Low density lipoprotein receptor as a candidate receptor for hepatitis C virus

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 microg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target.     

Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption

The initial binding of Hepatitis C virus (HCV) to the cell membrane is a critical determinant of pathogenesis. Two putative HCV receptors have been identified, CD81 and low-density lipoprotein receptor (LDLr). CD81 interacts in vitro with the HCV E2 envelope glycoprotein, and LDLr interacts with HCV present in human plasma. In order to characterize these potential receptors for HCV, virus from plasma, able to replicate in cell culture, was inoculated on Vero cells or human hepatocarcinoma cells. HCV adsorption was assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Anti-LDLr antibody, low and very low density lipoproteins inhibited significantly HCV adsorption, confirming the role of LDLr as HCV receptor. Only one out of the two anti-CD81 antibodies used in this study led to a partial inhibition of HCV binding. This study also highlights a role for glycosaminoglycans (GAGs) in HCV adsorption: treatment of virus with heparin led to 70% inhibition of attachment, as did desulfation of cellular GAGs. Treatment of Vero cells with heparin-lyase significantly inhibited virus attachment but by only 30%. These results demonstrate the complexity of the HCV binding step in which LDLr interacts strongly with HCV, whereas the interaction of HCV with GAGs and particularly with CD81 seem to be more moderate.     

Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-capture polymerase chain reaction.

A rapid and simple RNA-capture polymerase chain reaction assay (RCPA) for detection of hepatitis C virus (HCV) is described. The assay detects specifically the presence of (near) full length genomic RNA of HCV by capturing HCV-RNA at the 3' terminal end on magnetic beads, followed by cDNA synthesis and PCR with 5' end specific primers. Sera were obtained from 30 chimpanzees inoculated with non-A, non-B hepatitis material from various sources, 28-122 months after infection. The sera were tested for the presence of HCV-RNA by RCPA and for HCV antibodies by a Line ImmunoAssay (Inno-LIA HCV Ab). Both tests were compared and show a high degree of agreement. Screening of 30 chimpanzee sera revealed either clearing of the virus below detection level (22/30) or development of a HCV carrier state (8/30). Only 1 of 11 LIA-indeterminate samples was positive by RCPA. As the RCPA is more sensitive, it can be used to test for the presence of HCV in sera which are classified indeterminate by the LIA. The outcome of the infection seems to be independent of the nature of the inocula, suggesting that the individual immune response could determine either clearing of the virus or the development of chronic infection.     

Characterization of antibody response to hepatitis C virus protein E2 and significance of hypervariable region 1-specific antibodies in viral neutralization

Summary.  Antibodies directed against hypervariable region 1 (HVR1) within the viral glycoprotein E2 of hepatitis C virus (HCV) are postulated to neutralize virus. An in vitro infection/binding assay of human fibroblast cells was established in order to study neutralization of HCV. Occurrence of mutations in the nucleotide sequence of HVR1 as compared to the inoculum after infection of human fibroblasts suggested replication of HCV in these cells. The significance of HVR1-specific antibodies in sera of patients who were infected in a single-source outbreak by an HCV contaminated anti-D immunoglobulin (IgG) preparation was studied. Using immunoprecipitation and ELISA, HVR1-specific antibodies could be detected in most of the sera obtained early (? 1 year p.i.) and late (up to 14 years p.i.) in single patients. Further characterization of the HVR1-specific antibodies in patient sera by attachment studies of HCV to the human fibro-blasts suggested that HVR1-specific antibodies in sera obtained early p.i. can neutralize virus of the anti-D IgG preparation.     

