Hepatitis C Virus Genotype 1a Growth and Induction of Autophagy

ABSTRACT We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.

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Transforming Growth Factor β Acts as a Regulatory Molecule for Lipogenic Pathways among Hepatitis C Virus Genotype-Specific Infections

Journal of Virology ◽

10.1128/jvi.00811-19 ◽

2019 ◽

Vol 93 (18) ◽

Cited By ~ 2

Author(s):

Tapas Patra ◽

Reina Sasaki ◽

Keith Meyer ◽

Ratna B. Ray ◽

Ranjit Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Peroxisome Proliferator ◽

Virus Genotype ◽

Peroxisome Proliferator Activated Receptor ◽

Hcv Genotype ◽

Genotype 1A

ABSTRACT Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor β (TGF-β)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-β1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-β and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-β was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-β-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders. IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic hepatitis C virus (HCV) infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that transforming growth factor β (TGF-β) and peroxisome proliferator-activated receptor alpha (PPARα)-associated mechanistic pathways in hepatocytes infected with HCV genotype 2a and 3a differ from those in cells infected with genotype 1a. The results suggest that a targeted therapeutic approach for enhanced PPARα and lipolysis may reduce HCV genotype-associated lipid metabolic disorder in liver disease.

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Hepatitis C Virus Infection Induces the Beta Interferon Signaling Pathway in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.01695-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12375-12381 ◽

Cited By ~ 33

Author(s):

Tatsuo Kanda ◽

Robert Steele ◽

Ranjit Ray ◽

Ratna B. Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Signaling Pathway ◽

Human Hepatocytes ◽

Hcv Infection ◽

Oligoadenylate Synthetase ◽

Hcv Genotype ◽

Beta Interferon ◽

Genotype 1A

ABSTRACT Beta interferon (IFN-β) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-β signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-β and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-β and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-β and 2′,5′-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.

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Role of hepatitis C virus core antigen assay in hepatitis C care in developing country

Egyptian Liver Journal ◽

10.1186/s43066-021-00146-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ouafa Kallala ◽

Saoussen Kacem ◽

Imene Fodha ◽

Bruno Pozzetto ◽

Trabelsi Abdelhalim

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Developing Country ◽

Predictive Value ◽

Gold Standard ◽

Hcv Infection ◽

Core Antigen ◽

Hcv Rna ◽

Hcv Genotype ◽

Rna Levels

Abstract Background The World Health Organization (WHO) aims to achieve global hepatitis C elimination by 2030, defined as diagnosis of 90% of infected individuals and treating 80% of them. Current guidelines for the screening and diagnosis of hepatitis C infection denote using a relatively cheap screen with anti-hepatitis C virus (HCV) antibody immunoassay, followed by the much costlier molecular test for HCV RNA levels using polymerase chain reaction (PCR) assay to confirm active HCV infection. Simplification of the HCV evaluation algorithm to reduce the number of required tests could considerably expand the provision of HCV treatment especially in a developing country. This study investigates the performance of hepatitis C Core Antigen (HCV Ag) test by comparing HCV Ag results versus the results obtained with HCV ribonucleic acid (RNA) PCR which is considered the gold standard for the diagnosis of HCV infection. Results Among the 109 anti-HCV positive sera, 96 were positive for both HCV Ag (> 3 fmol/L) and HCV RNA (> 15 IU/mL); 8 were negative for both tests, while the remaining 5 were positive for HCV RNA only. Considering the HCV RNA as gold standard; the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HCV Ag test were found to be 95.05%, 100%, 100%, and 61.54%, respectively. HCV genotype was performed for 59 patients. The most common HCV genotype was genotype 1 (72.9%). Genotype 2 (15.3%) and genotype 3 (11.9%) were detected in the others samples. A high level of correlation was seen between HCV RNA and HCV Ag (r = 0.958, p < 0.001). The correlation for the samples that were genotyped 1 was significant (r = 0.966, p < 0.001). Conclusion In our study, it was found that there was strong correlation between HCV RNA levels and HCV Ag levels. So, it can be used for a one-step HCV antigen test to diagnose active HCV infection.

