丙型肝炎病毒F蛋白对p21基因的调节作用

目的研究丙型肝炎病毒（HCV）F蛋白对细胞p21基因转录、表达的影响。方法PCR扩增HCV1b来源的F基因，克隆至pcDNA3．1真核表达载体。F基因质粒转染HepG2细胞，48h后抽提细胞总RNA和总蛋白，实时定量PCR及Westem blot检测p21基因表达变化。结果HCVF蛋白在HepG2细胞内瞬时表达，相对于空载体对照（目标基因表达量为1），HCVF基因质粒转染细胞的p21转录水平为3．2，蛋白表达水平为1．4。结论HCVF蛋白增加p21转录和表达，其生物学效应值得进一步研究。     

Hepatitis C virus antibodies among risk groups in a South African area endemic for hepatitis B virus

The prevalence of anti-HCV was studied in a South African area endemic for hepatitis B virus. A total of 35,685 volunteer blood donors (22,034 whites, 9,218 Asians, 3,077 Africans, 1,356 coloureds), 71 haemophiliacs, 84 chronic dialysis patients, 100 antenatal attenders, 212 nurses, and 20 HIV-positive male homosexuals were tested for anti-HCV. Repeat positive second generation Ortho HCV EIA was used to determine HCV status for the blood donors; Abbott-II HCV EIA combined with a neutralisation test was used for the other risk groups. Antibody to hepatitis B core antigen (anti-HBc) was also tested in the haemophiliacs, nurses, and chronic dialysis patients. Seroprevalence for the blood donor population was 0.16, 0.34, 0.75, and 0.22% for whites, Asians, Africans, and coloureds, respectively. Of the risk groups tested, 39.4% of haemophiliacs and 4.8% of chronic dialysis patients were positive; of the remainder tested none was positive. Fifty percent of nurses, 47.9% of haemophiliacs, and 22.6% of dialysis patients had serological evidence of past exposure to hepatitis B virus (anti-HBc positive). These findings indicate a low prevalence of anti-HCV in the blood donor population, thus probably resulting in a low prevalence in groups exposed to blood and blood derivatives. The overall difference in prevalence between the race groups was significant (P < 0.0001). The high prevalence of hepatitis B virus compared to the low prevalence of HCV suggests that the main modes of transmission of the two viruses are probably different. © 1993 Wiley-Liss, Inc.     

肝细胞肝癌中丙型肝炎病毒C区、E区、NS3区、NS4区抗原的表达

本文报道研究丙型肝炎病毒抗原在肝细胞肝癌组织内的定位分布情况。以丙型肝炎病毒（ＨＣＶ）的Ｃ、Ｅ、ＮＳ３、ＮＳ４区四种单克隆抗体用免疫组化方法检测了１３９例肝细胞肝癌（ＨＣＣ）的肝脏标本，结果总的阳性率为１５．１％。２１例阳性标本中，Ｃ区单抗检测阳性占８０．９％（１７／２１），Ｅ区占３３．３％（７／２１），ＮＳ３、ＮＳ４区均占５７．１％（１２／２１），表明应用多区段单抗有助于提高ＨＣＶ抗原的检出率。阳性物质主要存在于胞浆中，呈细、粗颗粒及块状，３例出现膜及膜下型，１例核内有阳性反应。ＨＣＶ感染与ＨＣＣ的发生发展有一定的关系。     

Hepatitis C virus antibody in hepatocellular carcinoma in Taiwan.

The prevalence of antibody to hepatitis C virus (anti-HCV) was investigated in patients with hepatocellular carcinoma (HCC), and correlated with the clinical features. Anti-HCV was detected in 129 histology or aspiration cytology proven HCC patients and 54 healthy controls. Anti-HCV was examined by the HCV EIA (Abbott Laboratories). All healthy controls were anti-HCV-negative. Nineteen of 81 (23.5%) hepatitis B surface antigen (HBsAg)-positive HCC patients were positive for anti-HCV. Anti-HCV was found among 60.4% (29/48) of HCC patients without detectable HB-sAg. Forty-eight of 129 (37.2%) HCC patients were positive for anti-HCV. There was a significant difference in the prevalence of anti-HCV between patients with HBsAg (23.5%) and those without HBsAg (60.4%, P = 0.0001). However, irrespective of the status of HBsAg, there was no statistical difference in sex, age, routine liver function tests, alpha-fetoprotein concentration, or associated cirrhosis between patients with anti-HCV and those without. The results imply that hepatitis C virus may play a role in the pathogenesis of HCC.     