Intrahepatic HCV RNA loads in 37 HIV-HCV co-infected patients with controlled HIV infection

Serum and intrahepatic hepatitis C virus (HCV) RNA were measured in 37 HIV-HCV co-infected patients with controlled human immunodeficiency virus (HIV) infection and correlated with clinical, biological, and histological parameters. Thirty-seven interferon-naive patients underwent liver biopsy. HCV-induced activity (A) and fibrosis (F) were evaluated with METAVIR score. The 37 patients included had HIV plasma loads < 10,000 copies/ml, CD4(+) count > 250/microl. All the patients but two were receiving antiretroviral treatment. Liver tissue and sera were used for measurement of HCV RNA by the Cobas Amplicor HCV Monitor. All patients had serum and liver HCV RNA, and both levels were correlated (r = 0.47; P = 0.003). Intrahepatic HCV load did not depend on age, sex, duration of HCV infection, CD4(+), HCV genotype, or fibrosis. AST levels correlated with intrahepatic HCV load (r = 0.52; P = 0.001). Patients with METAVIR A1/A2 had significantly lower levels of liver HCV-RNA than were found in patients with METAVIR A3 (P = 0.026). Highly active antiretroviral therapy (HAART) including protease inhibitors(PI)-treated patients had significantly lower intrahepatic HCV load (P = 0.04). A weak but significant correlation between serum and liver HCV RNA was found. The amount of hepatic HCV RNA was correlated with AST levels, histological activity, but not with HCV genotype or fibrosis. The immune improvement associated with PI regimens could help reduce HCV load, supporting a protective effect of PI-induced immune restoration.     

Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins

The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL. HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity.     

Rapid hepatitis C virus clearance by antivirals correlates with immune status of infected patients

Altered immune parameters associated with hepatitis C virus (HCV) genotype 1b infection and their correlation with virus eradication in direct-acting antivirals (DAA)-treated patients were examined. Thirty-one HCV-infected patients were treated with DAAs for 12 weeks. Pre-DAA-treatment and post-DAA-treatment sera were analyzed for cytokines/chemokines using MILLIPLEX MAP. Serum complement level and antibody neutralization activity were measured separately. Sera from 11 spontaneously cleared HCV subjects were included for comparison. Rapid virological responders (RVR) or end-of-treatment responders (EOTR) were defined as patients with HCV RNA negative at week 4 or positive at week 4 and negative at week 12, respectively. HCV RNA eradication and a decrease in liver fibrosis-related cytokines after treatment were observed when compared with pretreatment sera from RVR and EOTR. In pretreatment sera, interferons and T-helper 1 or 2 cell-associated cytokines/chemokines were significantly higher among RVR as compared with EOTR. Furthermore, serum complement and virus neutralizing antibody levels were higher in pretreatment RVR sera. Eradication of HCV RNA by DAA decreased liver fibrosis-related cytokines. Pretreatment sera from RVR displayed an enhanced cytokine/chemokine, complement and virus neutralizing antibody response as compared with EOTR sera. Our results suggested that enhanced host immune status may play an additive role on HCV RNA clearance by DAA.     

A cross-sectional serum investigation of a clustering hepatitis C virus infection in Southwest China

Serum samples were collected in a village with a clustering hepatitis C virus (HCV) infection. HCV antibody, HCV RNA loads, liver function indexes, HCV envelope antibody, and neutralizing activity were assessed. Among 851 adult sera, 342 samples were positive for anti-HCV. Of these positive samples, 254 (74.3%) were HCV RNA positive (≥800 copies/mL). None of the 69 children's sera were positive for HCV antibody or RNA. Among the HCV antibody positive sera, alanine aminotransferase, and aspartate aminotransferase levels increased with the higher virus loads, but decreased when virus loads were higher than 1 × 10 ⁶ copies/mL. HCV envelope antibody and neutralizing antibody levels increased with viral load.     

Feasibility of freeze-dried sera for serological and molecular biological detection of hepatitis B and C viruses

We compared hepatitis B virus (HBV) surface antigen, anti-hepatitis C virus (HCV) antibody, and HCV RNA quantification in frozen and freeze-dried serum samples to assess the usefulness of freeze-dried sera for detection of HBV and HCV. The results indicated that freeze-dried sera as well as frozen sera can be useful for serological and molecular biological analyses of HBV and HCV.     

