体外培养人胎盘滋养层细胞内吞丙型肝炎病毒过程的电镜观察

目的 探讨丙型肝炎病毒（HCV）感染体外分离培养人胎盘滋养层细胞的可能机制，观察HCV感染滋养层细胞后在细胞内的具体定位。方法采用胰蛋白酶消化法及Percoll密度梯度分离法分离培养人胎盘组织中滋养层细胞后，以HCVRNA阳性血清对滋养层细胞进行体外感染实验，不同时间终止实验，制备电镜标本，透射电镜观察。结果HCV感染滋养层细胞的过程中有吞噬小泡形成，形态学上符合被覆小凹的特点；感染后HCV病毒样颗粒多定位于粗面内质网附近。结论HCV可以感染滋养层细胞，其感染过程可能是被覆小凹介导的内吞过程。本研究提供了HCV体外感染人胎盘滋养层细胞具体方式的形态学资料，对HCV宫内传播机制的进一步研究奠定了实验基础。     

HCV感染人滋养层细胞过程中抗体依赖性增强作用的初步研究

目的研究丙喇肝炎病毒（HCV）在感染人滋养层细胞的过程中是否存在抗体依赖性感染增强（ADE）作用，探讨HCV母婴传播的分子机制。方法将HCV阳性血清以4种不同方式感染体外培养人滋养层细胞，应用RT-PCR法对标本进行HCV RNA定性检测，并进一步评估不同浓度CD16单抗对感染过程的阻断作用。此外，应用免疫电镜技术观察滋养层细胞感染HCV病毒颗粒情。结果HCV RNA在全血清组、CD81单抗阻断组及LDL-R单抗阻断组多个标本均为阳性表达，且平均拷贝数较高，在FcγRⅢ（CD16）单抗阻断组多个标奉内未能检测到HCV RNA的表达，且阳性标本拷贝数较低；CD16单抗对感染的阻断作用随浓度的升高而增强；在全血清组、CD81单抗阻断组及LDL-R单抗阻断组多个标本细胞内可间断测得未脱壳的HCV病毒颗粒，CD16单抗阻断组细胞内未能检出。结论滋养层细胞膜CD81和LDL-R分子不参与HCV感染滋养层细胞的过程，而FcγRⅢ分子则与此过程密切相关。HCV感染人滋养层细胞过程中存在抗体依赖性增强作用。     

EBV转化B淋巴细胞中丙型肝炎病毒抗原表达的免疫组化研究

观察丙型肝炎患者外周血单个核细胞（PBMC）体外转化培养后其中是否仍有丙型肝炎病毒（HVC）存在。用EB病毒感染患者HCV阳性PBMC7并使其转化,获得永生化B细胞（EBVTB）。而后,用套式RT-PCR检测培养细胞和上清中的HCV RNA,用免疫组化SP法观察HCV抗原在细胞中的表达,HCV在传代细胞中持续存在,而培养细胞中的HCVRNA负链和培养上清中的HCV间断阳性,免疫组化发现HCVNS3、NS5和C抗原分布于胞细胞膜和核膜周围也可见阳性着色,细胞核阴性。HCV可以在体外EB病毒转化B细胞系中较长时间存在和复制。     

丙型肝炎病毒体外感染肝癌细胞株HepG2的初步研究

目的 研究人肝癌细胞株HepG2对丙型肝炎病毒（HCV）的易感性，建立细胞感染模型。方法 将人肝癌细胞株HepG2与慢性丙型肝炎患者血清共同温育6-8h，收集不同时相点标本，用逆转录聚合酶链反应（RT-PCR)法分别检测细胞内或上清液中正负链RNA，免疫组织化学法检测细胞内HCV NS3，NS5特异性抗原的表达情况，以及原位杂交法检测细胞内HCV负链RNA。结果 接种感染血清3-35d，可在细胞内或培养上清液中检出HCV正负链RNA；HCVNS3，NS5特异性抗原能在感染细胞内稳定表达，阳性物质位于胞浆中；原位杂交法证实细胞内存在负链RNA，也位于胞浆中，结论 人肝癌细胞株HepG2不但对HCV易感，而且可以稳定地支持HCV体外复制。     

