The molecular biology of hepatitis C virus

Hepatitis C virus (HCV) is one of the main causative agents for transfusion associated- and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive strand RNA of about 9400 nucleotides, as its genome, whose organization is similar to those of animal pestiviruses or human flaviviruses. In spite of the lack of the effective replication system in tissue culture cells, parts of the viral genome were expressed under the control of foreign promoters and the synthesized viral protein has been used for diagnostic assays.     

Hepatitis viruses: characterization and diagnostic techniques.

Two human hypatitis viruses have been identified and characterized, but one or more additional agents exist. Hepatitis B virus (HBV) is a complex 42-nm predominantly double-stranded DNA virus with distinct surface and core antigens and an endogenous DNA polymerase. Hepatitis A virus (HAV) is a 27-nm RNA virus with enterovirus-like properties. Progressively more sensitive and specific immunologic assays have been applied to the study of viral hepatitis and are available for routine diagnostic purposes. As a result we recognize distinct serologic response patterns to infection, new antigenic markers, biochemical-biophysical characteristics of the viruses, and their epidemiologic features. Recombinant DNA technology has permitted the cloning of HBV genetic material and gene products in E. coli, but the virus has not been cultivated in vitro. In contrast, successful in vitro cultivation of HAV has finally been accomplished. Application of sensitive serologic tests for HAV and HBV has revealed that "non-A, non-B" agents account for a substantial proportion of transfusion-associated hepatitis as well as hepatitis occurring in the absence of percutaneous exposure. These agents have been transmitted to chimpanzees, and several putative virus antigen-antibody systems have been described; however, a specific association between these virus antigens and non-A, non-B hepatitis has not been established.     

戊型肝炎病毒研究新进展

戊型肝炎病毒(hepatitis E virus,HEV)是引起全球多地区大规模流行和散发病例的急性病毒性肝炎的病原体,发展中国家尤为突出;我国是戊型肝炎(hepatitis E,HE)流行的高发区.HE是一种人畜共患病,猪是人HEV病毒的主要储库,因而HE已成为一个全球性的的公共卫生问题.对HEV的病毒学和基因组特征、临床表征、流行病学、主要抗原表位以及HEV诊断试剂和疫苗研制的进展进行了综述.     

Viral interference: A potential therapeutic method for chronic viral hepatitis?

Viral interference exists between different viral hepatitis. Acute Hepatitis C virus (HCV) super-infection on Hepatitis B virus (HBV) chronic carriers showed an inhibition of the HBV genome. And acute HBV super-infection on HCV chronic carriers indicated a similar interaction. In these cases, if the acute liver viral super-infection presents a self-limited course, the patients may be free from both viral infections or at least with undetectable underlying chronic viremia. The mechanism of viral interference is still undefined. Anyway this still leads to the hypothesis of using one hepatitis virus (live attenuated vaccine) to treat another hepatitis virus.     

Current Concepts of Viral Hepatitis and A Peek into the Future

New information has prompted revision of the conceptual framework for considering the epidemiology and virology of viral hepatitis. The means are now at hand to identify infections due to either Hepatitis A or B, as well as to implicate other etiologic agents in hepatitis. Immunologic evidence of variation in the antigens associated with Hepatitis B, and possibly in Hepatitis A, may explain some well known epidemiologic phenomena and has important implications in immune serum globulin prophylaxis. The ambiguous relationship of antigenemia and viremia in Hepatitis B is explored in relation to the hepatitis hazard of blood products, to trials of immune serum globulin, and to the potential role of the carrier-health worker in hepatitis transmission. The emerging concept of non-parenteral transmission of Hepatitis B is reviewed and future developments in the production of hepatitis vaccines and in experimental viral hepatitis in non-human primates is briefly discussed.     

