Direct detection of circulating hepatitis C virus RNA using probes from the 5'' untranslated region.

Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards.     

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase

BACKGROUND: Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT-elevated blood units. STUDY DESIGN AND METHODS: Testing was performed on 375 units of plasma, derived from an equivalent of 47,500 blood donations, with a highly sensitive hemi-nested PCR procedure. Using a triplet of primers directed at the conserved regions of HBV DNA and 5'-noncoding regions of HCV RNA, the hemi-nested PCR assay can reliably amplify 10 viral molecules to levels detectable in ethidium bromide-stained agarose gels. Pools of plasma from groups of four donors were screened with hemi-nested PCR. For any reactive pools, the plasma from individual donors was retested twice on different aliquots. RESULTS: Two of 375 units, both with midrange ALT elevation, were repeatedly reactive in hemi-nested PCR (one each for HBV DNA and HCV RNA). However, samples from the two suspect donors tested 9 and 5 months later revealed no seroconversion, elevated ALT, or viral genomes in hemi-nested PCR. CONCLUSION: The lack of confirmed HBV or HCV infection in this study representing an estimated 47,500 voluntary blood donations suggests that routine ALT testing for further prevention of posttransfusion hepatitis after exclusion of HBV- and/or HCV-seropositive blood may be superfluous.     

Detection of GB virus C/hepatitis G virus RNA by reverse-transcription polymerase chain reaction.

Recently, a novel RNA virus, designated GB virus C or hepatitis G virus (GBV-C/HGV) has been identified which may possibly be associated with human hepatitis. In this study, the nucleotide sequences of the partial nonstructural 5 (NS5) gene of GBV-C/HGV derived from sera of eight Chinese patients were determined. The overall degree of nucleotide conservation and the existence of regional highly conserved sequences make this part of the genome suitable for the development of diagnostic reagents. On the basis of sequence analysis, two sets of oligonucleotide primers were designed to establish a nested polymerase chain reaction (PCR) for detection of GBV-C/HGV RNA. The efficacy of three PCR methods (first, one stage PCR, second, nested PCR with primers from the NS5 region designed according to the prototype sequence and the third, our newly developed PCR) was compared in 133 Chinese patients with liver disease. The positive rates of these three methods were 8.3%, 11.3% and 18.0% respectively. The specificity of our PCR detecting system was verified by sequencing and restriction fragment length polymorphism (RFLP). In conclusion, because of the heterogeneity and geographic distribution character of GBV-C/HGV, it is necessary to assess the sequence variation among Chinese patients infected with GBV-C/HGV. This may allow to identify GBV-C/HGV RNA with high sensitivity and specificity.     

亚甲蓝光化学灭活对血浆丙型肝炎病毒基因组核酸降解的研究

目的研究丙型肝炎病毒(HCV)基因组核酸在亚甲蓝光化学法(methelene blue photochemistry,MB-P)灭活病毒前后的变化,在全基因组水平分析MB-P对基因组各结构区段的降解作用。方法含HCV的血浆中加入终浓度为1.0μmol/L的MB,经约30000Lux强度的荧光照射后,在不同作用时间点取样;将HCV全基因组序列分为互相重叠的8个区段,分别进行RT-PCR,分析基因组核酸的完整性;同时运用实时定量PCR(real time-PCR,RT-PCR)技术观察核酸降解的动力学变化。结果基因组各区段RT-PCR结果发现,经过不同的光照时间,HCV基因组各区段的稳定性不同,第2、4、5、6区段对MB-P作用较敏感,基因组5’端区段和3’端区段经MB-P作用后的稳定性高于基因组其它区段;RT-PCR结果显示,随着光照时间延长,可被检测到的病毒核酸拷贝数逐渐下降。结论MB-P灭活过程中HCV基因组核酸被降解,而且基因组不同区段对光化学作用的反应性不同,提示RNA降解可能是病毒灭活的重要机制;检测病毒核酸稳定性以监测病毒灭活具有一定临床实用价值。     

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.     

