HCV复制子系统的研究进展及其应用

丙型肝炎病毒(HCV)感染率占全世界人口的1%～2%[1],约占我国人口的3.2%[2],是急慢性肝病的主要致病因素之一,其转归和肝硬化及肝细胞癌有密切关系[3].在美国,HCV感染已经成为重症肝病晚期进行肝移植的最主要原因[4].由于其高流行性、病程的隐匿性和长期感染导致预后不良,丙型病毒性肝炎成为一个严重的医学和社会经济学问题.由于HCV基因组有显著的序列变异,并且HCV在体外自主复制水平极低,其唯一可感染的动物为黑猩猩,故缺少有效的细胞培养系统和经济的小动物模型来研究HCV复制及其机制.1999年Lohmann等在Science上首先报道了选择性双顺反子亚基因组HCV RNA复制子系统[5],此复制子在人类肝癌细胞株中能够自主复制,被公认为是HCV体外复制模型研究获得突破进展的里程碑.此后复制子系统在很多方面得到了发展并不断完善,其中报告基因复制子的发展能够方便、快捷的高流量筛选抗HCV药物.本文对HCV复制子系统的研究、发展及其应用进行阐述.     

着丝粒蛋白B在基因组未复制的有丝分裂期细胞中的表达

着丝粒蛋白Ｂ（ＣＥＮＰ－Ｂ）主要位于染色体着丝粒区域的中心域，是一种ＤＮＡ结合蛋白，与染色体着丝粒、动粒的结构形成与功能实施有关。本研究结果显示咖啡因可以诱导ＣＨＬ细胞发生ＭＵＧ细胞。利用核酸杂交技术证明了在ＣＨＬ细胞中存在ＣＥＮＰ－Ｂ基因，并研究了咖啡因诱导ＭＵＧ细胞形成过程中ＣＥＮＰ－Ｂ的基因表达。发现ＣＥＮＰ－Ｂ在ＭＵＧ细胞整个过程中均有表达，表现出表达持续性。这与我们在Ｈｅｌａ细胞中发现的     

1b型丙型肝炎病毒基因组不同区域基因变异对干扰素疗效的影响

本研究对14例基因型1b型慢性丙型肝炎患者血清HCV基因组中E2/NS1(含HVR1和HVR2)、NS5A区准种异质性进行了检测,对HVR区准种异质性程度与干扰素(IFN)疗效的关系、NS5A2209-2248区是否存在干扰素敏感决定区(ISDR)等热点问题进行了探讨,为1b型慢性丙型肝炎患者应用IFN抗病毒治疗时     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

儿童慢性乙肝病毒携带者T细胞亚群与HBV-DNA复制的关系

目的研究儿童慢性HBV携带者的T细胞亚群状态及与HBV-DNA复制的关系。方法设正常对照组（n=32）及儿童慢性HBV携带（ASC）组（n=104），ASC组中又根据HBV—DNA的定性分为HBV—DNA阳性（n=62）75t．阴性组（n=42）。血清HBV标志物和HBV—DNA分别经酶联免疫吸附试验（ELISA）和基因扩增（PCR）法检测，T细胞亚群采用碱性磷酸酶-抗碱性磷酸（AM）免疫组化染色法检测。结果ASC组CD3＋、CD4＋、CD4＋／CD8＋明显低于对照组，CD8＋明显高于对照组（P〈0．01）；其中HBV—DNA阳性组CD4＋、CD4＋／CD8＋明显低于HBV—DNA阴性组，CD8＋明显高于HBV—DNA阴性组（P〈0．01或0．05）。结论儿童慢性HBV携带者存在T细胞亚群功能紊乱。其中，尤以HBV—DNA阳性者为著。     

全基因组解析人类细胞的分泌过程

据2012年6月3日Simpson Jc（Nat Cell Biol,2012 Jun 3．doi．10．1038／ncb2510）报道,爱尔兰都柏林大学和欧洲分子生物学实验室（EMBL）的研究者共同努力,揭示了人类基因组所编码的15％蛋白质用于细胞来进行分泌的过程,这项研究使得研究者评定超过800万个单独细胞的功能成为可能。这是首次对人类细胞分泌过程的一个全基因组的评估。     

