庚型肝炎病毒（HGV/GBV-C）是1995年下半年由美国科学家发现的一种与人类肝炎有关的RNA病毒。它是一种呈世界性分布的血源性传播病毒。我国人群中也存在这种病毒的感染。病毒性肝炎是影响我国人群健康的重要因素。因此，开展庚型肝炎的流行病学研究及病原学研究，为进一步的研究和防治提供基本的资料和数据是十分重要的。本研究从病原学、流行病学和HGV的致病性等方面对我国庚型肝炎进行了较系统的研究。

1. 中国人庚型肝炎病毒（HGV）全基因cDNA克隆及序列测定

采用PCR基因游走技术从一例献血员血清标本中克隆测定中国株HGV的全基因序列。中国庚型肝炎病毒cDNA序列全长9128个核苷酸，编码一个长度为2870个氨基酸残基的多聚前体蛋白。5’端有一个369bp的非翻译区，3’端长度为147个核苷酸。基因组中G+C占59.93%(G占31.54%,C占27.39%),A为18.60%,T占22.47%。结构基因编码的膜蛋白有4个糖基化点，E1有一个（NLT），另三个在E2区（NVT、NRT和NGS）。HGV的“核心”区仅有45个氨基酸组成，表明其核心区可能是缺损的。HGV中国株与国外测定的几株核苷酸同源性均大于85%，氨基酸同源性大于94%。与国内测定的另一株HGV（HGU94695）的核苷酸和氨基酸同源性分别为88%和95%。目前世界上已测定的几株全序列间核苷酸同源性也大于80%，氨基酸同源性亦在90%以上，不同地区或国家存在的HGV株基因变异相对较小。

2. 我国部分地区几种人群中HGV的基因变异研究

为了了解我国人群中HGV感染状况及分析不同地理株HGV的基因型分布及基因变异的特征，本研究对我国北方和南方部分地区的某些人群进行了分子流行病学研究。用本实验建立的RT-PCR方法检测血清中的HGV RNA作为检测的指标。分别从10个不同省市采集各类肝炎病人标本和从4个地市采集自然人群标本。结果表明，HGV感染在我国分布广泛，不同地理区带均存在该型病毒感染。在自然人群中HGV RNA阳性率约为1.0%。在我国各型肝炎病人中的HGV RNA阳性率也只有7.8%，这表明HGV不是我国非甲非戊型肝炎的主要致病原。在我国，HGV传播感染的主要途径和来源是输血和献血及使用不合格的血制品。

用分子生物学技术克隆测定南方（广东、南京）和北方（河北、北京和辽宁）5株不同地理株HGV的E1区部分基因序列，分析其变异状况及基因型分布。结果显示，国内HGV不同进理株间的核苷酸同源性在85%以上，氨基酸同源性则在92%以上，基因变异相对较小。与HCV的变异相比，HGV基因变异的重要性相对较小。对一例HGV持续感染者进行回顾性随访研究，克隆和测定了该感染者血清中HGV E2区序列（91年、96年和97年共三次）。结果，91年至97年间核苷酸突变率小于10%，氨基酸变异率则更低。表明HGV的基因保守性大。

3. 庚型肝炎病毒的致病性研究

1．制备抗-HGV NS5蛋白的单克隆抗体：用原核表达系统（pQE-30）表达HGV NS5的部分基因区段进行表达。表达的蛋白抗原分子量14KD，用纯化的抗原包被，用ELISA间接法检测不同类型的血清标本（50份PCR检测HGV RNA阳性的病人血清标本、50份HCV单纯感染的血清标本和50份正常人血清标本）。该抗原与34%（17/50）的PCR检测HGV RNA阳性的病人血清标本发生反应；与3份HCV感染的血清标本和2份正常人血清标本有反应。结果表明表达的抗原与HGV感染者血清抗体具有良好的抗原反应性。用纯化的蛋白抗原作免疫原制备单克降抗体。

　　用上述重组表达的庚型肝炎病毒（HGV）NS-5区蛋白免疫小鼠，用免疫脾细胞与Sp2/0骨髓瘤细胞融合，成功地建立了5株分泌抗-HGV NS5区蛋白单克隆抗体（McAb）的杂交瘤细胞株。用间接ELISA方法检测表明，制备的McAb细胞株培养上清液效价1.28×10^-2-2.56×10^-2，腹水效价为6.4×10^-3–1.28×10^-5。经鉴定，这5株McAb与丙型肝炎病毒（HCV）C区、NS3区、NS5区的重组表达抗原、乙型肝炎表面抗原（HBsAg）和大肠杆菌菌体裂解蛋白抗原不发生交叉反应，而只与表达的HGV NS5区蛋白抗原产生特异性反应。

2．建立检测HGV感染的免疫组化方法：用制备的单克隆抗体建立的免疫组化法检测10例HGV感染者的肝组织标本、15例HCV单独感染者肝组织标本和阴性对照标本。结果，10例HGV感染者肝组织标本中，6例呈阳性反应，而HCV感染者和阴性对照标本均呈阴性反应。结果表明单克隆抗体具有很好的特异性。建立的免疫组化方法为庚型肝炎致病机理的研究及病理特征等方面的研究提供了有效的手段。