Association of hepatitis C virus in human sera with β-lipoprotein

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03–1.20 g/cm³). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm³. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19–1.20 g/cm³. HCV-RNA banding at low densities could be completely co-precipitated with anti- lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti- lipoprotein. In 3 sera no significant precipitation could be observed.     

Mycophenolate mofetil inhibits hepatitis C virus replication in human hepatic cells

Hepatitis C virus (HCV) infection is the most common indication for liver transplantation and the major cause of graft failure. A widely used immunosuppressant, cyclosporine A (CsA), for people who receive organ transplantation, has been recognized to have the ability to inhibit HCV replication both in vivo and in vitro. In this study, we investigated the effects of several other immunosuppressants, including mycophenolate mofetil (MMF), rapamycin and FK506, on HCV replication in human hepatic cells. MMF treatment of hepatic cells before or during HCV infection significantly suppressed full cycle viral replication, as evidenced by decreased expression of HCV RNA, protein and production of infectious virus. In contrast, rapamycin and FK506 had little effect on HCV replication. Investigation of the mechanism(s) disclosed that the inhibition of HCV replication by MMF was mainly due to its depletion of guanosine, a purine nucleoside crucial for synthesis of guanosine triphosphate, which is required for HCV RNA replication. The supplement of exogenous guanosine could reverse most of anti-HCV effect of mycophenolate mofetil. These data indicate that MMF, through the depletion of guanosine, inhibits full cycle HCV JFH-1 replication in human hepatic cells. It is of interest to further determine whether MMF is indeed beneficial for HCV-infected transplant recipients in future clinical studies.     

丙型肝炎病毒阳性血清体外感染人肝细胞的研究

目的 研究丙型肝炎病毒(HCV)抗体(Ab)阴性,HCV-RNA阳性血清建立体外感染肝细胞模型.方法 HCV Ab阴性,HCV-RNA阳性的窗口期血清与人肝细胞共同培养,用反转录-聚合酶链反应(RT-PCR)、免疫荧光染色、Western blot、共聚焦显微镜和透射电镜等方法检测细胞内HCV核酸复制、蛋白质表达及超微结构改变.结果 细胞与病毒共同培养7～45 d,细胞内和/或培养上清中可间断检出HCV正、负链RNA;细胞浆内有HCV 核心和NS3抗原的表达;细胞超微结构有改变,并于感染后第24天时观察到类似病毒样颗粒.结论 窗口期血清中的HCV能在人肝细胞7701中复制一段时间.     

Comparison of serum hepatitis C virus RNA and core antigen concentrations and determination of whether levels are associated with liver histology or affected by specimen storage time

An enzyme immunoassay has recently been developed for the hepatitis C virus (HCV) core antigen. To evaluate the possible association between core antigen and HCV RNA levels with regards to the change in liver histology over time as well as study the effect of duration of storage on viral load results, sequential sera were analyzed from 45 patients with chronic HCV infection who had undergone two or more liver biopsies. A relatively strong association was found between the core antigen and HCV RNA concentrations (r(s) = 0.8), with a core antigen level of 1 pg/ml corresponding to approximately 1,000 IU/ml. All 42 sera with detectable HCV RNA at the time of the second biopsy had core antigen concentrations above 1 pg/ml, and the three sera without detectable HCV RNA had concentrations below 1 pg/ml. No association was found between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necro-inflammatory grade, steatosis, genotype, alanine aminotransferase level, or alcohol consumption. A significant association was demonstrated between the storage time of the samples and both the HCV RNA and core antigen concentrations. The median log HCV RNA concentrations (international units/milliliter) were 3.92 for the sera obtained at the time of the first biopsy (median storage time, 13.0 years) and 4.41 for the sera obtained at the time of the second biopsy (median storage time, 6.6 years) compared to 5.96, the median for 102 different routine clinical patient samples.     

Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein

Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1–5% of cells in frozen sections of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160. © 1996 Wiley-Liss, Inc.     

Animal serum factor(s) causing adverse effects on RIAs of human enterovirus IgM.

The effects of animal sera used at various concentrations in dilution buffers for radioimmunoassays (RIAs) of human enterovirus-IgM were studied. The origin of the sera had no impact on the titres, but adverse effects on virus-specificity tests were noted when sera from some species were used. In attempts to block the binding of 35S-labelled virus by adding unlabelled virus, sera from cow, horse and lamb and from swine and man could usually not be used; instead of a blocking effect, an increase in bound labelled virus was noted in most cases. When fetal or newborn calf serum or sera from chicken were used, this phenomenon did not occur. The factor(s) causing the enhancement of virus binding could not be identified, but it was evident that it was not present in all sera from the same species and it was very probable that immunoglobulins were not involved.     

Binding of liver derived, low density hepatitis C virus to human hepatoma cells

HCV recovered from low density fractions of infected blood is associated with lipid and host apo-lipoproteins in lipo-viro-particles (LVP). It has been proposed that these particles are capable of binding and entering hepatocytes by viral glycoprotein independent mechanisms utilizing uptake pathways of normal host lipoproteins after binding to cell surface glycosaminoglycans (GAG), the low density lipoprotein receptor (LDL-r) or scavenger receptor B1 (SR-B1). In this study binding to human hepatoma cells of HCV low density RNA containing particles, semi-purified from macerates of infected human liver, is compared with that of normal host low density lipoprotein (LDL). Binding of both LDL and HCV low density RNA containing particles paralleled LDL-r but not SR-B1 expression on the recipient cells. Binding of both particle types was sensitive to suramin at 0 degrees C but less so at 37 degrees C suggesting that they both bind initially to GAG but, at 37 degrees C, are internalized or transferred to a suramin resistant receptor. Suramin resistant uptake of both particles was blocked in the presence of excess LDL or oxidized LDL. However, whilst LDL uptake was blocked by anti-apoB-100, HCV low density RNA uptake was enhanced by anti-apoB100 and further enhanced by a cocktail of anti-apo-B100 and anti-apoE. Pre-incubation of HCV low density RNA containing particles with antibodies to the E2 glycoprotein had little or no effect on uptake. These data indicate that whilst liver derived HCV RNA containing particles are taken up by HepG2 cells by a virus glycoprotein independent mechanism, the mechanism differs from that of LDL uptake.     

Multiplication and Isolation of Hepatitis C Virus in the NGUK-1 Rat Neurinoma Neural Cells

Hepatitis C virus (HCV) strains were isolated from human sera in vitro. NGUK-1 rat neurinoma neural cells [1] were selected as the experimental model of HCV. The cells were infected after attaining the confluence. The virus caused no cytopathic effect during its multiplication. After monolayer infection (24-96 h), culture fluid samples were tested for HCV RNA by classical PCR with electrophoretic detection of the reaction products and by quantitative real time PCR. All samples from infected culture were positive, control samples (intact cultures) were negative. Coefficient of correlations in quantitative PCR was 0.99. The results are reliable, conform to the normal values for this series of the test system, and indicate that HCV is replicated in continuous NGUK-1 cells. This in vitro model can be used for isolation of HCV.     

Co-amplification of specific sequences of HCV and HIV-1 genomes by using the polymerase chain reaction assay: a potential tool for the simultaneous detection of HCV and HIV-1.

A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti-HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01 and SN04; SN02 and SN03) based on the HCV prototype sequence of clones 37b and 81, and SK 38/39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization or Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the primer pairs, excluding the primer sequences. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to co-amplify specific sequences from two different viral genomes in the same reaction mixture offers the possibility of simultaneous detection and diagnosis of more than one viral agent in serum samples of infected individuals.