Detection of GB Virus-C/Hepatitis G Virus RNA in Serum by Reverse Transcription Polymerase Chain Reaction

Three PCR methods based on the GB virus-C/hepatitis G virus (GBV-C/HGV) 5′UTR and NS3 genomic region were used for the detection of GBV-C/HBV RNA in serum of 62 patients with chronic hepatitis C virus (HCV) infection. Ten of 62 (16%) patients were found to have GBV-C/HGV RNA, which was confirmed by sequence analysis of the 5′UTR PCR amplicon. All methods appear to be specific, but methods based on the 5′UTR appear to be more sensitive. J. Med. Virol. 52:92–96, 1997. © 1997 Wiley-Liss, Inc.     

利多组套式引物的测庚型肝炎病毒RNA

利用庚型肝炎病毒ＮＳ３－５区序列合成了四组套式引物，建立了灵敏，特异的 型肝炎病毒ＲＮＡ双扩增聚合酶链反应检测方法，用此方法检测了１０份庚型肝炎病毒抗体阳性患者血清及１０份阴性的健康人血清。前者不同组引物的检出率为ＮＳ３（１）引物９份了是性，ＮＳ３（２）引物８份阳性，ＮＳ４引物４份阳性，ＮＳ５（１）引物５份阳性，ＮＳ５（２）引物９份阳性；后者各组引物匀为阴性。结果表明，庚型肝炎病毒不同区域引物用于     

利用多组套式引物检测庚型肝炎病毒RNA

利用庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）ＮＳ３～５区序列合成了四组套式引物，建立了灵敏、特异的庚型肝炎病毒ＲＮＡ双扩增聚合酶链反应（ＰＣＲ）检测方法。用此方法检测了１０份庚型肝炎病毒抗体阳性患者血清及１０份阴性的健康人血清。前者不同组引物的检出率为ＮＳ３（１）引物９份阳性，ＮＳ３（２）引物８份阳性，ＮＳ４引物４份阳性，ＮＳ５（１）引物５份阳性，ＮＳ５（２）引物９份阳性；后者各组引物检测均为阴性。结果表明，庚型肝炎病毒不同区域引物用于ＲＴ－ＰＣＲ检出率差异较大。ＮＳ３（１）及ＮＳ５（２）这两组引物检出率最高，更适合用于ＲＴ－ＰＣＲ检测庚型肝炎病毒ＲＮＡ。     

Profiles of GBV-C/hepatitis G virus markers in patients coinfected with hepatitis C virus.

GBV-C/Hepatitis G virus (GBV-C/HGV) is a newly discovered viral agent, found widely among healthy blood donors and among individuals at risk of parenterally transmitted infections. GBV-C/HGV is found frequently in coinfection with HCV. A population of 109 HCV positive patients was examined for the presence of GBV-C/HGV RNA and antibodies to E2. Of the 109 patients, 23 (21%) had serum GBV-C/HGV RNA in serum, 39 (36%) had only antibodies to E2 and 8 (7%) were positive for both markers, with an overall prevalence of 64%. Different serologic and virological patterns were observed in GBV-C/HGV exposed patients according to their infection status. Active infection was characterized by positive RT/PCR signal with primers for both the 5'UTR and NS5 genomic regions, viremia levels above 10(4) copies/mL by real time quantitative RT/PCR and absence of detectable anti-E2. In the transition phase between active infection and recovery, GBV-C/HGV RNA was only detectable by RT/PCR using primers from the 5' untranslated region and viremia levels were below 10(4) copies/ml by quantitative PCR, with or without simultaneous presence of anti-E2 antibodies. Resolved infection was characterized by absence of detectable viremia and, in most patients, by the presence of anti-E2.     

