武汉地区散发性戊型肝炎分子流行病学及ORF3功能的研究
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摘要
戊型肝炎病毒(HEV)是通过粪-口途径传播的非甲非乙型肝炎的一个主要病因，在亚洲、非洲和拉丁美洲的一些发展中国家呈流行或散发流行，近期研究发现戊型肝炎散发发病率呈上升趋势。目前在中国戊型肝炎的发病率大约为10％-20％，其中大部分病例为青少年和成人，也有小部分为儿童和老人。尽管戊型肝炎引起的总体死亡率很低，但孕妇感染HEV后其死亡率可达20％。武汉地区，近年来戊型肝炎散发发病呈上升趋势，而目前临床上主要通过检测抗HEV-IgM和抗HEV-IgG来作为诊断指标。许多HEV抗原，包括合成肽、ORF3重组蛋白和大多数ORF2重组蛋白，对急性期血清有较好的活性，但对恢复期血清的反应性则较弱或结果不易判断。若用于血清流行病学研究会明显低估感染率，在临床诊断中无法判断既往感染与近期感染。虽然用于检测的抗原片断不断更新，检测方法多种多样，目前市场提供的抗HEV-IgM检测试剂盒仍由于灵敏度太低而无法满足临床诊断需要。因此，可靠的抗戊型肝炎病毒IgM以及IgG诊断试剂的研制，建立新的诊断方法是一个急待解决的问题。
来自世界不同地区的戊型肝炎病毒核苷酸序列不完全相同，根据戊型肝炎病毒核苷酸序列的同源性75％以上定义为同一个基因型，可将全球分离的HEV分为8个基因型，至少24个基因亚型。同一个体感染的戊型肝炎病毒核苷酸序列也不完全相同，其差异大约在2-15％，不超出病毒不同的基因型或亚型，称为准种。戊型肝炎病毒的异质性和临床特征对于理解其致病特点、建立特异性的珍断试剂盒以及制备相应的疫苗具有重要意义。
HEV基因序列有3个部分重叠的开放读码框(ORF)，ORF1主要编码与病毒RNA复制有关的非结构蛋白，ORF2为HEV的主要的结构基因编码区，编码衣壳蛋白，ORF3仅有369个核苷酸，与ORF1有1nt重叠，与ORF2有328nt重叠，产物含123个氨基酸，分子量为13.5kD。编码的产物与HEV的特异性免疫反应、病毒组装有关，参与调节MAPK细胞信号传导过程，与肿瘤激活基因101相互作用，ORF3的功能特点类似慢性肝炎的过程，其功能亦不明了。
为此，本课题对在武汉同济医院住院及门诊就诊的戊型肝炎病人进行了流行病学调查及基因分型，建立了用免疫吸附测定法(ELISA)检测病人血清中的抗-HEV IgA的方法，显示其在戊型肝炎诊断中的必要性。同时我们成功地构建了重组pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体，并且在HepG2细胞系上获得了稳定表达，最后我们探讨了他们对HepG2细胞系增殖和凋亡的影响，为进一步研究ORF3功能奠定了基础。
目的
1．研究武汉地区戊型肝炎病毒(HEV)流行病学特点及基因分型，了解我国戊型肝炎病毒异质性分布特点，寻求基因型别与疾病临床转归的关系。
2．建立酶联免疫吸附检测血清抗HEV-IgA的方法，试图提高HEV诊断的敏感性、特异性。
3．研究HEV开放读码框架3(ORF3)基因序列的准种特点，从基因水平寻找ORF3功能的基础。
4．构建表达戊型肝炎病毒(HEV)pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体；获得重组质粒稳定转染的HepG2细胞系，为HEV ORF3功能的研究作前期准备。
5．初步研究pEGFP-ORF3和pXF2RH-ORF3融合蛋白对HepG2细胞的增殖和凋亡作用。
方法
1．收集从2003年8月-2004年8月同济医院就诊的急性戊型肝炎患者916例，应用逆转录-套式聚合酶链反应法(RT-nested-PCR)，扩增HEV开放读码框架2(ORF2)的部分序列120份，选取其中25份进行测序，结果用Clustal X和Treeview软件比较武汉地区HEV序列与4个HEV主要代表株序列。
2．应用北京万泰公司商品化基因重组HEV ORF-2／ORF-3抗原预包被的酶标板，HRP标记羊抗人IgA建立检测血清抗HEV-IgA的方法。酶联免疫试验检测60例HEV RNA阳性戊型肝炎患者不同时间段血清抗HEV-IgA、抗HEV-IgM(捕获法)、抗HEV-IgG水平。
3．在经测序证实HEV血样本中，挑选2份标本，扩增其中HEV开放读码框架3(ORF3)的全部序列，克隆入pMD18-T Vector，其中1例挑选30个克隆进行测序。
4．利用PCR技术从HEV基因组中扩增出ORF3基因片断，EcolⅠ／BamHⅠ双酶切后分别连接到经同样酶切的pEGFP-N2和pXF2RH真核表达载体，转化DH5α菌株感受态细胞，获得阳性重组质粒pEGFP-ORF3和pXF2RH-ORF3。将阳性克隆用脂质体法转染HepG2细胞，G418筛选抗性克隆，在荧光显微镜下观察pEGFP-ORF3增强型绿荧光蛋白表达，SDS-PAGE、Western blot分析鉴定pXF2RH-ORF3融合蛋白的表达。
5．用MTT比色法测定细胞增殖情况，流式细胞仪碘化丙啶染色法检测细胞周期和细胞凋亡情况。
结果
1．25例散发性戊型肝炎病毒，核苷酸同源性在82.61-98.55％，与Ⅰ型(缅甸株)、Ⅱ型(墨西哥株)、Ⅲ型(美国株)、Ⅳ型(中国／台湾株)核苷酸同源性分别为76.52-81.74％、70.43-73.04％、76.52-81.16％和84.35-88.70％。系统进化树显示它们至少可分为三个亚型。
2．60例戊型肝炎患者血清抗HEV-IgA、抗HEV-IgM全部阳性。410例非戊型肝炎病人中，6例抗HEV-IgA阳性，7例抗HEV-IgM，除1例抗HEV-IgA、抗HEV-IgM同时阳性外(隐性感染)，其余均为单阳性，抗HEV-IgG和HEV RNA阴性。动态检测戊型肝炎患者血清HEV RNA、抗HEV-IgA、抗HEV-IgM和抗HEV-IgG，发现急性戊型肝炎血清HEV RNA持续阳性至病后2月(20±11d)，抗HEV-IgA、抗HEV-IgM阳性持续整个观察期，半数阳性率分别持续3个月和4个月，从病后第2个月起，抗HEV-IgA、抗HEV-IgM阳性率在同月相比，差异有显著意义。
3．2例ORF3区全基因组碱基序列同源性为90.43％～93.62％，来源于同一患者ORF3区全基因组不同克隆之间的碱基序列同源性为97.97％～99.71％，与Ⅳ型(中国／台湾株)核苷酸同源性为89.86％～99.71％。ORF3区可检出点突变、缺失突变、短序列的插入突变。
