Seroepidemiology of hepatitis C virus in Beijing, China

To investigate the seroprevalence of hepatitis C virus (HCV) in China we tested sera from healthy individuals without hepatitis and no history of parenteral blood exposure and from patients admitted to a hepatitis hospital in Beijing. Sera were tested for anti-HCV by first-generation enzyme immunoassay; selected positives were tested with two second-generation EIAs, one utilizing recombinant antigens and the other synthetic peptides. We found anti-HCV with the following frequencies: 10 of 164 (6%) individuals with no disease; 2 of 36 (5.5%) patients with acute non-A non-B hepatitis (NANBH); 26 of 39 (67%) patients with post-transfusion NANBH; 10 of 34 (29%) patients with chronic hepatitis negative for hepatitis B surface antigen (HBsAg); 3 of 30 (10%) patients with chronic HBsAg-positive hepatitis; 0 of 19 patients with acute HBsAg-positive hepatitis. Of 24 repeat-positive sera, 19 were positive by both and 4 by one second-generation tests. We conclude that hepatitis C infection is common in China, that it contributes substantially to the incidence of post-transfusion hepatitis, and that HCV plays a significant role in both acute and chronic hepatitis. Further studies are needed to extend these observations and to define the predominant routes of transmission of HCV in China.     

A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non-Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second-Generation Test

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non-fused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.     

A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity

A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity.     

Seroepidemiology of hepatitis C virus infection in Japan and HCV infection in haemodialysis patients

Abstract: Since January 1990, Japanese Red Cross Blood Centres have introduced hepatitis C virus screening with a first-generation ELISA. From April to December 1992, approximately 0.98% among 10905 489 blood donations screened by a second-generation assay were anti-HCV-positive in all Japan. Seropositivity of anti-HCV increased with the age and serum transminase value in both sexes. In blood donors having a history of transfusion, the anti-HCV reactive rate was 7.4%. The results of the study made by the Japanese Red Cross Non-A, Non-B Hepatitis Research Group show the effectiveness of implementation of HCV screening to prevent posttransfusion hepatitis. Consecutive haemodialysis patients with chronic renal failure are at risk for inflection by a variety of blood-borne agents transmitted within dialysis units. Because of their immunocompromised state, they frequently also have an unusual susceptibility to a variety of nosocomial infections, such as HBV, and HTLV-I. We tested the prevalence of anti-HCV in 1423 (848 males and 575 females) haemodialiysis patients from 18 hospitals in Kumamoto Prefecture, Japan using the Orhto first generation anti- HCV screening assay. There were 316 patients (22.2%) positive for HCV antibodies. The second-generation test was positive in most haemodialysis patients who were eractive to the firs-generation assay. The prevalence of HCV infection increased with the duration of haemodialysis, yet there was a high frequency of HCV seropositivity even wihtout blood transfusion. Acquisition of HCV in dialysis patients could be explained by HCV seropositivity even without blood (all haemodialysis are done with disposable kits, and needles), by secondary HCV infection after the immunodeficiency of haemodialysis, or by HCV infection of the kidney or glomerular deposition of immune HCV/anti-HCV complexes leading to chronic renal failure (as with HBV infection of the liver and kidney).     