初步探讨丙型肝炎病毒体外感染的致病机理

目的:初步探讨原代培养肝细胞对HCV的易感性,并在此基础上观察HCV感染后肝细胞形态学的变化。方法:用HCV RNA阳性血清与培养肝细胞共同孵育后,收集不同时相点标本,以逆转录聚合酶链反应(RT-PCR)、分别细胞内或上清中正负链RNA,免疫组化检测细胞内HCV NS3、NS5抗原的表达以及原位杂交检测细胞内HCV负链RNA,同时在电镜下观察细胞形态学变化。结果:接种感染血清后第3～5d,即可在细胞内或培养上清中检出HCV正负链RNA;感染肝细胞内可见HCV NS3、NS5抗原表达,阳性物质位于胞浆中;原位杂交法证实细胞内存在负链RNA,也位于胞浆中;光镜下未发现感染细胞形态学变化,电镜下发现胞浆内含有大量的脂肪空泡、多泡体,并可见疑似病毒样结构。结论:HCV不但能感染树鼩肝细胞,而且在体外能直接致细胞病变。     

激光共聚焦技术证实HCV NS5在人胎盘滋养层细胞的定位表达

目的胎盘屏障是营养物质以及某些药物、病原体、激素等从母体进入胎儿的必经之路。体外分离培养滋养层细胞是研究其功能及母体与胎儿之间物质交换的具体分子机制的细胞学基础。本研究的目的是探讨体外分离培养的人体胎盘滋养层细胞感染丙型肝炎病毒(HCV)后HCV NS5在滋养层细胞中的定位表达。方法采用胰蛋白酶消化法及Percoll密度梯度分离法分离培养人胎盘组织中滋养层细胞,以HCV RNA阳性血清对滋养层细胞进行体外感染试验,应用RT-PCR法定性及定量检测感染后细胞培养上清中HCV RNA,用激光共聚焦技术观察免疫荧光染色后HCV NS5在滋养层细胞中的定位。结果HCV感染后的细胞培养上清中可间断检测到HCV RNA,而对照组始终未检出;激光共聚焦观察显示HCV NS5主要定位于细胞核周围。结论感染HCV的人滋养层细胞中存在HCV NS5表达。此研究为进一步深入研究HCV的母婴传播分子机制奠定了实验基础。     

丙型肝炎病毒体外感染胎肝细胞的初步研究

目的：探索原代培养胎肝细胞对丙型肝炎病毒（HCV）的易感性,旨在建立较为稳定实用的细胞感染模型。方法：研究血清与培养肝细胞共同孵育6－8h后,收集不同时相点标本,用逆转录聚合酶链反应（RT－PCR）分别检测细胞内或上清液中正负链RNA,免疫组化检测细胞内HCV NS3,NS5特异性抗原的表达情况,以及原位杂交检测细胞内HCV负链RNA。结果：接种感染血清3d后,即可在细胞内或培养上清液中检出HCV正负链RNA,间断检出至感染后第17天,HCV NS3,NS5特异性抗原可在感染细胞内表达,阳性物质位于乐中,原位杂交法证实细胞内存在负链RNA,也位于胞浆中,结论：原代培养的胎肝细胞对HCV不但易感,而且稳定地支持HCV复制。     

丙型肝炎病毒对人B淋巴细胞系的感染及其形态学研究

目的：研究HCV对人B淋巴细胞的感染,建立HCV感染的丙型肝炎患者B淋巴细胞模型（CBCL）,并进行HCV的形态研究,方法：用EB病毒转化B细胞建立B细胞系,以逆转录多聚酶链反应（RT－PCR）,免疫组织化,原位杂交方法检测B细胞系上清液及细胞内的HCV抗原及HCVRNA,并通过电镜对HCV进行形态研究,结果：细胞系上清液中HCVRNA呈阳性,细胞内HCV抗原及HCVRNA均呈均呈阳性,电镜观察结果发现细胞内存在65nm和100nm圆形球病毒颗粒,并可见病毒芽生形成现象,结论：HCV可感染人B淋巴细胞并在其中复制,病毒在感染细胞胸质空泡部位合成和组装,以芽生方式进入胸质空泡形成病毒颗粒。     