4．真核表达载体转染HepG2细胞，经持续G418压力筛选和有限稀释法克隆化获得稳定转染的细胞系，细胞内表达增强型绿荧光蛋白，SDS-PAGE在28.5kD左右蛋白表达量明显高于对照组，Western blot在28.5kD左右有一条强的棕色条带，说明pEGFP-ORF3和pXF2RH-ORF3在HepG2细胞中得到表达。
5．pEGFP-ORF3和pXF2RH-ORF3转染组的HepG2细胞的增殖速度比未转染组和空载体转染组细胞轻度升高，差异无统计学意义(P＞0.05)。转染的肿瘤细胞株HepG2的细胞周期分布是G1=68.09％和67.1％，S=19.50％和19.0％，G2=12.41％和13.9％，凋亡率为0.83％和0.86％；未转染HepG2细胞周期分布是G1=72.15％，S=14.91％，G2=12.95％，凋亡率为1.41％，差异无统计学意义。流式细胞仪检测细胞凋亡峰不明显。
结论
1．武汉地区散发性戊型肝炎患者以HEVⅣ型感染为主。散发性戊型肝炎发病率呈上升趋势，发病年龄以30岁至59岁为主，男∶女之比约为3.3∶1，全年均可发病，3-6月为高峰季节。
2．成功建立检测血清抗HEV-IgA的方法，戊型肝炎患者抗HEV-IgA阳性率较抗HEV-IgM阳性率特异性强、持续时间长。联合HEV-IgM和抗HEV-IgA检测，可提高急性戊型肝炎诊断的敏感性。
3．多个克隆测序结果提示患者体内HEV呈现准种群共存，HEV-ORF3 C末端2个富含脯氨酸区多有点突变，点突变不影响脯氨酸表达。
4．在HepG2细胞中高表达pEGFP-ORF3和pXF2RH-ORF3融合蛋白，可获得大量融合蛋白。
5．转染pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体对HepG2细胞增殖速度与细胞凋亡无明显影响。
本研究的创新点
1．首次研究武汉地区戊型肝炎病毒(HEV)流行病学特点、基因分型和HEV开放读码框架3(ORF3)基因序列的准种特点。
2．初步建立了酶联免疫吸附检测血清抗HEV-IgA的方法。
3．首次在HepG2细胞表达HEV开放读码框架3(ORF3)基因编码融合蛋白，该蛋白没有引起HepG2细胞明显的凋亡，是否对细胞有增殖作用有待进一步研究。
研究意义
1．HEV病毒基因分型的研究可以加强对病毒起源及进化的认识；有助于分析戊型肝炎在全球范围内的地理分布特征，更好地了解HEV变异性和流行病学特征；了解其基因异质性与抗原异质性之间的关系，可以考虑建立不同基因型HEV感染的检测方法，提高检测特异性，进而避免因抗体特异性减弱或降低而导致的误诊。总之对病毒的临床治疗和预防都有着重要的意义。
2．戊型肝炎病毒(HEV)感染的诊断主要依赖于可靠的检测方法，目前临床常检测抗-HEV IgG及IgM。抗-HEV IgG出现早，检出率高，但持续时间长，发病后6～12个月大部分患者抗-HEV IgG仍然阳性，因此无法区分是急性戊型肝炎还是既往HEV感染。抗-HEV IgM出现早，发病三个月以后基本转阴，是急性戊型肝炎的特异指标，但检测的灵敏度不高(60％～70％)。因而在戊型肝炎诊断中有一定的局限性，抗HEV-IgA可作为戊型肝炎近期感染的辅助抗体，同时检测抗-HEV IgA，可提高诊断率和检测敏感性，还协助假阳性的判断，有利于正确诊断。
3．戊型肝炎病毒感染，临床症状显著者多是15-30岁年轻人，死亡率0.5-3.0％，孕妇死亡率高达20％。其发病机制可能有病毒直接损伤肝细胞和免疫损伤参与，详细机制不明。ORF3是胞外信号调节激酶(ERK)和应激活化蛋白激酶／C-Jun N端激酶等MAPK超家族激酶的底物，可能参与细胞的生长、凋亡过程。ORF3功能的研究有利于进一步了解HEV致病机制及确定治疗方案。
Hepatitis E virus (HEV) is one of major causes of epidemically and sporadically, enterically transmitted non-A, non-B hepatitis in many developing countries in Asia, Africa and Latin America. The cases of HEV have been gradually increasing recently. Now the epidemiology of HEV infection is about 10%-20% in China in which most common infections have been found in adolescents and young adults, but it also occurs to a lesser extent in children and elders. Although the overall mortality rate associated with HEV infection is very low, it is reported that the mortality rate is up to 20% in infected pregnant women. In the past few years there were a lot of updated detection against antigen fragments of HEV, but the commercial kit marketed have a poor sensitivity. The sporadical cases of HEV infection were increasing in the past few years in Wuhan, But nowadays only the immuneglobulin M (IgM) against HEV (anti-HEV IgM) and immuneglobulin G (IgG) against HEV (anti-HEV IgG) have mainly been used as diagnostic markers of HEV infection