Detection of genomic and intermediate replicative strands of hepatitis C virus in liver tissue by in situ hybridization.

Nonisotopic in situ hybridization using a digoxigenin-labeled cDNA probe to the 3' nonstructural region (NS5) of hepatitis C virus (HCV) was performed on liver tissue from 33 patients. The results were compared with PCR detection of HCV RNA performed on 24 of the biopsies. Nonisotopic in situ hybridization correlated well with PCR findings. Hybridization signals were detected, within the cytoplasm and nuclei/nucleoli of hepatocytes, mononuclear, and biliary epithelial cells. In patients with clinically and histologically defined chronic active hepatitis related to active HCV infection, HCV genome was frequently detected in biliary epithelium and correlated well with biliary damage, an otherwise uncommon finding in chronic active hepatitis due to other hepatotropic viruses. Further studies using sense and antisense probes synthesized from the 5' non-coding region of the HCV genome confirmed the localization of positive strand of HCV in the above cell populations. The replicative intermediate strand was also present in all cells, although less frequently observed, apart from biliary epithelium, where negative strand of HCV was undetectable. The findings of HCV genome in liver biopsies of two patients with no significant histological abnormalities may suggest that the damage seen in chronic HCV infection is immune mediated, although the cytopathic effect of the virus may also be important.     

丙型肝炎患者血清和外周血单个核细胞中HCV RNA检测及意义

目的 了解丙肝患者血清和外周血单个核细胞(PBMC)中HCV RNA存在情况及其临床意义。方法 应用套式PCR检测46例急性丙型肝炎(丙型肝炎以下简称丙肝)和42例慢性丙肝患者血清和PBMC中HCV RNA.结果 慢性丙肝患者PBMC中HCV RNA检出率显著高于急性丙肝患者(P<0.001);急、慢性丙肝患者血清和慢性丙肝患者PBMC中HCV RNA检出率显著高于ALT正常的抗-HCV阳性者(P<0.001);2例患者血清中HCV RNA阴性,PBMC中可测及,12例患者血清抗-HCV阴性,而血清HCV RNA阳性。结论 血清HCV RNA检测有助于抗-HCV阴性丙肝的诊断和早期诊断;丙肝的肝损害可能与HCV RNA血症有关;PBMC中HCV感染在丙肝的慢性化和慢性肝损害中可能起一定作用,PBMC中可贮存HCV RNA。     

一种竞争性RT-PCR测定HCV RNA方法的建立及其应用

目的　建立检测丙型肝炎病人血清HCVRNA水平的竞争性逆转录 聚合酶链式反应定量方法。方法　采用含HCV 5 非编码区 ( 5 NCR)缺失突变的重组质粒 5T1d ,将其目的基因亚克隆到体外转录载体pCDNAⅠ多克隆位点上 ,获得转录重组体 5T1d 。将其线性化后用SP6RNA聚合酶体外转录竞争性缺失突变模板RNA ,该模板定量后与血清HCVRNA一起作逆转录 聚合酶链式反应 (RT PCR) ,建立检测血清HCVRNA水平的竞争性定量方法。结果　检测 15例丙肝病人 2 0份血清 ,滴度为 6.6× 10 2 ～ 6.6× 10 6copies/ml。 结论　各类丙型肝炎患者血清病毒滴度均较高 ,经干扰素治疗后病毒水平下降 1～ 5个Log ,年龄小于 2 0岁者干扰素治疗效果较好。     

Risk factors in hepatitis C virus-infected blood donors

Risk factors of parenteral and nonparenteral exposure to hepatitis C virus (HCV) infection were studied in 125 blood donors in The Netherlands who were positive for anti-HCV on enzyme-linked immunosorbent assay (ELISA). Risk factors were related to confirmatory test results of four-antigen recombinant immunoblot assay (4-RIBA) and polymerase chain reaction (PCR) of the HCV 5' untranslated region. Twelve (10%) of the 125 anti-HCV C100 ELISA-positive blood donors were 4-RIBA positive. Eleven (92%) of 12 4-RIBA-positive blood donors were PCR positive, and all 113 remaining 4-RIBA-negative or -indeterminate donors were PCR negative. Eleven (92%) of 12 4-RIBA-positive blood donors had a risk factor of parenteral exposure, as compared to 17 (15%) of 113 4-RIBA-negative or -indeterminate donors. The prevalence of confirmed HCV infection among Amsterdam blood donors is calculated at 0.04 percent; parenteral exposure appears to be the major risk factor for HCV infection.     