The 5'-terminal sequence of the hepatitis C virus genome

The 5'-terminal sequence of the genome of hepatitis C virus (HCV) was determined for two distinct HCV strains in human and chimpanzee carriers. It had a 5'-noncoding region of at least 324 nucleotides, well preserved by the two strains with a high homology (99.1%), followed by 1348 nucleotides that continued to the documented sequence of prototype HCV spanning 7310 nucleotides (European Patent Application #88310922.5). Based on these results, HCV is considered to possess an uninterrupted open reading frame encoding at least 2886 amino acid residues. Two structural genes were postulated on the 5'-terminal sequence of the HCV genome. One gene in the upstream region, highly conserved by the two strains at the amino acid level and rich in basic amino acids such as arginine, appeared to encode the viral capsid protein. The other gene in the downstream region was divergent between the two strains at both nucleotide and amino acid levels. It coded for nine potential N-glycosylation sites, and was considered to encode the viral envelope protein. Disclosure of the 5'-terminal sequence of the HCV genome would facilitate its taxonomic classification, and contribute toward immunological diagnosis of infection and development of vaccines.     

GB virus C/Hepatitis G virus.

Hepatitis G virus (HGV) is a positive, single-strand RNA virus that has been classified in the family Flaviviridae. The 5'-untranslated region (UTR) of the HGV genome is lengthy and does not share any significant primary and secondary RNA structures with the 5'-UTR of hepatitis C virus (HCV). The internal ribosome entry site has extraordinarily weak activity. The HGV genome does not seem to encode a nucleocapside protein analogous to HCV. Blood-borne transmission is presumed to be the commonest mode of transmission of the virus. Current infection with HGV is diagnosed by detection of HGV RNA by the polymerase chain reaction (PCR), and past infection with HGV is detectable by testing anti-HGV E2. HGV is distributed worldwide, but its prevalence varies widely from one population to another. Although the prevalence of HGV in association with acute and chronic hepatitis is higher than that in the general population, further prospective studies are needed to demonstrate its relative significance in causing hepatitis and other disease. The major unresolved biological issue at the moment is its hepatotropsim and site of propagation. Recent progress demonstrates that HGV replicates in lymphocytes rather than hepatocytes. HGV may be pathogenic under special conditions, but does not influence carcinogenesis.     

新型二聚体突变荧光引物定量PCR方法的建立及其在检测HCV中的应用

目的 开发新型二聚体突变荧光引物技术,建立一种能广泛应用于临床检测HCv的实时PCR方法.方法 构建重组质粒pMD18-T-HCV 5′NCR作为标准品,设计二聚体突变荧光引物,优化定量PCR体系,并进行方法学评价.将本法应用于临床确诊的30份HCV阳性患者、30份其他病毒性肝炎患者和30份健康志愿者血清标本的检测,定量结果与商品化TaqMan HCv定量试剂盒的定量结果进行比较.结果 建立了利用二聚体突变荧光引物的实时PCR方法,检测的线性范围为20～109IU/ml;批内CV在1.37%～4.59%之间,批间CV在1.58%～4.81%之间;对所有其他病毒性肝炎患者和健康志愿者血清HCV的检测结果均阴性,检测特异性为100%(60/60);对临床确诊的HCV感染患者血清标本全部检出HCV阳性,定量结果与商品化TaqMan HCV定量试剂盒的定量结果具有很好的相关性,相关系数R2=0.9501.结论 建立的以二聚体突变荧光引物为平台的HCV实时PCR检测方法,具有快速、价廉、准确、结果可靠等特点,可为HCV感染的诊断、治疗监测和流行病学调查提供较好的技术支持.     

Transmission of serologically silent hepatitis B virus along with hepatitis C virus in two cases of posttransfusion hepatitis

The polymerase chain reaction (PCR) was used to investigate the presence of hepatitis B virus (HBV)-related DNA sequences in blood from three blood donors and two transfusion recipients who developed posttransfusion non-A, non-B hepatitis (NANBH). In the first case, the sole donor was positive for antibody to hepatitis B surface (HBs) and core (HBc) antigens and had elevated alanine aminotransferase (ALT) levels, while the recipient had no HBV serologic markers. Both the donor and the recipient had serologic markers of hepatitis C virus (HCV) and were found positive for HBV DNA and HCV RNA sequences by PCR. The second case involved two donors and one recipient. Serologic tests for conventional HBV markers were negative in all three individuals, but one of the donors had elevated ALT. HBV DNA sequences were detected by PCR in the serum of the recipient and of the donor with high ALT, but not in the serum of the donor with normal ALT. Anti-HCV was detected in the serum of the recipient and of the suspect donor but not in that of the donor with normal ALT. The sequences amplified in the S region and determined after cloning of PCR products for both donor-recipient pairs were indistinguishable from each other and identical to the sequence of the major HBV subtype of adw in the first case and ayw in the second case. Furthermore, for the second case, an identical single-point mutation was found in both the donor and the recipient. These data confirm the transmission of conserved HBV sequences together with HCV in posttransfusion NANBH.(ABSTRACT TRUNCATED AT 250 WORDS)     

