scholarly journals Modeling hepatitis C virus kinetics during liver transplantation highlights the role of the liver in virus clearance

2020 ◽  
Author(s):  
Louis Shekhtman ◽  
Miquel Navasa ◽  
Natasha Sansone ◽  
Gonzalo Crespo ◽  
Gitanjali Subramanya ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clearance Rate ◽  
Culture Medium ◽  
Viral Clearance ◽  
Huh7 Cells

AbstractWhile the liver, specifically hepatocytes, are widely accepted as the main source for hepatitis C virus (HCV) production, the role of the liver/hepatocytes in the clearance of circulating HCV remains largely unknown. Here we evaluated the function of the liver/hepatocytes in clearing virus from the circulation by investigating viral clearance during liver transplantation and from culture medium in vitro. Frequent HCV kinetic data during liver transplantation were recorded from 5 individuals throughout the anhepatic (AH) phase and for 4 hours after reperfusion (RP), along with recordings of fluid balances. Using mathematical modeling, the serum viral clearance rate, c, was estimated. Analogously, we monitored the clearance rate of HCV at 37°C from culture medium in vitro in the absence and presence of chronically infected Huh7 human hepatoma cells. During the AH phase, in 3 transplant cases viral levels remained at pre-AH levels, while in the other 2 cases HCV declined (half-life, t1/2~1h). Immediately post-RP, virus declined in a biphasic manner in Cases 1-4 consisting of an extremely rapid (median t1/2=5min) decline followed by a slower decline (HCV t1/2=67min). In Case 5, HCV remained at the same level post-RP as at the end of AH. Declines in virus level were not explained by adjusting for dilution from IV fluid and blood products. Consistent with what was observed in the majority of patients in the anhepatic phase, the t1/2 of HCV in cell culture was much longer in the absence of chronically HCV-infected Huh7 cells. Therefore, kinetic and modeling results from both in vivo liver transplantation cases and in vitro cell culture studies suggest that the liver plays a major role in clearing HCV from the circulation.

scholarly journals Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Cultures

2007 ◽  
Vol 81 (9) ◽  
pp. 4405-4411 ◽  
Author(s):  
Takanobu Kato ◽  
Takuya Matsumura ◽  
Theo Heller ◽  
Satoru Saito ◽  
Ronda K. Sapp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Culture Medium ◽  
Expression System ◽  
Core Antigen ◽  
Hcv Rna ◽  
Transfected Cells

ABSTRACT A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.

Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation

2005 ◽  
Vol 12 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Roberto J. Firpi ◽  
Haizhen Zhu ◽  
Giuseppe Morelli ◽  
Manal F. Abdelmalek ◽  
Consuelo Soldevila-Pico ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Sustained Virological Response ◽  
Virological Response

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

2008 ◽  
Vol 52 (6) ◽  
pp. 2097-2110 ◽  
Author(s):  
Pantxika Bellecave ◽  
Christian Cazenave ◽  
Julie Rumi ◽  
Cathy Staedel ◽  
Ophélie Cosnefroy ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Selection Procedure ◽  
Inhibitory Effect ◽  
Hcv Rna ◽  
Dna Aptamers ◽  
Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

Role of Hepatitis C Virus Infection on Lymphoproliferative Disorders after Non-liver Transplantation

2015 ◽  
Vol 6 (9) ◽  
pp. 899-907
Author(s):  
Hossein Khedmat ◽  
Mohsen Amini ◽  
Mohammad Ghamar-Chehreh ◽  
Aghdas Aghaei ◽  
Shahram Agah
Keyword(s):  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Lymphoproliferative Disorders ◽  
Hepatitis C Virus Infection

scholarly journals Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽  
2002 ◽  
Vol 297 (2) ◽  
pp. 298-306 ◽  
Author(s):  
I.Wayne Cheney ◽  
Suhaila Naim ◽  
Vicky C.H. Lai ◽  
Shannon Dempsey ◽  
Daniel Bellows ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Enzymatic Activity ◽  
Rna Replication ◽  
Viral Rna ◽  
Subgenomic Replicon ◽  
Replicon Cell ◽  
Ns5b Polymerase

scholarly journals 798 CELL CULTURE REPLICATION OF A GENOTYPE 1B HEPATITIS C VIRUS ISOLATE CLONED FROM A PATIENT WHO UNDERWENT LIVER TRANSPLANTATION

2011 ◽  
Vol 54 ◽  
pp. S320
Author(s):  
G. Koutsoudakis ◽  
S. Perez-del-Pulgar ◽  
M. Coto-Llerena ◽  
P. Gonzalez ◽  
J. Dragun ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Isolate ◽  
Genotype 1B

scholarly journals Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection

2016 ◽  
Vol 90 (16) ◽  
pp. 7456-7468 ◽  
Author(s):  
Vanessa M. Cowton ◽  
Allan G. N. Angus ◽  
Sarah J. Cole ◽  
Christina K. Markopoulou ◽  
Ania Owsianka ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Entry ◽  
Viral Life Cycle ◽  
Tryptophan Residue ◽  
Viral Glycoprotein ◽  
Cd81 Binding

ABSTRACTHepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.IMPORTANCEHepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

Suggested role of the Golgi apparatus and endoplasmic reticulum for crucial sites of hepatitis C virus replication in human lymphoblastoid cells infected in vitro

2003 ◽  
Vol 70 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Annalucia Serafino ◽  
Maria Beatrice Valli ◽  
Federica Andreola ◽  
Annalisa Crema ◽  
Giampietro Ravagnan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Golgi Apparatus ◽  
Virus Replication ◽  
Lymphoblastoid Cells

scholarly journals Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

2006 ◽  
Vol 80 (7) ◽  
pp. 3332-3340 ◽  
Author(s):  
Tetsuro Shimakami ◽  
Masao Honda ◽  
Takashi Kusakawa ◽  
Takayuki Murata ◽  
Kunitada Shimotohno ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Polymerase Activity ◽  
Subgenomic Replicon ◽  
Huh7 Cells ◽  
Hcv Ns5b

ABSTRACT We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

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