, thus, playing an important role in HCV related researches and drug discovery (3)(4)(5).…”

Section: Introductionmentioning

confidence: 99%

Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

Liu

Ali

et al. 2009

Cell Mol Immunol

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L-ficolin, one of lectin families, is a recently identified complement factor that initiates lectin pathway of complement. Little is known about its role in viral hepatitis. In the present study, we found that L-ficolin in serum from 103 patients with hepatitis C virus (HCV), were significantly higher than that in 150 healthy controls. We further found that L-ficolin expressions were significantly increased in vitro study by HCV JFH-1 infected human hepatocyte cell line Huh7.5.

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Section: Introductionmentioning

confidence: 99%

Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

Liu

Ali

et al. 2009

Cell Mol Immunol

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“…1a). To confirm the competency of this new plasmid, we electroporated Huh7.5.1 cells, which are known to well support HCV replication (13), with pRHIAG and observed expression of both fluorescent proteins in individual cells by fluorescent microscopy (Fig. 2a).…”

Section: New Reporter System To Evaluate Hcv Ires Activitymentioning

confidence: 99%

“…Huh7.5.1 (human hepatic cell line, (13)) and HEK293 (human embryo kidney cell line, (14)) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen), 100 lg/mL of kanamycin (SigmaAldrich), and non-essential amino acids (Invitrogen). Plasmid DNA was delivered to Huh7.5.1 and HEK293 cells by electropration with a Microporator MP-100 (Digital Bio, Seoul, Korea).…”

Section: Cell Culture and Electroporationmentioning

confidence: 99%

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

et al. 2011

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The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5 0 -end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs. ' 2011 International Society for Advancement of Cytometry Key terms hepatitis C virus; internal ribosome entry site; DsRed2; Azami Green; flow cytometry HEPATITIS C virus (HCV) is a major causative agent of liver cirrhosis and hepatocellular carcinoma (1). The HCV genome is a plus-strand RNA consisting of $9,600 nucleotides (nt) and has a single long open reading frame encoding a polyprotein of about 3,000 amino acids (2). Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5-7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.The HCV IRES activity has been studied, by using biochemical methods with luciferase genes as reporters (8-10), which have some flaws resulting from using cell lysates. In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid. To

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“…27 Transfection of Huh7 and Huh7.5.1 cells with the in vitro-transcribed fulllength JFH-1 genome or a recombinant chimeric genome with another genotype 2a isolate, J6, resulted in the secretion of viral particles that were infectious in cultured cells, in chimeric mice, and in chimpanzees. [28][29][30][31] The infectivity of cells could be neutralized with antibodies against the HCV entry receptor CD81, antibodies against E2, or immunoglobulins from chronically infected patients. Importantly, the replication of cell-cultured HCV in this system was inhibited by IFN-␣ as well as by several HCVspecific antiviral compounds.…”

Section: Cell-based In Vitro Hcv Systemsmentioning

confidence: 99%

Experimental models for hepatitis C viral infection #

Boonstra¹,

Laan²,

Vanwolleghem³

et al. 2009

Hepatology

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Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The majority of infected individuals develop a persistent infection, which is associated with a high risk of liver cirrhosis and hepatocellular carcinoma. Since its discovery 20 years ago, progress in our understanding of this virus has been suboptimal due to the lack of good model systems. However, in the past decade this has greatly accelerated with the development of various in vitro cell culture systems and in vivo small-animal models. These systems have made a major impact on the field of HCV research, and have provided important breakthroughs in our understanding of HCV infection and replication. Importantly, the in vitro cell culture systems and the small-animal models have allowed preclinical testing of numerous novel antiviral compounds for the treatment of chronic HCV infection. In this article, we give an overview of current models, discuss their limitations, and provide future perspectives for research directed at the prevention and cure of hepatitis C. (HEPATOLOGY 2009;50:1646-1655.) Hepatitis C Virus Pathology and BiologyThe hepatitis C virus (HCV) is considered a successful pathogen in establishing persistent infections by evading the immune system. Only a fraction of acutely infected individuals are able to clear the infection spontaneously, whereas about 80% of the infected individuals develop a chronic infection. 1,2 The symptoms are initially mild in these persistently infected patients, and it may take decades before the serious consequences of chronic HCV infection become apparent. Patients with chronic HCV are at increased risk for developing liver fibrosis, cirrhosis, and/or hepatocellular carcinoma. Currently, these longterm complications of chronic HCV infection are the leading indication for liver transplantation. 3,4 Because of the high incidence of new infections by blood transfusions in the 1980s before discovery of the virus, and because morbidity associated with chronic HCV infections generally takes decades to develop, it is expected that the burden of disease in the near future will rise dramatically.HCV is an enveloped flavivirus, with a positivestranded RNA genome of approximately 9600 nucleotides and is composed of a single open reading frame, which encodes a polyprotein precursor of approximately 3000 amino acids. The coding region is flanked by 5Ј and 3Ј noncoding regions, which are important for the regulation of genomic duplication as well as initiation of translation. The single polyprotein is cleaved by host and viral proteases into individual structural and nonstructural (NS) proteins (Fig. 1A). Replication of the HCV genome involves the synthesis of a full-length negative-stranded RNA intermediate, which in turn provides a template for the de novo production of positive-stranded RNA. Both these synthesis steps are mediated by the viral RNA-dependent RNA polymerase NS5B. 5-7 NS5B lacks proofreading abilities, and this leads to a high mutation rate and the generation of numerous quasispecies. ...

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Robust hepatitis C virus infectionin vitro

Cited by 1,614 publications

References 54 publications

Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

Experimental models for hepatitis C viral infection #

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