Changing seroepidemiology of hepatitis B, C, and D virus infections in high-risk populations

Needle-sharing and sexual contact are important transmission routes of hepatitis B, C, and D virus (HBV, HCV, HDV) infection. This study aimed to investigate the current status of these viral infections among high-risk populations including prostitutes and intravenous (i.v.) drug users, compared with the prevalence rate reported previously to examine the changing seroepidemiology. Of the 916 female prostitutes, 79 (9%) were positive for antibody to HCV (anti-HCV), 111 (12%) were positive for HBV surface antigen (HBsAg), and 5 (5%) had antibody to HDV (anti-HDV). The prevalence rate was significantly lower compared to that in 1989-1991 (12%, P = 0.037) for HCV infection, and to that in 1988 (59%) and 1996 (40%) (P < 0.0001) for HDV infection. Of the 494 i.v. drug users, 87 (18%) patients were HBsAg carriers and 12 (14%) were anti-HDV-positive. The prevalence rate of HDV infection was significantly lower than that reported in 1985 (79%, P < 0.0001). Among the 443 tested i.v. drug users, 182 (41%) were anti-HCV-positive, significantly lower than that in 1985 (53%, P = 0.026). Of the 263 male prostitutes, 11 (4%) were anti-HCV-positive, 45 (17%) were HBsAg-positive, and 7 (16%) were anti-HDV-positive. Of the 129 illegal immigrant prostitutes, 7 (5%) were anti-HCV-positive, 15 (12%) were HBsAg-positive and none were positive for anti-HDV. In conclusion, the findings indicate a declining prevalence of HCV and HDV infections among drug users and prostitutes over the past 16 years. Male prostitutes and immigrant prostitutes are new "high-risk" populations and may become a reservoir for disease transmission.     

逆转录病毒介导乙型肝炎病毒C基因在树突状细胞中的转移

目的：探讨逆转录病毒介导乙型肝炎病毒核心抗原(HBcAg)基因在骨髓来源的树突状细胞(DC)的转移效率，以及对DC成熟和功能的影响。方法：用含有HBV C基因的重组逆转录病毒载体，感染处于分裂期的小鼠骨髓祖细胞，感染后的骨髓细胞在GM—CSF和IL—4存在的条件下继续培养获得成熟的DC，用PCR、RT—PCR，分析目的基因的整合与转录；用Western blot和流式细胞仪，分析HBcAg的表达及基因转移效率，以及检测感染前后DC CD80和MHC—Ⅱ类分子的表达以及分泌IL—12能力的变化；通过将基因转移后的DC与淋巴细胞进行混合培养，检测其体外诱导CTL的能力。结果：逆转录病毒感染后并不影响骨髓来源的DC成熟，对其表面分子CD80和MHC-Ⅱ类分子的表达和分泌IL-12的能力没有影响。目的基因能整合到DC的基因组DNA中，并能转录和翻译。约28％的DC能表达HBcAg，感染后的DC体外能诱导T细胞应答。结论：逆转录病毒能有效地将HBV C基因转移到骨髓来源的DC中，对DC的成熟和功能无明显影响，并能诱导CTL应答，这将有助于开展以DC为基础的慢性乙肝的免疫治疗。     

乙型肝炎病毒X基因促TRAIL诱导Huh7细胞凋亡的机制

目的探讨乙型肝炎病毒(hepatitis B virus,HBV)X基因(HBX)促肿瘤坏死因子相关凋亡诱导配体(TNF relatedapoptosis inducing ligand,TRAIL)蛋白诱导人肝癌细胞Huh7凋亡的机制。方法应用脂质体转染法将pcDNA3.1-HBX、pcDNA3.1分别转染Huh7细胞,建立稳定表达HBX的细胞模型Huh7-HBX和空载体对照细胞模型Huh7-3.1;运用TRAIL蛋白分别作用Huh7-HBX细胞、Huh7-3.1及Huh7细胞后,采用CCK8、流式细胞术检测细胞增殖和凋亡情况;Western blot检测死亡受体4(deathreceptor 4,DR4)和死亡受体5(death receptor 5,DR5)的表达情况;分光光度法检测细胞caspase8和caspase3活性。结果 RT-PCR及Western blot证实成功构建细胞株Huh7-HBX;CCK8和流式细胞术检测结果均显示Huh7-HBX细胞的凋亡率明显高于Huh7-3.1及Huh7(P<0.05);Western blot显示Huh7-HBX细胞的DR4、DR5的表达量明显高于Huh7-3.1及Huh7细胞(P<0.05);caspase活性检测结果显示,Huh7-HBX细胞caspase8及caspase3的活性明显高于Huh7-3.1及Huh7(P<0.05)。结论 HBX能够通过上调Huh-7细胞表面死亡受体DR4、DR5的表达,进而促进TRAIL蛋白诱导的细胞凋亡,为进一步研究HBX的促凋亡机制提供实验依据。     