丙型病毒性肝炎母婴传播的现状、进展及未来

丙型肝炎病毒(HCV)可引起急性和慢性病毒性肝炎,可发展成肝纤维化、肝硬化,甚至肝细胞癌。HCV经典的传播途径为经血液或血液制品传播,但1992年后献血员HCV的筛检已使输血后肝炎大为减少。在发达国家,HCV传播途径正在发生改变,儿童非血液制品的丙肝日渐增多。母婴间宫内、分娩时及产后感染已成为当前及今后的重要研究课题。研究证实,HCV可经胎盘引起胎儿感染,宫内感染是HCV传播的一条重要途径。尽管人们对HCV母婴传播中所涉及的风险因素逐渐明确,但到目前为止对具体的传播机制和传播时机仍知之甚少。我们就丙型病毒性肝炎母婴传播的现状、进展及未来做简要综述。     

Hepatitis C virus: current understanding and prospects for future therapies.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide and the leading indication for liver transplantation. The hallmark of the disease is its propensity to evolve into chronicity, probably because viral heterogeneity allows the virus to escape immune-mediated neutralization. Treatment with interferon alpha (IFN-alpha) has been disappointing, but higher and more frequent doses, and combination therapies, including nucleoside analogs, might lead to improved suppression of HCV RNA levels. Molecular analysis of HCV before and during treatment has indicated that high viral RNA levels and the presence of HCV genotype 1 are independent predictors of poor treatment outcome. New antiviral agents in development include inhibitors of HCV replicative enzymes, such as protease, helicase and polymerase, as well as several genetic approaches, such as ribozymes and antisense oligonucleotides. The main hindrance to drug development for hepatitis C is the lack of a small animal model or a productive tissue culture system for assessing drug action.     

Existing and future therapeutic options for hepatitis C virus infection

Hepatitis C virus (HCV) infection is an important cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma and liver failure worldwide. Chronic hepatitis C virus infection is treated with interferon-a (IFN-alpha), pegylated interferon-alpha (PEG-IFNalpha) alone or in combination with ribavirin; however, a significant fraction of patients either fail to respond or relapse after cessation of therapy. Efforts to identify and develop highly specific and potent HCV inhibitors have intensified recently. Each of the virally encoded replication enzymes has been a focus of studies as well as viral receptors and the host immune system. This review summarizes recent progress in the search for novel anti-HCV agents.     

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.     

Immunopathogenesis of hepatitis C virus infection

Hepatitis C virus, a recently identified member of the family Flaviviridae, is an important cause of chronic viral hepatitis and cirrhosis. There are similarities in the nature of the immune response to this pathogen with immunity in other flavivirus and hepatotropic virus infections, such as hepatitis B. However, the high rate of viral persistence after primary hepatitis C infection, and the observation that neutralizing antibodies are not protective, would suggest that there are a number of important differences between hepatitis C, other flaviviruses, and hepatitis B. The phenomenon of quasispecies evolution and other viral factors have been proposed to contribute to immune evasion by hepatitis C virus. In the face of established persistent infection, virus-specific cytotoxic T lymphocytes may exert some control over viral replication. However, these same effectors may also be responsible for the progressive liver damage characteristic of chronic hepatitis C infection. The nature of protective immunity, including the role of innate immune responses early after hepatitis C exposure, remains to be defined.     

应用噬菌体随机肽库技术筛选戊型肝炎病毒中和表位模拟肽

以识别戊型肝炎病毒（HEV）构象依赖性中和表位的单克隆抗体8C11、8H3作为固相筛选分子,对噬菌体随机7肽库进行4轮筛选后,随机挑取单克隆噬菌体进行测序。合成优势7肽序列基因,将其插入HBcAg-AA78-83位置之中,进行原核表达,所获重组蛋白经蛋白印迹实验证实可与相应单抗结合,电镜下可见重组蛋白能形成与HBcAg相似的类病毒颗粒。化学合成单抗8H3筛选出的优势7肽,所获7肽经生物传感器结合实验证实与单抗8H3结合。这些结果提示用噬菌体7肽库可以筛选出部分模拟构象性表位的短肽,为亚单位疫苗的研制提供了新的思路。     