Antibodies to different virus antigens in patients with chronic hepatitis C

A total of 176 hospital patients with chronic hepatitis C (CHC), among them 110 males and 66 females, were examined. The spectrum of antibodies to four hepatitis C virus (HCV) proteins (core, NS3, NS4, NS5) and in 142 patients --IgM antibodies to HCV (anti-HCV IgM) were determined. In 92% of the CHC patients antibodies to core, NS3 and NS4 proteins were simultaneously detected. Differences in the detection of antibodies to HCV in males and females were not statistically reliable. In CHC patients aged up to 20 years anti-NS4 and anti-NS5 were less frequently detected. Among males of different age groups reliable differences in the detection rate of anti-NS5 were registered, while among females of different age groups no such differences were observed. With the increase of age these antibodies were detected somewhat more often. In females over 60 years anti-HCV IgM occurred more often than in males of the same age. The levels of alanine aminotransferase (ALT) were higher in persons with the presence of anti-NS5 and anti-HCV IgM than in persons with their absence. In all groups of CHC patients with biochemical activity and liver cirrhosis the detection rate of anti-HCV IgM was significantly higher than in patients with normal ALT activity. The antibody spectrum with the simultaneous absence of HCV IgM and anti-NS5, while found to contain antibodies to other HCV antigens, was registered significantly less frequently in patients with moderate and high CHC activity and the liver cirrhosis induced by HCV infection.     

Design and synthesis of HCV agents with sequential triple inhibitory potentials

The union of HCV-796, a potent selective HCV NS5B polymerase inhibitor, and Ribavirin, a molecule with activities against a wide spectrum of viruses, resulted in a class of new anti-HCV agents with a sequential triple inhibitory mechanism.     

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5''-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16^th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3–4 centuries, indicating a long epidemic history of HCV-2 in Ghana.     

丙型肝炎病毒(河北定州株)C33c基因的克隆、序列分析及在大肠杆菌中的表达

本研究通过RT-PCR从河北定州地区献血员血清中克隆了丙型肝炎病毒的C33c抗原基因,序列分析结果表明,该株的C33c基因和中国泰安株DNA同源性为91.0％,氨基酸序列同源性为92.2％;和HCV河北株的同源性分别为91.3％和96.2％;和日本JK1株的同源性分别为91.6％和94.8％。克隆的基因在大肠杆菌中得到表达和纯化,并可用作抗-HCV ELISA试剂的抗原组分。     

新型HCV EIA诊断试剂盒的研制

丙型肝炎病毒（ＨＣＶ）基因组结构区核壳蛋白（Ｃ）区抗原、膜蛋白Ｅ１和Ｅ２区抗原,以及非结构区ＮＳ３－ＮＳ５区抗原的区段,已经在原核细胞中获得有效的表达。同时,相应区段中的优势抗原表位肽也经化学合成法大规模地制备。ＨＣＶ基因组上各区段抗原性的分析发现,由Ｃ区和ＮＳ３区分别编码的Ｃ抗原和Ｃ３３ｃ抗原是ＨＣＶ基因组上两个优势抗原区段。其相应的抗体出现早（感染后６周可检出抗Ｃ３３ｃ抗体）,阳转率高（约９９％阳性检出率）,特异性和重复性均优于其它区段抗原。以中国人ＨＣＶ的Ｃ３３ｃ重组蛋白和分支状合成肽ＭＡＰ－Ｃ－１９为复合抗原,研制了适合我国抗ＨＣＶ抗体检测的新型丙型肝炎病毒酶免疫测定（ＨＣＶＥＬＡ）诊断试剂盒。它同当代美国Ａｂｂｏｔｔ／ＵＢＩＨＣＶＥＬＡ诊断试剂的符合率约９８％,同加拿大ＹＥＳ公司ＨＣＶＥＩＡ诊断试剂的符合率约９７．８％,阳性检出率提高了约２％,３次重复性达１００％,表明其特异性、敏感性和重复性均达到了当代第二代ＪＣＶＥＬＡ诊断试剂的水平。我国人群中抗ＨＣＶ抗体的分布情况为：正常人群的检出率１％－２％;外科类住院病人检出率约２８．８％;肝炎患者抗ＨＣＶ阳性率为３４．４％,慢活肝、肝硬化和重症肝炎患者     

Expression and purification of E2/NS1 protein of hepatitis C virus and detection of anti-E2/NS1 antibodies in chronic liver disease patients