Hepatitis C virus among blood donors: follow-up study

BACKGROUND: The exact significance of antibodies to hepatitis C virus (HCV) in blood donors remains unknown. Confirmatory tests of anti-HCV- reactive serum and HCV RNA by polymerase chain reaction (PCR) are used to refute a large proportion of false-positive results. STUDY DESIGN AND METHODS: Ninety-two blood donors who were anti-HCV reactive in a first-generation enzyme-linked immunosorbent assay (ELISA) were reevaluated 10 months later with a second-generation ELISA (ELISA-2) as well as with second-generation recombinant immunoblot assay (RIBA-2) and by PCR. RESULTS: Twenty-five (43.9%) of the 57 ELISA-2-positive donors were confirmed as positive by RIBA-2; of these, 84 percent were HCV RNA positive in PCR. Of the 57 who were still anti-HCV positive, 46 were followed up and tested again in the same manner 2 years after the first screening. At that time, the pattern was little changed: 94 percent of RIBA-2- and PCR-positive donors remained positive. Of RIBA-2- and PCR-positive blood donors, 62 percent had abnormal alanine aminotransferase levels in at least one of the three evaluations. Among the anti-HCV-positive donors confirmed by RIBA-2, 60 percent, versus 12.6 percent in the control group, had a significantly (p < 0.001) more frequent risk factor for HCV infection, due to parenteral exposure to blood. CONCLUSION: These data confirm a good correlation between RIBA-2 reactivity and the detection of HCV RNA in a population of anti-HCV- positive blood donors.     

HCV-RNA与HCV-cAg检测的相关性分析

目的探讨丙型肝炎病毒核心抗原(HCV-cAg)与HCV-RNA检测的相关性及其在丙型肝炎病毒感染诊断中的价值。方法采用双抗体夹心酶联免疫分析法(ELISA)检测HCV-cAg;采用实时荧光定量PCR法检测HCV-RNA,以了解两者检测方法的相关性。结果在160例HCV-RNA阳性血清中,HCV-cAg阳性148例,阳性符合率92.5%;在60例HCV-cAg阳性血清中,HCV-RNA阳性59例,阳性符合率98.3%。结论通过对HCV-RNA与HCV-cAg检测,说明HCV核心抗原与HCV-RNA检测可作为反映HCV复制的间接指标,预防窗口期感染。由于HCV-cAg在方法学上与HCV-RNA相比,具有方法简便、快速、价廉,所需设备简单,易于普及应用等优点,特别是在不具备HCV-RNA检测条件的基层医疗单位作为HCV感染检测的直接证据具有重要的意义。     

Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients

To establish the value of the second-generation recombinant immunoblot assay (RIBA-2) and cDNA polymerase chain reaction (cDNA PCR) for confirmation of hepatitis C virus (HCV) infection, anti-HCV reaction patterns and the presence of HCV RNA were examined in 610 blood donors and 255 non-A, non-B hepatitis patients who were positive or indeterminate in RIBA-2. Of RIBA-2-positive donors (n = 191) and patients (n = 224), 75.4 and 89.7 percent, respectively, were HCV RNA positive. The most frequently observed anti-HCV recognition patterns in HCV RNA-positive donors and patients were c22, c33c, and c100 and/or 5- 1-1 (67.3%, 57.7%) and c22, c33c (24.8%, 29.3%). Among subjects with a RIBA-2-indeterminate result, HCV RNA was detected more often in patients (n = 31) than in donors (n = 419): 67.7 and 2.1 percent, respectively. In viremic persons with single-band reactivity in RIBA-2, this reactivity was always directed against either c22 or c33c. HCV RNA was detected by cDNA PCR in none of 162 persons with only anti-c100 and/or anti-5-1-1 reactivity. Therefore, RIBA-2 reactivity against c100 in combination with 5-1-1 should not be considered positive but indeterminate. In RIBA-2-indeterminate persons, HCV RNA detection is necessary for reliable confirmation of HCV infection.     