clinically. Many HEV antigens, including synthetic peptides, recombination proteins of ORF3 and many recombination proteins of ORF2, have a better activity on acute sera, but as to chronic sera, it is just another case, or it is very hard to describe the diversity. If those reagents were used as epidemiologic study, the infection rate of HEV would be underestimated obviously, but as diagnostic criterias in clinic it could not tell the difference between the past infection and the current one so as not to satisfy with the need of diagnosis correctly. Therefore, it is an very important question to be solved to find a reliable diagnostic reagent kit on HEV infection including anti-HEV IgM and anti-HEV IgG..
There are many different nucleotide sequences of HEV according to the different places in the world. The recent reports of many different derived HEV strains provide a basis for identifying as the same genotypes in HEV with which around 75% nucleotide similarities between two genotypes. according to this criterion, Eight different genotypes have been found in the world, with which at least six subgenotypes had been identifacted. But the HEV strains in the same case are not always just the same one. There are a little difference among those strains with which the difference is about 2-5% of HEV full-length nucleotide sequence. It is termed viral quasispecies. The heterogenicity and Clinic feature of the hepatitis E virus (HEV) are very important to understand its pathopoiesis, to establish specific diagnostic assays and producing vaccine on HEV.
The HEV genome contains three open reading frames (ORFs). The ORF1 (about 5kb) is predicted to encode the viral nonstructural polyprotein, the ORF2 (about 2kb) encodes the viral major capsid protein, and the ORF3 encodes a protein of ～13.5kDa, a small protein, which is associated with the cytoskeletal and membrane fractions in expressing cells. Hepatitis-E was regarded a self-limited disease without chronic process for many years. But current research has indicated that there is a cis-reactive element in HEV ORF3. It is found ORF3 enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101. There is a long way to go before we understand the function of HEV ORF3.
Therefore, in our study, we first investigated the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan. Then, we established a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and reveal its change and significance of anti-HEV IgA in the sera of patients with hepatitis E. Meantime, we constructed a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtained a stable transfected HepG2 cell line. At last, we have investigated their effects of on HepG2.