EB病毒转化人外周血单个核细胞作为HCV复制模型的初步研究

目的　复制丙型肝炎病毒（ＨＣＶ） 感染的细胞模型。方法　用ＥＢ病毒感染其ＨＣＶ 阳性患者的外周血单个核细胞并使其转化，获得ＨＣＶ的外周永生化Ｂ细胞株。以后，每隔１ 个月应用套式逆转录聚合酶链反应（ＲＴＰＣＲ） 检测１ 次培养细胞和上清中的ＨＣＶＲＮＡ。结果　ＨＣＶ可以在传代细胞中持续存在１ 年，培养细胞中的ＨＣＶ负链和培养上清中的ＨＣＶ 则呈间断阳性。结论　ＨＣＶ 可以在该细胞株中较长时间存在、复制和分泌     

EB病毒转化人外周血单个核细胞作为HCV复制模型？…

目的 复制丙型肝炎病毒（ＨＣＶ）感染的细胞模型。方法 用ＥＢ病毒感染其ＨＣＶ阳性患者的外周血单个核细胞并使其转化，获得ＨＣＶ的外周永生化Ｂ细胞株。以后，每隔１个月应用套式逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测１次培养细胞和上清中的ＨＣＶ ＲＮＡ。结果 ＨＣＶ可以在传代细胞中持续存在１年，培养细胞中的ＨＣＶ负链和培养上清中的ＨＣＶ则呈间断阳性。结中以在该细胞株中较长时间存在、复制和分泌。     

High levels of subgenomic HCV plasma RNA in immunosilent infections

A genetic analysis of hepatitis C virus (HCV) in rare blood donors who remained HCV seronegative despite long-term high-level viremia revealed the chronic presence of HCV genomes with large in frame deletions in their structural genes. Full-length HCV genomes were only detected as minority variants. In one immunodeficiency virus (HIV) co-infected donor the truncated HCV genome transiently decreased in frequency concomitant with delayed seroconversion and re-emerged following partial seroreversion. The long-term production of heavily truncated HCV genomes in vivo suggests that these viruses retained the necessary elements for RNA replication while the deleted structural functions necessary for their spread in vivo was provided in trans by wild-type helper virus in co-infected cells. The absence of immunological pressure and a high viral load may therefore promote the emergence of truncated HCV subgenomic replicons in vivo.     

HCV replication in mononuclear cells stimulates anti-HCV-secreting B cells and reflects nonresponsiveness to interferon-α

Recently, it was demonstrated in chronic hepatitis C that the release of IgG and IgM anti-HCV antibodies by mononuclear cells (PBMCs) correlated with inflammatory activity, HCV persistence in serum, and negative outcome from antiviral therapy. Thus, persistent antigenic stimulation of the antibody-secreting B cells has been suggested. In this study, PBMCs were derived from 13 patients with chronic hepatitis C. Nucleic acids were extracted by the guanidine-thiocy-anate-method, and plus- and minus-stranded HCV-RNAs were determined using primers from the 5′-untranslated region of HCV. Simultaneously, unstimulated PBMCs were cultured for 8 days and anti-HCV antibodies were detected in the supernatants by EIA and RIBA. Seven patients (53.8%) had both plus- and minus-stranded HCV-RNA in PBMCs, while anti-HCV antibodies were secreted in vitro. One of 2 patients with plus- but not minus-stranded HCV- RNA in PBMCs was anti-HCV positive in vitro, whereas 4 patients without HCV-infected PBMCs were anti-HCV negative in vitro. Eight patients received antiviral therapy with interferon-α2b. Four nonresponders and 1 partial responder had plus- and minus-stranded HCV- RNA in PBMCs and anti-HCV secretion in vitro. On the other hand, 2 complete responders and another partial responder showed neither HCV infection of PBMCs nor anti-HCV secretion in vitro. In conclusion, infection of PBMCs by HCV is observed frequently in patients with chronic hepatitis C, and may be related to the high rate of nonresponders to antiviral treatment. The close correlation of anti-HCV secretion in vitro and viral replication suggests that the humoral response to HCV is triggered by viral antigens that are expressed on infected mononuclear cells. © 1995 Wiley-Liss, Inc.     

Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.     