肝病患者中庚型肝炎病毒感染的检测

目的探讨临床肝病病人中庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）感染情况及临床特点。方法应用庚型肝炎病毒基因组５’ＵＴＲ两对寡核苷酸作为引物，建立逆转录套式聚合酶链反应，检测１６９例不同肝病患者血清标本中ＧＢＶ－Ｃ／ＨＧＶＲＮＡ，并对其中１例ＰＣＲ扩增产物进行克隆及测序。结果１６９例各型肝病病人ＧＢＶ－Ｃ／ＨＧＶＲＮＡ总的检出率为９５％（１６／１６９）。在２９例有手术输血史患者中，３１０％（９／２９）ＧＢＶ－Ｃ／ＨＧＶＲＮＡ呈阳性，明显高于无手术输血史组（５％，Ｐ＜００１）。序列分析显示１株庚肝病毒５’ＵＴＲ部分基因片段与已知庚肝病毒株核苷酸同源性在８９１４％～９８９１％之间。结论ＧＢＶ－Ｃ／ＨＧＶ感染普遍存在于临床肝病患者中，病人感染ＧＢＶ－Ｃ／ＨＧＶ的临床表现未发现有特殊性，ＧＢＶ－Ｃ／ＨＧＶ可能不是非甲～戊型肝炎的主要致病因子。     

Detection of hepatitis G virus RNA in patients with hepatitis B,hepatitis C,and non-A-E hepatitis by RT-PCR using multiple primer sets

Hepatitis G virus(HGV)/GB virus C(GBV-C) is a newly identified virus associated with human hepatitis. The preliminary prevalence studies of HGV infection in Japan were entirely based on the detection of HGV RNA by RT-PCR. However, the selection of the different primer sets in such assay may influence sensitivity of the test because of the extensive genetic heterogeneity of HGV, and influence the estimation of the prevalence of HGV. To address this potential problem, we designed two primer sets from well conserved domains in the 5′NC and NS5 regions of HGV genome, and tested them together with the NS3-derived primer set in RT-PCR for their ability to detect HGV RNA in serial dilution of synthetic viral RNA templates. Subsequently, we used these three primer sets to detect HGV RNA in the sera of 371 Japanese patients with hepatitis B, hepatitis C, and non-A-E hepatitis. The results indicated that the primer set derived from the 5′NC region appeared to be most effective in detecting HGV RNA. The results also showed that only two out of the 126 patients (1.6%) with non-A-E hepatitis were positive for HGV RNA although the RNA were detected more frequently in patients with hepatitis B (2/38; 5.3%) and hepatitis C (17/207; 8.2%), suggesting that HGV is not a common causative agent for non-A-E hepatitis in Japan. J. Med. Virol. 52:385–390, 1997. © 1997 Wiley-Liss, Inc.     

Prospective follow-up of patients with GBV-C/HGV infection: specific mutational patterns, clinical outcome, and genetic diversity

An association between a specific mutational pattern within the nonstructural (NS)3 region of GB virus-C/hepatitis G virus (GBV-C/HGV) genome and fulminant hepatic failure has been suggested recently. The mutational pattern consists of 3-6 nucleotide mutations of which one is leading to an amino acid exchange. In the present study, patients with GBV-C/HGV mono-infection (n = 24) or GBV-C/HGV and HCV co-infection (n = 20) were investigated prospectively. In 6/44 patients (14%) the mutational pattern within GBV-C/HGV NS3 previously associated with fulminant hepatic failure was identified by direct sequence analysis of the NS3 region. All 44 patients were asymptomatic clinically and had normal liver functions at initial presentation and after a median follow-up of 2.2 years. In 22/24 patients with GBV-C/HGV mono-infection and all patients with GBV-C/HGV and HCV co-infection GBV-C/HGV RNA remained detectable at the end of the study period, whereas two patients infected with GBV-C/HGV alone became negative for GBV-C/HGV RNA and developed GBV-C/HGV anti-E2 antibodies indicating recovery from GBV-C/HGV infection. Aminotransferase levels remained elevated or became normal independent of the persistence of serum GBV-C/HGV RNA. The median rate of nucleotide substitutions in GBV-C/HGV mono-infected and HCV co-infected patients was 3.4 x 10(-3) and 3.2 x 10(-3) per site per year, respectively. In conclusion, the prevalence of the mutational pattern within NS3 region of GBV-C/HGV associated previously with fulminant hepatic failure is about 14% and not associated specifically with severe liver disease. Over a median follow-up of 2.2 years less than 5% of patients cleared spontaneously GBV-C/HGV and no correlation between viraemia and elevated liver enzymes was observed.     