3．HGV复制及致病性的研究：对22例HGV RNA阳性的临床病人进行临床特征分析。结果表明，HGV的感染以与其他肝炎病毒合并或重叠感染为主。与HBV、HCV、HEV及HAV等的合并感染占63.6%，单纯感染为36.4%。HGV感染者多以轻症肝炎出现。

　　用HGV NS5区McAb对10例庚肝患者肝组织进行免疫组化染色观察，6例单独HGV感染者肝组织呈特异阳性反应，阳性部位定位于胞浆，且周围有炎症细胞浸润，表明HGV NS5区抗原在肝细胞浆内表达，可能参与了HGV对肝细胞的损伤机制。

　　本次实验结果显示HGV抗原的表达和分布具有如下特点：（1）抗原阳性细胞数量少且分散，仅阳性细胞周围有炎症细胞浸润，提示HGV在肝组织内数量较少，对肝细胞损害较轻，这为研究庚型肝炎临床和病理特征提供了直观的形态学依据。（2）HGV复制部位：阳性反应部位定位于肝细胞胞浆中，呈胞浆均质型，未见明显核着色和膜型着色。（3）分布特点：阳性细胞数量分散，HGV NS5阳性着色颗粒表达于残存的肝细胞胞浆内，阳性细胞也可呈片簇状分布于汇管区周围。相邻肝细胞呈阴性反应。HGV可在肝组织内复制。

　　动物感染实验的免疫组化检测结果及原位杂交结果也表明HGV可以在肝组织内复制表达。由于缺乏肝外组织的材料，仍无法判别HGV的嗜肝性，但可以确定肝组织也是HGV复制表达的部位。

　　综上所述，HGV能在肝组织复制，能导致轻微的肝损伤，它是一部分肝炎的致病原。

　Complete cDNA Sequencing, Gene Variation and Pathogenicity of Hepatitis G Virus in Chinese

1. cDNA cloning and sequencing of HGV in Chinese

The complete genome of a HGV isolate was cloned and sequenced from the serum of a blood donor in Heibei province in China. The sequence of 9128bp contains a continuous open reading frame (ORF) that could encode a viral polyprotein of 2870 amino acids. The 5’ and 3’ untranslated regions are 367bp and 147bp respectively. Compared with reported HGV sequence, the Chinese HGV isolate shoes 85%~90% homology at the nucleotide level and 90%~94% homology at the amino acid level. The length of the “core” region is 45 amino acids which is similar to HGU36380, shorter than that of HGU44402, D87255.

2. Hepatitis G Virus infection and gene variation in various population in China

In order to understand the distribution of HGV infection and gene variation of Chinese HGV isolates, serum samples collected from 10 different provinces were detected by RT-PCR for HGV RNA. The results showed that HGV infection existed both in northern and southern China. The positive rate of HGV RNA was 7.6% (125/1564). The positive rate of HGV RNA was about 7.9% in patients with HCV infection, 6.9% in patients with hepatitis B, 1.0% in health population and only 7.8% in patients with hepatitis Non-A, Non-E. This showed that hepatitis G virus infection is not the major cause of Non-A, Non-E hepatitis in China. The partial gene 9E1) of 5 isolates was cloned and sequenced from different areas of china. The homology among them is about 85% at nucleotide acid level and over 90% at amino acid level. This showed that sequences of HGV isolates from different areas were conserved and no typical genotypes, unlike HCV, which can be divided into 6-7 typical genotypes.

　

3. Preliminary study on pathogenicity of HGV
1. Preparation of the monoclonal antibodies against hepatitis g virus NS5 protein

To analyze the clinical and pathological characteristics of patients with HGV infection and determine the site of HGV replication, 22 patients with hepatitis G virus infection were investigated and 10 out of the 22 patients were examined by liver biopsy. Among the 22 patients, 14 patients (63%) were co-infected with other hepatitis viruses, 8patients were infected by HGV alone. The patients with HGV infection alone have the mild elevated ALT level (average 100-200IU/ml). Lower than that of patients with co-infection of other hepatitis viruses. Ten patients with HGV infection, 15 patients with HCV infection alone and 78 patients with hepatitis Non-A to G were examined by liver biopsy and immunohistochemical method. Among 10 liver biopsy specimens of patients with HGV infection (8 out of them were infected by HGV alone), 6 specimens were positive for HGV NS5Ag, all of liver specimens from patients with HCV infection alone and patients with hepatitis Non-A to G were negative for the HGV NS5Ag immunohistochemically. The stain located mainly in cytoplasm of hepatocytes, and the positive cells distributed diffusely in pseudolobule of liver tissue. The results shows that HGV infection is associated with mild hepatitis, and that the virus can replicate in liver tissue, in situ hybridization results by Dig-labeled HGV gene probe showed strongly positive signals in liver biopsy samples from HGV-infected patients. These data suggested that HGV can replicated in hepatocytes and might be one causative agent of human hepatitis.

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