Glycoproteins on the surface of viral particles present the main target of neutralizing antibodies. The structural proteins of most Flaviviruses are known to elicit neutralizing antibodies and, thus, to help in both the natural resolution of the infection and the protection from challenge with homologous hepatitis C virus (HCV). Because such antigens are associated with the viral clearance in both humans and chimpanzees, we aimed to express the E2/NS1 protein of HCV and to study the role of anti-E2/NS1 antibodies in the natural resolution of HCV infection. The prevalence of anti-E2/NS1 antibodies to recombinant E2/NS1 protein was seen by Western blot in chronic liver disease patients (15 chronic hepatitis and 12 cirrhotic patients), who were positive for anti-HCV and negative for HBV infection. The study also included 2 negative controls (positive for HBV infection and negative for anti-HCV antibodies) and 2 healthy controls (negative for both HBV and HCV infection). Anti-E2/NS1 was present in 20% of the chronic hepatitis and 16% of the cirrhosis patients. None of the controls were positive for anti-E2/NS1 antibodies. Serum samples positive for anti-E2/NS1 antibodies were also positive for HCV RNA by RT/PCR. Accordingly, the presence of anti-E2/NS1 may have very little or no role in the natural resolution of HCV infection.     

Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain.

A hepatitis C virus (HCV) cDNA covering part of the nonstructural region, NS3, was amplified from the serum of 50 out of 76 French non-A, non-B hepatitis patients by the nested polymerase chain reaction (PCR). Determination of a 407-bp sequence from four such cases revealed the presence of two different virus genotypes, F1 and F2, which exhibited 19-20% sequence divergence. F1 was represented by three of the four isolates and showed a sequence homology of about 97.5% to the prototype American HCV isolate, but of only 79% to a reported Japanese isolate. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype American HCV. PCR products from the 50 samples were hybridized with labeled F1 and F2 fragments under stringent conditions; results indicated the F1-related strain(s) as the major HCV genotype. Furthermore, a total of 1477 bp of sequence has been determined for one of the isolates belonging to the F1 category. These results will have implications for the PCR detection of HCV infection and production of HCV vaccines, especially for European countries.     

一株丙型肝炎病毒缺陷株NS5A基因片段的序列分析

Ｑ丙型肝炎是由丙型肝炎病毒（ＨＣＶ）引起的一种严重的传染病。丙型肝炎病毒主要通过输血和应用不洁的血液制品传播,所以利用敏感、特异的检测方法筛选献血员对丙型肝炎的预防尤为重要。现在第二代ＨＣＶ检测试剂已大批应用,加入ＮＳ５Ａ抗原的第三代试剂也正在研制中...     

Hepatitis C virus infection from blood and blood products

Abstract: The addition of second-generation HCV epitopes in antibody detection assays has increased the sensitivity and specificity of blood donor testing, to prevent post-transfusion hepatitis non-A, non-B (PTH-NANB), later characterized as Hepatitis C. However, it is not clear whether all HCV infectious donors are detected by second generation anti-HCV testing. Prospective studies on PTH-NANB were left with some unresolved cases. The use of second-generation anti-HCV assays in blood banks presented a problem with a relatively large number of indeterminate reactivities in supplemental assays such as RIBA-2. These indeterminate reactivities may be solved by the use of polymerase chain reaction (PCR). PCR is more and more used as an extra confirmatory assay to resolve RIBA indeterminate results on blood donors. However, a European study on the proficiency of HCV PCR in different countries revealed that only a minority of the reference laboratories perform this test faultness. Lately, third generation RIBA was developed, which was originally designed to resolve RIBA-2 indeterminate cases. RIBA-3 was shown to be more sensitive and specific in early HCV infection and blood donors than RIBA-2. Third generation anti-HCV testing will become standard practice. Some questions, however, remain unanswered. Do we miss any rare HCV infectious donors, of other genotypes, with third-generation assays, based only on the type 1 sequence of HCV? Can we improve HCV detection in the early phase of infection? What is the role of sporadic HCV transmission? How can we standardize HCV nucleic acid detection methods?     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

Immune responses to plasmid DNA encoding the hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis. The genomic region encoding the virion-associated core protein is relatively conserved among HCV strains. To generate a DNA vaccine capable of expressing the HCV core protein, the genomic region encoding amino acid residues 1 to 191 of the HCV-1 strain was amplified and cloned into an eukaryotic expression vector. Intramuscular inoculation of recombinant plasmid DNA into BALB/c mice (H-2d) generated core-specific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity. Our results suggest that the HCV core polynucleotide warrants further investigation as a potential vaccine against HCV infection.     