Objective
1. To investigate the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan and understand the distribution feature of heterogenicity of HEV, diversify of HEV and the relation between genetypes and clinic outcome of the disease.
2. To establish a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and therefore increase the diagnostic specificity and sensitivity of HEV infection. 3. To study the quasispecies groups of hepatitis E virus ORF3 in the patients with hepatitis E infection in Wuhan and find the basis of the function of pORF3 on gene level.
4. To construct a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtain a stable transfected HepG2 cell line and supple basis to study the function of ORF3.
5. To investigate the effect of hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein on proliferation and apoptosis of HepG2 cell line.
Methods
1. Clinical data were elicited from patients' hospital records that are enrolled 916 cases clinically diagnosed as acute hepatitis during the period of August 2003 to August 2004, in which 120 patients were anti-HEV-IgM, IgG positive and of them the conserved genomic sequences of open reading frame 2 (345bp) in the HEV were detected using polymerase chain reaction, 25 of which have been cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysising of genotype Ⅰ, Ⅱ, Ⅲ, Ⅳ in HEV strains of Wuhan.
2. In this experiment, instead of Anti-HEV IgG detected in the later phase of HEV infection, a new method was established to assay Anti-HEV IgA, which can be detected in the different phase of the infection. And we compared Anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG methods in serial sera of 60 patients with HEV-RNA positive.
3. We choosed two samples from the sera that were confirmed positive by HEV sequenced. The conserved genomic sequences of open reading frame 2 (346 bp) and the whole ORF3 region in the HEV were detected using RT-nested-polymerase chain reaction. Which was cloned into pMD18-T Vector, and in one sample, 30 clones were sequenced.
4. The coding region of ORF3 gene of HEV was amplified by PCR and was digested by Ecol Ⅰ/BamH Ⅰ. This fragment was inserted into eukaryotic expression vector both pEGFP-N2 and pXF2RH with T4 ligase and transformed E. coli DH5α . The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectamine~(TM) 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The expression of fusion gene was analyzed by fluorescent microscopy, SDS-PAGE and Western blot, respectively.
5. MTT colorimetric assay was used to detect the proliferation activity of HepG2. Flow cytometry was used to observe the cell apoptosis of HepG2.
Results
1. 25 isolates shared the same genotype IV, with the similarity of nucleotides of 82.61-98.55%. Compared with other genotypes in HEV, They had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology at the nucleotide sequences in the HEV genotypes I -IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented 3 different subtypes at least but there was no significant difference found in epidemiology feature and liver function among the three subtypes.
2. The 60 patients with HEV-RNA positive had both anti-HEV IgA and anti-HEV IgM and 410 patients with HEV-RNA negative were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNAs were detectable in the serum until 20±lld. We used anti-HEV IgM and anti-HEV IgA assays to detect the HEV infection and found that positive results was during 90±15d and 120±23d separately, which demonstrates that the positive rates of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA (P<0.05).
3. The homology among the clones from one patient of ORF3 region was 97.97%～99.71%, while there was 89.86% ~ 99.71% at the nucleotide sequences compared with HEV genotype IV. The substitution, deletion and insertion of short sequence were found in 3 open reading frames.
4. Recombinant vector pEGFP-ORF3 and pXF2RH-ORF3 were successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected HepG2 cell membrane selected by G418 was confirmed by fluorescent microscopy, and pXF2RH-ORF3 approximately 28.5 kDa was conformed by SDS-PAGE and Western blot. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) the increase of growth inhibitory rate and apoptosis rate in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were not significant (P>0.05).
5. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) cell, proliferative capacity in HepG2/pEGFF-ORF3 (or pXF2RH-ORF3) was slightly increased (P>0.05). The Cell cycle in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were Gl= 68.09% and 67.1%, S=19.50% and 19.0%, G2=12.41% and 13.9%, apoptosis rates were 0.83% and 0.86%. The Cell cycle in HepG2 was G1=72.15%, S=14.91%, G2=12.95%, apoptosis rates 1.41%. The changes had not significant.
Conclusions
1. HEV sequences isolated from patients of Wuhan belong to HEV genotype IV with different subtypes. The infection rate of HEV is gradually increased in Wuhan, in which that of male patients are 3.3 times higher than female patients found in clinical investigation. Age from 30 years to 59 years and seasonality from March to June are susceptible factor for patients to hepatitis E infection.
2. The duration of anti-HEV IgA in the serum is longer than that of anti-HEV IgM and anti-HEV IgA assay is a good method to detect HEV infection. It is helpful to diagnose HEV after anti-HEV IgM had disappeared in the serum that demonstrated that Anti-HEV IgA with or without anti-HEV IgM is useful for serological diagnosis of hepatitis E.