多重定量PCR法同步检测HBV、HCV和HIV-1在血液筛查中的优化及应用

目的 初步探讨多重定量聚合酶链反应（PCR）同步检测乙型肝炎病毒（HBV） DNA,丙型肝炎病毒（HCV） RNA及人类免疫缺陷病毒（HIV）-1 RNA在血液筛查中的应用前景.方法 选择2012年8月至12月,于孝感市中心血站志愿献血的合格献血者血样中,经2次酶联免疫吸附法（ELISA）检测HBV表面抗原（HBsAg）、抗HCV及抗HIV-1,检测结果均呈阴性的4 800份血样为研究对象.采用全自动核酸混合提取仪对该4 800份血样进行核酸提取,然后利用多重定量PCR方法对血样中HBV、HCV及HIV-1进行同步扩增检测.采用中国药品和生物制品检定所提供的HBV DNA、HCV RNA及HIV-1 RNA标准参考品,检测多重定量PCR的灵敏度,并与单重定量PCR的灵敏度进行比较.在HBV、HCV及HIV-1 3者中任意1种病毒基因组浓度较高的条件下,对多重定量PCR检测另2种低浓度病毒基因组的能力进行评估.结果 增加多重定量PCR中c-MMLV逆转录酶和Hot Taq酶的用量,并适量加入单链结合蛋白（SSB）,可使其扩增效率提升至单重定量PCR扩增水平.本组4 800份血样中,经多重定量PCR检测出3份HBV DNA阳性样品,ELISA漏检率为0.062 5％,未发现HCV RNA和HIV-1 RNA阳性样品;多重定量PCR检测HBV DNA、HCV RNA及HIV-1 RNA在95％置信区间的灵敏度浓度分别为115 IU/mL、376 IU /mL和232 IU /mL;单重定量PCR检测HBV DNA、HCV RNA和HIV-1 RNA在95％置信区间的灵敏度浓度分别为51 IU /mL、94 IU /mL和78 IU/mL.结论 本研究初步建立了对献血者血液同时进行HBV DNA、HCV RNA及HIV-1 RNA检测的多重定量PCR检测方法;该检测体系经过进一步优化后,有望应用于临床大规模血液病毒筛查.     

Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.

To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.     

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。     

Partitioning of hepatitis C virus during Cohn-Oncley fractionation of plasma

Because of concern about the safety of immune globulins with respect to transmission of hepatitis C, the partitioning of hepatitis C virus (HCV) during alcohol fractionation of a plasma pool prepared exclusively from anti-HCV-reactive donations was examined. Quantitation of HCV RNA was accomplished by nested polymerase chain reaction (PCR) at limiting dilutions. One PCR unit was arbitrarily defined as the minimum amount of HCV RNA from which an amplified product could be detected. The starting plasma pool contained 1.4 x 10(5) PCR units per mL. Most of the HCV RNA was found in cryoprecipitate and in Cohn fractions I and III, but it was also detected in fraction II, which is used for immunoglobulin G preparations. A 3.4-percent solution of IgG prepared from this fraction II contained 30 PCR units per mL. The fractionation process leading to immune globulin resulted in overall reduction in HCV RNA by a factor of 4.7 x 10(4). Although the presence of HCV RNA in the final product does not necessarily imply the presence of infectious virus, this work suggests that the safety of immune globulins with respect to HCV transmission is not due solely to the partitioning of HCV away from the immunoglobulin fraction.     