Molecular Characterization of HCV 1b Intra-Familiar Infection Through Three Generations

Vertical transmission is an uncommon route of hepatitis C virus (HCV) infection. Little is known about the way of virus spread between relatives. Furthermore, the nucleotide sequence variability studies that can be used for the definition of cases of HCV transmission still need accurate standardization. In this study, we analyzed the HCV positive sera from subjects belonging to one family. Five out of seven individuals were positive both for anti-HCV and HCV-RNA. The epidemiological data, in our knowledge, excluded the possible risk of parenteral exposure to HCV for the members of the family. The genetic relatedness of the viruses infecting the members of this family was demonstrated by the phylogenetic analysis of sequences from E1 genome region. The analysis included the calculation of the genetic divergence specific index, based on the ratio of synonymous/non-synonymous mutations. By the analysis of this genome region, we demonstrated the occurrence of HCV transmission among family members. In 2 cases out of 3, Mother-to-Infant transmission was demonstrated that involved three generations of the family. Transmission by sexual route was absent. A method of sequence analysis of E1 HCV genome region is proposed as molecular approach for the definition of transmission cases of HCV.     

Multiple cyclophilins involved in different cellular pathways mediate HCV replication

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.     

Comparison of HCV core antigen and anti-HCV with HCV RNA results

Background

The measurement of anti-HCV antibodies using immunological methods and the confirmation of viral nuclear acid based on molecular methods is important in diagnosis and follow-up of the HCV infection.

Objectives

In this study, we aimed to analyse HCV core Antigen positivity among anti-HCV antibody positive sera to determine the significance of testing of HCV core Ag for the laboratory diagnosis of HCV infection, by considering the correlation between serum HCV core Ag and HCV RNA levels.

Methods

115 patients suspected of having hepatitis C and who were positive for anti-HCV antibody were investigated using chemiluminescent and molecular methods. Anti-HCV antibody, HCV core Ag and HCV RNA levels were detected by the Vitros ECiQ immunodiagnostic system, Architect i2000 system and RT-PCR, respectively.

Results

The sensitivity, specificity, positive and negative predictive values and accuracy rate of HCV core Antigen assay were detected as 86.5%(83/96), 100%(19/19), 100%(83/83), 59.4%(19/32), 88.7%(102/115) respectively.

Conclusion

HCV core Ag assay could be used for diagnosis of HCV infection as it is easy to perform, cost-effective, has high specificity and positive predictive value. However, it should be kept in mind that it may have lack of sensitivity and negative predictive value.     

Serotyping and genotyping of hepatitis C virus (HCV) strains in chronic HCV infection

Hepatitis C virus (HCV) genotypes can be established by methods based on PCR typing and serological typing. The accuracy of these methods depends on their sensitivity and specificity. These should be compared with the reference method, direct sequencing, and analysis of viral genomes. Among the serologic methods recently developed, the performance of a new serotyping assay (RIBA HCV 3.0 SIA, Chiron corporation, Emeryville) was assessed using a panel of 147 well-characterized French isolates from chronic hepatitis C patients. Definitive genotypes of the isolates were established by direct sequencing in 5′ NC and in some cases in NS-5B. HCV serotypes 1, 2, and 3 were determined by measuring type specific antibodies to core and NS-4 derived peptide antigens. Of the 147 sera, serotypic-specific antibodies were detected in 136 (sensitivity, 92.5%). The specificity of the RIBA SIA HCV serotyping assay was 92.6% (including samples with mixed results); without these, the specificity was 80.1%. Analysis of the 28 discrepant samples showed that (1) a different serotype was found in 18 samples including five for genotype 1, three for genotype 2, two for genotype 3, five for genotype 4, and three for genotype 5, and that (2) ten patients showed a reactivity with mixed serotypes, one had circulating antibodies to type 1 or 2, and nine had circulating antibodies to type 1 or 3. In summary, except for genotypes 4 and 5, the results of the test were well correlated (85.7%) with those of direct sequence genotyping. The former test is rapid and does not require the strict HCV RNA storage and preservation conditions of the latter. This new method may thus be considered as an alternative for HCV typing. However, although it is convenient, its lower sensitivity compared to the molecular typing method and the discrepant results limit its routine use in a clinical context. J. Med. Virol. 52:391–395, 1997. © 1997 Wiley-Liss, Inc.