3. There were HEV quasispecies groups in one patient with HEV infection. The substitution in two C-terminal proline-rich regions of HEV-ORF3 could not influence the translation of the proline and the substitutions in amino-acid sequence of viral protein are worthy of further study.
4. The stable transfected HepG2 cell line could express pEGFP-ORF3 and pXF2RH-ORF3 fusion protein.
5. Compared with HepG2/pEGFP-N2 (or pXF2RH), We found that there was no significance both in growth inhibitory rate and apoptosis rate in HepG2/ pEGFP-ORF3 (or pXF2RH-ORF3) (P>0.05).
来自世界不同地区的戊型肝炎病毒核苷酸序列不完全相同，根据戊型肝炎病毒核苷酸序列的同源性75％以上定义为同一个基因型，可将全球分离的HEV分为8个基因型，至少24个基因亚型。同一个体感染的戊型肝炎病毒核苷酸序列也不完全相同，其差异大约在2-15％，不超出病毒不同的基因型或亚型，称为准种。戊型肝炎病毒的异质性和临床特征对于理解其致病特点、建立特异性的珍断试剂盒以及制备相应的疫苗具有重要意义。
HEV基因序列有3个部分重叠的开放读码框(ORF)，ORF1主要编码与病毒RNA复制有关的非结构蛋白，ORF2为HEV的主要的结构基因编码区，编码衣壳蛋白，ORF3仅有369个核苷酸，与ORF1有1nt重叠，与ORF2有328nt重叠，产物含123个氨基酸，分子量为13.5kD。编码的产物与HEV的特异性免疫反应、病毒组装有关，参与调节MAPK细胞信号传导过程，与肿瘤激活基因101相互作用，ORF3的功能特点类似慢性肝炎的过程，其功能亦不明了。
为此，本课题对在武汉同济医院住院及门诊就诊的戊型肝炎病人进行了流行病学调查及基因分型，建立了用免疫吸附测定法(ELISA)检测病人血清中的抗-HEV IgA的方法，显示其在戊型肝炎诊断中的必要性。同时我们成功地构建了重组pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体，并且在HepG2细胞系上获得了稳定表达，最后我们探讨了他们对HepG2细胞系增殖和凋亡的影响，为进一步研究ORF3功能奠定了基础。
目的
1．研究武汉地区戊型肝炎病毒(HEV)流行病学特点及基因分型，了解我国戊型肝炎病毒异质性分布特点，寻求基因型别与疾病临床转归的关系。
2．建立酶联免疫吸附检测血清抗HEV-IgA的方法，试图提高HEV诊断的敏感性、特异性。
3．研究HEV开放读码框架3(ORF3)基因序列的准种特点，从基因水平寻找ORF3功能的基础。
4．构建表达戊型肝炎病毒(HEV)pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体；获得重组质粒稳定转染的HepG2细胞系，为HEV ORF3功能的研究作前期准备。
5．初步研究pEGFP-ORF3和pXF2RH-ORF3融合蛋白对HepG2细胞的增殖和凋亡作用。
方法
1．收集从2003年8月-2004年8月同济医院就诊的急性戊型肝炎患者916例，应用逆转录-套式聚合酶链反应法(RT-nested-PCR)，扩增HEV开放读码框架2(ORF2)的部分序列120份，选取其中25份进行测序，结果用Clustal X和Treeview软件比较武汉地区HEV序列与4个HEV主要代表株序列。
2．应用北京万泰公司商品化基因重组HEV ORF-2／ORF-3抗原预包被的酶标板，HRP标记羊抗人IgA建立检测血清抗HEV-IgA的方法。酶联免疫试验检测60例HEV RNA阳性戊型肝炎患者不同时间段血清抗HEV-IgA、抗HEV-IgM(捕获法)、抗HEV-IgG水平。
3．在经测序证实HEV血样本中，挑选2份标本，扩增其中HEV开放读码框架3(ORF3)的全部序列，克隆入pMD18-T Vector，其中1例挑选30个克隆进行测序。
4．利用PCR技术从HEV基因组中扩增出ORF3基因片断，EcolⅠ／BamHⅠ双酶切后分别连接到经同样酶切的pEGFP-N2和pXF2RH真核表达载体，转化DH5α菌株感受态细胞，获得阳性重组质粒pEGFP-ORF3和pXF2RH-ORF3。将阳性克隆用脂质体法转染HepG2细胞，G418筛选抗性克隆，在荧光显微镜下观察pEGFP-ORF3增强型绿荧光蛋白表达，SDS-PAGE、Western blot分析鉴定pXF2RH-ORF3融合蛋白的表达。
5．用MTT比色法测定细胞增殖情况，流式细胞仪碘化丙啶染色法检测细胞周期和细胞凋亡情况。
结果
1．25例散发性戊型肝炎病毒，核苷酸同源性在82.61-98.55％，与Ⅰ型(缅甸株)、Ⅱ型(墨西哥株)、Ⅲ型(美国株)、Ⅳ型(中国／台湾株)核苷酸同源性分别为76.52-81.74％、70.43-73.04％、76.52-81.16％和84.35-88.70％。系统进化树显示它们至少可分为三个亚型。
2．60例戊型肝炎患者血清抗HEV-IgA、抗HEV-IgM全部阳性。410例非戊型肝炎病人中，6例抗HEV-IgA阳性，7例抗HEV-IgM，除1例抗HEV-IgA、抗HEV-IgM同时阳性外(隐性感染)，其余均为单阳性，抗HEV-IgG和HEV RNA阴性。动态检测戊型肝炎患者血清HEV RNA、抗HEV-IgA、抗HEV-IgM和抗HEV-IgG，发现急性戊型肝炎血清HEV RNA持续阳性至病后2月(20±11d)，抗HEV-IgA、抗HEV-IgM阳性持续整个观察期，半数阳性率分别持续3个月和4个月，从病后第2个月起，抗HEV-IgA、抗HEV-IgM阳性率在同月相比，差异有显著意义。
3．2例ORF3区全基因组碱基序列同源性为90.43％～93.62％，来源于同一患者ORF3区全基因组不同克隆之间的碱基序列同源性为97.97％～99.71％，与Ⅳ型(中国／台湾株)核苷酸同源性为89.86％～99.71％。ORF3区可检出点突变、缺失突变、短序列的插入突变。
4．真核表达载体转染HepG2细胞，经持续G418压力筛选和有限稀释法克隆化获得稳定转染的细胞系，细胞内表达增强型绿荧光蛋白，SDS-PAGE在28.5kD左右蛋白表达量明显高于对照组，Western blot在28.5kD左右有一条强的棕色条带，说明pEGFP-ORF3和pXF2RH-ORF3在HepG2细胞中得到表达。