人滋养层细胞培养上清液及裂解液中丙型肝炎病毒的检测

目的应用改良的分离、纯化方法对孕早期人绒毛滋养细胞进行原代培养,利用HCV阳性血清对滋养细胞进行体外感染,探讨HCV在体外培养条件下对细胞的感染及HCV在细胞中的复制状态。方法采用胰蛋白酶消化法消化正常人妊娠孕早期胎盘组织,以35%、45%2个Percoll密度梯度进行分离纯化、并免疫组化鉴定,获得纯度较高的滋养层细胞,应用HCV阳性且高载量的血清进行体外感染,应用免疫荧光染色检测滋养层细胞中HCV NS5表达,通过细胞裂解液裂解感染后细胞,应用荧光定量PCR（RT-PCR）技术检测感染后细胞培养上清和细胞内的HCV RNA,以观察HCV的感染及复制情况。结果该法分离纯化的滋养层细胞生长良好,特异性角蛋白-7阳性,纯度高。原代滋养层细胞在与HCV阳性血清共同孵育后均未出现明显的细胞病变,感染后48 h在滋养层细胞细胞浆中可见HCV NS5表达,感染后分别于24、48、72、96、120 h收集的培养上清和感染后细胞内均可以检测到HCV RNA。排除了培养洗涤液HCV污染,进一步检测其细胞裂解液中HCV,结果检测HCV阳性。结论利用改良后人胎盘滋养层细胞的体外培养系统,HCV阳性血清能在体外感染原代培养的人滋养层细胞,HCV可在滋养层细胞中复制。     

肝细胞癌中丙型肝炎病毒的原位检测

目的了解丙型肝炎病毒在本地区肝细胞癌中存在的频度.方法66例首次住院手术肝癌标本(含癌及癌旁组织),其固定、包埋、切片均经无RNase处理.应用地高辛标记HCVcDNA重组基因探针进行原位杂交,检测HCV病毒核酸.免疫组化SABC法检测HCV不同基因区抗原,HCV-CP10,HCV-NS3,HCV-NS5在HCC中的表达.结果66例HCC中HCVRNA阳性50例,阳性率为75.8%,阳性信号大部分存在于胞质中,偶有核阳性,或核浆混合型.HCVCP10阳性42例,阳性率63.6%.HCVCP10的阳性表达定位于胞质,少数病例有明显的围核分布现象,未见明确的核阳性.HCVNS3阳性33例,阳性率50%.HCVNS3阳性信号以胞质型为主,少数为核、浆混合型.HCVNS5阳性48例,阳性率72.8%.HCVNS5阳性信号以胞质型为主,部分呈膜型,少数为核、浆混合型.HCVRNA及三种抗原在癌周组织中的表达明显强于癌组织者.阳性细胞在癌组织呈散在分布,少数呈灶性分布,偶尔呈弥漫分布.在癌周组织大部分呈灶性、弥漫性分布,也可呈散在分布.经x2检验原位杂交检测HCVRNA一般比免疫组化检测HCV不同基因区抗原阳性率高,敏感(HCVRNA与HCVCP10相比,x2=4.083,P<0.05;与HCVNS3相比,x2=13.479,P<0.005;与HCVNS5相比,x2=0.1,P>0.5).两种检测方法的结果又有联系(HCVRNA与HCVCP10之间,x2=21.038,P<0.005;与HCVNS3之间,x2=13.942,P<0.005;与HCVNS5之间,x2=29.838,P<0.005).结论原位杂交结合免疫组化更能准确反映HCC中HCV的感染情况.     

Post-translational modifications of hepatitis C viral proteins and their biological significance

Replication of hepatitis C virus(HCV)depends on the interaction of viral proteins with various host cellular proteins and signalling pathways.Similar to cellular proteins,post-translational modifications(PTMs)of HCV proteins are essential for proper protein function and regulation,thus,directly affecting viral life cycle and the generation of infectious virus particles.Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positivestranded RNA genome.The key modifications include the regulated intramembranous proteolytic cleavage of core protein,disulfide bond formation of core,glycosylation of HCV envelope proteins E1 and E2,methylation of nonstructural protein 3(NS3),biotinylation of NS4A,ubiquitination of NS5B and phosphorylation of core and NS5B.Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well.For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3,we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear.In this review,we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.     

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

Summary. As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV‐induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N‐terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b‐infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N‐terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.     