重型乙型肝炎病人中乙型肝炎病毒C基因启动子变异的研究

目的　了解重型乙型肝炎（ 乙肝） 病人血清乙肝病毒（ＨＢＶ）ＤＮＡＣ基因启动子（ＣＰ） 的变异。方法　对用聚合酶链反应（ＰＣＲ） 法扩增的血清ＨＢＶＤＮＡ 直接测序。结果　７ 例亚急性重型肝炎病人的ＨＢＶ 分离株ＣＰ区分别有２～１２ 个替代变异，１ 例病人有１１ｂｐ 的碱基插入。ＣＰ变异主要发生于ＣＰ的第１ 和第２ 个ＡＴ丰富区，ｎｔ１ ７６２ 和ｎｔ１ ７６４ 的替代变异见于７ 例亚急性重型肝炎病人的４ 例中，是ＣＰ变异的热点，其中３ 例ＨＢｅＡｇ 阴性，说明和ＨＢｅＡｇ 阴性表型相关。ＣＰ的第３ 个ＡＴ丰富区、ＨＢＶ逆转录起始位点（ＤＲ１） 和前Ｃ基因、前基因组转录起始位点未见变异。结论　重型肝炎病人的ＨＢＶＣＰ区存在较多的变异，ＣＰ变异主要发生于和前Ｃ基因相关的第１ 和第２ 个ＡＴ丰富区，可能和ＨＢｅＡｇ 阴性表型相关     

Changes in Hepatitis C Virus Genotype Distribution in Chronic Hepatitis C Infection Patients

Purpose: Identification of hepatitis C virus (HCV) genotypes is very important in the selection of antiviral treatment, dose adjustment of antiviral agents, determining the treatment duration and following-up of treatment response. We aimed to determine the distribution pattern of HCV genotypes in chronic hepatitis C infection (CHC) patients. Materials and Methods: We have included 106 CHC patients who were positive in the anti-HCV and HCV-RNA tests performed in our hospital during the 16-month period. Anti-HCV assays were performed on device using a chemiluminescent microparticle immunoassay, while HCV-RNA tests and HCV genotyping assays were performed by real-time polymerase chain reaction. Results: Of the 106 cases; genotype 1b was detected in 67.0%, genotype 3 was detected in 16.0%, genotype 1a was detected in 14.2% and genotype 2 was detected in 2.8% patients. Genotypes 4, 5 and 6 were not detected in our study group. There were no statistically significant differences between the gender and age groups according to the HCV genotype distribution. The genotype 3 detection rate (16%) was the highest rate among the studies compared with the other studies in our country. Conclusions: Events that cause social changes such as war and immigration and intense commercial and touristic activities affect and alter the HCV genotype distribution in HCV-infected patients. For this reason, further multicentre studies are required reflecting all the regions in order to determine the genotype distribution in HCV-infected patients at regular intervals.     

丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核细胞中的表达

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶＣ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的核心抗原。结果：用限制性内切酶酶切后，片段大小与计算值相符。Ｗｅｓｔｅｒｎｂｌｏｔ证实，表达抗原的Ｍｒ约为２２０００。结论：ＨＣＶＣ基因能够插入ｐｃＤＮＡ３真核表达载体，并使其在真核细胞中表达，为进一步ＨＣＶ基因疫苗的研制和探讨抗ＨＣＶ感染打下了基础。     

Hepatitis C virus infection

Hepatitis C virus (HCV) infection is a major public health problem. Up to 3% of the world's population is infected with HCV, and at least 200 000 adults in the UK carry the virus. Of those exposed to HCV, 80% become chronically infected, and at least 30% of carriers develop chronic liver disease, including cirrhosis and hepatocellular carcinoma. This review provides an overview of selected features of the molecular biology and pathogenesis of HCV infection, and thereafter discusses in detail the epidemiology of HCV, the hepatic and extra-hepatic diseases caused by the virus, and the current treatment options for both acute and chronic virus infection. The special cases of healthcare workers, prison inmates and individuals coinfected with human immunodeficiency virus and HCV are considered in detail.     