5．pEGFP-ORF3和pXF2RH-ORF3转染组的HepG2细胞的增殖速度比未转染组和空载体转染组细胞轻度升高，差异无统计学意义(P＞0.05)。转染的肿瘤细胞株HepG2的细胞周期分布是G1=68.09％和67.1％，S=19.50％和19.0％，G2=12.41％和13.9％，凋亡率为0.83％和0.86％；未转染HepG2细胞周期分布是G1=72.15％，S=14.91％，G2=12.95％，凋亡率为1.41％，差异无统计学意义。流式细胞仪检测细胞凋亡峰不明显。
结论
1．武汉地区散发性戊型肝炎患者以HEVⅣ型感染为主。散发性戊型肝炎发病率呈上升趋势，发病年龄以30岁至59岁为主，男∶女之比约为3.3∶1，全年均可发病，3-6月为高峰季节。
2．成功建立检测血清抗HEV-IgA的方法，戊型肝炎患者抗HEV-IgA阳性率较抗HEV-IgM阳性率特异性强、持续时间长。联合HEV-IgM和抗HEV-IgA检测，可提高急性戊型肝炎诊断的敏感性。
3．多个克隆测序结果提示患者体内HEV呈现准种群共存，HEV-ORF3 C末端2个富含脯氨酸区多有点突变，点突变不影响脯氨酸表达。
4．在HepG2细胞中高表达pEGFP-ORF3和pXF2RH-ORF3融合蛋白，可获得大量融合蛋白。
5．转染pEGFP-ORF3和pXF2RH-ORF3融合蛋白的真核表达载体对HepG2细胞增殖速度与细胞凋亡无明显影响。
本研究的创新点
1．首次研究武汉地区戊型肝炎病毒(HEV)流行病学特点、基因分型和HEV开放读码框架3(ORF3)基因序列的准种特点。
2．初步建立了酶联免疫吸附检测血清抗HEV-IgA的方法。
3．首次在HepG2细胞表达HEV开放读码框架3(ORF3)基因编码融合蛋白，该蛋白没有引起HepG2细胞明显的凋亡，是否对细胞有增殖作用有待进一步研究。
研究意义
1．HEV病毒基因分型的研究可以加强对病毒起源及进化的认识；有助于分析戊型肝炎在全球范围内的地理分布特征，更好地了解HEV变异性和流行病学特征；了解其基因异质性与抗原异质性之间的关系，可以考虑建立不同基因型HEV感染的检测方法，提高检测特异性，进而避免因抗体特异性减弱或降低而导致的误诊。总之对病毒的临床治疗和预防都有着重要的意义。
2．戊型肝炎病毒(HEV)感染的诊断主要依赖于可靠的检测方法，目前临床常检测抗-HEV IgG及IgM。抗-HEV IgG出现早，检出率高，但持续时间长，发病后6～12个月大部分患者抗-HEV IgG仍然阳性，因此无法区分是急性戊型肝炎还是既往HEV感染。抗-HEV IgM出现早，发病三个月以后基本转阴，是急性戊型肝炎的特异指标，但检测的灵敏度不高(60％～70％)。因而在戊型肝炎诊断中有一定的局限性，抗HEV-IgA可作为戊型肝炎近期感染的辅助抗体，同时检测抗-HEV IgA，可提高诊断率和检测敏感性，还协助假阳性的判断，有利于正确诊断。
3．戊型肝炎病毒感染，临床症状显著者多是15-30岁年轻人，死亡率0.5-3.0％，孕妇死亡率高达20％。其发病机制可能有病毒直接损伤肝细胞和免疫损伤参与，详细机制不明。ORF3是胞外信号调节激酶(ERK)和应激活化蛋白激酶／C-Jun N端激酶等MAPK超家族激酶的底物，可能参与细胞的生长、凋亡过程。ORF3功能的研究有利于进一步了解HEV致病机制及确定治疗方案。
Hepatitis E virus (HEV) is one of major causes of epidemically and sporadically, enterically transmitted non-A, non-B hepatitis in many developing countries in Asia, Africa and Latin America. The cases of HEV have been gradually increasing recently. Now the epidemiology of HEV infection is about 10%-20% in China in which most common infections have been found in adolescents and young adults, but it also occurs to a lesser extent in children and elders. Although the overall mortality rate associated with HEV infection is very low, it is reported that the mortality rate is up to 20% in infected pregnant women. In the past few years there were a lot of updated detection against antigen fragments of HEV, but the commercial kit marketed have a poor sensitivity. The sporadical cases of HEV infection were increasing in the past few years in Wuhan, But nowadays only the immuneglobulin M (IgM) against HEV (anti-HEV IgM) and immuneglobulin G (IgG) against HEV (anti-HEV IgG) have mainly been used as diagnostic markers of HEV infection clinically. Many HEV antigens, including synthetic peptides, recombination proteins of ORF3 and many recombination proteins of ORF2, have a better activity on acute sera, but as to chronic sera, it is just another case, or it is very hard to describe the diversity. If those reagents were used as epidemiologic study, the infection rate of HEV would be underestimated obviously, but as diagnostic criterias in clinic it could not tell the difference between the past infection and the current one so as not to satisfy with the need of diagnosis correctly. Therefore, it is an very important question to be solved to find a reliable diagnostic reagent kit on HEV infection including anti-HEV IgM and anti-HEV IgG..