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Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00308-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3670-3681 ◽

Cited By ~ 84

Author(s):

Fiona McPhee ◽

Jacques Friborg ◽

Steven Levine ◽

Chaoqun Chen ◽

Paul Falk ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Clinical Studies ◽

Replication Capacity ◽

Replication Complex ◽

Hcv Genotype ◽

Genotype 1A ◽

Genotype 1B ◽

Ns3 Protease

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

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NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C

Intervirology ◽

10.1159/000489307 ◽

2018 ◽

Vol 61 (1) ◽

pp. 1-8

Author(s):

Deborah D’Aliberti ◽

Irene Cacciola ◽

Cristina Musolino ◽

Giuseppina Raffa ◽

Roberto Filomia ◽

...

Keyword(s):

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Hepatitis C ◽

Liver Tissue ◽

Serum Samples ◽

Virus Genotype ◽

Genotype 1A ◽

Treatment Naïve

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In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04619-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1505-1511 ◽

Cited By ~ 73

Author(s):

Warren Kati ◽

Gennadiy Koev ◽

Michelle Irvin ◽

Jill Beyer ◽

Yaya Liu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Clinical Isolates ◽

Genotype 1 ◽

Subgenomic Replicon ◽

Hcv Genotype ◽

Genotype 1A ◽

Treatment Naïve ◽

Hcv Genotype 1 ◽

Hcv Ns5b

ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

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A large healthcare-associated outbreak of hepatitis C virus genotype 1a in a clinic in Korea

Journal of Clinical Virology ◽

10.1016/j.jcv.2018.07.006 ◽

2018 ◽

Vol 106 ◽

pp. 53-57 ◽

Cited By ~ 7

Author(s):

Yoon-Seok Chung ◽

Ju-yeon Choi ◽

Myung Guk Han ◽

Kye Ryeong Park ◽

Su-Jin Park ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genotype ◽

Genotype 1A ◽

Healthcare Associated

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Serologic, Virologic, and Histologic Characteristics of Chronic Phase Hepatitis C Virus Disease in Children Infected by Transfusion

PEDIATRICS ◽

10.1542/peds.94.6.919 ◽

1994 ◽

Vol 94 (6) ◽

pp. 919-922

Author(s):

Suguru Matsuoka ◽

Katsuyoshi Tatara ◽

Yasunobu Hayabuchi ◽

Yoshiyuki Taguchi ◽

Kazuhiro Mori ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Acute Infection ◽

Chronic Phase ◽

Hcv Infection ◽

Core Antigen ◽

Hcv Rna ◽

Histologic Evidence ◽

Group A ◽

Group B

Objective. We studied the time course of hepatic dysfunction, seropositivity to hepatitis C virus (HCV) antibodies, viremia, and histologic evidence of hepatic injury to evaluate the course of HCV infection in children infected by blood transfusion. Patients and methods. Twenty-nine patients (ages 4 to 18 years) who underwent open-heart surgeries for congenital heart disease were grouped into three categories based on alterations in serum alanine aminotransferase (ALT) levels: Group A, acute infection; Group B, subacute infection; and Group C, chronic infection. Results. In Group C, all 13 patients had detectable HCV RNA in serum. In contrast, all patients in Group A had no detectable HCV RNA. In Group B, one of nine patients had detectable HCV RNA and two of four patients examined had persistent chronic hepatitis by histologic criteria. Antibodies directed against C100-3 antigen or core-antigen were more useful than second-generation HCV antibody assays in determining the relationship between viremia and immunologic response. Infection with HCV genotype II and the presence of higher HCV RNA copy numbers were associated with histologic evidence of hepatic damage. Conclusion. An abnormal ALT value is frequently associated with viremia, and biochemically resolved acute infection reflects clearance of HCV. However, a normal ALT does not always reflect an absence of hepatocyte damage and HCV replication in patients with subacute disease. The measures outlined in this study are useful indicators of disease activity during the chronic phase of post-transfusion HCV infection.

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