A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Vi ral Receptors Neutralizing Antibodies and Lipoproteins.

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H386 and R408), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Vi ral Receptors Neutralizing Antibodies and Lipoproteins.

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H386 and R408), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions

Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV

Neutrophil and Monocyte Function in Patients with Chronic Hepatitis C Undergoing Antiviral Therapy with Regimens Containing Protease Inhibitors with and without Interferon

Real-life data showed an increased incidence of bacterial infections in patients with advanced liver disease receiving a protease inhibitor (PI)-containing antiviral regimen against hepatitis C (HCV). However, the causes of this event are unknown. We hypothesized that PIs might impair innate immune responses through the inhibition of proteases participating in the anti-bacterial functions of neutrophils and monocytes. The aims of the study were to assess phagocytic and oxidative burst capacity in neutrophils and monocytes obtained from patients receiving a PI containing-antiviral regimen, and to determine cytokine secretion after neutrophil stimulation with flagellin. Forty patients with chronic HCV (80% with cirrhosis) were enrolled in the study, 28 received triple therapy (Group A) with pegylated-interferon and ribavirin for 4 weeks followed by the addition of a PI (telaprevir, boceprevir or simeprevir), and 12 patients received an interferon-free regimen (Group B) with simeprevir and sofosbuvir. Phagocytosis and oxidative burst capacity were analyzed by flow cytometry at baseline, week 4, and week 8 of therapy. In neutrophils from Group A patients, oxidative burst rate and oxidative enzymatic activity per cell significantly decreased throughout the study period (p = 0.014 and p = 0.010, respectively). Pairwise comparisons showed a decrease between baseline and week 4 and 8 of therapy. No differences were observed after the introduction of the PI. The oxidative enzymatic activity per cell in monocytes significantly decrease during the study period (p = 0.042) due to a decrease from baseline to week 8 of therapy (p = 0.037) in patients from Group A. None of these findings were observed in Group B patients. Cytokine secretion did not significantly change during the study in both groups. In conclusion, our data suggest that the use interferon (rather than the PI) has a deleterious effect on neutrophil and monocyte phagocytic and oxidative burst capacity in this cohort of patients with HCV-related advanced liver fibrosis