There are many different nucleotide sequences of HEV according to the different places in the world. The recent reports of many different derived HEV strains provide a basis for identifying as the same genotypes in HEV with which around 75% nucleotide similarities between two genotypes. according to this criterion, Eight different genotypes have been found in the world, with which at least six subgenotypes had been identifacted. But the HEV strains in the same case are not always just the same one. There are a little difference among those strains with which the difference is about 2-5% of HEV full-length nucleotide sequence. It is termed viral quasispecies. The heterogenicity and Clinic feature of the hepatitis E virus (HEV) are very important to understand its pathopoiesis, to establish specific diagnostic assays and producing vaccine on HEV.
The HEV genome contains three open reading frames (ORFs). The ORF1 (about 5kb) is predicted to encode the viral nonstructural polyprotein, the ORF2 (about 2kb) encodes the viral major capsid protein, and the ORF3 encodes a protein of ～13.5kDa, a small protein, which is associated with the cytoskeletal and membrane fractions in expressing cells. Hepatitis-E was regarded a self-limited disease without chronic process for many years. But current research has indicated that there is a cis-reactive element in HEV ORF3. It is found ORF3 enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101. There is a long way to go before we understand the function of HEV ORF3.
Therefore, in our study, we first investigated the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan. Then, we established a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and reveal its change and significance of anti-HEV IgA in the sera of patients with hepatitis E. Meantime, we constructed a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtained a stable transfected HepG2 cell line. At last, we have investigated their effects of on HepG2.
Objective
1. To investigate the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan and understand the distribution feature of heterogenicity of HEV, diversify of HEV and the relation between genetypes and clinic outcome of the disease.
2. To establish a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and therefore increase the diagnostic specificity and sensitivity of HEV infection. 3. To study the quasispecies groups of hepatitis E virus ORF3 in the patients with hepatitis E infection in Wuhan and find the basis of the function of pORF3 on gene level.
4. To construct a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtain a stable transfected HepG2 cell line and supple basis to study the function of ORF3.
5. To investigate the effect of hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein on proliferation and apoptosis of HepG2 cell line.