High prevalence of GBV-C hepatitis G virus infection in a rural South African population

A novel virus, GBV-C/hepatitis G virus (GBV-C/HGV), has been cloned and characterised recently. GBV-C/HGV global epidemiology and risk factors for acquisition are currently unclear. We aimed to establish the determinants of this infection in a rural South African (SA) population. The study population included two samples, namely a community-based sample, and consenting persons from a nonspecialist outpatient department in the same district. A questionnaire regarding demographic details and putative risk factors was administered; blood samples were taken on which a polymerase chain reaction (PCR) was performed for both 5′NCR and NS5a regions of GBV-C/HGV using commercially available primers and probes. Two hundred and forty-nine people were studied with a mean GBV-C/HGV prevalence of 10.4%. Outpatient department and community prevalences differed significantly (18.0% and 6.3%, respectively, P = 0.004). GBV-C/HGV infection was associated with excessive alcohol consumption (P = 0.02; OR, 4.18) and a lack of waterborne sewerage (P = 0.04). PCR amplification of the NS5a region of all but two South African GBV-C/HGV positive samples showed poor reactivity. The prevalence of GBV-C/HGV in rural SA appears to be higher than that reported from Europe and North America. Infection appeared to be associated with excess alcohol intake and a history of previous blood transfusions. The discrepant NS5a and 5′NCR PCR sensitivity in this study raises the possibility of genetic differences in southern African GBV-C/HGV. J. Med. Virol. 53:225–228, 1997. © 1997 Wiley-Liss, Inc.     

High prevalence of GB virus C/hepatitis G virus infection in different risk groups of HIV-infected patients

Objectives: To investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA and anti-E2 antibodies in different risk groups of HIV-infected patients compared to that in healthy blood donors, and to study the effects of possible interactions between HIV and GBV-C/HGV on the carrier state and hepatic changes.
Methods: Sera from 100 consecutive unselected HIV-infected outpatients and from 100 healthy blood donors were screened for GBV-C/HGV viremia and anti-E2 antibodies. Anti-E2 antibodies were detected using an immunoassay developed by Boehringer Mannheim according to the manufacturer's instructions. GBV-C/HGV RNA was extracted from sera and reverse transcribed. The resulting cDNA was amplified with a PCR developed in the laboratory with primers derived from the 5' noncoding region of the viral genome and detected with a specific capture probe. This procedure was validated by a French multicenter quality control group.
Results: Thirty-one of the 100 HIV-infected patients and 8% of the healthy blood donors displayed anti-E2 antibodies. Four HIV-infected patients and one healthy blood donor were found to be GBV-C/HGV viremic. When analyzed by risk factor for the acquisition of HIV, no differences in the prevalence of anti-E2 antibodies were found between intravenous drug users and homosexual and heterosexual patients.
Conclusions: We found a high prevalence of GBV-C/HGV infection in the HIV-infected population, irrespective of the risk group factor for HIV infection, suggesting that the sexual route is as effective as the parenteral route for the acquisition of GBV-C/HGV. No biological alteration could be attributed to GBV-C/HGV, even in the viremic patients. HIV-infected patients were able to clear GBV-C/HGV viremia and to mount a humoral immune response.     