Hepatitis C virus: Quantitation and distribution in liver

The optimal method for viral quantitation and the most appropriate site for determining viral load in patients with chronic hepatitis C virus (HCV) infection are unknown. We developed a method for measuring HCV RNA in the liver with the following features: 1) efficient extraction of RNA from tissue (89% of RNA recovered); 2) accurate amplification using branched DNA with strong concordance between a single sample tested on multiple occasions either in the same or in different runs; 3) good sensitivity (95%) and specificity (100%). HCV RNA was detected in as little as 2 mg of tissue, and viral load determined in a needle biopsy was representative of viral load in other parts of the liver. Within individual livers, 68% of the samples quantitated were within 1.5-fold of the geometric mean, and 95% were within 2.2-fold of the geometric mean. The mean ratio of virus in the liver and serum was 103, range 17.4–286. A delay of 30 minutes before freezing the liver tissue resulted in a reduction in the measured viral load in some, but not all instances. A sensitive, specific, and reproducible method for quantitating HCV RNA in the liver has been developed. Measurement of viral load at one site was representative of viral load at other sites. While hepatic HCV RNA levels are consistently greater than serum levels, the ratio of liver to serum viral load varies widely. The clinical use of measurement of viral load in the liver remains to be defined. J. Med. Virol. 51:217–224, 1997. © 1997 Wiley-Liss, Inc.     

Hepatitis C virus E2 and NS5A region variability during sequential treatment with two interferon-alpha preparations

To determine the pattern and significance of the HCV genetic heterogeneity before and during treatment with recombinant-2b or lymphoblastoid alpha-interferon, hypervariable region 1 (HVR-1) and NS5A quasispecies were characterised by cloning and sequencing in 12 HCV-1b-infected subjects. Patients were either responder-relapsers or non-responders to treatment. Extensive amino acid sequence analysis was applied to reveal the significance of HCV variation at key sites within HVR-1 and NS5A regions. Genetic complexity, genetic diversity, and the non-synonymous to synonymous substitution ratios of HVR-1 quasispecies decreased during treatment in responder-relapser patients only, and more markedly so following lymphoblastoid alpha-interferon. In non-responders, the HVR-1 quasispecies broadened. Amino acids G406 and Q409, which represent a major viral epitope, were highly conserved throughout treatment. Responder-relapser patients had a higher mutation frequency in NS5A than non-responders. Lymphoblastoid alpha-interferon promoted the selection of intermediate Interferon Sensitivity Determining Region (ISDR) sequences, whereas recombinant-2b alpha-interferon favoured maintenance or selection of conserved ISDR sequences. Variability upstream of the ISDR was associated with treatment response, but the amino acid substitutions conferring higher replicative ability to in vitro HCV replicons were absent in in vivo isolates. In conclusion, the pattern of HVR-1 quasispecies evolution correlates with the clinical response, and the conservation of specific amino acids may be useful for immune targeting in vivo. In responder-relapser patients, the initial HVR-1 evolution resembles that found in sustained responders. Variability within the entire NS5A, as opposed to a single region (ISDR), may have a role in influencing alpha-interferon treatment outcome. A differential effect of different alpha-interferon preparations on HCV quasispecies kinetics may exist.     

An Ultrastructural Study of Six Cases of Chronic Active C Hepatitis. A Comparison with Chronic Active B Hepatitis

Liver biopsies from six patients affected by chronic active hepatitis (CAH) induced by hepatitis C virus (HCV) have been investigated ultra-structurally and their features compared with those of five cases of CAH induced by hepatitis B virus (HBV). Clusters of deeply packed nuclear inclusions (18 to 22 nm in diameter) were found in patients with HCV-CAH. They were irregularly round and ill-delimited. These inclusions were distinct from the regular round and well-delimited nuclear inclusions associated with HBV. HCV-associated nuclear inclusions were similar to a peculiar type of intranuclear particle described in non-A, non-B hepatitis before the HCV disease had been recognized as a distinct entity. These inclusions have never been hitherto reported in infections caused by HBV or other known hepatotropic viruses. Together, these data suggest that the occurrence of these inclusions can be related to HCV activity in hepatocytes. Changes in the microarchitecture of the liver cell and its microenvironment were similar in HCV- and HBV-CAH. They were heterogeneous in different cases and did not display a clear correlation with the severity of the disease.