HCV核心抗原酶联免疫检测与HCV—RNA定量检测敏感性的比较

[目的]比较ELISA法测定总HCV—cAg与荧光定量PCR法测定HCV—RNA两种检测方法对诊断HCV感染的敏感性,分析HCV—cAg ELISA法诊断试剂的重要意义。[方法]ELISA法测定血清样本中的HCV抗体,改进型ELISA法测定血清样本中的HCV—cAg,实时荧光定量PCR法测定血清样本中的HCV—RNA,阳性样本经重复实验确认。[结果]通过用HCV抗体检测试剂对门诊病人或献浆员进行筛查,获得HCV抗体阳性血清样本264例。对这些病例别进行ELISA法测定总HCV—cAg实验和荧光定量PCR法测定HCV—RNA实验,测到HCV—cAg阳性病例101例,HCV—RNA阳性病例115例。经统计学分析,两种检测方法的敏感性差别不太大（0．01〈P〈0．05）。[结论]HCV—cAgELISA法检测和HCV—RNA定量检测具有相近的敏感性,日存某种稗序上HCV—cAg ELISA法榆测可以作为HCV—RNA定量榆测的有效替代。     

献血者HCV RNA实时荧光定量PCR方法的建立

目的建立快速检测献血者中丙型肝炎病毒(hepatitis C virus,HCV)感染的方法。方法利用体外转录制备的HCV RNA转录体进行病毒RNA抽提方法及抽提效率的比较;将RNA转录体与正常血清进行不同数目的混合,对最佳混合标本数进行了摸索;建立HCV荧光定量PCR方法(fluorescence quantitative PCR,FQ-PCR),并对献血者进行混合标本HCV核酸检测。结果确定了比较理想的病毒RNA抽提方法;用于HCV核酸检测的混合标本数为24例;建立的荧光定量PCR方法能最低检测出10个copies/ml;采取24例混合血标本方法,FQ-PCR检测HCV RNA,576例ELISA检测HCV阴性的献血者未检测出HCV RNA。结论初步建立了献血者HCV感染的混合标本荧光定量PCR检测方法。     

A cell-based model of HCV-negative-strand RNA replication utilizing a chimeric hepatitis C virus/reporter RNA template

The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric 'HCV-like' RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5' and 3' untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5' HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4alpha that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.     

Hepatitis C virus RNA testing by nested PCR in blood preparations in Yugoslavia

Patients receiving any kind of human blood preparations are in permanent danger of any infection including hepatitis C (HCV) infection. Testing for the presence of HCV in blood preparations is one of the steps towards safe medical treatment. One of the approaches for this testing is a detection of HCV nucleic acid. In this paper we describe a simple method for isolation of HCV RNA from blood preparations and control of HCV RNA presence in 19 intravenous and intramuscular products, manufactured in the National Blood Transfusion Institute in Belgrade. RT-PCR was performed according the rules saving RNA. Primers were located in 5' conserved region. Seven out of 19 batches of gamma-globulin, albumin, anti-tetanus and anti-rabies immunoglobulin preparations were found to be HCV RNA positive. For the time being, the PCR method is too expensive for routine HCV RNA testing of hundreds of blood donors per day. Serological screening test of blood donors and nested PCR testing for HCV RNA in blood preparations could be an efficient combination of tests in prevention of posttransfusion hepatitis C.     

Serum hepatitis C virus (HCV) RNA: a reliable tool for evaluating HCV- related liver disease in anti-HCV-positive blood donors with persistently normal alanine aminotransferase values