Nucleic acid-amplification testing for hepatitis B in cornea donors

Careful donor selection and implementation of tests of appropriate sensitivity and specificity are of paramount importance for minimizing the risk of transmitting infectious diseases from donors to corneal allograft recipients. Reported cases of viral transmission with corneal grafts are very unusual. Nevertheless potential virus transmission through the engraftment cannot be ruled out. According to European Guideline 2006/17/EC, screening for antibodies for Hepatitis B core antigen (anti HBc) is mandatory, and when this test is positive, some criteria must be established before using corneas. Despite the continuous progress in screening tests, donors carrying an occult hepatitis B infection (OBI) can cause transplant-transmitted hepatitis B. To date, Nucleic Acid Testing (NAT) is not an obligatory assay in corneal tissue setting neither in our country nor in the rest of European countries. Herein, we report three cornea donors that were rejected with the diagnosis of OBI through the testing of sensitive NAT and the serological profile of Hepatitis B virus. The aim of this report is to emphasize the need to include NAT in new reviews of EU Tissues and Cells Directives in order to increase level of security in tissue donation as well as not to reject a high number of donors with isolated profile of anti HBc in geographical areas with high prevalence of Hepatitis B, that could be rejected without a true criterion of Hepatitis B infection.     

消化道传播病毒性肝炎研究进展

病毒性肝炎是由多种不同肝炎病毒引起的,以肝脏损害为主要表现,具有广泛流行性和严重传染性的一类疾病,严重危害人类健康,是我国目前重大的公共卫生问题之一。迄今鉴定出的具有明确致病性的肝炎病毒主要是甲型肝炎病毒(HAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)和戊型肝炎病毒(HEV),分别引起甲、乙、丙、丁、戊型肝炎。病毒性肝炎按传播途径的不同可以分为两类,一类是经肠道外传播的病毒性肝炎,包括乙、丙、丁型肝炎;另一类是经肠道(即消化道)传播的肝炎病毒,包括甲肝和戊肝,其发病有季节性,可呈暴发流行。本文旨在对经消化道传播的病毒型肝炎(甲肝、戊肝)的病原学、流行病学特征及其影响因素、控制和预防作一综述,以期对其流行和科学防控研究提供参考。     

Hepatitis A

Hepatitis A is a disease of worldwide distribution which occurs in endemic and epidemic form and is transmitted primarily by person-to-person contact through the fecal-oral route. Common source epidemics due to contamination of food are relatively common, and water-borne epidemics have been described less frequently. The presumed etiologic agent of hepatitis A has now been visualized by immune electron microscopic (IEM) techniques in early acute-illness-phase stools of humans with hepatitis A as well as in chimpanzees experimentally infected with material known to contain hepatitis A virus. In addition, several new serologic tests for the detection of antibody against hepatitis A virus have been described. These include complement fixation and immune adherence techniques. Current data suggest that hepatitis A is caused by a single viral agent lacking the morphologic heterogeneity of hepatitis B viral components and that there may be relative antigenic homogeneity between strains of virus recovered from various parts of the world. Serologic studies to date also indicate that hepatitis A virus is not a major contributing cause in post-transfusion hepatitis.     

Cellular defense systems against oxidative and ER stresses: mechanisms of regulation and influence of hepatitis C virus

Hepatitis C virus is one of the most spread and dangerous human pathogens. In most cases hepatitis C develops into chronic diseases which in many cases escape antiviral therapy and is associated with contributed to progression of various virus-associated organ damage and disorders including liver fibrosis, steatosis and hepatocellular carcinoma. Many of these diseases are currently linked to oxidative and ER stresses induced by the viral proteins. At the same time, hepatitis C virus disturbs systems protecting cells from these stresses, thus avoiding their effect on processes of the virus life cycle. Here, we have analyzed recent data on mechanisms of the cellular defense system functioning in infected and uninfected cells. In addition, major data on the hepatitis C virus genome structure and main functions of the virus proteins have been summarized briefly.     

Why do human hepatitis viruses replicate so poorly in cell cultures?

The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.     

The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents.     

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.     

A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus.

A putative core protein derived from hepatitis C virus was expressed in E. coli. More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein. Western blotting with this E. coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.