Hepatitis C virus‐specific CD8+ T cell frequencies are associated with the responses of pegylated interferon‐α and ribavirin combination therapy in patients with chronic hepatitis C virus infection

Aim: Hepatitis C virus (HCV)‐specific cytotoxic T lymphocytes (CTLs) play critical roles in elimination of the HCV‐infected hepatocytes. However, the mechanism of HCV elimination by pegylated interferon‐α (peg‐IFNα) plus ribavirin is not fully understood. We examined HCV‐specific CTL responses during this combination therapy. Methods: CD8+ T cells were isolated from 16 HCV infected patients treated by this combination therapy and were subjected to IFN‐γ enzyme‐linked immunospot (ELISPOT) assay. Results: The numbers of IFN‐γ spots against HCV Core or NS3 protein‐derived peptides in HCV patients before treatment were similar to those in healthy donors, and those in HCV patients significantly increased 4 weeks after the initiation of combination therapy. All HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses in pre‐treated patients were not associated with ALT levels and HCV viral loads of HCV patients before treatment. And those in pre‐treated patients were similar between sustained virologic responder (SVR) patients and non‐SVR patients. Significant increase of HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses between before and 4 weeks after this combination therapy were observed in SVR patients, but not in non‐SVR patients. Conclusions: These results demonstrated that significant increase of HCV‐specific CD8+ T cells at 4 weeks after the initiation of IFN treatment might be associated with the elimination of HCV. Our findings suggest that the reactivity against HCV Core and NS3 proteins‐derived peptides might be useful in predicting the clinical outcome of the combination therapy of peg‐IFNα and ribavirin.     

Antiviral therapy: inhibition of Hepatitis C Virus expression by RNA interference directed against the NS5B region of the viral genome

Background: Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-a) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells). Methods: We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay. Results: Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells. Conclusions: Synthetic siRNA against NS5B-HCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.     

Ethanol inhibition: the humoral and cellular immune response to hepatitis C virus NS5 protein after genetic immunization

BACKGROUND: The combination of chronic hepatitis C virus (HCV) and ethanol may increase viral replication, impair cellular immunity, and result in severe and progressive liver disease. Because HCV nonstructural proteins play a major role in viral elimination, we examined the cellular and humoral immune responses after genetic immunization against NS5 in a chronic ethanol mouse model. METHODS: Mice were fed an ethanol or isocaloric pair-fed control liquid diet and were immunized with HCV NS5-expression plasmid. RESULTS: The humoral and cellular arms of the immune system were significantly impaired in ethanol-fed mice. Abstinence partially reversed the inhibitory effects on antibody levels and the CD4+ proliferative immune response but did not restore the CD8+ cytotoxic T-cell response to this HCV nonstructural protein. Furthermore, we determined whether murine interleukin-2 coadministration with the NS5 expression plasmid would reverse the inhibitory effects of chronic ethanol consumption; again, partial restoration was observed for B-cell and CD4+ T-cell activity, but not for cytotoxic T cells. CONCLUSIONS: These results suggest that the high rate of chronic HCV infection in alcoholics may be due to ethanol's effects on antiviral immune responses.     

Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling

Hepatitis C virus (HCV) is a major human pathogen that infects 170 million people. A hallmark of HCV is its ability to establish persistent infections reflecting the evasion of host immunity and interference with alpha/beta-IFN innate immune defenses. We demonstrate that disruption of retinoic acid-inducible gene I (RIG-I) signaling by the viral NS3/4A protease contributes to the ability of HCV to control innate antiviral defenses. RIG-I was essential for virus or HCV RNA-induced signaling to the IFN-beta promoter in human hepatoma cells. This signaling was disrupted by the protease activity of NS3/4A, which ablates RIG-I signaling of downstream IFN regulatory factor 3 and NF-kappaB activation, attenuating expression of host antiviral defense genes and interrupting an IFN amplification loop that otherwise suppresses HCV replication. Treatment of cells with an active site inhibitor of the NS3/4A protease relieved this suppression and restored intracellular antiviral defenses. Thus, NS3/4A control of RIG-I supports HCV persistence by preventing IFN regulatory factor 3 and NF-kappaB activation. Our results demonstrate that these processes are amenable to restoration through pharmacologic inhibition of viral protease function.     

Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes

Background

In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.

Objectives

To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.

Materials and Methods

The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.

Results

RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.

Conclusions

These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.     

Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis C virus (HCV) infection. The cell cultures were inoculated with a serum containing HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigens showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.