BACKGROUND: The significance of hepatitis C virus (HCV) antibodies in blood donors with persistently normal alanine aminotransferase (ALT) levels requires evaluation. STUDY DESIGN AND METHODS: The serum and the liver were assayed for HCV RNA. Liver histology was analyzed in 14 HCV- positive subjects who had repeatedly normal ALT values over a follow-up period of at least 3 months. RESULTS: HCV RNA was not detected in liver and serum, and liver histology showed minimal changes in more than one- half of the subjects (8/14), even if third-generation recombinant immunoblot assay was positive; this demonstrated that HCV can be eradicated spontaneously. Moderately histopathological liver lesions were usually found in HCV RNA-positive subjects (6/14), but one subject had active disease that required interferon therapy; this shows that chronic hepatitis may be present in HCV-positive individuals despite repeatedly normal transaminase values. HCV genotypes other than 1b (II) were usually identified, and the presence or absence of serum and liver HCV RNA correlated completely in all 14 patients. CONCLUSION: Serum HCV RNA should be assayed in those HCV-positive donors having repeatedly normal transaminase activity; if it is positive, indicating an ongoing HCV infection, a liver biopsy should be performed to measure the degree of the liver disease and determine the appropriate antiviral therapy.     

Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction.

Hepatitis C virus (HCV) RNA was detected in the sera of patients with non-A, non-B chronic liver disease by polymerase chain reaction (PCR). RNA was extracted from the serum, reverse transcribed to cDNA, and amplified by PCR. With this method, 30 patients with non-A, non-B chronic liver disease and 10 healthy subjects were tested. HCV RNA was detected in 13 of 16 (81%) anti-HCV-positive patients and also in 7 of 14 (50%) anti-HCV-negative patients, but in none of 10 anti-HCV-negative healthy subjects. Specificity of this method was confirmed by direct sequencing of amplified cDNA segment. The nucleotide sequences (37 nucleotides) obtained from 15 patients showed only 68-78% homology compared with the prototype HCV nucleotide sequence. In addition, of 15 nucleotide sequences, there were 12 different types. But the translated amino acid sequences (12 amino acids) showed 83-100% homology compared with the prototype HCV amino acid sequence. These data suggest the majority of anti-HCV-positive patients are carriers of HCV. But to detect all the viremic patients, the anti-HCV antibody testing may be insufficient. Direct detection of HCV RNA may be useful in the study of virus replication and its association with various liver diseases.     

Correlation of viral RNA, alanine aminotransferase, and histopathology in hepatitis C virus-associated hepatitis.

BACKGROUND: Hepatitis C virus (HCV) infection is usually monitored by the level of alanine aminotransferase (ALT) and histopathological changes in liver biopsy specimens. However, accumulating data indicate these parameters are not always correlated with disease progression or the response of HCV infection to therapy. MATERIALS AND METHODS: Using the Amplicor PCR Monitor Test Kit (Roche Diagnostic Systems, Branchburg, NJ), HCV RNA level was measured in 38 patients with positive anti-HCV antibodies and in 21 of those patients after interferon treatment. The grade and stage of histological changes on hematoxylin and eosin-stained sections of liver biopsy specimens were evaluated on a scale of 1 to 4. In each case, the HCV RNA level was compared with the histological grade or stage and level of ALT and statistically analyzed by Student's t-test. RESULTS: ALT level did not correlate with pretreatment and posttreatment levels of HCV RNA or histopathological changes. However, there was a statistically significant correlation between HCV RNA and histological grade (P,.05). CONCLUSION: HCV RNA measurement is a better means of determining and monitoring HCV infection than either ALT level or histopathological characteristics and may provide insight into hepatic injury caused by HCV infection even without an invasive liver biopsy.     

IgM antibody response to the hepatitis C virus core protein in intravenous drug users

To verify whether a solid-phase enzyme immunoassay for serum IgM antibodies to the hepatitis C virus (HCV) core protein (IgM anti-HCVcore) might be proposed as a surrogate test for serum HCV RNA, we studied 86 anti-HCV antibody-positive intravenous drug users. Serum HCV RNA was demonstrated by RT-PCR with primers derived from the 5' non-coding and the core region. IgM anti-HCVcore antibodies were found in 62/86 (72%) subjects; circulating HCV RNA was detected by the 5' noncoding assay in 53/86 samples (62%) and by the core region assay in 35/86 samples (41%). IgM anti-HCVcore reactivity was associated with core HCV RNA seropositivity (p < 0.05) but not with 5' noncoding HCV RNA seropositivity (p = NS). Patients infected by HCV type 1a were more-often positive for IgM anti-HCVcore (p < 0.05) and for core HCV RNA (p = 0.005) than patients infected by other HCV genotypes. IgM anti-HCVcore reactivity was significantly more common in subjects positive for core HCV RNA (p < 0.005) and in subjects aged > 30 years (p < 0.05). In conclusion, the IgM anti-HCVcore assay frequently tests positive in intravenous drug users, particularly when infected by HCV 1a, but is not a surrogate of testing for serum HCV RNA.     