GBV-C/HGV infection in hepatitis C virus-infected deferred Swedish blood donors

Sera from 62 hepatitis C virus (HCV)-infected Swedish blood donors were tested by a nested polymerase chain reaction using primers targeting the 5′-noncoding region of the GB virus-C/hepatitis G (GBV-C/HGV) genome and an enzyme-linked immunosorbent assay that detects antibodies to the envelope protein E2 of GBV-C/HGV (anti-E2). Fourteen (22%) and 21 (34%) of the 62 blood donors were found to be GBV-C/HGV RNA and anti-E2 positive, respectively. None of the blood donors was positive for both GBV-C/HGV RNA and anti-E2. Thus, 35 of 62 (56%) HCV-infected donors had been exposed to GBV-C/HGV infection. At sequencing of the 14 GBV-C/HGV isolates, 12 were identified as subtype 2a and 2 as subtype 2b. One of 7 (14%) donors with mild liver disease such as steatosis and nonspecific reactive hepatitis had been exposed to GBV-C/HGV vs. 34 of 55 (62%) with chronic hepatitis with or without cirrhosis (P = 0.04). All other differences in histology were small between HCV and dual HCV GBV-C/HGV-infected donors. In conclusion, more than half of HCV-infected Swedish blood donors in this study were positive for either GBV-C/HGV RNA or anti-E2. GBV-C/HGV viremia and seropositivity were mutually exclusive. J. Med. Virol. 54:75–79, 1998. © 1998 Wiley-Liss, Inc.     

从MT2、HeLa细胞DNA及人和黑猩猩PBMC DNA中扩增GBV-C核苷酸

目的　检测MT2和HeLa细胞DNA、6例人和 1例黑猩猩PBMCDNA中是否存在与GBV C同源的核苷酸序列。方法　直接以上述DNA为模板、采用GBV C 5′ NCR和NS3区引物的直接套式PCR(dPCR)、核苷酸序列分析、引物介导原位扩增 (PRINS)NDA序列特异荧光标记技术。结果　从MT2和HeLa细胞DNA、4例人PBMCDNA标本中获得 5′ NCR引物扩增片段 ,从MT2和HeLa细胞DNA、5例人和黑猩猩PBMCDNA标本中获得NS3区引物扩增片段。这些扩增产物的核苷酸序列与GBV C同源性分别为 74 2 9%～ 77 14%和 73 80 %～ 79 15 %。PRINS检测结果显示 ,dPCR阳性的PBMC及其染色体上有荧光着色。结论　MT2和HeLa细胞DNA、人和黑猩猩PBMCDNA中存在与GBV C 5′ NCR和 或NS3区同源性较高的核苷酸序列 ,这些序列可能位于dPCR阳性的PBMC染色体上。     

用逆转录—套式聚合酶链反应检测我国不同临床型肝？…

目的 为了研究庚型肝炎病毒（ＨＧＶ）在我国的感染状况。方法 概括已发表的ＨＧＶ的５‘端非编码区（５’－ＵＴＲ区）及螺旋酶区（ＮＳ３区）两段高度保守的基因序列分别设计两套引物，用逆转录－套式聚合酶链式反应检测ＨＧＶＲＮＡ。结果 从北京、秦皇岛、河南等地采集各种肝病患者及职业献血员血清３５４份，ＨＧＶＲＮＡ阳性９７份，阳性率为２２．３％。其中已确定的临床型肝炎／肝病患者２５４例，ＨＧＶＲＮＡ阳性者为５     

Follow-up of four HIV-infected individuals after administration of hepatitis C virus and GBV-C/hepatitis G virus contaminated intravenous immunoglobulin: Evidence for HCV but not for GBV-C/HGV transmission

In 1994, hepatitis C virus (HCV) infection was transmitted to four HIV seropositive patients attending the Department of Angiology, University Clinics, Frankfurt am Main, by the administration of Gammagard®. The patients were suffering from thrombocytopenia and received betweeen 20 and 30 g of the contaminated lot 93F21AB11. GBV-C/HGV RNA could be amplified from the Gammagard® lot 93F21AB11 using 5′NCR and NS5 primer pairs. All the four patients were negative in the GBV-C/HGV RT-PCR prior to therapy and until the end of the follow-up period. GBV-C/HGV IgG antibodies to the putative envelope (E2) were detected using the E2 HGV-env kit (Boehringer-Mannheim, Germany) in Gammagard® lot 93F21AB11 and in one patient before donation of immunoglobulin. Anti-E2 seroconversion was observed in one recipient, the other two patients remained anti-E2 seronegative until the end of the observation period. It is concluded that there is no direct evidence for transmission of GBV-C/HGV by contaminated intravenous immunoglobulin since GBV-C/HGV RNA was not detected in the recipients up to 1 year after administration. J. Med. Virol. 53:25–30, 1997. © 1997 Wiley-Liss, Inc.