Prevalence of hepatitis G in patients on chronic hemodialysis

Hepatitis G virus (HGV) is a newly described RNA virus from the family of flaviviridae. It is closely related to the hepatitis C Virus (HCV) but is more common than HCV among healthy blood donors. The pathogenicity of HGV in immunosuppressed patients such as those undergoing hemodialysis is unclear. We measured the incidence of HGV in 105 patients undergoing hemodialysis in a chronic outpatient hemodialysis facility. HGV-RNA was detected using a RT-PCR method with primers directed against the 5' non-coding region and the NS5a gene of HGV. Nine (8.6%) patients were HGV RNA positive, eleven (10.5%) were anti-HCV positive, three (2.9%) were positive for hepatitis B surface antigen. Four patients were positive for both HGV and HCV; three of them had normal liver enzymes while one showed elevated ALT levels but no other signs of exacerbation of preexisting hepatitis. The prevalence of HGV among dialysis patients is comparable to that of HCV. The transmission route for HCV is nosocomial transmission during dialysis, whereas HGV shows both ways of transmission: blood transfusion mediated by a high prevalence of HGV among healthy blood donors and nosocomial transmission. HGV appears to play a minor role in acute hepatitis, even in immunosuppressed patients.     

Absence of hepatitis C virus RNA in ambiguous results of mass blood screening for hepatitis C virus antibodies

The purpose of the study was to elucidate whether hepatitis C virus (HCV) RNA was present in the blood of patients from a Moscow therapeutic-and-prophylactic institution with uncertain results of a study of anti-HCV that were obtained using the algorithm developed for mass screening. The two-stage scheme of testing the serum for anti-HCV, which was obligatory in accordance with the Order of the Ministry of Health of the Russian Federation, was supplemented by a study the samples having a low optical density samples and those with controversial results in the test systems with the expanded spectrum of detectable anti-HCV, the instructions of which comprises criteria for an uncertain result. Sera with uncertain results of anti-HCV tests were assessed by two polymerase chain reaction (PCR) techniques for HCV RNA. The cDNA fragments complementary to HCV RNA were detected in the sera obtained from two elderly persons in PCR when a signal was recorded in agarose gel. The other real-time PCR failed to detect RNA in none sera with an uncertain result as to anti-HCV.     

经宫内传播的丙型肝炎病毒基因分型

目的：探讨经宫内传播的丙型肝炎病毒(hepatitis C virus，HCV)在母亲与子代间的基因型别一致性，进一步确定HCV母婴传播的分子学基础。方法：选择6对母亲与胎儿抗-HCV与HCV RNA(ribonucileic acid)均阳性者为研究对象，以PCR技术检测母亲与胎儿HCV RNA的基因型别。结果：6对母亲与胎儿的HCVRNA基因型均一致，其中5对基因型别属Ⅱ型，1对属Ⅲ型。结论：母亲与胎儿所携的HCV基因型别一致。     

Hepatitis C virus and GB virus C/hepatitis G virus viremia in Swedish blood donors with different alanine aminotransferase levels

BACKGROUND: Hepatitis C virus (HCV) is a known blood-borne hepatotropic virus for which antibody screening of blood donors is universally practiced. The newly identified GB virus C (GBV-C) and its strain variant hepatitis G virus (HGV) are of unknown pathogenic significance, and screening of blood donors for this agent has not yet been implemented. Polymerase chain reaction (PCR) is the most sensitive method for detecting HCV viremia and is the only method presently available for the diagnosis of GBV-C/HGV infection. STUDY DESIGN AND METHODS: RNA extracts of sera from 577 anti-HCV-negative blood donors (393 with elevated alanine aminotransferase [ALT] levels, 184 with normal ALT levels) were tested with nested PCR for HCV and GBV-C/HGV directed at the 5'-noncoding regions of the two viruses. RESULTS: One donor with elevated ALT was HCV PCR positive. This donor was anti-HCV negative when recruited to the study but subsequently developed anti- HCV. Of the 19 donors with GBV-C/HGV viremia in the series as a whole, 16 belonged to the group with elevated ALT levels and 3 to the group with normal ALT levels; the group difference in prevalence was nonsignificant (4.1% [16/393] vs. 1.6% [3/184; p = 0.20]). Phylogenetic analysis showed 16 of the GBV-C/HGV isolates to be classifiable as subtype 2a and three as subtype 2b. At follow-up 3 to 5 years later, 11 of 18 donors were still viremic. CONCLUSION: There was no significant difference in GBV-C/HGV viremia in the group with elevated ALT levels and the group with normal ALT levels. The frequency and subtype distribution in the present series were similar to those in other Western countries.     

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.