丙型肝炎病毒载量与核心抗原及丙氨酸转氨酶检测的比较研究

目的研究实时荧光定量PCR定量检测丙型肝炎病毒(HCV)RNA载量与HCV核心抗原和丙氨酸氨基转移酶(ALT)检测的相关性及符合性。方法逆转录-PCR荧光探针法定量检测94例怀疑HCV感染患者的血清HCV RNA,同时用酶联免疫吸附试验(ELISA)检测HCV核心抗原,全自动生化分析仪检测ALT水平。结果 94份样本中HCV RNA阳性率为56.4%(53/94),核心抗原阳性率为53.2%(50/94),经统计学分析,两种方法的阳性率差异无统计学意义(P>0.05),符合率为84.9%(45/53);ALT异常率为62.3%(33/53),并随HCV RNA载量的升高而增加。结论 HCV RNA定量检测及HCV核心抗原检测结合ALT结果分析有助于临床了解HCV在体内的复制水平和肝脏的炎性反应状态,指导临床用药及观察疗效。     

HBV和HCV重叠感染患者生化免疫指标变化及临床意义

目的了解HBV和HCV重叠感染情况及其对肝功能及血清病毒载量的影响。方法常规检测3816例临床血清样本中的HBV及HcV血清标志物以及肝功能等指标,实时荧光定量PCR检测病毒载量。结果3816例送检样本中HBsAg阳性率8．9％（341／3816）,抗HCV抗体阳性率1．2％（46／3816）,其中HCVRNA检测阳性15例,现症感染率为0．39%。3816例样本中,HBV／HCV重叠感染15例,单纯HBV感染326例,单纯HCV感染31例。单纯HBV感染患者血清转氨酶（ALT和AST）及总胆红素（TBil）水平均明显高于重叠感染患者和单纯HCV感染患者。HBV／HCV重叠感染组患者HBVDNA栽量低于单纯HBV感染组,两组比较,差异有统计学意义（P〈0．05）;HBV／HCV重叠感染组患者HCVRNA载量也低于单纯HCV感染组,但两组比较,差异无统计学意义（P〉O．05）。结论重庆地区HBV／HCV感染情况与我国一般人群分布基本一致,重叠感染患者肝功能血清学指标改变低于单纯HBV感染患者。     

Antiproliferative effect of hepatitis C virus on mitogen-stimulated peripheral blood mononuclear cells: potential role in viral persistence in Egyptian patients

OBJECTIVES: We aimed to study the effect of hepatitis C virus (HCV) and sera of chronic HCV patients on phytohemagglutinin (PHA)-stimulated normal donor PBMCs and to study the effect of chronic HCV infection on some cytokine profile. SUBJECTS AND METHODS: (3)H-Thymidine uptake was utilized to study effect of pelleted virus and patients sera on PBMCs proliferation in vitro. The study included 337 Egyptian chronic liver patients from Ain Shams University Hospitals and 90 healthy control subjects. The patients' group included chronic hepatitis C (250 subjects), and other chronic liver diseases (87 subjects). All subjects' sera were subjected to RT-PCR for HCV RNA detection, IL-4, IL-1beta, and TNF-alpha measurement by EIA, and biochemical measurement of ALT and albumin. RESULTS: Treatment of PHA-stimulated normal donor PBMCs with pelleted virus led to decrease (dose response) in their rate of proliferation. This was partially reversed after addition of HCV RNA positive patients' sera. HCV RNA positive patients were significantly higher in IL-4 and ALT, and lower in IL-1beta and albumin than HCV RNA negative patients. CONCLUSION: HCV infection suppresses early immune response. This leads to increased IL-4 Th2 cytokine.