Methods
1. Clinical data were elicited from patients' hospital records that are enrolled 916 cases clinically diagnosed as acute hepatitis during the period of August 2003 to August 2004, in which 120 patients were anti-HEV-IgM, IgG positive and of them the conserved genomic sequences of open reading frame 2 (345bp) in the HEV were detected using polymerase chain reaction, 25 of which have been cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysising of genotype Ⅰ, Ⅱ, Ⅲ, Ⅳ in HEV strains of Wuhan.
2. In this experiment, instead of Anti-HEV IgG detected in the later phase of HEV infection, a new method was established to assay Anti-HEV IgA, which can be detected in the different phase of the infection. And we compared Anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG methods in serial sera of 60 patients with HEV-RNA positive.
3. We choosed two samples from the sera that were confirmed positive by HEV sequenced. The conserved genomic sequences of open reading frame 2 (346 bp) and the whole ORF3 region in the HEV were detected using RT-nested-polymerase chain reaction. Which was cloned into pMD18-T Vector, and in one sample, 30 clones were sequenced.
4. The coding region of ORF3 gene of HEV was amplified by PCR and was digested by Ecol Ⅰ/BamH Ⅰ. This fragment was inserted into eukaryotic expression vector both pEGFP-N2 and pXF2RH with T4 ligase and transformed E. coli DH5α . The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectamine~(TM) 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The expression of fusion gene was analyzed by fluorescent microscopy, SDS-PAGE and Western blot, respectively.
5. MTT colorimetric assay was used to detect the proliferation activity of HepG2. Flow cytometry was used to observe the cell apoptosis of HepG2.
Results
1. 25 isolates shared the same genotype IV, with the similarity of nucleotides of 82.61-98.55%. Compared with other genotypes in HEV, They had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology at the nucleotide sequences in the HEV genotypes I -IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented 3 different subtypes at least but there was no significant difference found in epidemiology feature and liver function among the three subtypes.
2. The 60 patients with HEV-RNA positive had both anti-HEV IgA and anti-HEV IgM and 410 patients with HEV-RNA negative were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNAs were detectable in the serum until 20±lld. We used anti-HEV IgM and anti-HEV IgA assays to detect the HEV infection and found that positive results was during 90±15d and 120±23d separately, which demonstrates that the positive rates of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA (P<0.05).
3. The homology among the clones from one patient of ORF3 region was 97.97%～99.71%, while there was 89.86% ~ 99.71% at the nucleotide sequences compared with HEV genotype IV. The substitution, deletion and insertion of short sequence were found in 3 open reading frames.
4. Recombinant vector pEGFP-ORF3 and pXF2RH-ORF3 were successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected HepG2 cell membrane selected by G418 was confirmed by fluorescent microscopy, and pXF2RH-ORF3 approximately 28.5 kDa was conformed by SDS-PAGE and Western blot. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) the increase of growth inhibitory rate and apoptosis rate in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were not significant (P>0.05).
5. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) cell, proliferative capacity in HepG2/pEGFF-ORF3 (or pXF2RH-ORF3) was slightly increased (P>0.05). The Cell cycle in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were Gl= 68.09% and 67.1%, S=19.50% and 19.0%, G2=12.41% and 13.9%, apoptosis rates were 0.83% and 0.86%. The Cell cycle in HepG2 was G1=72.15%, S=14.91%, G2=12.95%, apoptosis rates 1.41%. The changes had not significant.
Conclusions
1. HEV sequences isolated from patients of Wuhan belong to HEV genotype IV with different subtypes. The infection rate of HEV is gradually increased in Wuhan, in which that of male patients are 3.3 times higher than female patients found in clinical investigation. Age from 30 years to 59 years and seasonality from March to June are susceptible factor for patients to hepatitis E infection.
2. The duration of anti-HEV IgA in the serum is longer than that of anti-HEV IgM and anti-HEV IgA assay is a good method to detect HEV infection. It is helpful to diagnose HEV after anti-HEV IgM had disappeared in the serum that demonstrated that Anti-HEV IgA with or without anti-HEV IgM is useful for serological diagnosis of hepatitis E.
3. There were HEV quasispecies groups in one patient with HEV infection. The substitution in two C-terminal proline-rich regions of HEV-ORF3 could not influence the translation of the proline and the substitutions in amino-acid sequence of viral protein are worthy of further study.
4. The stable transfected HepG2 cell line could express pEGFP-ORF3 and pXF2RH-ORF3 fusion protein.
5. Compared with HepG2/pEGFP-N2 (or pXF2RH), We found that there was no significance both in growth inhibitory rate and apoptosis rate in HepG2/ pEGFP-ORF3 (or pXF2RH-ORF3) (P>0.05).
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