A 3D structural model and dynamics of hepatitis C virus NS3/4A protease (genotype 4a,strain ED43) suggest conformational instability of the catalytic triad: implications in catalysis and drug resistivity

Egypt has the highest prevalence of hepatitis C virus (HCV) infection worldwide with a frequency of 15%. More than 90% of these infections are due to genotype 4, and the subtype 4a (HCV-4a) predominates. Moreover, due to the increased mobility of people, HCV-4a has recently spread to several European countries. The protease domain of the HCV nonstructural protein 3 (NS3) has been targeted for inhibition by several drugs. This approach has had marked success in inhibiting genotype 1 (HCV-1), the predominant genotype in the USA, Europe, and Japan. However, HCV-4a was found to resist inhibition by a number of these drugs, and little progress has been made to understand the structural basis of its drug resistivity. As a step forward, we sequenced the NS3 HCV-4a protease gene (strain ED43) and subsequently built a 3D structural model threaded through a template crystal structure of HCV-1b NS3 protease. The model protease, HCV-4a, shares 83% sequence identity with the template protease, HCV-1b, and has nearly identical rigid structural features. Molecular dynamics simulations predict similar overall dynamics of the two proteases. However, local dynamics and 4D analysis of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) indicate conformational instability of the catalytic site in HCV-4a NS3 protease. These results suggest that the divergent dynamics behavior, more than the rigid structure, could be related to the altered catalytic activity and drug resistivity seen in HCV-4a.     

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.     

Identification of novel inhibitors of HCV RNA-dependent RNA polymerase by pharmacophore-based virtual screening and in vitro evaluation

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis where no effective treatment is available. The HCV NS5B with RNA-dependent RNA polymerase (RdRp) activity is a key target for the treatment of HCV infection. Here we report novel NS5B polymerase inhibitors identified by virtual screening and in vitro evaluation of their inhibitory activities. On the basis of a newly identified binding pocket of NS5B, distinct from the nucleotide binding site but highly conserved among various HCV isolates, we performed virtual screening of compounds that fit this binding pocket from the available chemical database of 3.5 million compounds. The inhibitory activities of the in silico selected 119 compounds were estimated with in vitro RdRp assay. Three compounds with IC50 values of about 20 μM were identified, and their kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate. Furthermore, the single-point mutations of the conserved residues in the binding pocket of NS5B resulted in the significant decrease of the RdRp activity, indicating that the binding pocket presented here might be important for the therapeutic intervention of HCV. These novel inhibitors would be useful for the development of effective anti-HCV agents.     

HCV感染者抗HCV抗体产生的不均匀性与B细胞优势克隆

本文采用抗原捕捉ＥＬＩＳＡ方法检测了ＨＣＶ感染者血清中抗－ＨＣＶＩｇＧ抗体轻链Κ和λ的比值,发现所检测的抗ＨＣＶ－ＮＳ４、抗ＨＣＶ－ＣＰ１和抗ＨＣＶ－ＣＰ２抗体轻链的表达呈现明显的偏斜,６５例抗ＨＣＶ阳性者中６３例（占９６．９％）,至少一种抗ＨＣＶ抗体К／λ偏离了正常１∶１的比值,尤以λ链较多,分别占６５．６％、８９．９％和７０．２％,但任何一个ＨＣＶ感染者血清抗－ＨＣＶ抗体既可能是Κ链占优势,也