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目的自从建立起甲、乙、丙、丁、戊5种肝炎病毒的病原学诊断之后,仍有少部分肝炎患者的病因得不到明确,因此不少学者试图探索是否还有新型肝炎病毒的存在,并进行了大量的流行病学和实验诊断的研究,认为的确存在可经肠道外传播并引起人类肝炎的致病因子.1995年 Simons et al 成功地克隆出2株黄病毒 RNA 序列,称之为 GBV-A,GBV-B.继而从一名西非患者血清中克隆出另一黄病毒 RNA 序列,称之为 GBV-C.1996年初美国另一研究小组从一名慢性输血后肝炎患者血清中克隆出一株黄病毒 RNA 序列,称之为 HGV.进一步的研究后认为 GBV-C,HGV 为同一病毒的不同分离株.动物实验发现,GBV-B 可单独引起动物发病,而 GBV-A 单独感染时未能引起发病.本文以此为依据,对 GBV-B 感染者的临床与免疫病理加以研究,试图探讨 GBV-B 对人体肝脏的致病性及其在人体内的分布状态.方法根据 GBV-B NS5区的核苷酸和氨基酸序列,通过计算机软件对其抗原位点进行了预测,并分析其亲水性、键流动性、电荷状态及抗原表位分布等特性,合成一条30个氨基酸多肽.探讨合成肽最佳包被量之后,成功地建立了间接 ELISA 法,用来检测抗-GBV-B.应用此方法检测各型肝炎及其他高危人群血清标本1286份.同时应用 RT-PCR 检测血清中 GBV-B RNA及免疫组化检测肝炎患者肝组织相关病毒抗原.结果经鉴定合成肽纯度在95%以上,氨基酸分析的实际值与理论值基本一致.将合成肽与牛血清清蛋白(BSA)偶联免疫白兔获得高效价抗-GBV-B NS5区的多克隆抗体(PcAb).批内、批间变异系数分别为6.06%和7.65%(均<10%);中和抑制试验结果证明我们建立的 ELISA 法具有较好的特异性.血清抗-GBV-B 检测结果表明各型肝炎患者血清抗-GBV-B 阳性率为10.56%;其中非甲～非戊型肝炎患者血清抗体阳性率为8.57%;肝炎高危人群如献血员、血液透析者及静脉药瘾者血清抗-GBV-B 阳性率分别为2.90%,8.62%及13.44%.随机抽取抗-GBV-B 阳性和抗-GBV-B 阴性血清标本各32例进行RT-PCR 检测 GBV-B RNA,结果64例均为阴性.应用抗-GBV-B 多克隆抗体为试剂,对42例肝炎患者肝组织进行免疫组化研究,均未检测出 GBV-B 相关抗原.结论提示 GBV-B 可能不是人类肝炎病毒,对人体肝脏的致病性轻微,其抗体在不同人群中出现可能是一种短暂的感染,其意义有待进一步研究.
Objective Since the etiological diagnosis of five hepatitis A, B, C, D and E hepatitis viruses has not been established yet, the etiology of a small number of hepatitis patients remains unclear. Therefore, many scholars are trying to find out whether there is a new type of hepatitis virus , And carried out a large number of epidemiological and experimental diagnosis of the study that does exist there may be transmitted by the extraintestinal tract and cause of human hepatitis risk factors.In 1995 Simons et al successfully cloned two flavivirus RNA sequences, Was GBV-A, GBV-B. Then another flavivirus RNA sequence was cloned from a West African patient serum, which was called GBV-C.1996. Cloned a flavivirus RNA sequence, called HGV.Further study that GBV-C, HGV different isolates of the same virus.Animal experiments found that GBV-B can cause animal disease alone, and GBV-A alone B on the basis of this, the clinical and immunopathology of GBV-B infection were studied in order to explore the pathogenicity of GBV-B to human liver and its distribution in human.Methods According to GBV-B NS5 area The nucleotide and amino acid sequence were predicted by computer software, and its amino acid sequence was predicted and its hydrophilicity, bond mobility, charge status and epitope distribution were analyzed to synthesize a 30 amino acid polypeptide. After the optimal amount of peptide was coated, an indirect ELISA was successfully established for the detection of anti-GBV-B. This method was used to detect 1286 serum samples from various hepatitis and other high-risk groups. -B RNA and immunohistochemistry were used to detect the liver tissue associated virus antigen in patients with hepatitis.Results The purity of the synthesized peptide was above 95%, and the actual value of amino acid analysis was consistent with the theoretical value.Compared with bovine serum albumin (BSA) The polyclonal antibody (PcAb) with high titers of anti-GBV-B NS5 region was obtained from the co-immunized rabbits.The intra-assay and inter-assay coefficients of variation were 6.06% and 7.65% (all <10% The results of ELISA showed that the positive rate of anti-GBV-B in serum was 10.56%. The positive rate of serum anti-GBV-B was 8.57%; hepatitis high-risk groups such as offer The positive rates of serum anti-GBV-B in patients, hemodialysis patients and intravenous drug addicts were 2.90%, 8.62% and 13.44%, respectively. Thirty-two cases of anti-GBV-B positive and anti-GBV- GBV-B RNA was detected by RT-PCR, and the results were negative in 64 cases.Using anti-GBV-B polyclonal antibody as an agent, the immunohistochemistry of liver tissue in 42 patients with hepatitis was not detected, and no GBV-B related antigen .ConclusionsIt is suggested that GBV-B may not be human hepatitis virus, and pathogenic to human liver is minor. Its antibody may appear to be a transient infection in different populations, and its significance needs further study.